Describes self with respect to goals that morph and thus change interpretation of one's self, rather than a static sense of an origin story.

Learn more about Ultima Thule here--the farthest object to be explored by the New Horizons spacecraft.

L sound matches with "Leave" and "b(l)ack" "p(l)ume" and "lie"

L sound matches with "Leave" and "b(l)ack" "p(l)ume" and "sou(l)"

L sound matches with "Leave" and "b(l)ack" "lie" and "sou(l)"

L sound matches with "b(l)ack" and "p(l)ume" "lie" and "sou(l)"

(L)中若想表示处理4中数据流A与处理6中数据流B经处理5的到数据流C

Den ganzen Absatz lang Angst machen und dann am Ende zugeben, dass alles Stuss ist.

Doch wissenschaftliche Studien fehlen bislang, die diesen Zusammenhang zweifelsfrei bestätigen.

Euthemistischer geht es kaum. Da hier keine Quellen angegeben sind bin ich selber man auf die Suche gegangen. Es gibt keine Beweise. Es gibt keine wissenschaftliche Grundlage. Die Aussage

Wir denken das, sind uns aber nicht sicher. stimmt schlicht nicht.

Hier einige Studien:

Hypothese: Milchkonsum erniedrigt das Brustkrebsrisiko: van't Veer, P., Dekker, J. M., Lamers, J. W., Kok, F. J., Schouten, E. G., Brants, H. A., ... & Hermus, R. J. (1989). Consumption of fermented milk products and breast cancer: a case-control study in The Netherlands. Cancer research, 49(14), 4020-4023

Biffi, A., Coradini, D., Larsen, R., Riva, L., & Di Fronzo, G. (1997). Antiproliferative effect of fermented milk on the growth of a human breast cancer cell line.

Mai, X. M., Becker, A. B., Sellers, E. A. C., Liem, J. J., & Kozyrskyj, A. L. (2007). Infrequent milk consumption plus being overweight may have great risk for asthma in girls. Allergy, 62(11), 1295-1301.

Fussman, C., Todem, D., Forster, J., Arshad, H., Urbanek, R., & Karmaus, W. (2007). Cow's milk exposure and asthma in a newborn cohort: repeated ascertainment indicates reverse causation. Journal of Asthma, 44(2), 99-105.

Tatsächlich sind die Studien, die ich gefunden habe, eher der Meinung Milchkonsum reduziere das Risiko auf Brustkrebs und Asthma (bei übergewichtigen Mädchen). Natürlich sind auch diese Studien nicht als Beweis, sondern mehr als ein Hinweis oder Puzzelstück zu behandeln.

Good news first. You have an absolutely fantastic argument here. Grapes of Wrath is not a Western story, It is an American story. The problem here is that you don't prove that argument at all. I know it sounds like a lot of work to totally reshape your argument, but I think the non-Stagecoach parts of this paper could very well be repurposed to that end if you give yourself enough time.

I really enjoyed the poem towards the end because it began to play out more like a story. In the end it was like a wish or a dream of escape. Then she is brought abruptly back to reality. The desperation and burning desire that is present in wanting to experience life pushes through the poem and the end makes it all the more passionate when you are brought back from this fired up drive to this calm and quiet existence.

Curator: @Jmenke

Resource used:

RRID:IMSR_JAX:006373

SciCrunch record: RRID:IMSR_JAX:006373

Alternate resolvers: SciCrunch xml N2T identifiers.org

What is this?

prog no heaven

computer science theology

This paper describes the use of the phylogenetic marker that is used to barcode fungal species. One of the reasons that the yeast symbiotic partner was missed is because this barcode doesn't work for that species.

Genomic data is used to show that the two main species of Bryoria fungi examined in this work are conspecific, that is they are genomically identical to one another.

T-distribution Stochastic Neighbor Embedding(t-SNE)

之前介绍的所有方法都存在相同的弊病：

similar data are close, but different data may collapse，亦即，相似（label）的点靠的确实很近，但不相似(label)的点也有可能靠的很近。

t-SNE 的原理

\(x \rightarrow z\)

t-SNE 一样是降维，从 x 向量降维到 z. 但 t-SNE 有一步很独特的标准化步骤：

一， t-SNE 第一步：similarity normalization

这一步假设我们已经知道 similarity 的公式，关于 similarity 的公式在【第四步】单独讨论，因为实在神妙。

这一步是对任意两个点之间的相似度进行标准化，目的是尽量让所有的相似度的度量都处在 [0,1] 之间。你可以把他看做是对相似度进行标准化，也可以看做是为求解KL散度做准备 --- 求条件概率分布。

compute similarity between all pairs of x: \(S(x^i, x^j)\)

我们这里使用 Similarity(A,B) 来近似 P(A and B), 使用 \(\sum_{A\neq B}S(A,B)\) 来近似 P(B)

\(P(A|B) = \frac{P(A\cap B)}{P(B)} = \frac{P(A\cap B)}{\sum_{all\ I\ \neq B}P(I\cap B)}\)

\(P(x^j|x^i)=\frac{S(x^i, x^j)}{\sum_{k\neq i}S(x^i, x^k)}\)

假设我们已经找到了一个 low dimension z-space。我们也就可以计算转换后样本的相似度，进一步计算 \(z^i\) \(z^j\) 的条件概率。

compute similarity between all pairs of z: \(S'(z^i, z^j)\)

\(P(z^j|z^i)=\frac{S(z^i, z^j)}{\sum_{k\neq i}S(z^i, z^k)}\)

Find a set of z making the two distributions as close as possible:

\(L = \sum_{i}KL(P(\star | x^i)||Q(\star | z^i))\)

二， t-SNE 第二部：find z

我们要找到一组转换后的“样本”， 使得转换前后的两组样本集（通过KL-divergence测量）的分布越接近越好：

衡量两个分布的相似度：使用 KL 散度(也叫 Infomation Gain)。KL 散度越小，表示两个概率分布越接近。

\(L = \sum_{i}KL(P(\star | x^i) || Q(\star | z^i))\)

find zi to minimize the L.

这个应该是很好做的，因为只要我们能找到 similarity 的计算公式，我们就能把 KL divergence 转换成关于 zi 的相关公式，然后使用梯度下降法---GD最小化这个式子即可。

三，t-SNE 的弊端

1. 需要计算所有两两pair的相似度
2. 新点加入，需要重新计算他与所有点之间的相似度
3. 由于步骤2导致的后续所有的条件概率\(P\ and\ Q\) 都需要重新计算

因为 t-SNE 要求我们计算数据集的两两点之间的相似度，所以这是一个非常高计算量的算法。同时新数据点的加入会影响整个算法的过程，他会重新计算一遍整个过程，这个是十分不友好的，所以 t-SNE 一般不用于训练过程，仅仅用在可视化中，即便在可视化中也不会仅仅使用 t-SNE，依旧是因为他的超高计算量。

在用 t-SNE 进行可视化的时候，一般先使用 PCA 把几千维度的数据点降维到几十维度，然后再利用 t-SNE 对几十维度的数据进行降维，比如降到2维之后，再plot到平面上。

四，t-SNE 的 similarity 公式

之前说过如果一种 similarity 公式：计算两点(xi, xj)之间的 2-norm distance（欧氏距离）：

\(S(x^i, x^j)=exp(-||x^i - x^j||_2)\)

一般用在 graph 模型中计算 similarity。好处是他可以保证非常相近的点才会让这个 similarity 公式有值，因为 exponential 会使得该公式的结果随着两点距离变大呈指数级下降。

在 t-SNE 之前有一个算法叫做 SNE 在 z-space 和 x-space 都使用这个相似度公式。

similarity of x-space: \(S(x^i, x^j)=exp(-||x^i - x^j||_2)\) similarity of z-space: \(S'(z^i, z^j)=exp(-||z^i - z^j||_2)\)

t-SNE 神妙的地方就在于他在 z-space 上采用另一个公式作为 similarity 公式, 这个公式是 t-distribution 的一种（t 分布有参数可以调，可以调出很多不同的分布）：

\(S(x^i, x^j)=exp(-||x^i - x^j||_2)\) \(S'(z^i, z^j)=\frac{1}{1+||z^i - z^j||_2}\)

可以通过函数图像来理解为什么需要进行这种修正，以及这种修正为什么能保证x-space原来近的点, 在 z-space 依旧近，原来 x-space 稍远的点，在 z-space 会拉的非常远：

也就是说，原来 x-space 上的点如果存在一些 gap（similarity 较小），这些 gap 就会在映射到 z-space 后被强化，变的更大更大。

2. 綫性相加（combinations），伸展（span）和單位矢量 l 綫性代數的本質 第二章

本节介绍三个相互依存的概念：单位向量，span，线性无关。

基于单位向量和数字的向量的表示

• basis vector \(\hat{i}\)
• basis vector \(\hat{j}\)
• adding together two scaled vectors

是一种新的看待线性代数的观点，非常重要的三个知识点，至此向量的表示可以变成在各个单位向量做放缩然后取和，或者，单位向量的线性组合：

\((-5)\hat{i} + (2)\hat{j}\)

可以表示为：

$$ \begin{vmatrix} -5 \\ 2 \end{vmatrix} $$

what if we choose different basis vectors?

虽然不论使用什么方向的两个单位向量，其线性组合始终可以覆盖全部二维空间，但是我们仍然得到了同一个向量的两个不同的表示：

although \((3.1)\hat{i} + (-2.9)\hat{j} = \(-0.8)\hat{i}+(1.3)\hat{j}\) 但是该向量的实际表示却完全不同：

$$ \begin{vmatrix} -0.8 \\ 1.3 \end{vmatrix} \neq \begin{vmatrix} 3.1 \\ -2.9 \end{vmatrix} $$

所以这里需要给出一种关于线性代数的数字表示法\([3.1, -2.9]\)的一个基本条件：每当使用这种表示法时都必须明确单位向量是什么。

span of vectors

可以想象的是：

• 如果两个单位向量之间存在夹角那么他们的线性组合形成的向量一定可以覆盖整个平面；
• 如果两个单位向量处在同一个方向（相同or相反）那么他们的线性组合形成的向量只能覆盖这条直线
• 如果两个单位向量都是 \(\vec{0}\)，那么他们的线性组合形成的向量都是\(\vec{0}\)

引入概念span

The "span" of \(\vec{v}\) and \(\vec{w}\) is the set of all their linear combinations:

\(a\vec{v} + b\vec{w}\)

let \(a\) and \(b\) vary over all linear numbers.

两个向量的 span 与另一个表述是等价的，仅仅通过加法和乘法两种操作可以产生的所有向量

Vectors VS. Points

【tips】如果仅仅考虑一个向量，经常将向量想象成带箭头线段；如果考虑一堆向量的集合，经常将向量想象成点。

• 那么两个同方向的向量的span就形成一条直线
• 那么两个不同方向的向量的span就形成一个平面
• 那么三个不同方向的向量的span就形成一个体

Redundant and Linearly dependent

任何时候如果你有多个向量，但是去掉其中一个或几个，前者和后者的span没有减少（span is essencially a set --- set of all possible linear combination）

\(span(\vec{v},\vec{w},\vec{u})=span(\vec{v},\vec{w})\)

那么就可以说这个向量与其他向量是 Linear dependent （线性相关）， 或者说这个（可以去掉的）向量可以表示为其他向量的线性组合, 因为这个可以去掉的向量处在其他向量的span中。

\(redundant\ \vec{u} \in span(\vec{v}, \vec{w})\)

或者说，他对扩大span(set of linear combination of vectors)没有作用。

由此衍生出另一个概念：Linearly independent

Linearly independent

\(\vec{u} \neq a\vec{v} + b\vec{w},\ for\ all\ values\ of\ a\ and\ b\)

如果某个单位向量无法通过其他单位向量的任何一种系数的线性组合来得到，那么就说这个向量与其他向量都是线性无关的

basis vector

有了之前的 span linearly dependent 两个概念，下面才能正式定义第三个概念：何为 basis vector ？

The basis of a vector space is a set of linearly independent vectors that span the full space

Unsupervised Learning: Word Embedding

why Word Embedding ?

Word Embedding 是 Diemension Reduction 一个非常好，非常广为人知的应用。

1-of-N Encoding 及其弊端

apple = [1 0 0 0 0]

bag = [0 1 0 0 0]

cat = [0 0 1 0 0]

dog = [0 0 0 1 0]

elephant = [0 0 0 0 1]

这个 N 就是这个世界上所有的单词的数量，或者你可以自己创建 vocabulary, 那么这个 N 就是 vocabulary 的 size.但是这样向量化的方式，太众生平等了，原本有一定关系的单词，比如 cat 和 dog 都是动物，这根本无法从 [0 0 1 0 0] 和 [0 0 0 1 0] 中看出任何端倪。

解决这件事情的一个方法是 Word Class

Word Class 及其弊端

先把所有的单词 cluster 成簇，然后用簇代替 1-of-N encoding 来表示单词。

• cluster 1: dog, cat, bird;
• cluster 2: ran, jumped, walk
• cluster 3: flower, tree, apple

虽然 Word Class 保留了单词的词性信息使得同类单词聚在一起，但是不同词性之间的关系依旧无法表达：名词 + 动词 + 名词/代词, 这种主谓宾的关系就没法用 Word Class 表示。

这个问题可以通过 Word Embedding 来解决

Word Embedding 把每个单词的 1-of-N encoding 向量都 project 到一个低维度空间中（Dimension Reduction），这个低维度空间就叫做 Embedding. 这样每个单词都是低维度空间中的一个点，我们希望：

相同语义的单词，在该维度空间中也相互接近

不但如此，当 Embedding 是二维空间（能够可视化）时，所有的点及其原本单词画在坐标图上之后，很容易就可以看到这个低维度的空间的每个维度（x,y轴）都带有具体的某种含义. 比如，dim-x 可能表示生物，dim-y 可能表示动作。

How to find vector of Embedding space?

为什么 autoencoder 无法做出 Word Embedding?

Word Embedding 本质上是【非监督降维】，我们之前学习的方法最直接的就是使用 autoencoder, 但用在这里很显然是无效的。因为你的输入是 1-of-N encoding 向量，在这种向量表示下每个输入样本都是 independent 的，也就是单个样本之间没有任何关系 --- 毫无内在规律的样本，你怎么可能学出他们之间的关系呢？（ps, 本来无一物，何处惹尘埃。）

你是什么，是由你的圈子（context）决定的

我们的目的是让计算机理解单词的意思，这个完全不可能通过常规语言交流达此目的，所以需要曲径通幽，你只能通过其他方法来让计算机间接理解。最常用的间接的方法就是：understand word by its context.

• 马英九 520 宣誓就职
• 蔡英文 520 宣誓就职

及其肯定不知道马英九和蔡英文到底是什么，但是只要他读了【含有‘马英九’和‘蔡英文’的】大量的文章，知道马英九和蔡英文的前后经常出现类似的文字（similar context）,机器就可以推断出“马英九”和“蔡英文”是某种相关的东西。

How to exploit the context?

• count based
• predition based

count based

这种方法最经典的做法叫做 Glove Vector https://nlp.stanford.edu/projects/glove/

代价函数与 MF 和 LSA 的一模一样，使用 GD 就可以解，目标是找到越是经常共同出现在一篇文章的两个单词(num. of occur)，越是具有相似的word vector(inner-product)。

这种【越是。。。越是。。。】的表达方式，就可以使用前一个“越是”的量作为后一个“越是”的目标去优化。

越是 A，越是 B ===> \(L = \sum(A-B)^2\)

if two words \(w_i\) and \(w_j\) frequently co-occur(occur in the same document), \(V(w_i)\) and \(V(w_j)\) would be close to each other.

• \(V(wi)\) ：word vector of word wi

核心思想类似 MF 和 LSA:

\(V(w_i) \cdot V(w_j) \Longleftrightarrow N_{i,j}\)

\(L = \sum_{i,j}(V(w_i)\cdot V(w_j) - N_{i,j})^2\)

find the word vector \(V(wi)\) and \(V(w)\), which can minimize the distance between inner product of thses two word vector and the number of times \(w_i\) and \(w_j\) occur in the same document.

prediction based 做法

prediction based 获取 word vector 是通过训练一个 单层NN 用 \(w_{i-1}\) 预测 \(w_i\) 单词的出现作为样本的数据和标签(\(x=w_{i-1}, y=w_i\))，选取第一层 Hiden layer 的输入作为 word vector.

【注意】：上面不是刚说过 autoencoder 学不到任何东西么，那是因为 autoencoder 的input 和 output 都是单词 \(w_i\)，亦即（\(x=w_i, y=w_i\)），但是这里 prediction-based NN 用的是下一个单词的 1-of-encoding 作为label.

本质是用 cross-entropy 作为loss-fn训练一个 NN，这个 NN 的输入是某个单词的 1-of-encoding, 输出是 volcabulary-size 维度的（概率）向量--- 表示 volcabulary 中每个单词会被当做下一个单词的概率。

$$ Num.\ of\ volcabulary\ = 10w $$

$$ \begin{vmatrix} 0 \\ 1 \\ 0 \\ 0 \\ 0 \\.\\.\\. \end{vmatrix}^{R^{10w}} \rightarrow NN \rightarrow \begin{vmatrix} 0.01 \\ 0.73 \\ 0.02 \\ 0.04 \\ 0.11 \\.\\.\\. \end{vmatrix}^{R^{10w}} $$

• take out theinput of the neurons in 1st layer
• use it to represent a word \(w\)
• word vector, word embedding feature: \(V(w)\)

训练好这个 prediction-based NN 之后，一个新的词汇进来就可以直接用这个词汇的 1-of-N encoding 乘以第一层隐含层的权重，就可以得到这个单词的 word vector.

$$ x_i = 1-of-N\ encoding\ of\ word\ i $$

$$ W = weight\ matrix\ of\ NN\ between\ input-layer\ and\ 1st-hidden-layer $$

$$ WordVector_{x_i} = W^Tx_i $$

prediction-based 背后原理

【注意】虽然这里的例子不是单词，但意会一下即可。

• 马英九 520 宣誓就职
• 蔡英文 520 宣誓就职

因为 “马英九” 和 “蔡英文” 后面跟的都是 “520宣誓就职”，所以用在 prediction-based NN 中

• 马英九 520 宣誓就职 \((x='Mayingjing', y='520 swear the oath of office')\)
• 蔡英文 520 宣誓就职 \((x='Caiyingwen', y='520 swear the oath of office')\)

也就是说给 prediction-based NN 输入 马英九 or 蔡英文的时候 NN 会“努力”把两个不同的输入 project 到同一个输出上。那么之后的每一层都会做这件事情，所以我们可以使用第一层的输入来做为 word vector.

更好的 prediction-based NN

• 用前 10 个单词预测下一个

一般情况下我们不会只用前一个单词去预测下一个单词(\(w_{i-1} \Rightarrow w_i\))，并抽取 1st layer's input 作为 word vector, 我们会使用前面至少10个词汇来预测下一个词汇(\([w_{i-10}, w_{i-9}, ..., w_{i-1}] \Rightarrow w_i\))

• 前10个词汇的相同 1-of-N encoding 位应该使用相同的 w 权重

如果使用不同的权重，（以前两个预测下一个为例）

• “马英九宣誓就职时说xxxx”
• “宣誓就职时马英九说xxxx”

[w1:马英九] + [w2:宣誓就职时] => [w3:说xxxx]

[w1:宣誓就职时] + [w2:马英九] => [w3:说xxxx]

如果使用不同的权重，那么 [w1:马英九] + [w2:宣誓就职时] 和 [w1:宣誓就职时] + [w2:马英九] 就会产生不同的 word vector.

具体过程描述如下：

• |V| : volcabulary size, for 1-of-N encoding

• |z| : word vector size, the dimension of embedding space

• the length of \(x_{i-1}\) and \(x_{i-2}\) are both |V|.

• the length of \(z\) is |z|
• \(z = W_1x_{i-2} + W_2x_{i-1}\)
• the weight matrix \(W_1\) and \(W_2\) are both |Z|*|V| matrix
• \(W_1 = W_2 = W \rightarrow z = W(x_{i-2} + x_{i-1})\)
• we get \(W\) after NN trained
• a new word's vector is \(wordvector_{w_i} = W(1ofNencdoing_{w_i})\)

实作时，我们如何保证权重共享呢 --- \(W_1 = W_2\)？

How to make wi equal to wj?

Given wi and wj the same initialization, and the same update per step.

\(w_0 = w_0\)

\(w_i \leftarrow w_i - \eta \frac{\partial C}{\partial w_i} - \eta \frac{\partial C}{\partial w_i}\)

\(w_i \leftarrow w_i - \eta \frac{\partial C}{\partial w_i} - \eta \frac{\partial C}{\partial w_i}\)

扩展 prediction-based 方法

• Continuous bag of word(CBOW) model

\([w_{i-1}, w_{i+1}] \Rightarrow w_i\)

• Skip-gram

\(w_i \Rightarrow [w_{i-1}, w_{i+1}]\)

word embedding 用于关系推导和问题回答

关系推导

有了 word vector， 很多关系都可以数字化（向量化）

• 包含关系
• 因果关系
• 等等

两类词汇的 word vector 的差值之间存在某种平行和相似（相似三角形or多边形）性，可以据此产生很多应用。

\(WordVector_{China} - WordVector_{Beijing} // WordVector_{Spain} - WordVector_{Madrid}\)

for \(WordVector_{country} - WordVector_{capital}\), if a word A \(\in\) country, and word B \(\in\) capital of this country, then \(A_0 - B_0 // A_1 - B_1 // A_2 - B_2 // . ..\)

问题回答

问题回答也是这个思路---利用word vector差值是相互平行的。

\(V(Germany) - V(Berlin) // V(Italy) - V(Rome)\)

\(vector = V(Berlin) + V(Italy) - V(Rome)\)

\(find\ a\ word\ from\ corpus\ whose\ word\ vector\ closest\ with\ 'vector'\)

中英文混合翻译

学习 word embedding prediction-based NN 的网络原理（单层; 前后单词为一个样本；取第一层输入）可以实现更进一步的应用。

先获取中文的 word vector, 然后获取英文的 word vector, （这之后开始使用 prediction-based NN 的原理）然后 learn 一个 NN 使得他能把相同意思的中英文 word vector 投影到某个 space 上的同一点，这之后提取这个网络的第一层 hidden layer 的输入权重，就可以用来转换其他的英文和中文单词到该 space 上，通过就近取意的方法获取该单词的意思。

对图像做 word embedding

这里也是学习 word embedding prediciton-based NN 的网络原理，他可以实现扩展型图像识别，一般的图像识别是只能识别数据集中已经存在的类别，而通过 word embedding 的这种模式，可以实现对图像数据集中不存在（但是在 word 数据集中存在）的类别也正确识别（而不是指鹿为马，如果image dataset 中原本没有 ‘鹿’ 的话，普通的图像识别会就近的选择最'像'的类别，也就是他只能在指定的类别中选最优的）。

先通过大量阅读做 word vector, 然后训练一个 NN 输入为图片，输出为（与之前的 word vector）相同维度的 vector, 并且使得 NN 把与 word(eg, 'dog') 相同类型的image(eg, dog img) project 到该word 的 word vector 附近甚至同一点上。

如此面对新来的图片比如 '鹿.img', 输入给这个 NN 他就会 project 到 word vector space 上的 “鹿” 周围的某一点上。

对 document 做 embedding

1. word sequences with different lengths -> the vector with the same length
2. the vector representing the meaning of the word sequence

如何把 document 变成一个 vector 呢？

首先把 document 用 bag of word(a vector) 来表示，然后将其输入给一个 auto encoder , autoencoder 的 bottle-neck layer 就是这篇文章的 embedding vector。

这里与使用 autoencoder 无法用来做 word embedding 的道理是一样的，只不过对于 word embedding 来说 autoencoder 面对的是完全相互 independent 的 1-of-N encoding, 其本身就无规律可言，所以 autoencoder 不可能学到任何东西，所以没有直接规律，就寻找间接规律 通过学习 context 来判断单词的语义。

这里 autoencoder 面对的是 document(bag-of-word), bag of word 中包含的信息仅仅是单词的数量 （大概是这样的向量\([22, 1, 879, 53, 109, ....]\)）不论 bag-of-word(for document) 还是 1-of-N encoding(for word) 都是语义缺乏的编码方式。所以想通过这种编码方式让NN萃取有价值的信息是不可能的。

还是那个原则没有直接规律，就寻找间接规律：

• 1-of-N encoding, lack of words relationship, what we lack, what we use NN to predict, we discover(construct) some form of data-pair (x -> y) who can represent the "relationship" to train a NN , and the BY-PRODUCT is the weight of hiden layer --- a function(or call it matrix) who can project the word to word vector(a meaningful encoding)

• bag-of-word encoding, lack of words ordering(another relationship), using （李宏毅老师没有明说，只列了 reference）

white blood cells destroying an infection

an infection destroying white blood cells

they have same bag-of-words, but vary differentmeaning.

Matrix Factorization

有时候你会有两种 object, 他们之间由某种共通的 latent factor 去操控。

比如我们都会喜欢动漫人物，但我们不是随随便便就喜欢的，而是说在我们自己的性格和动漫人物的性格背后隐含着某个相同的“属性列表”

每个人根据自己的【特点】都可以看成是【属性列表】中的一个向量或是高维空间中的一个点（向量），每个动漫人物也可以根据其【特点】看成是【属性列表】中的一个向量或高维空间中的一个点（向量）。

如果这两个向量很相似的话，那么两者 match，就会产生【喜欢】

假设：

• 已知 \(r_{person}\) 和 \(r_{idiom}\) 是这两个向量
• 求：人和动漫人物之间的匹配度（喜欢程度）。

现在：

• 已知 任何动漫人物之间的匹配度（喜欢程度）
• 求：向量\(r_{person}\) 和 \(r_{idiom}\)

【注意】这个 latent vector 的数目是试出来的，一开始我们并不知道。

比如 latent attribute = ['傲娇', '天然呆']

person A : \(r^A = [0.7, 0.3]\)

character 1: \(r^1 = [0.7, 0.3]\)

\(r^A \cdot r^1 = 0.58\)

• # of Otaku = M
• # of characters = N
• # of latent factor(a vector) = K (means vector r is K dim)

$$ X \in R^{M*N} \approx \begin{vmatrix} r^A \\ r^B \\r^C\\.\\.\\. \end{vmatrix}^{M*K} * \begin{vmatrix} r^1 & r^2 & r^3 &.&.&. \end{vmatrix}^{K*N} $$

这个问题可以直接使用 SVD 来解决，虽然SVD分解得到的是三个矩阵，你可以视情况将中间矩阵合并给前面的或后面的

MF 处理缺值问题 --- Gradient Descent

上面的是理想情况：table X 中所有的值都完备；

现实情况是：tabel X 通常是缺值的，有很多 Missing value. 那该如何是好呢？

使用 GD，来做，目标函数是：

\(L = \sum_{(i,j)}(r^i \cdot r^j - n_{ij})^2\)

通过对这个目标函数最小化，就可以求出 r.

然后就可以用这些 r 来求出 table 中的每一个值。

more about MF

MF 的求值函数（table X 的计算函数，我们之前一直假设他是两个 latent vector 的匹配程度）可以考虑更多的因素。他不仅仅可以表示匹配程度。

从： \(r^A \cdot r^1 \approx 5\) 到更精确的： \(r^A \cdot r^1 + b_A + b_1\approx 5\)

• \(b_A\): 表示他对动漫多感兴趣
• \(b_1\): 表示这个动漫的推广力度如何

如此新的 GD 优化目标就变成：

\(L = \sum_{(i,j)}(r^i \cdot r^j + b_i + b_j - n_{ij})^2\)

也可以加 L1 - Regularization, 比如 \(r^i, r^j\) 是 sparse 的---喜好很明确，要么天然呆，要么就是傲娇的，不会有模糊的喜好。

MF for Topic analysis

MF 技术用在语义分析就叫做 LSA(latent semantic analysis):

• character -> document
• otakus -> word
• table item -> term frequency of word in this document

注意：通常我们在做 LSA 的时候还会加一步操作，term frequency always weighted by inverse document frequency, 这步操作叫做 TF-IDF.

也就是说，你用作 \(L = \sum_{(i,j)}(r^i \cdot r^j + b_i + b_j - n_{ij})^2\) 中的 \(n_{ij}\) 不是原始的某篇文章中的某个单词的出现次数，而是出现次数乘以包含这个单词的文章数的倒数亦即，

(n_{ij} = \frac{TF}{IDF})\

如此当我们通过 GD 找到 latent vector 时，这个向量的每一个位表示的是 topics(财经，政治，娱乐，游戏等)

la salud mental y todos los cargos asociados al estrés toxico.

important info on relapse rates for other behavior

This paper answers the research question “Can species physiologically adapt to climate warming?” They analyzed data from numerous other studies that described the thermal tolerances of species worldwide. Main findings of this study are that tolerance to heat was mostly conserved within closely related species, whereas tolerance to the cold was a trait that could vary.

Trait-based analyses conducted by Kerr et al. (2015) correspond to these results. The upper thermal limits of bumblebees were found to be an ancestral trait, since it was conserved across closely related species.

It is now clear that climate change has affected species, as shown by endless amounts of papers in the primary literature. This paper estimates sizeable species extinctions by 2050 linked to climate change. They do this with species distribution modeling. This technique uses known species distributions and climatic conditions, and estimates how the distribution could change in the future according to models of future climate. Authors used optimistic and pessimistic estimates of future climate data (since, we do not know how much humans will cut down on activities that aggravate climate change), and even took into account the capacity for some species to move to and colonize other places. They estimated extinctions of 15% to 37% of the species included in their study. Studies that look at future impacts of climate change yield uncertain results since these cannot be verified.

The IPCC (Intergovernmental Panel on Climate Change) put together a report summarizing climate literature. Refer to the PDF of the synthesis of this report here if you are interested in finding out more information on climate change: https://www.ipcc.ch/pdf/assessment-report/ar4/wg1/ar4-wg1-spm.pdf

Elizabeth, I think you have a great start to your paper. The introduction is well thought out and sounds very good. The citations flow well together. The only thing I might suggest on changing is adding more of your own thoughts and words to the paper.

B A L A N C E

It's interesting to see that in an age of technological progression and innovation, the minority is focused on creation and media.

I really like this quote because it does show that the community we share is more than just the people we know face to face, but its all around us. It kinda connects back to infinitive groups

The paper by N. G. Hairston, F. .E. Smith, and L. B. Slobodkin, "Community structure, Population Control, and Competition," observed what and how organisms are limited by their respective resources. Producers may be limited by an array of variables: light, water, and nutrients. These limiting resources can cause a change in competition throughout the trophic levels. In this paper, the limiting resources are the nutrients from guano. As the foxes prey and decrease the number of seabirds visiting the island, the dispersal of guano also decreases on the island.

This paper demonstrates how bird and butterfly communities have changed over time, and while they shifted through time, they failed to keep up with climate change. They show that northward shifts for bird communities shifted by 37 km, and butterfly communities shifted by 114 km. The climate shifted much faster than this, leaving lags of 212 km for birds, and 135 km for butterflies. These are the distances that birds and butterflies would need to travel to reach temperatures similar to that of their historical range shifts.

infdo

This recommendation is offically updated to state the following:

Insulin-treated patients with hypoglycemia unawareness or an episode of level 2 (<54 mg/dL [3.0 mmol/L]) hypoglycemia should be advised to raise their glycemic targets to strictly avoid hypoglycemia for at least several weeks in order to partially reverse hypoglycemia unawareness and reduce risk of future episodes. A

Reason for Change: Alignment of terminology/definitions will minimize confusion for practitioners. To align hypoglycemia definitions between a consensus report (reference below) and the Standards of Care, hypoglycemia has been re-categorized into 3 levels as outlined in the annotation to table 6.3.

References:

Agiostratidou G, Anhalt H, Ball D, et al. Standardizing Clinically Meaningful Outcome Measures Beyond HbA1c for Type 1 Diabetes: A Consensus Report of the American Association of Clinical Endocrinologists, the American Association of Diabetes Educators, the American Diabetes Association, the Endocrine Society, JDRF International, The Leona M. and Harry B. Helmsley Charitable Trust, the Pediatric Endocrine Society, and the T1D Exchange. Diabetes Care 2017;40:1622-1630.

American Diabetes Association. 6. Glycemic targets: Standards of Medical Care in Diabetes—2018 [web annotation]. Diabetes Care 2018;41(Suppl. 1):S55–S64. Retrieved from https://hyp.is/wmtWGjwnEeiOWY_FhVG-zA/care.diabetesjournals.org/content/41/Supplement_1/S55.

Annotation published April 11, 2018.

Annotation approved by PPC: March 10, 2018.

Suggested Citation: American Diabetes Association. Standards of Medical Care in Diabetes—2018 Abridged for Primary Care Providers [web annotation]. Clinical Diabetes 2018;36(1):14-37. Retrieved from https://hyp.is/GRjHQj2iEei1dkdhu5hlMw/clinical.diabetesjournals.org/content/36/1/14.

This recommendation is officially updated to state the following:

Glucagon should be prescribed for all individuals at increased risk of level 2 hypoglycemia, defined as blood glucose <54 mg/dL (3.0 mmol/L), so it is available should it be needed. Caregivers, school personnel, or family members of these individuals should know where it is and when and how to administer it. Glucagon administration is not limited to health care professionals. E

Reason for Change: Alignment of terminology/definitions will minimize confusion for practitioners. To align hypoglycemia definitions between a consensus report (reference below) and the Standards of Care, hypoglycemia has been re-categorized into 3 levels as outlined in the annotation to table 6.3.

References:

Agiostratidou G, Anhalt H, Ball D, et al. Standardizing Clinically Meaningful Outcome Measures Beyond HbA1c for Type 1 Diabetes: A Consensus Report of the American Association of Clinical Endocrinologists, the American Association of Diabetes Educators, the American Diabetes Association, the Endocrine Society, JDRF International, The Leona M. and Harry B. Helmsley Charitable Trust, the Pediatric Endocrine Society, and the T1D Exchange. Diabetes Care 2017;40:1622-1630.

American Diabetes Association. 6. Glycemic targets: Standards of Medical Care in Diabetes—2018 [web annotation]. Diabetes Care 2018;41(Suppl. 1):S55–S64. Retrieved from https://hyp.is/wmtWGjwnEeiOWY_FhVG-zA/care.diabetesjournals.org/content/41/Supplement_1/S55.

Annotation published April 11, 2018.

Annotation approved by PPC: March 10, 2018.

Suggested Citation: American Diabetes Association. Standards of Medical Care in Diabetes—2018 Abridged for Primary Care Providers [web annotation]. Clinical Diabetes 2018;36(1):14-37. Retrieved from https://hyp.is/bRFwXD2hEeidg9OXP8lExw/clinical.diabetesjournals.org/content/36/1/14.

This section is officially updated to state the following:

"Level 1 hypoglycemia in hospitalized patients is defined as a blood glucose <70 mg/dL (3.9 mmol/L) and level 2 hypoglycemia as glucose values <54 mg/dL (3.0 mmol/L)."

Reason for Change: Alignment of terminology/definitions will minimize confusion for practitioners. To align hypoglycemia definitions between a consensus report (reference below) and the Standards of Care, hypoglycemia has been re-categorized into 3 levels as outlined described in the annotation to table 6.3.

References:

Agiostratidou G, Anhalt H, Ball D, et al. Standardizing Clinically Meaningful Outcome Measures Beyond HbA1c for Type 1 Diabetes: A Consensus Report of the American Association of Clinical Endocrinologists, the American Association of Diabetes Educators, the American Diabetes Association, the Endocrine Society, JDRF International, The Leona M. and Harry B. Helmsley Charitable Trust, the Pediatric Endocrine Society, and the T1D Exchange. Diabetes Care 2017;40:1622-1630.

American Diabetes Association. 6. Glycemic targets: Standards of Medical Care in Diabetes—2018 [web annotation]. Diabetes Care 2018;41(Suppl. 1):S55–S64. Retrieved from https://hyp.is/wmtWGjwnEeiOWY_FhVG-zA/care.diabetesjournals.org/content/41/Supplement_1/S55.

Annotation published April 11, 2018. Annotation approved by PPC: March 10, 2018.

Suggested Citation: American Diabetes Association. Standards of Medical Care in Diabetes—2018 Abridged for Primary Care Providers [web annotation]. Clinical Diabetes 2018;36(1):14-37. Retrieved from https://hyp.is/ZrydmD2iEeiE8GuxzoHaoA/clinical.diabetesjournals.org/content/36/1/14.

On 2015 Feb 11, Marco Ventura commented:

I do not understand what is the novelty of this study. Very similar studies have been already published about the comparative genomics of the genus Bifidobacterium. Just as a remark for the authors and for the potential readers that are not from this field: see the following papers published in August 2014:

• Milani, C., Lugli, G.A., Duranti, S., Turroni, F., Bottacini, F., Mangifesta, M., Sanchez, B., Viappiani, A., Mancabelli, L., Taminiau, B., Delcenserie, V., Barrangou, R., Margolles, A., van Sinderen, D., and Ventura, M. 2014. Genome encyclopaedia of type strains of the genus Bifidobacterium. Appl. Environ. Microbiol. 80(20):6290-302.

• Lugli, G.A., Milani, C., Turroni, F., Duranti, S., Ferrario, C., Viappiani, A., Mancabelli, L., Mangifesta, M., Taminiau, B., Delcenserie, V., van Sinderen, D. and Ventura, M. 2014. Investigation of the evolutionary development of the genus Bifidobacterium by comparative genomics. Appl. Environ. Microbiol. 80(20):6383-94.

Enjoy the reading

On 2016 Feb 05, James Murray commented:

The superoxide dependent nitrogenase described in this (Ribbe M, 1997) paper is extremely unlikely to exist.

The paper describes the purification of the components of an oxygen-tolerant nitrogenase, not homologous to the known nif,vnf, or anf-type, from Streptomyces thermoautotrophicus UBT1, a thermophilic carboxydotroph.

Results published in February 2016 (MacKellar D, 2016) show that three independent isolates of S. thermoautotrophicus, including the original UBT1 strain, do not grow in the absence of combined nitrogen and are incapable of incorporating isotopically labelled dinitrogen into biomass, nor do they contain the claimed superoxide dependent nitrogenase genes. The N-terminal sequences assigned to nitrogenase components in Ribbe M, 1997, and the full DNA gene sequences in the PhD thesis of Carla Hofmann-Findeklee (2000, KF951061.1, KF951060.1, KF951059.1, KF956113.1) are found at near-identity in Bacillus schlegelii DSM9132 (recently renamed to Hydrogenibacillus schlegelii, "SdnMSL-like" sequences in KT861421.1), a non-diazotrophic thermophilic carboxydotrophic organism isolated in the Meyer lab (Krüger & Meyer, 1984) and known to be cultured in the Meyer laboratory in 1994 (Hänzelmann, 1994). The independently isolated B. schlegelii DSM2000 strain also has these sequences at near-identity. The closest relatives to these sequences are to Firmicutes and not Actinomycetes like S. thermoautotrophiucs. The four "nitrogenase" sequences are easily identified as encoding a superoxide dismutase ("st2", sdnO), and a three-subunit aerobic carbon monoxide dehydrogenase ("st1", sdnMSL).

Ribbe M, 1997 relies on an ammonia production assay to determine the nitrogenase activity. This assay is known to have a high background due to environmental ammonia and protein deamination. Incorporation of isotopically labelled dinitrogen is usually considered the gold standard for the identification of a nitrogenase enzyme. No incorporation of isotopically labelled nitrogen into ammonia is shown using the claimed biochemical nitrogenase preparation. The cells were grown in media with 1.5 g/l ammonium chloride, so there was no selection for diazotrophy. No published demonstration of the superoxide dependent nitrogenase has occurred outside the Meyer laboratory.

The nitrogenase scheme described in Ribbe M, 1997 is chemically and biologically implausible. There is no known ATPase domain, as required by the proposed reaction scheme, in any of the described proteins. The known nitrogenase types require the highly reducing ferredoxin or flavodoxin as reductants. Superoxide is an unlikely electron donor for a nitrogenase, as it is not as reducing as even NADPH or NADH, and is reactive and toxic. No other biologically productive use of superoxide as an electron donor is known. An aerobic reduction of nitrogen to ammonia is unknown, and unlikely, as under the highly reducing conditions, oxygen would most probably be reduced in preference to nitrogen. The rate of activity described is too low to be that of a biological enzyme supporting diazotrophic growth, as it would take the proposed nitrogenase over 100 hours just to replace the nitrogen in the enzyme itself, which is also incompatible with the claimed rate of diazotrophic growth of S. thermoautotrophicus (Gadkari D, 1992).

To summarize:

• Recent evidence suggests that three independently isolated strains of S. thermoautotrophicus are not diazotrophic.

• If the Meyer laboratory did contaminate their S. thermoautotrophicus culture with a strain of B. schlegelii (such as the DSM9132 strain), we would observe the N-terminal sequences presented here and the DNA gene sequences also produced in the Meyer laboratory.

• The extremely low activity "nitrogenase" was described based on a problematic ammonia production assay.

• A superoxide-dependent aerobic nitrogenase is chemically and biologically implausible.

Declaration: I am an author on the MacKellar D, 2016 paper, but this comment is entirely my own.

References:

Bernd Krüger and Ortwin Meyer. Thermophilic bacilli growing with carbon monoxide. Archives of Microbiology, 139(4):402–408, 1984.

Petra Hänzelmann. Isolierung und Charakterisierung von Kohlenmonoxid-Dehydrogenase aus dem obligat thermophilen Bakterium Bacillus schlegelii. Diplomarbeit thesis, University of Bayreuth, 1994.

Carla Hofmann-Findeklee. Molekularbiologische Untersuchung der Strukturgene des aeroben N2-fixierenden Systems von Streptomyces thermoautotrophicus sowie funktionelle Charakterisierung von rekombinantem SdnO. PhD thesis, University of Bayreuth, 2000.

On 2018 Jan 21, Tom Kindlon commented:

References:

1 Chalder T, Berelowitz G, Pawlikowska T, Watts L, Wessely S, Wright D, Wallace EP: Development of a Fatigue Scale. J Psychosom Res 1993, 37:147-153.

2 Neu D, Hoffmann G, Moutrier R, Verbanck P, Linkowski P, Le Bon O. Are patients with chronic fatigue syndrome just 'tired' or also 'sleepy'? J Sleep Res. 2008 Dec;17(4):427-31. doi: 10.1111/j.1365-2869.2008.00679.x.

3 Goudsmit, EM., Stouten, B and Howes, S. Fatigue in myalgic encephalomyelitis. Bulletin of the IACFS/ME, 2008, 16, 3, 3-10. https://web.archive.org/web/20140719090603/http://www.iacfsme.org/BULLETINFALL2008/Fall08GoudsmitFatigueinMyalgicEnceph/tabid/292/Default.aspx

4 Goldsmith LP, Dunn G, Bentall RP, Lewis SW, Wearden AJ. Correction: Therapist Effects and the Impact of Early Therapeutic Alliance on Symptomatic Outcome in Chronic Fatigue Syndrome. PLoS One. 2016 Jun 1;11(6):e0157199. doi: 10.1371/journal.pone.0157199. eCollection 2016. https://doi.org/10.1371/journal.pone.0157199.s001 (CSV form: https://www.mediafire.com/file/rvh3brmgoaznude/Goldsmith+2015+FINE+data+journal.csv )

5 A dataset from the PACE trial. Released following a freedom of information request. https://sites.google.com/site/pacefoir/pace-ipd_foia-qmul-2014-f73.xlsx Readme file: https://sites.google.com/site/pacefoir/pace-ipd-readme.txt.

6 Morriss RK, Wearden AJ, Mullis R. Exploring the validity of the Chalder Fatigue scale in chronic fatigue syndrome. J Psychosom Res. 1998 Nov;45(5):411-7.

7 Loge JH, Ekeberg O, Kaasa S. Fatigue in the general Norwegian population: normative data and associations. J Psychosom Res 1998; 45: 53-65. CrossRef | PubMed

8 Van Kessel K, Moss-Morris R, Willoughby, Chalder T, Johnson MH, Robinson E, A randomized controlled trial of cognitive behavior therapy for multiple sclerosis fatigue, Psychosom. Med. 2008; 70:205-213.

On 2018 Jan 19, Andrea Messori commented:

Eight gene therapies are already supported by a published clinical trial

by Andrea Messori

HTA Unit, ESTAR

50135 Firenze, Italy

The study by Rangarajan and co-workers shows that gene therapies can be successful for a disease condition where the deficient factor is coded for by a very large gene. As of December 31, 2017, a published clinical trial is already available for the following 8 gene therapies:

-Voretigene neparvovec (LUXTURNA, Spark Therapeutics): trial by Russell et al 2017; approved by FDA [2].

-Tisagenlecleucel (KYMRIAH, Novartis): trial by Oncologic Drugs Advisory Committee[3]; approved by FDA [4].

-Axicabtagene ciloleucel (YESCARTA, Gilead): trial by Neelapu et al [5]; approved by FDA [6].

-STRIMVELIS (GSK): trial by Cicalese et al 2016; approved by EMA [7].

-Gene therapy with a high-specific-activity factor IX variant: trial by George et al. 2017 [9].

-Valoctocogene roxaparvovec: trial by Rangarajan et al 2017 [10].

-Single-dose gene-replacement therapy Spinal Muscular Atrophy: trial by Mendell et al 2017 [11].

-Hematopoietic stem-cell gene therapy for cerebral adrenoleukodystrophy: trial by Eichler et al 2017 [12].

References

1) Russell S, Bennett and co-workers for Cerebral Adrenoleukodystrophy J, Wellman JA, Chung DC, Yu ZF, Tillman A, Wittes J, Pappas J, Elci O, McCague S, Cross D, Marshall KA, Walshire J, Kehoe TL, Reichert H, Davis M, Raffini L, Georg Spinal Muscular Atrophy e LA, Hudson FP, Dingfield L, Zhu X, Haller JA, Sohn EH, Mahajan VB, Pfeifer W, Weckmann M, Johnson C, Gewaily D, Drack A, Stone E, Wachtel K, Simonelli F, Leroy BP, Wright JF, High KA, Maguire AM. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017 Aug 26;390(10097):849-860. doi: 10.1016/S0140-6736(17)31868-8.

2) FDA. FDA approves hereditary blindness gene therapy. Nat Biotechnol. 2018 Jan 10;36(1):6. doi: 10.1038/nbt0118-6a.

3) Novartis. Oncologic Drugs Advisory Committee Briefing Document. Tisagenlecleucel (CTL019) for the Treatment of Pediatric and Young Adult Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia. https://www.fda.gov/downloads/advisorycommittees /committeesmeetingmaterials/drugs /oncologicdrugsadvisorycommittee/ucm566168.pdf , July 12, 2017. Accessed September 11, 2017.

4) Bach PB, Giralt SA, Saltz LB. FDA Approval of Tisagenlecleucel: Promise and Complexities of a $475 000 Cancer Drug. JAMA. 2017 Nov 21;318(19):1861-1862. doi:10.1001/jama.2017.15218.

5) Neelapu SS, Locke FL, Bartlett NL, Lekakis LJ, Miklos DB, Ja for a totalcobson CA, Braunschweig I, Oluwole OO, Siddiqi T, Lin Y, Timmerman JM, Stiff PJ, Friedberg JW, Flinn IW, Goy A, Hill BT, Smith MR, Deol A, Farooq U, McSweeney P, Munoz J, Avivi I, Castro JE, Westin JR, Chavez JC, Ghobadi A, Komanduri KV, Levy R, Jacobsen ED, Witzig TE, Reagan P, Bot A, Rossi J, Navale L, Jiang Y, Aycock J, Elias M, Chang D, Wiezorek J, Go WY. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med. 2017 Dec 28;377(26):2531-2544. doi: 10.1056/NEJMoa1707447.

6) FDA. U.S. Food & Drug Administration. YESCARTA (axicabtagene ciloleucel), Axicabtagene Ciloleucel (YESCARTA, Gilead). https://www.fda.gov/BiologicsBloodVaccines/CellularGeneTherapyProducts/ApprovedProducts/ucm581222.htm

7) Cicalese MP, Ferrua F, Castagnaro L, Pajno R, Barzaghi F, Giannelli S, Dionisio F, Brigida I, Bonopane M, Casiraghi M et al. Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency. Blood 2016;128: 45 – 54.

8) Aiuti A, Roncarolo MG, Naldini L. Gene therapy for ADA-SCID, the first marketing approval of an ex vivo gene therapy in Europe: paving the road for the next generation of advanced therapy medicinal products. EMBO Mol Med. 2017 Jun;9(6):737-740. doi: 10.15252/emmm.201707573. PubMed PMID: 28396566; URL https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5452047/pdf/EMMM-9-737.pdf

9) George LA, Sullivan SK, Giermasz A, Rasko JEJ, Samelson-Jones BJ, Ducore J, Cuker A, Sullivan LM, Majumdar S, Teitel J, McGuinn CE, Ragni MV, Luk AY, Hui D, Wright JF, Chen Y, Liu Y, Wachtel K, Winters A, Tiefenbacher S, Arruda VR, van der Loo JCM, Zelenaia O, Takefman D, Carr ME, Couto LB, Anguela XM, High KA. Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant. N Engl J Med. 2017 Dec 7;377(23):2215-2227. doi: 10.1056/NEJMoa1708538.

10) Rangarajan S, Walsh L, Lester W, Perry D, Madan B, Laffan M, Yu H, Vettermann C, Pierce GF, Wong WY, Pasi KJ. AAV5-Factor VIII Gene Transfer in Severe Hemophilia A. N Engl J Med. 2017 Dec 28;377(26):2519-2530. doi:10.1056/NEJMoa1708483.

11) Mendell JR, Al-Zaidy S, Shell R, Arnold WD, Rodino-Klapac LR, Prior TW, Lowes L, Alfano L, Berry K, Church K, Kissel JT, Nagendran S, L'Italien J, Sproule DM, Wells C, Cardenas JA, Heitzer MD, Kaspar A, Corcoran S, Braun L, Likhite S, Miranda C, Meyer K, Foust KD, Burghes AHM, Kaspar BK. Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. N Engl J Med. 2017 Nov 2;377(18):1713-1722. doi: 10.1056/NEJMoa1706198.

12) Eichler F, Duncan C, Musolino PL, Orchard PJ, De Oliveira S, Thrasher AJ, Armant M, Dansereau C, Lund TC, Miller WP, Raymond GV, Sankar R, Shah AJ, Sevin C, Gaspar HB, Gissen P, Amartino H, Bratkovic D, Smith NJC, Paker AM, Shamir E, O'Meara T, Davidson D, Aubourg P, Williams DA. Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy. N Engl J Med. 2017 Oct 26;377(17):1630-1638. doi: 10.1056/NEJMoa1700554.

On 2017 Nov 23, Franck Ramus commented:

This study seems severely underpowered, which is surprising for such a frequent disorder as dyslexia, and given that we are in the midst of a replication crisis (e.g., Button et al. 2013; Ramus et al. 2017).

With 12 participants per group, this study had 29% chance to observe a moderate group difference (d=0.6). Here, the significant result is due to a huge group difference (d=1.28) in the left V5/MT-LGN connectivity. Even larger than the corresponding behavioural difference in RAN letters (d=1.27) and numbers (d=0.95) (from Table S1). How plausible is it that there should be such a large brain difference between two groups of dyslexic and control individuals, even larger than the cognitive symptoms that this brain difference is presumed to underlie?

Similarly, with 12 dyslexic individuals, only huge correlations greater than 0.576 could be significant. Luckily this study observed a correlation of 0.588 between left V5/MT-LGN connectivity and RAN (using a one-tailed test and correcting for two tests), but not with reading comprehension. But what about the other behavioural variables, spelling and reading speed? Are they not core symptoms of dyslexia, even more so than RAN? Do they not rely on visual abilities? Were the a priori predictions so specific to RAN and reading comprehension, that correlations with spelling and reading speed were not even tested? If those predictions had been preregistered, this might be credible. Alternatively, were those correlations tested, but not taken into account in the correction for multiple tests? (not even mentioning correlations within the control group, or across the two groups)

Button, K. S., Ioannidis, J. P. A., Mokrysz, C., Nosek, B. A., Flint, J., Robinson, E. S. J., & Munafò, M. R. (2013). Power failure: why small sample size undermines the reliability of neuroscience. Nature Reviews Neuroscience, 14(5), 365‑376. https://doi.org/10.1038/nrn3475 Ramus, F., Altarelli, I., Jednoróg, K., Zhao, J., & Scotto di Covella, L. (2017). Neuroanatomy of developmental dyslexia : pitfalls and promise. Neuroscience & Biobehavioral Reviews. https://doi.org/10.1016/j.neubiorev.2017.08.001

On 2017 Aug 29, Marcus de Jong commented:

With great interest, but also with significant apprehension, we read the article by Yamanaka et al. recently published on-line ahead of print in Neurosurgical Review, entitled "Trilateral retinoblastoma: A systematic review of 211 cases."

We have had a long-time interest in retinoblastoma, including trilateral retinoblastoma, and we became worried about the fact that the authors allegedly have included 211 patients with trilateral retinoblastoma in their statistical analysis. We have reason to believe that this number, and consequently, the presented results may not be correct. In 2014, we published a systematic review and meta-analysis on trilateral retinoblastoma in Lancet Oncology, based on strict adherence to the PRISMA Statement (de Jong et al. [1]). We also contacted authors to resolve any equivocal issues. In particular, we meticulously matched patients between reports to prevent including any patients twice in our analysis. Many patients with trilateral retinoblastoma appear in the literature two or more times over the years; in the extreme case, one patient features in five different reports over 12 years. Resolving duplications and consolidating the sequential reports by each patient, we ended up with 174 unique individuals with trilateral retinoblastoma (see the attached table from the online supplement of our article).

Naturally, a number of trilateral retinoblastoma cases included in Yamanaka et al.'s paper have been published after acceptance of our paper. These are Andrade et al. [2] with 1 case, De Ioris et al. [3] with 2 new cases, and Pham et al. [4] with 3 cases, amounting to a total difference of 6 unique patients.

However, we included several articles that Yamanaka et al. have not ascertained during their research: De Jong et al. [5], Duncan et al. [6], Dunst et al. [7], Gururangan et al. [8], Jin et al. [9], Lim et al. [10], Mauger et al. [11], Onadim et al. [12], Popovic et al. [13], White et al. [14], and Zimmerman et al. [15]. These amount to a total of 18 unique cases.

We became very concerned after noting that, correcting for new cases and articles not included by Yamanaka et al., the difference in the number of patients between the two articles is no less than 49 (our 174 - 18 not included by Yamanaka et al. = 156 in De Jong et al. [1] versus their 211 - 6 newly reported = 205 in Yamanaka et al. [16]). Because Yamanaka et al. have not provided a patient-by-patient table of their cases unlike we did, we are limited to this numerical comparison and cannot verify case by case their unique cases.

In summary, we have reason to believe that Yamanaka et al. have not accounted for the fact that trilateral retinoblastoma patients have often been published in more than one paper. Although the results of the meta-analysis by Yamanaka et al., in general, show results that appear largely similar to those that what we published, the unexplained and large difference in the number of patients suggests to us that most, if not all, percentages and p-values from the meta-analysis by Yamanaka et al. do not reflect reality. There is also no way of knowing whether a particular statistic is correct or not.

To solve this issue, we ask Yamanaka et al. to clarify this crucial aspect of their meta-analysis, ideally by providing a patient-by-patient list of the 211 cases to verify their uniqueness to be attached to their paper. Hopefully the authors can provide a valid explanation that completely resolves our concerns. Otherwise, we are afraid that the statistical results of this paper will be misleading and cannot be trusted, and then perhaps the paper in the present form should be retracted in order to correct the literature and to avoid wrong interpretations in clinical practice of managing children with trilateral retinoblastoma.

Marcus de Jong,

on behalf of the authors of de Jong et al. [1]

References

1.de Jong MC, Kors WA, de Graaf P, et al (2014) Trilateral retinoblastoma: A systematic review and meta-analysis. Lancet Oncol 15:1157–1167. doi: 10.1016/S1470-2045(14)70336-5

2.Andrade GC de, Pinto NP de C, Motono M, et al (2015) Trilateral retinoblastoma with unilateral eye involvement. Rev Assoc Med Bras 61:308–10. doi: 10.1590/1806-9282.61.04.308

3.De Ioris MA, Valente P, Randisi F, et al (2014) Baseline central nervous system magnetic resonance imaging in early detection of trilateral retinoblastoma: pitfalls in the diagnosis of pineal gland lesions. Anticancer Res 34:7449–54.

4.Pham TTH, Siebert E, Asbach P, et al (2015) Magnetic resonance imaging based morphologic evaluation of the pineal gland for suspected pineoblastoma in retinoblastoma patients and age-matched controls. J Neurol Sci 359:185–192. doi: 10.1016/j.jns.2015.10.046

5.de Jong MC, Moll AC, Göricke S, et al (2016) From a Suspicious Cystic Pineal Gland to Pineoblastoma in a Patient with Familial Unilateral Retinoblastoma. Ophthalmic Genet 37:116–8. doi: 10.3109/13816810.2014.929717

6.Duncan JL, Scott IU, Murray TG, et al (2001) Routine neuroimaging in retinoblastoma for the detection of intracranial tumors. Arch Ophthalmol 119:450–2. 7.Dunst J, Fellner E, Erhardt J (1990) Trilaterales Retinoblastom mit spinalen Metastasen. Rofo 153:343–4. doi: 10.1055/s-2008-1033391

8.Gururangan S, McLaughlin C, Quinn J, et al (2003) High-dose chemotherapy with autologous stem-cell rescue in children and adults with newly diagnosed pineoblastomas. J Clin Oncol 21:2187–91. doi: 10.1200/JCO.2003.10.096

9.Jin J, Tang H-F, Zhou Y-B (2006) Trilateral retinoblastoma: a case report. World J Pediatr 2:151–153.

10.Lim FPM, Soh SY, Iyer JV, et al (2013) Clinical profile, management, and outcome of retinoblastoma in Singapore. J Pediatr Ophthalmol Strabismus 50:106–12.

11.Mauger TF, Makley TA, Davidorf FH, Rogers GL (1992) Retinoblastoma, microphthalmia, coloboma, and neuroepithelioma of the pineal body. Ann Ophthalmol 24:290–4.

12.Onadim Z, Woolford AJ, Kingston JE, Hungerford JL (1997) The RB1 gene mutation in a child with ectopic intracranial retinoblastoma. Br J Cancer 76:1405–9.

13.Popovic MB, Balmer A, Maeder P, et al (2006) Benign pineal cysts in children with bilateral retinoblastoma: a new variant of trilateral retinoblastoma? Pediatr Blood Cancer 46:755–61.

14.White L, Johnston H, Jones R, et al (1993) Postoperative chemotherapy without radiation in young children with malignant non-astrocytic brain tumours. A report from the Australia and New Zealand Childhood Cancer Study Group (ANZCCSG). Cancer Chemother Pharmacol 32:403–6.

15.Zimmerman L (1985) Trilateral retinoblastoma. In: Blodi F (ed) Retinoblastoma. Churchill Livingstone, New York, pp 185–210

16.Yamanaka R, Hayano A, Takashima Y (2017) Trilateral retinoblastoma: A systematic review of 211 cases. Neurosurg Rev. doi: 10.1007/s10143-017-0890-4

Also posted here: https://pubpeer.com/publications/50D5D3EFEA81DCD2421841D84FDA8D#

On 2017 Dec 05, Joseph M Barnby commented:

This letter to the editor was originally submitted to JMIR uHealth and mHealth. The letter was later withdrawn as we became aware it was eligible to have an APC applied to it; in spite of what we understood the JMIR APC policy to be regarding letters to the editor. Neither of our institutions at the time of publication were able to cover the relevant fees. We have posted our reviewed letter here and invited Clare Killikelly to post her submitted response.

Authors:

Mr J M Barnby - Department of Neuroimaging, Institute of Psychiatry, Psychology and Neuroscience, King's College London, SE5 8AF. (Corresponding Author; joe.barnby@kcl.ac.uk)

Dr S A J Fonseca - Division of Psychiatry, University College London, WC1E 6BT.

The Editor JMIR mHealth and uHealth

On 20th July 2017, your journal published a useful and wide-ranging systematic review of mobile and web-based technologies for people with psychosis [1] – a field of mental health which has huge potential to shape the way service users are able to take control of their treatment. Authors found service user aid in design, length of intervention, and social support were all important factors in whether a participant would stop using a digital intervention.

Part of the authors’ summary was that, a) symptom severity may not have a significant effect on drop-out, and b) continuing to develop these interventions alongside users is vital. However, we believe that point a) is not clearly supported by the evidence presented in the article, and point b) misses out (or doesn’t plainly state) a crucial aspect of the technology.

We believe concluding that symptom severity doesn’t have a significant effect on drop-out is premature. The authors reviewed 20 studies that used web-based or mobile technologies and found 6 that measured the impact of symptom severity, chronicity, and duration on drop-out [2, 3, 4, 5, 6, 7]. However, the review only presents the results of those who stayed in the trials, and we believe it’s safe to assume that participants did not drop out randomly. As there were several individuals who declined to take part or dropped out of the study, and since we have no data for this group, we cannot assume that symptom severity does not affect drop-out. In fact, we would argue that it is likely this group were more symptomatic than those who took part. We believe the authors should have stated this more clearly and commented on it in their conclusions about drop-out, including suggesting ways to test this.

The authors only mention that intensity, frequency, and duration of interventions are all vital to adherence, but don’t specifically discuss the role of User Experience/User Interface (UXUI) – a role in software design which tries to make interaction with content as smooth and intuitive as possible. This is a separate but related concept to ‘co-production’ – the involvement of service users in intervention design. Commercial mobile-phone and web-based software is continuously developing more sophisticated and visually pleasing versions. It’s reasonable to hypothesise that better UXUI may result in less drop-out of interventions. Testing this hypothesis may give insight into how a more pleasing user experience may affect drop-out, and how extra investment into UXUI with user input may improve symptom control through better engagement. Indeed, research has suggested this approach might be useful in other areas of healthcare [9] and proposed it might be an important aspect to health intervention design [10]. UXUI focus is increasingly more relevant as mainstream software designers find more ways to keep users engaged, and we believe this highlights that digital interventions may have to compete for attention to meet the expectations of the users they wish to benefit.

In future research aimed at improving user adherence we suggest testing, a) whether simple, text-based designs are as effective as visually pleasing well-designed interfaces when the content is constant (for example, computerised cognitive behavioural therapy), and b) if gamification of therapeutic content improves engagement.

We would like to thank the authors for their current review in this important and emerging area of mental health research, and hope that these comments serve to constructively build upon the discussion.

Yours sincerely, Joseph M Barnby & Dr J Andres S Fonseca

References:

[1] Killikelly C, He Z, Reeder C, Wykes T. Improving Adherence to Web-Based and Mobile Technologies for People With Psychosis: Systematic Review of New Potential Predictors of Adherence. JMIR Mhealth Uhealth 2017; 5(7):e94

[2] van der Krieke L, Emerencia A, Boonstra N, Wunderink L, de JP, Sytema S. A web-based tool to support shared decision making for people with a psychotic disorder: randomized controlled trial and process evaluation. J Med Internet Res 2013 Oct 07;15(10):e216

[3] Ben-Zeev D, Brenner C, Begale M, Duffecy J, Mohr D, Mueser K. Feasibility, acceptability, and preliminary efficacy of a smartphone intervention for schizophrenia. Schizophr Bull 2014 Nov;40(6):1244-1253

[4] Palmier-Claus J, Ainsworth J, Machin M, Dunn G, Barkus E, Barrowclough C, et al. Affective instability prior to and after thoughts about self-injury in individuals with and at-risk of psychosis: a mobile phone based study. Arch Suicide Res 2013;17(3):275-287.

[5] Schlosser D, Campellone T, Kim D, Truong B, Vergani S, Ward C, et al. Feasibility of PRIME: a cognitive neuroscience-informed mobile app intervention to enhance motivated behavior and improve quality of life in recent onset schizophrenia. JMIR Res Protoc 2016 Apr 28;5(2):e77

[6] Kimhy D, Vakhrusheva J, Khan S, Chang RW, Hansen MC, Ballon JS, et al. Emotional granularity and social functioning in individuals with schizophrenia: an experience sampling study. J Psychiatr Res 2014 Jun; 53: 141-148

[7] Hartley S, Haddock G, Vasconcelos ES, Emsley R, Barrowclough C. An experience sampling study of worry and rumination in psychosis. Psychol Med 2014 Jun;44(8):1605-1614.

[8] Gleeson J, Lederman R, Wadley G, Bendall S, McGorry P, Alvarez-Jimenez M. Safety and privacy outcomes from a moderated online social therapy for young people with first-episode psychosis. Psychiatr Serv 2014 Apr 01;65(4):546-550

[9] Boulos MN, Gammon S, Dixon MC, MacRury SM, Fergusson MJ, Rodrigues FM, Baptista TM, Yang SP. Digital games for type 1 and type 2 diabetes: underpinning theory with three illustrative examples. JMIR Serious Games. 2015 Jan;3(1).

[10] Wilhide III CC, Peeples MM, Kouyaté RC. Evidence-based mHealth chronic disease mobile app intervention design: development of a framework. JMIR research protocols. 2016 Jan;5(1).

On 2017 Oct 22, George Kunos commented:

We read with interest the above article (Varga B, 2017), comparing the pharmacological properties of a series of cannabinoid receptor 1 (CB1R) blockers, including first generation brain-penetrant compounds and more recently introduced peripherally restricted compounds in various tests, including their anti-obesity effects in a mouse diet-induced obesity (DIO) model. The complete lack of effect of the peripheral CB1R inverse agonist JD-5037 in the DIO mouse model was of particular interest to us, as we have earlier documented its high anti-obesity efficacy in DIO mice (Tam J, 2012, Cinar R, 2014) and in a mouse model of Prader-Willi syndrome (Knani I, 2016), as well as its anti-diabetic efficacy in a rat model of type 2 diabetes (Jourdan T, 2013, Jourdan T, 2014). In these as well as in more recent studies (Tam J, 2017, Hinden L, 2018, Udi S, 2017), JD-5037 was dissolved in 4% DMSO/1% Tween-80 in PBS (vehicle #1) for administration by oral gavage (see Jourdan T, 2013), whereas Varga et al. used a 5% Tween-80 solution (vehicle #2). Because in our hands the inclusion of DMSO was critical for keeping this highly lipophilic compound in solution, we compared the oral bioavailability and peripheral target engagement of JD-5037 dissolved in these 2 vehicles. Using vehicle #1, the peak plasma concentration of JD-5037 measured by LC-MS/MS 1 h after oral administration to lean, male C57Bl6/J mice was 1076 ± 208 ng/mL (1840 nM), similar to values we published earlier (Tam J, 2012). In contrast, using vehicle #2, the plasma level of JD-5037 was 0.38 ± 0.02 ng/mL (0.67 nM), more than 1,000-fold lower and barely detectable. JD-5037 is 99.6% protein-bound in plasma (Tam J, 2012), so its calculated free concentration was 7.4 nM using vehicle #1 versus 2.7 pM using vehicle #2, the latter value being 2 orders of magnitude below the binding Kd of JD-5037 for CB1R (0.4 nM), which predicts no significant CB1R occupancy. We further verified this by using the upper GI motility test as a measure of peripheral CB1R occupancy (Tam J, 2012). In vehicle-treated mice, an oral charcoal bolus traveled 54 ± 3% of the length of the small intestine in 30 minutes, whereas In mice treated with a maximally effective dose of the CB1R agonist ACEA, the charcoal bolus traveled only 25 ± 3%, indicating a 54% inhibition. The inhibitory effect of ACEA was completely blocked by pretreatment with a single dose of 3 mg/kg JD-5037 in vehicle #1 (53 ± 3%), in agreement with our published data (Tam J, 2012). In contrast, the same dose of JD-5037 administered in vehicle #2 was completely without effect, the distance traveled by the charcoal bolus (29 ± 2%) being the same as with ACEA alone. Thus, the negative findings of Varga et al. can be attributed to lack of absorption and a consequent lack of peripheral CB1R occupancy by JD-5037, due to the use of an inappropriate vehicle. Such pitfalls are avoidable by verifying bioavailability and target engagement, which is a basic requirement when testing the in vivo efficacy of novel compounds.

George Kunos, Joseph Tam^1, Resat Cinar, Tony Jourdan; National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

¹ Current address: School of Pharmacy, The Hebrew University, Jerusalem, Israel

On 2017 Jul 11, Martine Crasnier-Mednansky commented:

The cya crp* mutant strain CA-8404 isolated by L. Soll (Sabourin D, 1975), which has been widely used for transduction of its crp* gene (also used in the present work), was finally characterized by Karimova G, 2004 as containing three mutations in the crp gene. Karimova G, 2004 further reported this CRP* was indeed capable of responding to cAMP and therefore was still sensitive to Carbon Catabolite Repression (CCR). Of interest to the present study, and any other studies aimed at releasing CCR in Escherichia coli, Karimova G, 2004 also characterized a novel CRP* with two mutations which totally relieved CCR as compared to the three-mutation CRP*.

Here cAMP-dependent CCR may not be the main culprit for preventing xylose utilization by the Escherichia coli wild type W strain (the Waksman’s strain). Generally, an increase in cAMP upon glucose depletion allows utilization of less-preferred sugar like xylose. Figure S7-A indicates that upon glucose exhaustion, E. coli W was still unable to use xylose after 96 hours, even though it could use xylose quite efficiently in the absence of glucose (Figure 2-D). In addition, the CRP* isolated by the authors (G141D) for specifically increasing xylose catabolism (Figure 1), and which doubled xylose utilization in the parent strain XW043, did not improve E. coli W xylose consumption at all in the presence of glucose (Figure 4-A). Thus, based on current knowledge, the inability of the Waksman’s strain to use xylose in the presence of a large excess glucose does not appear to relate to cAMP-dependent CCR. Interestingly, E. coli B, unlike W, is unable to use glucose fully when grown in excess glucose, and the typical increase in cAMP does not occur after cessation of growth (figure 2 in Peterkofsky A, 1971). Thus, if some glucose remains unused in the medium, cells may fail to use xylose.

On 2017 Jun 24, Sin Hang Lee commented:

Jorge Cervantes proposed a new theory to argue against the existence of chronic Lyme disease with persistent infection [1]. According to this theory, “after antibiotic eradication of Bb, its DNA is able to persist in anatomical locations that coincide with sites of inflammation.” He assumed that the free, naked and water-soluble DNA molecules released from the dying Borrelia burgdorferi spirochetes remain in the extracellular matrix of the patient’s tissues. However, under section “3. Borrelia DNA persistence” of his article, the references cited did not test for free borrelial DNA at all, and therefore do not back up his theory. For examples, in the reference by Li et al. [2], the authors concluded that the DNA detected was in moribund or dead B burgdorferi cells, not in free form. In the reference by Schmidt et al. [3] and the reference by Aberer et al. [4], borrelial DNA was detected in the pellet of patients’ urine samples after centrifugation at 14,000 x g and 36,000 × g, respectively. Since soluble free DNA molecules cannot be pelleted by such a low centrifugal force, the borrelial DNA detected by these authors must be still bound to bacterial cells or cell fragments in the urine. In the reference by Kubanek et al. [5], the authors actually showed by electron microscopy that the tissues tested positive for borrelial DNA clearly contained borrelial bacteria, not free DNA.

Free, extracellular, naked bacterial DNA is very prone to decay. Foreign DNA experimentally introduced into a mammal is degraded and eliminated from the host’s blood within 48 hours [6]. But the stability of extracellular DNA still depends on its form or even on the sequence of its nucleotide bases. Circular plasmid DNA is more stable in vitro than a segment of linear chromosomal DNA after release from the bacterial cell. Even DNase I does not cleave DNA randomly although not base nor sequence specific. Extracellular bacterial 16S rDNA is known to be degraded much more rapidly in the environment than those bound to cell fragments [7]. Borrelial 16S rDNA extracted by ammonium hydroxide stored in TE buffer is stable, but is degraded rapidly in human serum at room temperature (unpublished personal observation). DNA sequencing-confirmed detection of borrelial 16S rDNA in the pellet of serum or plasma samples derived from patients’ venous blood constitutes solid molecular evidence of spirochetemia in Lyme borreliosis [8, 9]. Whether spirochetemia in chronic Lyme disease needs to be treated with prolonged antibiotics is an important heath care issue which should be further discussed. To push an elusive DNA-binding AMP treatment of chronic Lyme disease can only direct the attention away from the real issue of how to define Lyme disease, acute or chronic, as an emerging infectious disease, like Ebola and Zika, for proper patient management. There is no evidence that free naked borrelial DNA has been demonstrated in any patient samples.

The author should also cite a reference to back up his claim that human macrophages can remove extracellular Bb DNA. The reference by Brencicova and Diebold [10], cited by the author, clearly stated “Endosomal TLR are situated in the membrane of the endolysosomal compartment of APC and sample the content of these compartments for the presence of nucleic acid agonists. Pathogens or dead cells gain access to the compartment by endocytosis. Alternatively, infection-induced autophagy can shuttle viral nucleic acids and antigens into the endolysosomal compartment and allow for recognition of replicating virus within infected cells by endosomal TLR.” Free DNA was not mentioned.

References [1] Cervantes J. Doctor says you are cured, but you still feel the pain. Borrelia DNA persistence in Lyme disease. Microbes Infect 2017 Jun 15. pii: S1286-4579(17)30090-4. doi: 10.1016/j.micinf.2017.06.002. [Epub ahead of print] Review. [2] Li X, McHugh GA, Damle N, Sikand VK, Glickstein L, Steere AC. Burden and viability of Borrelia burgdorferi in skin and joints of patients with erythema migrans or lyme arthritis. Arthritis Rheum 2011;63: 2238-47. [3] Schmidt B, Muellegger RR, Stockenhuber C, Soyer HP, Hoedl S, Luger A, et al. Detection of Borrelia burgdorferi-specific DNA in urine specimens from patients with erythema migrans before and after antibiotic therapy. J Clin Microbiol 1996;34:1359-63. [4] Aberer E, Bergmann AR, Derler AM, Schmidt B. Course of Borrelia burgdorferi DNA shedding in urine after treatment. Acta Derm Venereol 2007;87(1):39-42. [5] Kubanek M, Sramko M, Berenova D, Hulinska D, Hrbackova H, Maluskova J, et al. Detection of Borrelia burgdorferi sensu lato in endomyocardial biopsy specimens in individuals with recent-onset dilated cardiomyopathy. Eur J Heart Fail 2012;14:588-96. [6] Schubbert R, Renz D, Schmitz B, Doerfler W. Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA. Proc Natl Acad Sci U S A. 1997;94:961-6. [7] Corinaldesi C, Danovaro R, Dell'Anno A. Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments. Appl Environ Microbiol 2005;71:46-50. [8] Lee SH, Vigliotti JS, Vigliotti VS, Jones W, Shearer DM. Detection of borreliae in archived sera from patients with clinically suspect Lyme disease. Int J Mol Sci. 2014;15:4284-98. [9] Lee SH. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report. Int Med Case Rep J. 2016;9:101-6. [10] Brencicova E, Diebold SS. Nucleic acids and endosomal pattern recognition: how to tell friend from foe? Front Cell Infect Microbiol 2013;3:37.

On 2017 Jul 24, Frank M Sacks commented:

On behalf of the authors, I respond to comments by Hilda Bastian about the American Heart Association Presidential Advisory on Dietary Fats and Cardiovascular disease Sacks FM, 2017.

The comprehensive advisory includes, (i) Clinical trials that tested the effects of dietary saturated fat compared to unsaturated fat or carbohydrate on cardiovascular disease (CVD) events, e.g. heart attack, (ii) Clinical trials that tested the effects of dietary fats on lipid risk factors, e.g. LDL-cholesterol, (iii) Prospective epidemiological studies on dietary fats and carbohydrates and CVD, and (iv) Animal models of diet and atherosclerosis. Thus, it reflects the “totality of evidence”. The confluence of findings provides a very strong scientific case for the recommendation that dietary saturated fat be replaced with unsaturated fat, especially polyunsaturated fat.

Recent systematic reviews and meta-analyses Mozaffarian D, 2010, Chowdhury R, 2014, Hooper <PMID: 26068959 used well accepted methodologies, and included trials published up to 2009, 2013, and 2014. Only a small number of clinical trials evaluated direct effects of dietary fat on CVD. Most of these studies, and all that have an impact on the overall findings, were conducted years ago, and are well known. Contrary to the Bastian’s comments, there are no more recent trials on this topic. These 3 meta-analyses each confirm the beneficial effect of replacing saturated with polyunsaturated fat. The similarity of findings lends robustness to the overall conclusions of the report. The meta-analyses and all the individual trials are discussed critically in detail in the advisory.

Because the topic of the advisory is the effect of dietary fats on CVD, coconut oil is well within its scope. Coconut oil is currently rated as a healthy oil by 72% of the American public, despite its composition derived from 98% saturated fats, which increase the blood level of LDL-cholesterol, a cause of atherosclerosis and CVD. The meta-analysis by Mensink reports the quantitative effects on LDL-cholesterol of the saturated fats that are in coconut oil, mainly lauric, myristic, and palmitic acids. Each of these increase LDL-cholesterol compared to carbohydrate and more so when compared to the unsaturated fats. This is sufficient to warn the public about anticipated adverse effects of coconut oil on CVD.

Some studies tested coconut oil itself, and found that it increases LDL-cholesterol as would be predicted by its saturated fat content. These studies were identified and summarized in the systematic review by Eyres L, 2016 which used rigorous, well-accepted methodology. The criteria for inclusion of an article in the systematic review were well conceived. Eyres et al. concluded, “Overall, the weight of the evidence from intervention studies to date suggests that replacing coconut oil with cis unsaturated fats would alter blood lipid profiles in a manner consistent with a reduction in risk factors for cardiovascular disease.” Bastian implies that this systematic review is composed of weak studies and omitted several studies that would affect the conclusion of the advisory to avoid eating coconut oil. This is not true. Eyres et al. identified eight studies; all were controlled clinical trials that used valid nutritional protocols and statistical analyses. All reported higher LDL-C levels when coconut oil was consumed compared to unsaturated oils, including olive, corn and soybean oils, statistically significantly in 7 of them. Together, these trials included populations from the US, Sri Lanka, New Zealand, Pacific Islands, and Malaysia, demonstrating generalizability. There is no objective scientific reason to disparage them. The only substantive criticisms mentioned by Bastian are a short duration and small sample. These criticisms are unwarranted. Effects of diet on blood lipids, especially LDL-cholesterol, are established quickly, by 2 weeks. A small sample, with careful dietary control and execution, can yield a well-powered trial with valid results. In summary, the 8 trials in the Eyres et al. systematic review provide strong evidence that coconut oil increases LDL-C levels compared with unsaturated oils.

What about the 7 studies named by Bastian that were not included in the systematic review? McKenney JM, 1995 reported that coconut oil increased LDL-cholesterol significantly by 12% compared with canola oil in 11 patients with hypercholesterolemia. In a second study in 17 patients treated with lovastatin, LDL-C increased nonsignificantly in the coconut oil period. Thus, the results of this small study would add to the overall effects of coconut oil shown in the other studies to increase LDL-cholesterol. Ganji V, 1996 reported that coconut oil increased LDL-cholesterol compared to soybean oil in 10 normal participants. Assunção ML, 2009 reported no difference in the effects of coconut and soybean oils on LDL-cholesterol levels. However, LDL-cholesterol levels increased during the soybean oil period, clearly an anomolous result. Cardoso DA, 2015 conducted a nonrandomized study comparing coconut oil, 13 mL per day, with no supplemental oil. Because there is no control for the coconut oil, it is unclear how to interpret the lack of difference in LDL-cholesterol between the groups. de Paula Franco E, 2015 conducted a sequential study of a calorie-reduced diet followed by coconut flour, 26 g per day. This study was not randomized and did not have a control group. Enns reported in her Ph.D. degree dissertation at the University of Manitoba the results of a randomized trial that compared a 2:1:1 mix of butter, coconut oil, and high-linoleic safflower oil, 25 g per day, with canola oil, 25 g per day. This trial did not claim to be a study on the effects of coconut oil. Finally, Shedden reported in her M.S. degree thesis at Arizona State University the results of a placebo-controlled randomized trial of coconut oil, 2 g per day. This miniscule amount of coconut oil did not affect LDL-cholesterol. In summary, among the 7 studies cited by Bastian not in the Eyers review, 4 would appropriately be excluded as result of being non-randomized, uncontrolled, using a very small amount, not including a control group or not even being a trial of coconut oil. Among the 3 randomized trials, McKenney et al., Ganji et al. and Assuncao et al., the first two found that coconut oil increased LDL-cholesterol levels. The trial of Assuncao et al. would likely fail an outlier test because it is the only one among 12 studies in which LDL-C levels is lower on coconut than soybean oil. Given the differences in study designs, populations, and localities, the results of coconut oil trials are remarkably uniform showing that it increases LDL-cholesterol levels, an established cause of cardiovascular disease.

Bastian employs a tactic in common with some other critics of good nutritional science, namely, to a) disparage and misrepresent high quality studies that show harmful effects of saturated fat; b) promote and misrepresent seriously flawed and irrelevant studies that report the opposite; and c) cite meta-analyses with faulty designs often based on inclusion of flawed studies. We offer a challenge to those who assert health benefits to coconut oil, or saturated fat, in general. Produce well-designed and executed studies that show that there are beneficial effects on a bona fide health outcome or a recognized surrogate, e.g., LDL-cholesterol.

Frank M. Sacks, for the authors.

On 2017 Jun 18, Raphael Stricker commented:

Chronic Lyme Disease Treatment: Science versus Anecdotes.

Lorraine Johnson, Raphael B. Stricker, MD.

Lymedisease.org, PO Box 1352, Chico, CA 95927; ILADS, PO Box 341461, Bethesda, MD 20827

lorrainejohnson@outlook.com; rstricker@usmamed.com

The article by Marzec et al. published in MMWR purports to show the dangers of treatment in patients diagnosed with chronic Lyme disease (1). Recent reports from the Centers for Disease Control and Prevention (CDC) indicate that more than 300,000 new cases of Lyme disease are diagnosed each year in the USA (2). The MMWR article from the CDC describes five anecdotal cases of treatment complications in these patients while ignoring the significant morbidity related to denial of treatment for chronic Lyme disease (2,3). The resultant biased report raises scientific and ethical issues about the CDC's role in promoting the best care for patients with tickborne diseases.

The MMWR piece resulted from anecdotal reports gathered by Dr. Christina Nelson of the CDC. The article notes that the information was gathered because “clinicians and state health departments periodically contact CDC concerning patients who have acquired serious bacterial infections during treatments for chronic Lyme disease.” However, an ethics complaint filed against Dr. Nelson by the Lyme disease patient advocacy group LymeDisease.org suggests that these adverse event reports were in fact specifically solicited by Dr. Nelson via emails distributed in 2014 (4). Dr. Nelson asked clinicians from the Infectious Diseases Society of America (IDSA) to provide anecdotal evidence of harm to patients from intravenous antibiotic therapy related to Lyme disease, and she apparently offered coauthorship of her article as an incentive to describe these adverse events. She did not ask for consequences of failing to treat these patients, nor did she solicit commentary from practitioners who treat chronic Lyme disease according to the guidelines of the International Lyme and Associated Diseases Society (ILADS).

The risk of any medical treatment is extremely context-sensitive. A crucial question is whether the risks of treatment are warranted given the potential benefits, the availability of other treatment options, the severity of the patient's presentation, and the risk tolerance of the individual patient. By asking for an assessment of treatment risks only, Dr. Nelson is framing the issue in a manner that excludes the other half of the equation in a risk/benefit assessment. She is also ignoring an issue that is critical to patients who suffer a profoundly diminished quality of life due to their illness, namely the risk of not treating (5,6). Moreover, by failing to mention that these adverse event reports were rare and specifically solicited, she implies that these rare occurrences are a common concern. In reality, studies of the risks and benefits associated with intravenous antibiotic treatment for Lyme disease indicate that the risks of adverse events are no greater than the risks of intravenous therapy in other unrelated diseases (7,8).

By asking the question only of those on one side of the controversy, Dr. Nelson is further demonstrating favoritism and a lack of impartiality on the part of the CDC. Accordingly, Dr. Nelson's solicitation of anecdotal adverse events for case studies of Lyme disease is a highly inappropriate partisan act of favoritism toward the IDSA viewpoint at the expense of critical stakeholders - Lyme disease patients and their treating physicians - and an attack on the ILADS viewpoints.

References 1. Marzec NS, Nelson C, Waldron PR, et al. Serious bacterial infections acquired during treatment of patients given a diagnosis of chronic Lyme disease - United States. MMWR Morb Mortal Wkly Rep. 2017 Jun 16;66(23):607-609. 2. Stricker RB, Johnson L. Lyme disease: Call for a ‘‘Manhattan Project’’ to combat the epidemic. PLoS Pathog. 2014;10(1): e1003796. 3. Stricker RB, Fesler MC. Chronic Lyme disease: A working case definition. Chronic Dis Int. 2017; 4(1): 1025. 4. Leland DK. TOUCHED BY LYME: CDC ignores ethics, attacks “chronic Lyme”. Available at https://www.lymedisease.org/touchedbylyme-cdc-ignores-ethics/. Accessed June 16, 2017. 5. Johnson L, Aylward A, Stricker RB. Healthcare access and burden of care for patients with Lyme disease: a large United States survey. Health Policy. 2011;102: 64–71. 6. Johnson L, Wilcox S, Mankoff J, Stricker RB. Severity of chronic Lyme disease compared to other chronic conditions: a quality of life survey. Peer J. 2014;2:e322. 7. Stricker RB, Green CL, Savely VR, Chamallas SN, Johnson L. Safety of intravenous antibiotic therapy in patients referred for treatment of neurologic Lyme disease. Minerva Med. 2010;101:1–7. 8. Stricker RB, Delong AK, Green CL, et al. Benefit of intravenous antibiotic therapy in patients referred for treatment of neurologic Lyme disease. Int J Gen Med. 2011; 4: 639–646. Disclosure: RBS and LJ are members of the International Lyme and Associated Diseases Society (ILADS) and directors of LymeDisease.org. They have no financial or other conflicts to declare.

On 2017 Sep 12, David Keller commented:

Pre-Existing Rheumatoid Arthritis Should Increase, Not Reduce, the Risk of Incident Parkinson Disease

The authors of this editorial ask "Would tamping down the immune system be a good thing for PD (Parkinson disease) symptoms, or would activation of the immune system be advantageous?"[1] They cite epidemiologic studies which showed "a higher risk of PD in patients with type 1 diabetes mellitus (T1DM) and Crohn disease (CD), among others [2]", in conflict with studies that showed a decreased risk of PD in patients with rheumatoid arthritis (RA). So, does the presence of RA increase or decrease the incidence of PD?

Genome-wide association studies (GWAS) found "enrichment" of loci associated with Parkinson disease conditional on the presence of loci associated with each of 7 autoimmune diseases, to varying degrees. The graphs in Figure 1 [3] demonstrate that for all 7 autoimmune diseases, the greater the population of SNPs associated with autoimmunity, the greater the enrichment of PD SNPs. The authors designate this as "leftward" deviation of the curves, although mathematically it is really UPWARD deviation from the straight line representing the null hypothesis. So, in genetic studies, all seven of the tested autoimmune diseases (T1DM, CD, Ulcerative Colitis, Celiac Disease, Psoriasis, Multiple Sclerosis and Rheumatoid Arthritis) were genetically associated with increased risk of incident PD.

How can genetic risk of PD increase directly with the genetic risk of RA, yet epidemiological studies demonstrate an inverse association of established RA disease on the incidence of PD? The authors of one such epidemiological study did not believe their own results, and hypothesized that "the decreased risk [of incident PD] among patients with RA might be explained by underdiagnosis of movement disorders such as PD in this patient group, or by a protective effect of treatment with anti-inflammatory drugs over prolonged periods." [4] In other words, early signs of PD, such as bradykinesia, could be masked in RA patients, in whom slow movement might be attributed to pain or joint destruction, and ibuprofen use could have further confounded their results.

The nonsteroidal antiinflammatory drugs (NSAID) have been studied extensively, and the only one which significantly reduces the risk of incident PD is ibuprofen.[5] A study by Sung and colleagues [6] concluded that pre-existing RA reduces the risk of incident PD, but they corrected their data for the use of any NSAID, rather than the use of ibuprofen, introducing systematic errors in their results, and potentially invalidating their conclusions. [7]

Can a destructive autoimmune disease like RA reduce the risk of incident PD, in contrast to 6 other autoimmune diseases, which raise risk for PD? Or, do symptom masking and the protective effects of ibuprofen explain the reduction in incident PD seen in patients with RA? In an unpublished reply to these arguments, Sung's group wrote: "[Keller's] criticism focuses on the issue whether [any] non-aspirin NSAID or ibuprofen only, has the truly protective effect against the development of PD", and agreed that "ibuprofen was associated with decreased risk of PD, but not aspirin or other NSAIDs" and concluded that "ibuprofen use should be considered as an important covariable in future correlational research in PD." [8]

References

1: McFarland NR, McFarland KN, Golde TE. Parkinson Disease and Autoimmune Disorders-What Can We Learn From Genome-wide Pleiotropy? JAMA Neurol. 2017 Jul 1;74(7):769-770. doi: 10.1001/jamaneurol.2017.0843. PubMed PMID: 28586798.

2: Lin JC, Lin CS, Hsu CW, Lin CL, Kao CH. Association Between Parkinson's Disease and Inflammatory Bowel Disease: a Nationwide Taiwanese Retrospective Cohort Study. Inflamm Bowel Dis. 2016 May;22(5):1049-55. doi: 10.1097/MIB.0000000000000735. PubMed PMID: 26919462.

3: Witoelar A, Jansen IE, et al. for the International Parkinson’s Disease Genomics Consortium. Genome-wide Pleiotropy Between Parkinson Disease and Autoimmune Diseases. JAMA Neurol. 2017;74(7):780–792. doi:10.1001/jamaneurol.2017.0469

4: Rugbjerg K, Friis S, Ritz B, Schernhammer ES, Korbo L, Olsen JH. Autoimmune disease and risk for Parkinson disease: a population-based case-control study. Neurology. 2009 Nov 3;73(18):1462-8. doi: 10.1212/WNL.0b013e3181c06635. Epub 2009 Sep 23. PubMed PMID: 19776374; PubMed Central PMCID: PMC2779008.

5: Gao X, Chen H, Schwarzschild MA, Ascherio A. Use of ibuprofen and risk of Parkinson disease. Neurology. 2011 Mar 8;76(10):863-9. doi: 10.1212/WNL.0b013e31820f2d79. Epub 2011 Mar 2. PubMed PMID: 21368281; PubMed Central PMCID: PMC3059148.

6: Sung YF, Liu FC, Lin CC, Lee JT, Yang FC, Chou YC, Lin CL, Kao CH, Lo HY, Yang TY. Reduced Risk of Parkinson Disease in Patients With Rheumatoid Arthritis: A Nationwide Population-Based Study. Mayo Clin Proc. 2016 Oct;91(10):1346-1353. doi: 10.1016/j.mayocp.2016.06.023. PubMed PMID: 27712633.

7: Keller DL, Only ibuprofen is associated with reduced PD risk - controlling for use of any NSAID introduces error. PubMed Commons Comment, accessed on 9/12/2017 at the following URL: https://www.ncbi.nlm.nih.gov/pubmed/27712633#cm27712633_34408

8: Sung YF, Lin CL, Kao CH, and Yang TY. Reply to Keller's unpublished letter to Mayo Clinic Proceedings. Received by email on November 22, 2016.

On 2017 Aug 29, Andreas Lundh commented:

Comment on Association of streptococcal throat infection with mental disorders

A recent Danish register-based cohort study(1) concludes that “individuals with a streptococcal throat infection had elevated risks of mental disorders, particularly OCD and tic disorders.” However, some methodological issues need consideration.

Firstly, the choice of exposure has a risk of misclassification. Exposure (i.e. Group A Streptococcal (GAS) throat infection) was the combination of a rapid antigen test performed by the patient’s general practitioner and subsequent antibiotic prescription. In Denmark use of rapid antigen test and antibiotic prescription are guided by modified Centor criteria(2,3) where symptomatic patients at high risk of GAS throat infection are not tested, but instead receive empirical antibiotic treatment. This leads to patients being misclassified as unexposed since they are never tested.

Secondly, the choice of outcome has a similar risk of misclassification. Psychiatric diagnoses are identified from national databases that only contain hospital information. Psychiatric patients that are not treated in hospitals (e.g. treated by primary care psychiatrists) are misclassified as not having had the outcome. Misclassification seems likely as only 0.1% and 0.2%, respectively, had a diagnosis of OCD or tics in the study period.

Thirdly, the analytical strategy has a risk of bias. The authors compared patients that had received both a rapid antigen test and antibiotics with a group that was never tested. GAS throat infection will in most cases resolve spontaneously and many patients will never contact their general practitioner for testing. The group tested therefore likely differs from the group not being tested and represents a group with certain healthcare seeking behavior. This is substantiated by the findings that risk of mental disorders seems to increase with number of tests and regardless of whether the tests are negative or positive. A more reasonable analysis that avoids confounding by test indication would be to compare the group of tested patients prescribed antibiotics with the group of tested patients without prescribed antibiotics. This comparison weakens the association and it is no longer statistically significant for tics.

Instead of describing this as a possible source of bias the authors conclude that nonstreptococcal throat infection was also associated with increased risk of mental disorders, a theory that was not part of the original study hypothesis. Another interpretation is that these associations can be explained by a certain healthcare seeking behavior of patients and parents leading to an increased probability of receiving an antigen test, being prescribed an antibiotic and being treated in hospital.

References

1) Orlovska S, Vestergaard CH, Bech BH, Nordentoft M, Vestergaard M, Benros ME. Association of Streptococcal Throat Infection With Mental Disorders: Testing Key Aspects of the PANDAS Hypothesis in a Nationwide Study. JAMA Psychiatry 2017;74:740-6.

2) Bjerrum L, Gahrn-Hansen B, Hansen MP, Córdoba G, Aabenhus R, Monrad RN. [Airway infections – diagnosis and treatment. Clinical guideline for general practitioners]. Copenhagen: Danish College of General Practitioners; 2014.

3) Choby BA. Diagnosis and treatment of streptococcal pharyngitis. Am Fam Physician 2009; 79:383-90.

On 2017 Jun 12, John Sotos commented:

West describes angiogenesis, erythropoiesis, and vasoconstriction as clinical sequelae arising from chronically hypoxic tissues at altitude. Another, similar, effect is solid-organ hyperplasia.

In the late 1960s a Peruvian medical student observed several-fold enlargement of the carotid bodies in Andean altitude dwellers (1,2). The degree of enlargment increased with time spent at altitude and, in animal models, reversed after restoration of normoxia(3). Interestingly, this hyperplasia is mediated via endothelin signalling, not by hypoxia-inducible-factors(4).

Tissue hyperplasia may also occur in hypoxic patients at sea level. For example, even before the Peruvian discovery, hyperplasia -- and sometimes malignancy -- of adrenal chromaffin cells, i.e. pheochromocytomas, were described in adults having uncorrected cyanotic congenital heart disease(5). Carotid body cells and adrenal chromaffin cells have similar lineage (from the neural crest) and function (oxygen sensing).

(1) Arias-Stella J. Human carotid body at high altitudes. (Abstract). American Journal of Pathology. 1969; 55: 82a.

(2) Heath D. The carotid bodies in chronic respiratory disease. Histopathology. 1991; 18: 281-283.

(3) Kay JM, Laidler P. Hypoxia and the carotid body. J Clin Pathol Suppl (R Coll Pathol). 1977; 11: 30-44.

(4) Platero-Luengo A, González-Granero S, Durán R, Díaz-Castro B, Piruat J, García-Verdugo JM, Pardal R, López-Barneo J. An O2-Sensitive Glomus Cell-Stem Cell Synapse Induces Carotid Body Growth in Chronic Hypoxia. Cell. 2014; 156, 291–303.

(5) Folger GM, Roberts WC, Mehrizi A, Shah KD, Glancy DL, Carpenter CCJ, Esterly JR. Cyanotic Malformations of the Heart with Pheochromocytoma: A Report of Five Cases. Circulation. 1964; 29: 750-757.

On 2017 Aug 06, John Greenwood commented:

(cross-posted from Pub Peer, comment numbers refer to that discussion but content is the same)

To address your comments in reverse order -

Spatial vision and spatial maps (Comment 19):

We use the term “spatial vision” in the sense defined by Russell & Karen De Valois: “We consider spatial vision to encompass both the perception of the distribution of light across space and the perception of the location of visual objects within three-dimensional space. We thus include sections on depth perception, pattern vision, and more traditional topics such as acuity." De Valois, R. L., & De Valois, K. K. (1980). Spatial Vision. Annual Review of Psychology, 31(1), 309-341. doi:doi:10.1146/annurev.ps.31.020180.001521

The idea of a "spatial map” refers to the representation of the visual field in cortical regions. There is extensive evidence that visual areas are organised retinotopically across the cortical surface, making them “maps". See e.g. Wandell, B. A., Dumoulin, S. O., & Brewer, A. A. (2007). Visual field maps in human cortex. Neuron, 56(2), 366-383.

Measurement of lapse rates (Comments 4, 17, 18):

There really is no issue here. In Experiment 1, we fit a psychometric function in the form of a cumulative Gaussian to responses plotted as a function of (e.g.) target-flanker separation (as in Fig. 1B), with three free parameters: midpoint, slope, and lapse rate. The lapse rate is 100-x where x is the asymptote of the curve. It accounts for lapses (keypress errors etc) when performance is otherwise high - i.e. it is independent of the chance level. In this dataset it is never about 5%. However its inclusion does improve estimate of slope (and therefore threshold) which we are interested in. Any individual differences are therefore better estimated by factoring out individual differences in lapse rate. Its removal does not qualitatively affect the pattern of results in any case. You cite Wichmann and Hill (2001) and that is indeed the basis of this three-parameter fit (though ours is custom code that doesn’t apply the bootstrapping procedures etc that they use).

Spatial representations (comment 8):

We were testing the proposal that crowding and saccadic preparation might depend on some degree of shared processes within the visual system. Specific predictions for shared vs distinct spatial representations are made on p E3574 and in more detail on p E3576 of our manuscript. The idea comes from several prior studies arguing for a link between the two, as we cite, e.g.: Nandy, A. S., & Tjan, B. S. (2012). Saccade-confounded image statistics explain visual crowding. Nature Neuroscience, 15(3), 463-469. Harrison, W. J., Mattingley, J. B., & Remington, R. W. (2013). Eye movement targets are released from visual crowding. The Journal of Neuroscience, 33(7), 2927-2933.

Bisection (Comments 7, 13, 15):

Your issue relates to biases in bisection. This is indeed an interesting area, mostly studied for foveal presentation. These biases are however small in relation to the size of thresholds for discrimination, particularly for the thresholds seen in peripheral vision where our measurements were made. An issue with bias for vertical judgements would lead to higher thresholds for vertical vs. horizontal judgements, which we don’t see. The predominant pattern in bisection thresholds (as with the other tasks) is a radial/tangential anisotropy, so vertical thresholds are worse than horizontal on the vertical meridian, but better than horizontal thresholds on the horizontal meridian. The role of biases in that anisotropy is an interesting question, but again these biases tend to be small relative to threshold.

Vernier acuity (Comment 6):

We don’t measure vernier acuity, for exactly the reasons you outline (stated on p E3577).

Data analyses (comment 5):

The measurement of crowding/interference zones follows conventions established by others, as we cite, e.g.: Pelli, D. G., Palomares, M., & Majaj, N. J. (2004). Crowding is unlike ordinary masking: Distinguishing feature integration from detection. Journal of Vision, 4(12), 1136-1169.

Our analyses are certainly not post-hoc exercises in data mining. The logic is outlined at the end of the introduction for both studies (p E3574).

Inclusion of the authors as subjects (Comment 3):

In what way should this affect the results? This can certainly be an issue for studies where knowledge of the various conditions can bias outcomes. Here this is not true. We did of course check that data from the authors did not differ in any meaningful way from other subjects (aside from individual differences), and it did not. Testing (and training) experienced psychophysical observers takes time, and authors tend to be experienced psychophysical observers.

The theoretical framework of our experiments (Comments 1 & 2):

We make an assumption about hierarchical processing within the visual system, as we outline in the introduction. We test predictions that arise from this. We don’t deny that feedback connections exist, but I don’t think their presence would alter the predictions outlined at the end of the introduction. We also make assumptions regarding the potential processing stages/sites underlying the various tasks examined. Of course we can’t be certain about this (and psychophysics is indeed ill-poised to test these assumptions) and that is the reason that no one task is linked to any specific neural locus, e.g. crowding shows neural correlates in visual areas V1-V4, as we state (e.g. p E3574). Considerable parts of the paper are then addressed at considering whether some tasks may be lower- or higher-level than others, and we outline a range of justifications for the arguments made. These are all testable assumptions, and it will be interesting to see how future work then addresses this.

All of these comments are really fixated on aspects of our theoretical background and minor details of the methods. None of this in any way negates our findings. Namely, there are distinct processes within the visual system, e.g. crowding and saccadic precision, that nonetheless show similarities in their pattern of variations across the visual field. We show several results that suggest these two processes to be dissociable (e.g. that the distribution of saccadic errors is identical for trials where crowded targets were correctly vs incorrectly identified). If they’re clearly dissociable tasks, how then to explain the correlation in their pattern of variation? We propose that these properties are inherited from earlier stages in the visual system. Future work can put this to the test.

On 2017 Apr 11, Yu-Chen Liu commented:

The authors appreciate the insightful feedbacks and agree with prospect that hypothesis derived from small RNA-seq data analysis deserve examination in skeptical views and further experimental validation. Regarding the skeptical view of Prof. Witwer on this issue, whether a specific sequence were indeed originate from plant can be validated through examining the 2’-O-methylation on their 3’ end (Chin, et al., 2016; Yu, et al., 2005). The threshold of potential copy per cell for plant miRNAs to affect human gene expression was also discussed in previous researches (Chin, et al., 2016; Zhang, et al., 2012).

Some apparent misunderstandings are needed to be clarified:

In the commentary of Prof. Witwer:

“A cross-check of the source files and articles shows that the plasma data evaluated by Liu et al were from 198 plasma samples, not 410 as reported. Ninomiya et al sequenced six human plasma samples, six PBMC samples, and 11 cultured cell lines 19. Yuan et al sequenced 192 human plasma libraries (prepared from polymer-precipitated plasma particles). Each library was sequenced once, and then a second time to increase total reads.”

Authors’ response:

First of all, the statement "410 samples" within the article was meant to the amount of runs of small RNA-seq run conducted in the referred researches. Whether multiple NGS runs conducted on same plasma sample should be count as individual experiment replicates is debatable. The analysis of each small RNA-seq run was conduct independently. The authors appreciate the kind comments for the potential confusion that can be made in this issue.

In the commentary of Prof. Witwer:

“Strikingly, the putative MIR2910 sequence is not only a fragment of plant rRNA; it has a 100% coverage, 100% identity match in the human 18S rRNA (see NR 003286.2 in GenBank; Table 3). These matches of putative plant RNAs with human sequences are difficult to reconcile with the statement of Liu et al that BLAST of putative plant miRNAs "resulted in zero alignment hit", suggesting that perhaps a mistake was made, and that the BLAST procedure was performed incorrectly.”

Authors’ response:

The precursor sequences of the plant miRNAs, including the stem loop sequences (precursor sequences) were utilized in the BLAST sequence alignment in this work. The precursor sequence of peu-MIR2910, “UAGUUGGUGGAGCGAUUUGUCUGGUUAAUUCCGUUAACGAACGAGACCUCAGCCUGCUA” was used. The alignment was not performed merely with the mature sequence, “UAGUUGGUGGAGCGAUUUGUC”. The stem loop sequences, as well as the alignment of the sequences against the plant genomes, was taken into consideration by using miRDeep2 (Friedländer, et al., 2012). As illustrated in the provided figures, sequencing reads were mapped to the precursor sequences of MIR2910 and MIR2916. As listed in the table below, a lot of sequencing reads can be aligned to other regions within the precursor sequences except the sequencing reads aligned to mature sequences. For instance, in small RNA-seq data of DRR023286, 5369 reads were mapped to peu-MIR2910, and 4010 reads were mapped to the other regions in the precursor sequences.  

miRNA | Run |Total reads | on Mature | on precursor

peu-MIR2910 | DRR023286 | 9370 | 5369 | 4010

peu-MIR2910 | SRR2105454 | 3013 | 1433 | 1580

peu-MIR2914 | DRR023286 | 1036 | 19 | 1017

peu-MIR2916 |SRR2105342 | 556 | 227 | 329

(Check the file MIR2910_in_DRR023286.pdf, MIR2910_in_SRR2105454.pdf, MIR2914_in_DRR023286 and MIR2916_in_SRR2105342.pdf)

The pictures are available in the URL:

https://www.dropbox.com/sh/9r7oiybju8g7wq2/AADw0zkuGSDsTI3Aa_4x6r8Ua?dl=0

As described in the article, all reported reads mapped onto the plant miRNA sequences were also mapped onto the five conserve plant genomes. Within the provided link a compressed folder file “miRNA_read.tar.gz” is available. Results of the analysis through miRDeep2, were summarized in these pdf files. Each figure file was named according to the summarized reads, sequence run and the mapped plant genome. For example, reads from the run SRR2105181 aligned onto both Zea mays genome and peu-MIR2910 precursor sequences are summarized in the figure file “SRR2105181_Zea_mays_peu-MIR2910.pdf”.

In the commentary of Prof. Witwer:

“Curiously, several sequences did not map to the species to which they were ascribed by the PMRD. Unfortunately, the PMRD could not be accessed directly during this study; however, other databases appear to provide access to its contents.”

Authors’ response:

All the stem loop sequences of plant miRNAs were acquired from the 2016 updated version of PMRD (Zhang, et al., 2010), which was not properly referred. The used data were provided in the previously mentioned URL.

In the commentary of Prof. Witwer:

“Counts were presented as reads per million mapped reads (rpm). In contrast, Liu et al appear to have reported total mapped reads in their data table. Yuan et al also set an expression cutoff of 32 rpm (log2 rpm of 5 or above). With an average 12.5 million reads per sample (the sum of the two runs per library), and, on average, about half of the sequences mapped, the 32 rpm cutoff would translate to around 200 total reads in the average sample as mapped by Liu et al.”

Authors’ response:

Regarding the concern of reads per million mapped reads (rpm) threshold, the author appreciate the kind remind of the need to normalize sequence reads count into the unit in reads per million mapped reads (rpm) for proper comparison between samples of different sequence depth. However the comparison was unfortunately not conducted in this work. Given the fact that the reads were mapped onto plant genome instead of human genome, the normalization would be rather pointless, considering the overall mapped putative plant reads only consist of ~3% of the overall reads. On the other hand, the general amount of cell free RNA present in plasma samples was meant to be generally lower than within cellar samples (Schwarzenbach, et al., 2011).

Reference

Chin, A.R., et al. Cross-kingdom inhibition of breast cancer growth by plant miR159. Cell research 2016;26(2):217-228.

Friedländer, M.R., et al. miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic acids research 2012;40(1):37-52.

Schwarzenbach, H., Hoon, D.S. and Pantel, K. Cell-free nucleic acids as biomarkers in cancer patients. Nature Reviews Cancer 2011;11(6):426-437.

Yu, B., et al. Methylation as a crucial step in plant microRNA biogenesis. Science 2005;307(5711):932-935.

Zhang, L., et al. Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell research 2012;22(1):107-126.

Zhang, Z., et al. PMRD: plant microRNA database. Nucleic acids research 2010;38(suppl 1):D806-D813.

On 2017 Jun 12, Bastian Fromm commented:

Summary The author describes the results of a combined smallRNA sequencing and blasting approach in Taenia ovis. Specifically RNA was retrieved from Tov metacercaria and then mapped to the genome of T. solium. Mapping reads are then blasted against miRBase and so the author describes 34 miRNAs as present in Tov.

Major problems

1. The author uses miRBase as reference for cestode miRNAs although it is very outdated (last update 2014). The author should rather have used available literature for comparisons (1-9).
2. Consequently (?) the author fails to acknowledge his results in the light of standard work in the field of miRNA evolution in flatworms (10-12) and to draw conclusions about the completeness of his predictions.
3. The approach of mapping a smallRNA sequencing library of a given species against another is problematic and I cannot understand why no the author does not at least try to use classical PCR to confirm loci.

Minor problems 1) page 3 line 61 author should make sentence more clear. It looks like author removed all reads that had adapter sequences. Recommendation 1) Author should get all available PRE-sequences for cestodes and ma his Tov reads with liberal settings to them and report results.

1. Jiang, S., Li, X., Wang, X., Ban, Q., Hui, W. and Jia, B. (2016) MicroRNA profiling of the intestinal tissue of Kazakh sheep after experimental Echinococcus granulosus infection, using a high-throughput approach. Parasite, 23, 23.
2. Kamenetzky, L., Stegmayer, G., Maldonado, L., Macchiaroli, N., Yones, C. and Milone, D.H. (2016) MicroRNA discovery in the human parasite Echinococcus multilocularis from genome-wide data. Genomics, 107, 274-280.
3. Macchiaroli, N., Cucher, M., Zarowiecki, M., Maldonado, L., Kamenetzky, L. and Rosenzvit, M.C. (2015) microRNA profiling in the zoonotic parasite Echinococcus canadensis using a high-throughput approach. Parasit Vectors, 8, 83.
4. Jin, X., Guo, X., Zhu, D., Ayaz, M. and Zheng, Y. (2017) miRNA profiling in the mice in response to Echinococcus multilocularis infection. Acta tropica, 166, 39-44.
5. Bai, Y., Zhang, Z., Jin, L., Kang, H., Zhu, Y., Zhang, L., Li, X., Ma, F., Zhao, L., Shi, B. et al. (2014) Genome-wide sequencing of small RNAs reveals a tissue-specific loss of conserved microRNA families in Echinococcus granulosus. BMC genomics, 15, 736.
6. Cucher, M., Prada, L., Mourglia-Ettlin, G., Dematteis, S., Camicia, F., Asurmendi, S. and Rosenzvit, M. (2011) Identification of Echinococcus granulosus microRNAs and their expression in different life cycle stages and parasite genotypes. International journal for parasitology, 41, 439-448.
7. Ai, L., Xu, M.J., Chen, M.X., Zhang, Y.N., Chen, S.H., Guo, J., Cai, Y.C., Zhou, X.N., Zhu, X.Q. and Chen, J.X. (2012) Characterization of microRNAs in Taenia saginata of zoonotic significance by Solexa deep sequencing and bioinformatics analysis. Parasitology research, 110, 2373-2378.
8. Wu, X., Fu, Y., Yang, D., Xie, Y., Zhang, R., Zheng, W., Nie, H., Yan, N., Wang, N., Wang, J. et al. (2013) Identification of neglected cestode Taenia multiceps microRNAs by illumina sequencing and bioinformatic analysis. BMC veterinary research, 9, 162.
9. Ai, L., Chen, M.-X., Zhang, Y.-N., Chen, S.-H., Zhou, X.-N. and Chen, J.-X. (2014) Comparative analysis of the miRNA profiles from Taenia solium and Taenia asiatica adult. African Journal of Microbiology Research, 8, 895-902.
10. Fromm, B., Worren, M.M., Hahn, C., Hovig, E. and Bachmann, L. (2013) Substantial Loss of Conserved and Gain of Novel MicroRNA Families in Flatworms. Molecular biology and evolution, 30, 2619-2628.
11. Cai, P., Gobert, G.N. and McManus, D.P. (2016) MicroRNAs in Parasitic Helminthiases: Current Status and Future Perspectives. Trends Parasitol, 32, 71-86.
12. Fromm, B., Ovchinnikov, V., Hoye, E., Bernal, D., Hackenberg, M. and Marcilla, A. (2016) On the presence and immunoregulatory functions of extracellular microRNAs in the trematode Fasciola hepatica. Parasite immunology.

On 2017 Jun 08, Christian Gluud commented:

Response to Søren Dinesen Østergaard's critique

Søren Dinesen Østergaard (SDØ) [1] criticizes our systematic review on selective serotonin reuptake inhibitors (SSRI) for patients with major depressive disorder [2] for using Hamilton’s depression rating scale (HDRS)17 instead of HDRS6. SDØ refer to four studies ‘documenting’ his claims [3-6].

Two of the studies relate to duloxetine and desvenlafaxine, which are dual action drugs and not SSRIs [3, 4]. The third study is a meta-analysis assessing fluoxetine versus placebo [5]. The results show a mean effect size of the SSRI of -0.30 (95% confidence interval (CI) -0.39 to -0.21) when using HDRS17 and an effect size of -0.37 (95% CI -0.46 to -0.28) when using HDRS6. The difference of 0.07 corresponds to 0.7 HDRS17 points assuming a standard deviation of 10 points. The fourth study is a patient-level analysis of 18 industry-sponsored placebo-controlled trials regarding paroxetine, citalopram, sertraline, or fluoxetine [6]. The authors report a mean effect size of the SSRIs of -0.27 when using HDRS17 and an effect size of -0.35 when using HDRS6 [6]. The difference of 0.08 corresponds to 0.8 HDRS17 points assuming a standard deviation of 10 points. Hence, the absolute effect size difference between the two scales seems less than 1 HDRS point. The National Institute for Clinical Excellence (NICE) recommended a difference of 3 points on the HDRS17 for 'a minimal effect' [7-9]. However, the required minimal clinical relevant difference is probably much larger than this figure. One study showed that a mirtazapine-placebo mean difference of up to 3.0 points on the HDRS corresponds to ‘no clinical change’ [10]. Another study showed that a SSRI-placebo mean difference of 3.0 points is undetectable by clinicians, and that a mean difference of 7.0 HDRS17 points is required to correspond to a rating of ‘minimal improvement’ [11].

Moreover, none of the meta-analyses [5, 6] take into account risks of systematic errors (‘bias’) [12-14] or risks of random errors [15]. Hence, there are risks that the two meta-analyses may overestimate the beneficial effects of SSRIs.

Other studies have shown that HDRS17 and HDRS6 largely show similar results [16,17]. It cannot be concluded that HDRS6 is a better assessment scale than HDRS17, just considering the psychometric validity of the two scales. If the total score of HDRS17 is affected by some of the adverse effects of SSRIs, then this might in fact better reflect the actual summed clinical effects of SSRIs than HDRS6 ignoring these effects. Until scales are validated against patient-centred clinically relevant outcomes, such scales are merely non-validated surrogate outcomes [18].

National and international medical agencies [19-21] all recommend HDRS17 for assessing depressive symptoms. We need access to all individual patient data from all randomised clinical trials to compare the effects of antidepressants on HDRS6 to HDRS17 [22].

SDØ states “no conflicts of interest“. We are aware that SDØ has received substantial support from ‘Lundbeckfonden’, its main objective being to maintain and expand the activities of the Lundbeck Group, one of the companies producing and selling SSRIs [23]. We think it would have been fair to declare this.

Janus Christian Jakobsen, Kiran Kumar Katakam, Naqash Javaid Sethi, Jane Lindshou, Jesper Krogh, and Christian Gluud.

Conflicts of interest:
 None known.

Copenhagen Trial Unit, Centre for Clinical Intervention Research, Rigshospitalet, Copenhagen, Denmark

References 1. Ostergaard SD: Do not blame the SSRIs: blame the Hamilton Depression Rating Scale. Acta Neuropsychiatrica 2017:1-3. 2. Jakobsen JC, Katakam KK, Schou A, et al: Selective serotonin reuptake inhibitors versus placebo in patients with major depressive disorder. A systematic review with meta-analysis and Trial Sequential Analysis. BMC Psychiatr 2017, 17(1):58. 3. Bech P, Kajdasz DK, Porsdal V: Dose-response relationship of duloxetine in placebo-controlled clinical trials in patients with major depressive disorder. Psychopharmacology 2006, 188(3):273-280. 4. Bech P, Boyer P, Germain JM, et al: HAM-D17 and HAM-D6 sensitivity to change in relation to desvenlafaxine dose and baseline depression severity in major depressive disorder. Pharmacopsychiatry 2010, 43(7):271-276. 5. Bech P, Cialdella P, Haugh MC, et al: Meta-analysis of randomised controlled trials of fluoxetine v. placebo and tricyclic antidepressants in the short-term treatment of major depression. Br J Psychiatr 2000, 176:421-428. 6. Hieronymus F, Emilsson JF, Nilsson S, Eriksson E: Consistent superiority of selective serotonin reuptake inhibitors over placebo in reducing depressed mood in patients with major depression. Mol Psychiatr 2016, 21(4):523-530. 7. Fournier JC, DeRubeis RJ, Hollon SD, et al: Antidepressant drug effects and depression severity: a patient-level meta-analysis. JAMA 2010, 303(1):47-53. 8. Mathews M, Gommoll C, Nunez R, Khan A: Efficacy and safety of vilazodone 20 and 40 Mg in major depressive disorder: a randomized, double-blind, placebo-controlled trial. Int Clin Psychopharmacol 2015, 30. 9. Kirsch I, Deacon BJ, Huedo-Medina TB, et al: Initial severity and antidepressant benefits: a meta-analysis of data submitted to the Food and Drug Administration. PLoS medicine 2008, 5(2):e45. 10. Leucht S, Fennema H, Engel R, et al: What does the HAMD mean? J Affect Disord 2013, 148(2-3):243-248. 11. Moncrieff J, Kirsch I: Empirically derived criteria cast doubt on the clinical significance of antidepressant-placebo differences. Cont Clin Trials 2015, 43:60-62. 12. Hróbjartsson A, Thomsen ASS, Emanuelsson F, et al: Observer bias in randomized clinical trials with measurement scale outcomes: a systematic review of trials with both blinded and nonblinded assessors. CMAJ : Canadian Medical Association Journal = Journal de l'Association Medicale Canadienne 2013, 185(4):E201-211. 13. Lundh A, Lexchin J, Mintzes B, Scholl JB, Bero L: Industry sponsorship and research outcome. Cochrane Database Syst Rev 2017, Art. No.: MR000033. DOI: 10.1002/14651858.MR000033.pub3.(2):MR000033. 14. Savovic J, Jones HE, Altman DG, et al: Influence of reported study design characteristics on intervention effect estimates from randomized, controlled trials. Ann Intern Med 2012, 157(6):429-438. 15. Wetterslev J, Jakobsen JC, Gluud C: Trial Sequential Analysis in systematic reviews with meta-analysis. BMC Medical Research Methodology 2017, 17(1):39. 16. Hooper CL, Bakish D: An examination of the sensitivity of the six-item Hamilton Rating Scale for Depression in a sample of patients suffering from major depressive disorder. J Psychiatry Neurosci 2000, 25(2):178-184. 17. O'Sullivan RL, Fava M, Agustin C, Baer L, Rosenbaum JF: Sensitivity of the six-item Hamilton Depression Rating Scale. Acta psychiatrica Scandinavica 1997, 95(5):379-384. 18. Gluud C, Brok J, Gong Y, Koretz RL: Hepatology may have problems with putative surrogate outcome measures. J Hepatol 2007, 46(4):734-742. 19. Sundhedsstyrelsen (Danish Health Agency): Referenceprogram for unipolar depression hos voksne (Guideline for unipolar depression in adults). http://wwwsstdk/~/media/6F9CE14B6FF245AABCD222575787FEB7ashx 2007. 20. European Medicines Agency: Guideline on clinical investigation of medicinal products in the treatment of depression. EMA/CHMP/185423/2010 Rev 2 previously (CPMP/EWP/518/97, Rev 1) 2013. 21. U.S. Food and Drug Administration: https://www.fda.gov/ohrms/dockets/AC/07/briefing/2007-4273b1_04-DescriptionofMADRSHAMDDepressionR(1).pdf. 22. Skoog M, Saarimäki JM, Gluud C, et al.: Transaprency and Registration in Clinical Research in the Nordic Countries. Nordic Trial Alliance, NordForsk. 2015:1-108. 23. Lundbeck Foundation. http://www.lundbeckfonden.com/about-the-foundation.25.aspx.

On 2016 Dec 07, Raphael Stricker commented:

Serological Test Sensitivity in Late Lyme Disease.

Raphael B. Stricker, MD<br> Union Square Medical Associates San Francisco, CA rstricker@usmamed.com

Cook and Puri have written an excellent review of the sorry state of commercial two-tier testing for Lyme disease (1). Unfortunately the authors failed to address the myth of high serological test sensitivity in late Lyme disease.

In the review, Figure 4 and Table 7 show a mean two-tier serological test sensitivity of 87.3-95.8% for late Lyme arthritis, neuroborreliosis and Lyme carditis. However, this apparently high sensitivity is based on circular reasoning: in order for patients to be diagnosed with these late conditions, they were required to have clinical symptoms AND POSITIVE SEROLOGICAL TESTING. Then guess what, they had positive serological testing! This spurious circular reasoning invalidates the high sensitivity rate and should have been pointed out by the authors of the review.

As an example, the study by Bacon et al. (2) contains the following language: "For late disease, the case definition requires at least one late manifestation AND LABORATORY CONFIRMATION OF INFECTION, and therefore the possibility of selection bias toward reactive samples cannot be discounted" (emphasis added). Other studies of late Lyme disease using spurious circular reasoning to prove high sensitivity of two-tier serological testing have been discussed elsewhere (3-5).

In the ongoing controversy over Lyme disease, it is important to avoid propagation of myths about the tickborne illness, and insightful analysis of flawed reasoning is the best way to accomplish this goal.

References 1. Cook MJ, Puri BK. Commercial test kits for detection of Lyme borreliosis: a meta-analysis of test accuracy. Int J Gen Med 2016;9:427–40. 2. Bacon RM, Biggerstaff BJ, Schriefer ME, et al. Serodiagnosis of Lyme disease by kinetic enzyme-linked immunosorbent assay using recombinant VlsE1 or peptide antigens of Borrelia burgdorferi compared with 2-tiered testing using whole-cell lysates. J Infect Dis. 2003;187:1187–99. 3. Stricker RB, Johnson L. Serologic tests for Lyme disease: More smoke and mirrors. Clin Infect Dis. 2008;47:1111-2. 4. Stricker RB, Johnson L. Lyme disease: the next decade. Infect Drug Resist. 2011;4:1–9. 5. Stricker RB, Johnson L. Circular reasoning in CDC Lyme disease test review. Pubmed Commons comment on: Moore A, Nelson C, Molins C, Mead P, Schriefer M. Current guidelines, common clinical pitfalls, and future directions for laboratory diagnosis of Lyme disease, United States. Emerg Infect Dis. 2016;22:1169-77.

Disclosure: RBS is a member of the International Lyme and Associated Diseases Society (ILADS) and a director of LymeDisease.org. He has no financial or other conflicts to declare.

On 2017 Feb 14, Mark Schiffman commented:

Prophylactic HPV vaccines consist of the major coat protein L1 assembled into macromolecular structures – virus like particles (VLPs) that mimic the geometry and morphology of the wild type virus coat or capsid but do not contain full length HPV DNA genomes. VLPs, with their repeat crystalline array of L1 pentamers as in the wild type virus, are intrinsically immunogenic(1) eliciting high antibody titres with or without adjuvant(2). The safety profile of the licensed vaccines was assessed extensively in randomized clinical trials (RCTs)(3-5). In the 10 years since the first 2 commercial vaccines Gardasil and Cervarix were licensed, the safety profile has been intensively monitored in the post-licensure setting by robust pharmacovigilance using both passive and active surveillance (3, 6). These studies, which collectively have included millions of subjects, provide no evidence whatsoever to support the speculation that HPV vaccines by virtue of their protein content, adjuvants or any other element within the formulation –could induce, trigger or exacerbate auto-immune disorders, thromboembolic events, demyelinating diseases or other chronic conditions.<br> The Global Advisory Committee on Vaccine Safety of the World Health Organisation has reviewed the safety data for HPV vaccines on several occasions http://www.who.int/vaccine_safety/committee/topics/hpv/en/ GACVS stated in 2014: “In summary, the GACVS continues to closely monitor the safety of HPV vaccines and, based on a careful examination of the available evidence, continues to affirm that its benefit-risk profile remains favorable. The Committee is concerned, however, by the claims of harm that are being raised on the basis of anecdotal observations and reports in the absence of biological or epidemiological substantiation. While the reporting of adverse events following immunization by the public and health care providers should be encouraged and remains the cornerstone of safety surveillance, their interpretation requires due diligence and great care. As stated before, allegations of harm from vaccination based on weak evidence can lead to real harm when, as a result, safe and effective vaccines cease to be used. To date, there is no scientific evidence that aluminium-containing vaccines cause harm, that the presence of aluminium at the injection site (the MMF “tattoo”) is related to any autoimmune syndrome, and that HPV DNA fragments are responsible for inflammation, cerebral vasculitis or other immune-mediated phenomena.” http://www.who.int/vaccine_safety/committee/topics/hpv/GACVS_Statement_HPV_12_Mar_2014.pdf Efficacy against vaccine type high grade cervical intraepithelial neoplasia CIN3 (the well-established precursor of cervical cancer(7)) has been demonstrated for the vaccines in the relevant RCTs (8-10). Cervical cancer cannot be used for ethical reasons as an end point in clinical trials (7). With regard to screening, contrary to the misleading comments by Dr. Lee, there is a large and authoritative body of evidence (including many RCTs) showing that any of the approved HPV tests is substantially more sensitive for detection of CIN2, CIN3, or cancer than cytology (11). The figure he quotes of 58% sensitivity is the result of a controversial application of “verification bias adjustment” in detection of CIN2 or worse, in a single trial. Large systematic reviews have consistently reported much higher sensitivity of HPV testing compared to cytology (12). The sensitivity of HPV testing is not at issue; rather specificity is a concern. As we emphasized in the article, HPV testing does require a secondary triage method to identify persistent infection and cancer precursors that require treatment, because HPV is very common and most infections “clear”. There are several choice of triage strategy prior to treatment; HPV typing and cytology or its analogues are most often proposed. Automated methods will soon be available. Carcinogenic human papillomavirus infections are a global public health problem, >80 % of the annual ≥530,000 cervical cancer cases occur in resource poor countries in which the disease is often incurable (13). Whatever preventive measures are adopted, evaluating the impact of interventions to control infection and disease requires a global perspective; from this perspective the promise of HPV vaccination and HPV testing are overwhelmingly supported by highly credible data. • Mark Schiffman • , John Doorbar • , Nicolas Wentzensen • , Silvia de Sanjosé • , Carole Fakhry • , Bradley J. Monk • , Margaret A. Stanley • & Silvia Franceschi  

1. Bachmann MF, Zinkernagel RM. The influence of virus structure on antibody responses and virus serotype formation. Immunology today. 1996;17(12):553-8.
2. Harro CD, Pang YY, Roden RB, Hildesheim A, Wang Z, Reynolds MJ, et al. Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine. J Natl Cancer Inst. 2001;93(Feb 21. 4): 284-492.
3. Vichnin M, Bonanni P, Klein NP, Garland SM, Block SL, Kjaer SK, et al. An Overview of Quadrivalent Human Papillomavirus Vaccine Safety: 2006 to 2015. Pediatr Infect Dis J. 2015;34(9):983-91.
4. Moreira ED, Jr., Block SL, Ferris D, Giuliano AR, Iversen OE, Joura EA, et al. Safety Profile of the 9-Valent HPV Vaccine: A Combined Analysis of 7 Phase III Clinical Trials. Pediatrics. 2016.
5. Descamps D, Hardt K, Spiessens B, Izurieta P, Verstraeten T, Breuer T, et al. Safety of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine for cervical cancer prevention: A pooled analysis of 11 clinical trials. Hum Vaccin. 2009;5(5).
6. Angelo MG, Zima J, Tavares Da Silva F, Baril L, Arellano F. Post-licensure safety surveillance for human papillomavirus-16/18-AS04-adjuvanted vaccine: more than 4 years of experience. Pharmacoepidemiol Drug Saf. 2014;23(5):456-65.
7. Pagliusi SR, Teresa Aguado M. Efficacy and other milestones for human papillomavirus vaccine introduction. Vaccine. 2004;23(Dec 16. 5):569-78.
8. Future II Study Group. Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. The New England journal of medicine. 2007;356(19):1915-27.
9. Lehtinen M, Paavonen J, Wheeler CM, Jaisamrarn U, Garland SM, Castellsague X, et al. Overall efficacy of HPV-16/18 AS04-adjuvanted vaccine against grade 3 or greater cervical intraepithelial neoplasia: 4-year end-of-study analysis of the randomised, double-blind PATRICIA trial. Lancet Oncol. 2012;13(1):89-99.
10. Joura EA, Giuliano AR, Iversen OE, Bouchard C, Mao C, Mehlsen J, et al. A 9-valent HPV vaccine against infection and intraepithelial neoplasia in women. The New England journal of medicine. 2015;372(8):711-23.
11. Ronco G, Dillner J, Elfström KM, Tunesi S, Snijders PJ, Arbyn M, Kitchener H, Segnan N, Gilham C, Giorgi-Rossi P, Berkhof J, Peto J, Meijer CJ; International HPV screening working group.. Efficacy of HPV-based screening for prevention of invasive cervical cancer: follow-up of four European randomised controlled trials. Lancet. 2014 Feb 8;383(9916):524-32. Erratum in: Lancet. 2015 Oct 10;386(10002):1446.<br>
12. Arbyn M, Ronco G, Anttila A, Meijer CJLM, Poljak M, Ogilvie G et al. Evidence Regarding Human Papillomavirus Testing in Secondary Prevention of Cervical Cancer. Vaccine. 2012; 30 Suppl 5:F88-99.
13. Plummer M, de Martel C, Vignat J, Ferlay J, Bray F, Franceschi S. Global burden of cancers attributable to infections in 2012: a synthetic analysis. Lancet Glob Health. 2016 Sep;4(9):e609-16. doi: 10.1016/S2214-109X(16)30143-7.

On 2017 May 11, Jean-Pierre Bayley commented:

Should we screen carriers of maternally inherited SDHD mutations?

Jean-Pierre Bayley (1), Jeroen C Jansen (2), Eleonora P M Corssmit (3) and Frederik J Hes (4) 1. Department of Human Genetics, 2. Department of Otorhinolaryngology, 3. Department of Endocrinology and Metabolic Diseases, 4. Department of Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands

We wish to comment on the above paper by Burnichon and colleagues: Burnichon N, et al. Risk assessment of maternally inherited SDHD paraganglioma and phaeochromocytoma. J Med Genet. 2017; 54:125-133. 3

In this paper a prospective study is presented that identified and described development of pheochromocytoma in a carrier of an SDHD mutation. Although at first sight not an uncommon occurrence in carriers of these mutations, this case is unusual because the mutation was inherited via the maternal line. This is now only the third reported case of confirmed phaeochromocytoma development following maternal transmission of an SDHD mutation. (1-3) The patient in question was identified amongst a cohort of 20 maternal mutation carriers who underwent imaging surveillance. Based on the identification of one patient in this cohort (5%), the authors make recommendations for the clinical care of carriers of a maternally inherited SDHD mutation. They advise targeted familial genetic testing from the age of 18 in families with SDHD mutations, and that identified carriers undergo imaging and biochemical workup to detect asymptomatic tumours. If the first workup is negative, the authors suggest that patients be informed about paraganglioma-phaeochromocytoma (PPGL) symptoms and recommend an annual clinical examination and blood pressure measurement, with a new workup indicated in case of symptoms suggestive of PPGL. Although this paper is a meaningful contribution to the literature, we are concerned that the authors base their subsequent clinical recommendations on a relatively small cohort. In a recent study, we described one confirmed case of maternal transmission and concluded that “we consider the increase in risk represented by these reports to be negligible.” (2)

Two reasons underlie this statement. Firstly, the somatic rearrangements underlying the maternal cases identified to date are far more complex (loss of the paternal wild-type SDHD allele by mitotic recombination, followed by loss of the recombined paternal chromosome containing the paternal 11q23 region and the maternal 11p15 region) than the molecular events seen in paternal cases (loss of whole chromosome 11). Secondly, our conclusions were based, implicitly, on many previous studies at our centre over the past three decades in which we described various aspects of the large SDHD cohort collected by us over that period. Genetic aspects of this cohort, and 601 patients with paternally transmitted SDHD mutations, were described by Hensen and co-workers in 2012. (4) As all previous studies suggest that mutations are equally transmissible via the paternal or maternal line, our identification of a single maternal case amongst this cohort suggests that the penetrance of maternally transmitted mutations is very low. Using the calculation employed by Burnichon and colleagues and assuming that at least 600 maternal mutation carriers are alive in the Netherlands, we arrive at an estimate of 0.17% (1/601 = 0.17%), rather than their figure of 5%. In addition to our own cohort, 1000’s of SDHD mutation carriers have been identified world-wide. Assuming that 1 in 20 maternally transmitted mutations result in tumours, many more maternally inherited cases would have come to our attention, even without surveillance.

In our opinion the question of management of maternally inherited SDHD mutations comes down to a risk-benefit analysis. The most obvious implication of the recommendations made by Burnichon and colleagues in our patient population would be the institution of surveillance, with all the attendant practical, financial and psychological burdens for 600 carriers of maternally inherited SDHD mutations in order to identify a single case. Furthermore, SDHD-associated PPGL mortality rates and survival in a Dutch cohort of SDHD variant carriers was not substantially increased compared with the general population. (5) In practice, carriers of maternally inherited SDHD mutations at our centre are not advised to undergo surveillance. Instead, we reassure them that their risk of developing PPGL is exceptionally low (described three times worldwide), but that they should be aware, more so than the general population, of symptoms that are suggestive of paraganglioma or phaeochromocytoma. Many families have been in our care for over 25 years and in that time we have found no evidence to suggest that this policy should be revised.

NB. A version of this comment has been posted on the Journal of Medical Genetics website and has been commented on in turn by Burnichon and colleagues.

References

1.Yeap PM, Tobias ES, Mavraki E, Fletcher A, Bradshaw N, Freel EM, Cooke A, Murday VA, Davidson HR, Perry CG, Lindsay RS. Molecular analysis of pheochromocytoma after maternal transmission of SDHD mutation elucidates mechanism of parent-of-origin effect. J Clin Endocrinol Metab 2011;96:E2009-E2013.

2.Bayley JP, Oldenburg RA, Nuk J, Hoekstra AS, van der Meer CA, Korpershoek E, McGillivray B, Corssmit EP, Dinjens WN, de Krijger RR, Devilee P, Jansen JC, Hes FJ. Paraganglioma and pheochromocytoma upon maternal transmission of SDHD mutations. BMC Med Genet 2014;15:111.

3.Burnichon N, Mazzella JM, Drui D, Amar L, Bertherat J, Coupier I, Delemer B, Guilhem I, Herman P, Kerlan V, Tabarin A, Wion N, Lahlou-Laforet K, Favier J, Gimenez-Roqueplo AP. Risk assessment of maternally inherited SDHD paraganglioma and phaeochromocytoma. J Med Genet 2017;54:125-33.

4.Hensen EF, van DN, Jansen JC, Corssmit EP, Tops CM, Romijn JA, Vriends AH, Van Der Mey AG, Cornelisse CJ, Devilee P, Bayley JP. High prevalence of founder mutations of the succinate dehydrogenase genes in the Netherlands. Clin Genet 2012;81:284-8.

5.van Hulsteijn LT, Heesterman B, Jansen JC, Bayley JP, Hes FJ, Corssmit EP, Dekkers OM. No evidence for increased mortality in SDHD variant carriers compared with the general population. Eur J Hum Genet 2015;23:1713-6.

On 2017 Feb 13, David Reardon commented:

This analysis of perinatal psychiatric episodes by Munk-Olsen T, 2016¹ is flawed by the failure to examine the effects of prior pregnancy losses. Numerous studies have shown that prior fetal loss, either from miscarriage, stillbirth, or induced abortion, increases the risk psychiatric disorders during and after subsequent pregnancies.^2-7 There is even a dose effect, with multiple losses associated with elevated rates compared to a single loss.²

Notably, the heightened risk of mental illness following miscarriage and abortion have also been confirmed by several of Munk-Olsen’s own studies.^8-10 Unfortunately, while abortion was used as a control variable in two cases, the effects were not described.^8,10

In light of the literature, Munk-Olsen T, 2016's conclusion that it is not possible to “predict which women will become ill postpartum”¹ is an overstatement. There is strong evidence that prior fetal loss is risk factor.

It is strongly recommended that the authors of this most recent study¹ should publish a reanalysis showing the effects of prior pregnancy loss relative to (a) one or more abortions and (b) one or more miscarriages or other natural losses. These results could lead to improved screening to identify women who may benefit from additional care.

Editors and peer reviewers should be alert to the recommendation that all studies relative to the intersection between mental and reproductive health should always consider the effects of prior pregnancy loss.^11-13 In particular, both the Royal College of Psychiatrists¹⁴ and the American Psychological Association¹⁵ have lamented the lack of high quality studies examining the statistical associations between abortion and mental health. Record linkage studies from national data sets, such as that examined by Munk-Olsen, can help to fill this gap of knowledge . . . but only if they include analyses examining these effects.

References

1) Munk-Olsen T, Maegbaek ML, Johannsen BM, et al. Perinatal psychiatric episodes: a population-based study on treatment incidence and prevalence. Transl Psychiatry. 2016;6(10):e919. doi:10.1038/tp.2016.190.

2) Giannandrea SAM, Cerulli C, Anson E, Chaudron LH. Increased risk for postpartum psychiatric disorders among women with past pregnancy loss. J Womens Health (Larchmt). 2013;22(9):760-768. doi:10.1089/jwh.2012.4011.

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5) Räisänen S, Lehto SM, Nielsen HS, Gissler M, Kramer MR, Heinonen S. Risk factors for and perinatal outcomes of major depression during pregnancy: a population-based analysis during 2002-2010 in Finland. BMJ Open. 2014;4(11):e004883. doi:10.1136/bmjopen-2014-004883.

6) Montmasson H, Bertrand P, Perrotin F, El-Hage W. Facteurs prédictifs de l’état de stress post-traumatique du postpartum chez la primipare. J Gynécologie Obs Biol la Reprod. 2012;41(6):553-560. doi:10.1016/j.jgyn.2012.04.010.

7) McCarthy F, Moss-Morris R, Khashan A, et al. Previous pregnancy loss has an adverse impact on distress and behaviour in subsequent pregnancy. BJOG An Int J Obstet Gynaecol. 2015;122(13):1757-1764. doi:10.1111/1471-0528.13233.

8) Munk-Olsen T, Bech BH, Vestergaard M, Li J, Olsen J, Laursen TM. Psychiatric disorders following fetal death: a population-based cohort study. BMJ Open. 2014:1-6. doi:10.1136/bmjopen-2014-005187.

9) Meltzer-Brody S, Maegbaek ML, Medland SE, Miller WC, Sullivan P, Munk-Olsen T. Obstetrical, pregnancy and socio-economic predictors for new-onset severe postpartum psychiatric disorders in primiparous women. Psychol Med. 2017:1-15. doi:10.1017/S0033291716003020.

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On 2017 Jul 02, Suzy Chapman commented:

ICD-11 Beta draft: Rationale for Proposal for Deletion of proposed new category: Bodily distress disorder

March 8, 2017

Full text:

http://wp.me/pKrrB-4dc

References:

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5 American Psychiatric Association. (2013). Somatic Symptom and Related Disorders. In Diagnostic and statistical manual of mental disorders (5th ed.). Washington, DC: Author.

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7 Lam TP, Goldberg DP, Dowell AC, Fortes S, Mbatia JK, Minhas FA, Klinkman MS. Proposed new diagnoses of anxious depression and bodily stress syndrome in ICD-11-PHC: an international focus group study. Fam Pract. 2013 Feb;30(1):76-87. doi: 10.1093/fampra/cms037. Epub 2012 Jul 28. [PMID: 22843638]

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10 Medically Unexplained Symptoms, Somatisation and Bodily Distress: Developing Better Clinical Services, Francis Creed, Peter Henningsen, Per Fink (Eds), Cambridge University Press, 2011.

11 Frances Creed and Per Fink. Presentations, Research Clinic for Functional Disorders Symposium, Aarhus University Hospital, May 15, 2014.

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13 Chalder, T. An introduction to “medically unexplained” persistent physical symptoms. Presentation, Department of Psychological Medicine, King’s Health Partners, 2014. [Accessed 27 February 2017]

14 Schumacher S, Rief W, Klaus K, Brähler E, Mewes R. Medium- and long-term prognostic validity of competing classification proposals for the former somatoform disorders. Psychol Med. 2017 Feb 9:1-14. doi: 10.1017/S0033291717000149. [PMID: 28179046]

15 Fink P, Toft T, Hansen MS, Ornbol E, Olesen F. Symptoms and syndromes of bodily distress: an exploratory study of 978 internal medical, neurological, and primary care patients. Psychosom Med. 2007 Jan;69(1):30-9. [PMID: 17244846]

16 Carroll L. Alice’s Adventures in Wonderland. 1885. Macmillan.

On 2016 Oct 11, Alem Matthees commented:

References for the above comment

1) Hawkes N. Freedom of information: can researchers still promise control of participants' data? BMJ. 2016 Sep 21;354:i5053. doi: 10.1136/bmj.i5053. PMID: 27654128. http://www.bmj.com/content/354/bmj.i5053

2) White PD, Goldsmith KA, Johnson AL, Potts L, Walwyn R, DeCesare JC, Baber HL, Burgess M, Clark LV, Cox DL, Bavinton J, Angus BJ, Murphy G, Murphy M, O'Dowd H, Wilks D, McCrone P, Chalder T, Sharpe M; PACE trial management group. Comparison of adaptive pacing therapy, cognitive behaviour therapy, graded exercise therapy, and specialist medical care for chronic fatigue syndrome (PACE): a randomised trial. Lancet. 2011 Mar 5;377(9768):823-36. doi: 10.1016/S0140-6736(11)60096-2. Epub 2011 Feb 18. PMID: 21334061. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065633/

3) Confidentiality: NHS Code of Practice. November 2003. https://www.gov.uk/government/publications/confidentiality-nhs-code-of-practice

4) General Medical Council (2009). Confidentiality guidance: Research and other secondary uses. http://www.gmc-uk.org/guidance/ethical_guidance/confidentiality_40_50_research_and_secondary_issues.asp

5) Queen Mary University of London. PACE trial protocol: Final version 5.2, 09.03.2006. A1.6-A1.7 [Full Trial Consent Forms] https://www.whatdotheyknow.com/request/203455/response/508208/attach/3/Consent forms.pdf

6) Appeal to the First-tier Tribunal (Information Rights), case number EA/2015/0269: http://informationrights.decisions.tribunals.gov.uk/DBFiles/Decision/i1854/Queen Mary University of London EA-2015-0269 (12-8-16).PDF

7) Tracking switched outcomes in clinical trials. http://compare-trials.org/

8) White PD, Chalder T, Sharpe M, Johnson T, Goldsmith K. PACE trial authors' reply to letter by Kindlon. BMJ. 2013 Oct 15;347:f5963. doi: 10.1136/bmj.f5963. PMID: 24129374. http://www.bmj.com/content/347/bmj.f5963

9) Goldsmith KA, White PD, Chalder T, Johnson AL, Sharpe M. The PACE trial: analysis of primary outcomes using composite measures of improvement. Queen Mary University of London. 8 September 2016. http://www.wolfson.qmul.ac.uk/images/pdfs/pace/PACE_published_protocol_based_analysis_final_8th_Sept_2016.pdf

10) Wikipedia. Multiple comparisons problem. Accessed 02 October 2016. https://en.wikipedia.org/wiki/Multiple_comparisons_problem

11) Matthees A, Kindlon T, Maryhew C, Stark P, Levin B. A preliminary analysis of ‘recovery’ from chronic fatigue syndrome in the PACE trial using individual participant data. Virology Blog. 21 September 2016. http://www.virology.ws/wp-content/uploads/2016/09/preliminary-analysis.pdf

12) Tuller D. Trial By Error, Continued: Questions for Dr. White and his PACE Colleagues. Virology Blog. 4 January 2016. http://www.virology.ws/2016/01/04/trial-by-error-continued-questions-for-dr-white-and-his-pace-colleagues/

13) Walwyn R, Potts L, McCrone P, Johnson AL, DeCesare JC, Baber H, Goldsmith K, Sharpe M, Chalder T, White PD. A randomised trial of adaptive pacing therapy, cognitive behaviour therapy, graded exercise, and specialist medical care for chronic fatigue syndrome (PACE): statistical analysis plan. Trials. 2013 Nov 13;14:386. doi: 10.1186/1745-6215-14-386. PMID: 24225069. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226009/

14) White PD, Goldsmith K, Johnson AL, Chalder T, Sharpe M. Recovery from chronic fatigue syndrome after treatments given in the PACE trial. Psychol Med. 2013 Oct;43(10):2227-35. doi: 10.1017/S0033291713000020. PMID: 23363640. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776285/

15) Beth Smith ME, Nelson HD, Haney E, et al. Diagnosis and Treatment of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Rockville (MD): Agency for Healthcare Research and Quality (US); 2014 Dec. (Evidence Reports/Technology Assessments, No. 219.) July 2016 Addendum. Available from: http://www.ncbi.nlm.nih.gov/books/NBK379582/

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17) Kennedy A. Authors of our own misfortune?: The problems with psychogenic explanations for physical illnesses. CreateSpace Independent Publishing Platform. 4 September 2012. ISBN-13: 978-1479253951. https://www.amazon.com/Authors-our-own-misfortune-explanations/dp/1479253952

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19) Higgins PTJ, Altman DG, Sterne JAC; on behalf of the Cochrane Statistical Methods Group and the Cochrane Bias Methods Group. Chapter 8: Assessing risk of bias in included studies. Version 5.1.0 [updated March 2011]. http://handbook.cochrane.org/chapter_8/8_assessing_risk_of_bias_in_included_studies.htm

20) Schulz KF, Grimes DA. Blinding in randomised trials: hiding who got what. Lancet. 2002 Feb 23;359(9307):696-700. PMID: 11879884. http://www.who.int/rhl/LANCET_696-700.pdf

21) Boot WR, Simons DJ, Stothart C, Stutts C. The Pervasive Problem With Placebos in Psychology: Why Active Control Groups Are Not Sufficient to Rule Out Placebo Effects. Perspect Psychol Sci. 2013 Jul;8(4):445-54. Doi: 10.1177/1745691613491271. PMID: 26173122. http://pps.sagepub.com/content/8/4/445.long

22) Button KS, Munafò MR. Addressing risk of bias in trials of cognitive behavioral therapy. Shanghai Arch Psychiatry. 2015 Jun 25;27(3):144-8. doi: 10.11919/j.issn.1002-0829.215042. PMID: 26300596. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526826

23) Wood L, Egger M, Gluud LL, Schulz KF, Jüni P, Altman DG, Gluud C, Martin RM, Wood AJ, Sterne JA. Empirical evidence of bias in treatment effect estimates in controlled trials with different interventions and outcomes: meta-epidemiological study. BMJ 336 (7644): 601–5. DOI:10.1136/bmj.39465.451748.AD. PMID 18316340. PMC 2267990. http://www.bmj.com/cgi/content/full/336/7644/601

24) Kindlon TP. Objective measures found a lack of improvement for CBT & GET in the PACE Trial: subjective improvements may simply represent response biases or placebo effects in this non-blinded trial. BMJ Rapid Response. 18 January 2015. http://www.bmj.com/content/350/bmj.h227/rr-10

25) Knoop H, Wiborg J. What makes a difference in chronic fatigue syndrome? Lancet Psychiatry. 2015 Feb;2(2):113-4. doi: 10.1016/S2215-0366(14)00145-X. Epub 2015 Jan 28. PMID: 26359736. https://www.ncbi.nlm.nih.gov/pubmed/26359736

26) johnthejack (Peters J). Using public money to keep publicly funded data from the public. 29 June 2016. https://johnthejack.com/2016/06/29/using-public-money-to-keep-publicly-funded-data-from-the-public/

27) Coyne JC. Further insights into war against data sharing: Science Media Centre’s letter writing campaign to UK Parliament. 31 January 2016. https://jcoynester.wordpress.com/2016/01/31/further-insights-into-the-war-against-data-sharing-the-science-media-centres-letter-writing-campaign-to-uk-parliament/

On 2017 Feb 06, GARRET STUBER commented:

*This review was completed as part of a graduate level circuits and behavior course at UNC-Chapel Hill. The critique was written by students in the class and edited by the instructor, Garret Stuber.

Comments and critique

Written by Li et al., this paper investigated a class of oxytocin receptor interneurons (OxtrINs) on which the same group first characterized in 2014 [1]. OxtrINs are a subset of somatostatin positive interneurons in the medial prefrontal cortex (mPFC) that seem to be important for sociosexual behaviors in females, specifically during estrus and not diestrus. To complement their previous story, here the authors concluded that OxtrINs in males regulate anxiety-related behaviors through the release of corticotropin releasing hormone binding protein (Crhbp). While we agree that these neurons could be mediating sexually dimorphic behaviors, it is unclear how robust these differences really are.

We had some technical issues with this paper. First, it is unclear exactly how many mice were allotted to each experimental group, and it would have been useful to see individual data in each of the behavioral experiments, so that we can better understand some of the variability in the authors’ graphs. Even among different experiments, there were variable sizes of n (e.g. Fig. 5F-H, “n = 8-14 mice per group”). There was also no mention of how many cells per animal were tested for each brain slice experiment; instead, we received total numbers of cells tested per group. This paper did not include the complementary female data to Fig. 4F-G and Fig. 5A-B, the experiments pairing blue light with Crhr1 antagonist or Crhbp antagonist. We would have appreciated seeing this data adjacent to that for the males. In addition, there was no mentioned control for the optogenetic experiments. The authors only compared responses between light on and light off trials. Typically in optogenetic approaches, a set of control mice are also implanted with optic fibers and flashed with blue light in the absence of virus to test whether the light alone influences behavior. Incidentally, there is evidence that blue light influences blood flow, which may affect neuronal activity [2]. It was also unclear during the sociosexual behavioral testing whether the males were exposed to females in estrus or diestrus. In all, lack of detailed sample sizes and controls made it difficult to assess how prominent these sex differences were.

These issues aside, knocking out endogenous Oxtr in their targeted interneuron population was a key experiment, as it demonstrated that oxytocin signaling in OxtrINs is important in anxiety-related behaviors in males, but not in females regardless of the estrus stage. They did this using a floxed Oxtr mouse and deleted OxtR using a Cre-inducible virus, allowing for temporal and cell-type-specific control of this deletion, and subsequently measured the resulting phenotype using an elevated plus maze and open field task. The authors also validated that changes in exploration were not due to hyperactivity. We think these experiments are convincing.

TRAP profiling, which the same research group pioneered in 2014 [3], provided a set of genes enriched in OxtrINs. TRAP targets RNAs while they are translated into proteins, so we think their results here are particularly relevant. Moreover, the authors provided a list of genes enriched in sex-specific OxtrINs, a useful resource for those interested in gene expression differences in males and females. Once they identified Crhbp, an inhibitor of Crh, they hypothesized that OxtrINs were releasing Crhbp to modulate anxiogenic behaviors in males. The authors next measured Crh levels in the paraventricular nucleus of the hypothalamus and found that Crh levels are higher in females than males. They thus concluded Crh levels were driving sex differences associated with OxtrINs. We wonder whether Crh levels are also higher in the female mPFC, but we agree here too.

To demonstrate that Crhbp expressed by OxtrINs is important in modulating anxiety-like behaviors in males, the authors targeted Crhbp mRNA using Cre-inducible viral delivery of an shRNA construct and subsequently tested anxiety-related behaviors. They found that knocking down Crhbp was anxiogenic in males and not in females. This was a critical experiment, but the shRNA constructs targeting Crhbp were validated solely in a cell line. It would have been more appropriate to perform a western blot on mPFC punches of adult mice, showing whether this lentiviral construct knocked down Crhbp expression in the mouse brain prior to behavioral testing. In fact, it also would have been useful to see a quantification of the shRNA transfection rate, as well as its specificity in vivo. As stated above, we also do not know the distribution of behavioral responses here either. Without these pieces of information, it is difficult to assess how reliable or robust their knockdown was.

The authors concluded that sexually dimorphic hormones act through the otherwise sexually monomorphic OxtrINs to regulate anxiety-related behaviors in males and sociosexual behaviors in females. We agree that OxtrINs interact with oxytocin and Crh to bring about sex-specific phenotypes, but we also think that using additional paradigms testing anxiety and social behaviors, such as a predator odor, novelty-suppressed feeding or social grooming, could shed more light on the nuances of mPFC circuitry. In addition, the authors suggested that OxtrINs are sexually monomorphic because they are equally abundant in males and females. The authors’ TRAP data however suggested that OxtrINs of males and females have different gene expression profiles (Table S2), thus indicating that these interneurons may form different connections in each sex that mediate the electrophysiological and behavioral differences we see in this study.

It would be interesting to overexpress Crhbp in female mice, preferably in a cell-type-specific manner, to see whether female mice would demonstrate the anxiety-like behavior seen in males. If the Crh:Crhbp balance is in fact mediating this sexually dimorphic behavior through OxtrINs, we would expect that doing these manipulations may “masculinize” the females’ behavior. Regardless, we believe that this study opens opportunities for future work into how oxytocin and Crh release from the hypothalamus may act together to coordinate behavior. It will also be interesting to see if single-cell RNA sequencing could provide insight into whether OxtrINs can be further divided into sexually dimorphic subtypes. As the authors pointed out, understanding the dynamics of Crh and oxytocin in the mPFC will be important for gender-specific therapy and treatment.

[1] Nakajima, M. et al. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons. Cell. 159, 295-305 (2014).

[2] Rungta, R. L. et al. Light controls cerebral blood flow in naïve animals. Nature Communications. 8, 14191 (2017).

[3] Heiman, M. et al. Cell-type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nature Protocols. 9, 1282-1291 (2014).

On 2017 May 16, Michael Stillman commented:

I read this article with great interest. And with significant concern.

A sweeping review by the Department of Health and Human Services' Office for Human Research Protections of Dr. Harkema's spinal cord injury research program (https://www.hhs.gov/ohrp/compliance-and-reporting/determination-letters/2016/october-17-2016-university-louisville/index.html accessed May 16, 2017) documented numerous instances of sloppy methodologies and potential frank scientific misconduct. This report included evidence of: a) missing source documents, leading to an inability to verify whether protocols had been followed or captured data was valid; b) multiple instances of unapproved deviations from experiments protocols; c) participants having been injured while participating in translational research experiments; d) a failure to document and adjudicate adverse events and to report unanticipated problems to the IRB; and e) subjects being misled about the cost of participating in research protocols. Dr. Harkema's conduct was so concerning that the National Institute of Disability, Independent Living, and Rehabilitation Research (NIDILRR) prematurely halted and defunded one of her major research projects (http://kycir.org/2016/07/11/top-u-of-l-researcher-loses-federal-funding-for-paralysis-study/ accessed May 26, 2017).

I approached the editors of "Journal of Neurotrauma" with reports from both Health and Human Services (above) and University of Louisville's IRB and asked them three questions: a) were they adequately concerned with this study's integrity to consider a retraction; b) were they adequately concerned to consider publishing a "concerned" letter to the editor questioning the study's integrity and reliability; and c) were they interested in reviewing adverse events associated with the experiments. Their response: "no," "no," and "no."

I call on the editorial board of "Journal of Neurotrauma" to carefully inspect all documents and data sets related to this work. I would further expect them to review all adverse events reports, and to demand evidence that they've been reviewed and adjudicated by an independent medical monitor or study physician. Short of this, this work remains specious.

On 2016 Sep 30, Paul Brookes commented:

I submitted a response to this opinion piece to the journal (Circ. Res.), but unfortunately was informed that they do not accept or publish correspondence related to this type of article. So, here's my un-published letter, which raises a number of issues with the article...

A recent Circ. Res. viewpointLoscalzo J, 2016 discussed the complex relationships between redox biology and metabolism in the setting of hypoxia, with an emphasis on the use of biochemically correct terminology. While there is broad agreement that the field of redox biology is often confounded by use of inappropriate methods and language Kalyanaraman B, 2012,Forman HJ, 2015, concern is raised regarding some ideas on reductive stress in the latter part of the article.

In discussing the fate of glycolytically-derived NADH in hypoxia, the reader is urged to “Remember that while redirecting glucose metabolism to glycolysis decreases NADH production by the TCA cycle and decreases leaky electron transport chain flux, glycolysis continues to produce NADH". First, glucose undergoes glycolysis regardless of cellular oxygenation status; this simply happens at a faster rate in hypoxia. As such, glucose is not redirected but rather its product pyruvate is. Second, regardless a proposed lower rate of NADH generation by the TCA cycle (which may not actually be the case Chouchani ET, 2014,Hochachka PW, 1975) NADH still accumulates in hypoxic mitochondria because its major consumer, the O2-dependent respiratory chain, is inhibited. It is clear that both NADH consumers and producers can determine the NADH/NAD+ ratio, and in hypoxia the consumption side of the equation cannot be forgotten.

While the field is in broad agreement that NADH accumulates in hypoxia, the piece goes on to claim that “How the cell handles this mounting pool of reducing equivalents remained enigmatic until recently.” This is misleading. The defining characteristic of hypoxia, one that has dominated the literature in the nearly 90 years since Warburg's seminal work Warburg O, 1927, is the generation of lactate by lactate dehydrogenase (LDH), a key NADH consuming reaction that permits glycolysis to continue. Lactate is “How cells handle the mounting pool of reducing equivalents.”

Without mentioning lactate, an alternate fate for hypoxic NADH is proposed, based on the recent discovery that both LDH and malate dehydrogenase (MDH) can use NADH to drive the reduction of 2-oxoglutarate (α-ketoglutarate, α-KG) to the L(S)-enantiomer of 2-hydroxyglutarate (L-2-HG) under hypoxic conditions Oldham WM, 2015,Intlekofer AM, 2015. We also found elevated 2-HG in the ischemic preconditioned heart Nadtochiy SM, 2015, and recently reported that acidic pH – a common feature of hypoxia – can promote 2-HG generation by LDH and MDH Nadtochiy SM, 2016.

While there can be little doubt that the discovery of hypoxic L-2-HG accumulation is an important milestone in understanding hypoxic metabolism and signaling, the claim that L-2-HG is “a reservoir for reducing equivalents and buffers NADH/NAD+” is troublesome on several counts. From a quantitative standpoint, we reported the canonical activities of LDH (pyruvate + NADH --> lactate + NAD+) and of MDH (oxaloacetate + NADH --> malate + NAD+) are at least 3-orders of magnitude greater than the rates at which these enzymes can reduce α-KG to L-2-HG Nadtochiy SM, 2016. This is in agreement with an earlier study reporting a catalytic efficiency ratio of 10⁷ for the canonical vs. L-2-HG generating activities of MDH Rzem R, 2007. Given these constraints, we consider it unlikely that the generation of L-2-HG by these enzymes is a quantitatively important NADH sink, compared to their native reactions. It is also misleading to refer to the α-KG --> L-2-HG reaction as a "reservoir for reducing equivalents", because even though this reaction consumes NADH, it is not clear whether the reverse reaction regenerates NADH. Specifically, the metabolite rescue enzyme L-2-HG-dehydrogenase uses an FAD electron acceptor and is not known to consume NAD+ Nadtochiy SM, 2016,Rzem R, 2007,Weil-Malherbe H, 1937.

Another potentially important sink for reducing equivalents in hypoxia that was not mentioned, is succinate. During hypoxia, NADH oxidation by mitochondrial complex I can drive the reversal of complex II (succinate dehydrogenase) to reduce fumarate to succinate Chouchani ET, 2014. This redox circuit, in which fumarate replaces oxygen as an electron acceptor for respiration, was first hinted at over 50 years ago SANADI DR, 1963. Importantly (and in contrast to L-2-HG as mentioned above), the metabolites recovered upon withdrawal from a fumarate --> succinate "electron bank" are the same as those deposited.

Although recent attention has focused on the pathologic effects of accumulated succinate in driving ROS generation at tissue reperfusion Chouchani ET, 2014,Pell VR, 2016, the physiologic importance of hypoxic complex II reversal as a redox reservoir and as an evolutionarily-conserved survival mechanism Hochachka PW, 1975 should not be overlooked. Quantitatively, the levels of lactate and succinate accumulated during hypoxia are comparable Hochachka PW, 1975, and both are several orders of magnitude greater than reported hypoxic 2-HG levels.

While overall the article makes a number of important points regarding reductive stress and the correct use of terminology in this field, we feel that the currently available data do not support a quantitatively significant role for L-2-HG as a hypoxic reservoir for reducing equivalents. These quantitative limitations do not diminish the potential importance of L-2-HG as a hypoxic signaling molecule Nadtochiy SM, 2016,Su X, 2016,Xu W, 2011.

Paul S. Brookes, PhD.

On 2016 Dec 22, Holger Schunemann commented:

Error in author listing; the correct citation for this article is http://www.bmj.com/content/354/bmj.i3507: BMJ. 2016 Jul 20;354:i3507. doi: 10.1136/bmj.i3507. When and how to update systematic reviews: consensus and checklist. Garner P, Hopewell S, Chandler J, MacLehose H, Akl EA, Beyene J, Chang S, Churchill R, Dearness K, Guyatt G, Lefebvre C, Liles B, Marshall R, Martínez García L, Mavergames C, Nasser M, Qaseem A, Sampson M, Soares-Weiser K, Takwoingi Y, Thabane L, Trivella M, Tugwell P, Welsh E, Wilson EC, Schünemann HJ

On 2017 Dec 26, Kevin Kavanagh commented:

We have expressed concerns in a previous letter and PubMed Common’s posting regarding the article by Kelly, et al,(1) where the efficacy of the evaluated product may have been overstated.(2, 3) In the authors reply, data was presented which presents less than a 30% reduction(4) as opposed to a 42% which is stated in the article and advertised by the company.(1,5) To our knowledge the peer-review record has not been corrected. In addition, we have concerns regarding at least the appearance of an undeclared conflict-of-interest between one of the article’s authors, Connie Steed, and the company in question, DebMed.(6)

It has come to the authors’ attention that the editor in charge of adjudicating the above concerns may also have a conflict-of-interest with DebMed and with one of the authors of the manuscript in question. Significant concerns regarding the conflicts of interest of the Editor Elaine Larson have arisen because of the following associations:

• Co-Author with Connie Steed (one of the authors in the manuscript in question) and Paul Alpert (Vice President of Patient Safety Strategy for DebMed) in an article published in Feb 2011.(7). Conflicts-of-Interest stated the following “Elaine Larson has received research funding from Deb Worldwide Healthcare, Inc.”

• Co-Author with Paul Alpert (Vice-President of Patient Safety Strategy for DebMed) in an article published in Jan 2013.(8)

• Co-Author with Paul Alpert (Vice-President of Patient Safety Strategy for DebMed) in an article published in Feb 2014.(9)

• Connie Steed, RN is listed as the 2016 Secretary for the Association for Professionals in Infection Control and Epidemiology, Inc., which has as its official publication the American Journal of Infection Control(10) and provides this Journal as a benefit of their membership.(11)

We feel that because of the above, the appearance of a conflict of interest exists which may have clouded the decision making and inhibited the correction of the potential research integrity problems in the article in question.

References

(1) Kelly, J.W., Blackhurst, D., McAtee, W., and Steed, C. Electronic hand hygiene monitoring as a tool for reducing health care-associated methicillin-resistant Staphylococcus aureus infection. Am J Infect Control. 2016; 44: 956–95 Kelly JW, 2016

(2) Kavanagh KT, Saman DM. Comment Regarding: Electronic hand hygiene monitoring as a tool for reducing health care–associated methicillin-resistant Staphylococcus aureus infection. American Journal of infection Control. December 01 2016 http://www.ajicjournal.org/article/S0196-6553(16)30904-X/fulltext

(3) Kavanagh KT, Saman DM. Comment on PMID: 27908437: Response to Letter Regarding Manuscript “Electronic Hand Hygiene Monitoring as a Tool for Reducing Nosocomial Methicillin-resistant Staphylococcus aureus Infection” In: PubMed Commons [Internet]. Bethesda (MD): National Library of Medicine; 2016 Dec. 16. Available from: https://www.ncbi.nlm.nih.gov/pubmed/27908437#cm27908437_34333

(4) Kelly JW, Blackhurst D, McAtee W, Steed C. Response to Letter Regarding Manuscript "Electronic Hand Hygiene Monitoring as a Tool for Reducing Nosocomial Methicillin-resistant Staphylococcus aureus Infection". Am J Infect Control. 2016 Dec 1;44(12):1763. doi>: 10.1016/j.ajic.2016.08.009. http://www.ajicjournal.org/article/S0196-6553(16)30812-4/fulltext Kelly JW, 2016

(5) DebMed: Discover how one facility was able to reduce MRSA HAIs by up to 42%. Last accessed on Dec. 21, 2017 from http://info.debmed.com/mrsa-study-flyer

(6) Hospital Reduces MRSA Rates by 42% with electronic hand hygiene measurement. Infection Control Today. July 8, 2016. http://www.infectioncontroltoday.com/news/2016/07/hospital-reduces-mrsa-rates-by-42-with-electronic-hand-hygiene-measurement.aspx

(7) Connie Steed, MSN, RN, CIC J. William Kelly, MD Dawn Blackhurst, DrPH Sue Boeker, BSN, RN, CIC Thomas Diller, MD, MMM Paul Alper, BA Elaine Larson, RN, PhD, FAAN, CIC Hospital hand hygiene opportunities: Where and when (HOW2)? The HOW2 Benchmark Study. American Journal of Infection Control. Feb 2011 39(1):19-26. Steed C, 2011

(8) Buet A, Cohen B, Marine M, Scully F, Alper P, Simpser E, Saiman L, Larson E. Hand hygiene opportunities in pediatric extended care facilities. J Pediatr Nurs. 2013 Jan;28(1):72-6. doi: 10.1016/j.pedn.2012.04.010. Epub 2012 Jun 1. Buet A, 2013

(9) Conway LJ, Riley L, Saiman L, Cohen B, Alper P, Larson EL. Implementation and impact of an automated group monitoring and feedback system to promote hand hygiene among health care personnel. Jt Comm J Qual Patient Saf. 2014 Sep;40(9):408-17. Conway LJ, 2014

(10) American Journal of Infection Control Home Page. Accessed on Dec. 26, 2017 from https://www.journals.elsevier.com/ajic-american-journal-of-infection-control

(11) Association for Professionals in Infection Control and Epidemiology, Inc. Web Posting. Accessed on Dec. 26, 2017 from http://www.ajicjournal.org/article/S0196-6553(15)01270-5/pdf

Comment also posted on PubPeer

On 2016 Jun 18, Raphael Stricker commented:

Circular Reasoning in CDC Lyme Disease Test Review

Raphael B. Stricker, MD; Lorraine Johnson, JD, MBA

Previous studies have shown that commercial two-tier serological testing has a sensitivity of about 46% in later-stage Lyme disease in the USA [1]. Commercial two-tier Lyme testing in Europe demonstrates the same poor test sensitivity [2]. The Table in the latest Centers for Disease Control and Prevention (CDC) review by Moore et al. cites three studies allegedly showing that two-tier Lyme testing in later-stage (“non-cutaneous”) Lyme disease has a sensitivity of 87-96% [3]. These numbers will undoubtedly be used to support two-tier testing as a valid diagnostic tool for Lyme disease. Therefore it is important to understand the circular reasoning that produced these inflated and misleading numbers.

Analysis of the three studies cited in the CDC review reveals the following:

+1. Branda et al [4]: Two-tier Lyme test sensitivity 87% (55 patients). The Methods section of this article contains the following language: "All patients categorized as having Lyme disease met the CDC surveillance criteria for the diagnosis.” The reference for this statement [5] contains the CDC surveillance criteria for the diagnosis of Lyme disease. The portion of the CDC surveillance criteria relevant for later Lyme disease is set forth below.

Clinical case definition:

1. Erythema migrans, or
2. At least one late manifestation, as defined below, and laboratory confirmation of infection (emphasis added).

Laboratory criteria for diagnosis:

1. Isolation of Borrelia burgdorferi from clinical specimen, or
2. Demonstration of diagnostic levels of IgM and IgG antibodies to the spirochete in serum or CSF, or
3. Significant change in IgM or IgG antibody response to B. burgdorferi in paired acute- and convalescent-phase serum samples.

This surveillance case definition was developed for national reporting of Lyme disease; it is NOT appropriate for clinical diagnosis (emphasis added).

Comment: Although the Branda et al. study does not say how many later-stage Lyme patients were culture-positive, presumably most were included based on positive serology. Patients who had positive serology as part of the study entry criteria would be expected to have positive serology on the same outcome measure. Circular reasoning.

+2. Molins et al [6]: Two-tier Lyme test sensitivity 96% (46 patients). The Methods section of this article contains the following language: "Lyme disease serology or results from two-tiered testing did not play a role in patient enrollment except for inclusion of late-stage Lyme arthritis patients (emphasis added).”

Comment: Once again, patients with later-stage Lyme disease had to have positive serology in order to be included in the study, and then they had positive serology. Circular reasoning.

+3. Wormser et al [7]: Two-tier Lyme test sensitivity 94% (142 patients). This study was a cost analysis article based on another study [8] that contains the following language: "Lyme arthritis was defined as the presence of joint swelling that was clinically compatible with Lyme arthritis in conjunction with serologic evidence of borrelial infection demonstrated by at least a positive WCS ELISA (emphasis added).”

Comment: Once again, patients with later-stage Lyme disease had to have positive serology in order to be included in the study, and then they had positive serology. Circular reasoning.

Conclusions: Based on circular reasoning, the latest CDC analysis perpetuates the myth that two-tier testing is sensitive for later-stage Lyme disease. The comment in the CDC surveillance guidelines that this testing is NOT appropriate for clinical diagnosis is being ignored by the CDC.

References

[1] Stricker RB, Johnson L. Lyme disease diagnosis and treatment: Lessons from the AIDS epidemic. Minerva Med. 2010;101:419–25.

[2] Ang CW, Notermans DW, Hommes M, Simoons-Smit AM, Herremans T. Large differences between test strategies for the detection of anti-Borrelia antibodies are revealed by comparing eight ELISAs and five immunoblots. Eur J Clin Microbiol Infect Dis. 2011;30:1027-32.

[3] Moore A, Nelson C, Molins C, Mead P, Schriefer M. Current guidelines, common clinical pitfalls, and future directions for laboratory diagnosis of Lyme disease, United States. Emerg Infect Dis. 2016 Jul;22(7). doi: 10.3201/eid2207.151694.

[4] Branda JA, Linskey K, Kim YA, Steere AC, Ferraro MJ. Two-tiered antibody testing for Lyme disease with use of 2 enzyme immunoassays, a whole-cell sonicate enzyme immunoassay fol¬lowed by a VlsE C6 peptide enzyme immunoassay. Clin Infect Dis. 2011;53:541–7.

[5] Wharton M, Chorba TL, Vogt RL, Morse DL, Buehler JW. Case definitions for public health surveillance. MMWR Recomm Rep 1990; 39:1–43.

[6] Molins CR, Sexton C, Young JW, Ashton LV, Pappert R, Beard CB, et al. Collection and characterization of samples for establishment of a serum repository for Lyme disease diagnostic test development and evaluation. J Clin Microbiol. 2014;52:3755–62.

[7] Wormser GP, Levin A, Soman S, Adenikinju O, Longo MV, Branda JA. Comparative cost-effectiveness of two-tiered testing strategies for serodiagnosis of Lyme disease with noncutaneous manifestations. J Clin Microbiol. 2013;51:4045–9.

[8] Wormser GP, Schriefer M, Aguero-Rosenfeld ME, Levin A, Steere AC, Nadelman RB, et al. Single-tier testing with the C6 peptide ELISA kit compared with two-tier testing for Lyme disease. Diagn Microbiol Infect Dis. 2013;75:9–15.

Disclosure: RBS and LJ are members of the International Lyme and Associated Diseases Society (ILADS) and directors of LymeDisease.org. They have no financial or other conflicts to declare.

On 2016 May 27, Hans Morreau commented:

Massive Chromosomal Loss with Subsequent Whole Genome Doubling is seen in a Wide Variety of Rare Tumor Types:

The authors Zheng et al. performed an impressive molecular characterization of 91 cases of adrenal cortical carcinoma (ACC). The integrated analysis is the way to understand the behaviour of this rare disease far better and hopefully will lead to better treatment options. They conclude that there is a subset of ACCs showing massive chromosomal loss with subsequent whole genome doubling (WGD). The authors state that this chromosomal loss leads to a hypodiploid karyotype. Such a phenomenon “was only matched by chromophobe renal cell carcinoma”. The latter is partly correct. In 2012 we published the occurrence near-haploidisation with or without subsequent endoreduplication (whole-genome doubling) in oncocytic follicular thyroid carcinoma (FTC-OV) and anaplastic thyroid carcinoma (ATC) derived from FTC-OV (Corver et al., 2012). In combined SNP array analysis and DNA content analysis the genomes of these lesions were seen as near-homozygous genomes (NHG) with DNA indices of 0.6-1.4 depending on the absence or presence of endoreduplication. Wagle et al. independently confirmed our observations in the New England Journal of Medicine with the description of one ATC patient who showed a spectacular treatment response upon giving the mTOR inhibitor everolimus. The patient’s ATC derived from FTC-OV and high density SNP analysis clearly identified NHG in the tumor. The phenomenon of NHG with or without endoreduplication is similar to the pattern that is seen by Zheng et al, although the terminology to describe this is slightly different. In 2014 we also showed that in a subset of ACC and parathyroid carcinoma NHG and endoreduplication can be seen, especially in tumors with oncocytic metaplasia (Corver et al., 2014). As seen by Zheng et al the “allelic states” (Corver et al., 2008) in ACC indicated the presence of more chromosomal breakpoints than seen in FTC. In fact similar observations of NHG or widespread chromosomal loss with endoreduplication of the complete genome has been described in peripheral chondrosarcomas (Bovee et al., 2000) and a subset of childhood acute lymphoblastic leukemia (Holmfeldt et al., 2013) and other uncommon cancers (Mandahl et al., 2012). It is intriguing what is eventually responsible for the widespread chromosomal loss with or without endoreduplication. In our model we proposed a stepwise process that might be related to metabolic processes, something that still needs to be proven. It might now be a step forward in understanding the underlying biology of endoreduplication/WGD with the observation of Zheng et al. that TERT expression is higher in the WGD group of ACCs. In conclusion the combined analysis of different tumor types with massive chromosomal loss with subsequent WGD might lead to further insights underlying this remarkable process.

References:

Zheng S, Cherniack AD, Dewal N, Moffitt RA, Danilova L, Murray BA, Lerario AM, Else T, Knijnenburg TA, Ciriello G, et al. (2016). Comprehensive Pan-Genomic Characterization of Adrenocortical Carcinoma. Cancer Cell. 29(5):723-36.

Corver, W. E., Ruano, D., Weijers, K., den Hartog, W. C., van Nieuwenhuizen, M. P., de Miranda, N., van Eijk, R., Middeldorp, A., Jordanova, E. S., Oosting, J., et al. (2012). Genome haploidisation with chromosome 7 retention in oncocytic follicular thyroid carcinoma. PLoS ONE 7, e38287.

Wagle N, Grabiner BC, Van Allen EM, Amin-Mansour A, Taylor-Weiner A, Rosenberg M, Gray N, Barletta JA, Guo Y, Swanson SJ, et al.(2014) Response and acquired resistance to everolimus in anaplastic thyroid cancer. N Engl J Med. 371(15):1426-33.

Corver, W. E., van, W. T., Molenaar, K., Schrumpf, M., van den Akker, B., van, E. R., Ruano, N. D., Oosting, J., and Morreau, H. (2014). Near-haploidization significantly associates with oncocytic adrenocortical, thyroid, and parathyroid tumors but not with mitochondrial DNA mutations. Genes Chromosomes Cancer 53, 833-844.

Corver, W. E., Middeldorp, A., Ter Haar, N. T., Jordanova, E. S., van Puijenbroek, M., van Eijk, R., Cornelisse, C. J., Fleuren, G. J., Morreau, H., Oosting, J., and van Wezel, T. (2008). Genome-wide allelic state analysis on flow-sorted tumor fractions provides an accurate measure of chromosomal aberrations. Cancer Res 68, 10333-10340.

Bovee, J. V., van Royen, M., Bardoel, A. F., Rosenberg, C., Cornelisse, C. J., Cleton-Jansen, A. M., and Hogendoorn, P. C. (2000). Near-haploidy and subsequent polyploidization characterize the progression of peripheral chondrosarcoma. Am J Pathol 157, 1587-1595.

Holmfeldt, L., Wei, L., Diaz-Flores, E., Walsh, M., Zhang, J., Ding, L., Payne-Turner, D., Churchman, M., Andersson, A., Chen, S. C., et al. (2013). The genomic landscape of hypodiploid acute lymphoblastic leukemia. Nat Genet 45, 242-252.

Mandahl, N., Johansson, B., Mertens, F., and Mitelman, F. (2012). Disease-associated patterns of disomic chromosomes in hyperhaploid neoplasms. Genes Chromosomes Cancer 51, 536-544.

Hans Morreau, also on behalf of Willem Corver and Tom van Wezel, Dept of Pathology, Leiden University Medical Center The Netherlands Email: j.morreau@lumc.nl.

Note: This comment was also posted on the website of Cancer Cell attached to the manuscript of Zheng et al 2016. This will however not be visible in the PubMed domain. I do not have conflicts of interest to declare.

On 2016 Apr 12, Martin Hofmeister commented:

Do not forget published reviews

I thank Dr Mat Eil Ismail et al, for their interesting article "Preoperative physiotherapy and short-term functional outcomes of primary total knee arthroplasty", published in the March 2016 issue of the Singapore Medical Journal (SMJ). There is one aspect worth mentioning. In my opinion, systematic reviews of the past five years should be included in an original article consistent with good scientific practice. The reviews of Kwok et al, Jordan et al, Simmons et al and the Australian Agency for Clinical Innovation about the evidence of preoperative physiotherapy on outcomes following total knee arthroplasty are not mentioned in the discussion (1-4). The results of the SMJ study reconfirm the above-mentioned reviews: No statistically significant effect in patient key outcomes (1-4). I refer readers to the latest meta-analysis "Does preoperative rehabilitation for patients planning to undergo joint replacement surgery improve outcomes?" that can be found in the February 2016 issue of the BMJ Open (5).

REFERENCES

1) Kwok IH, Paton B, Haddad FS. Does Pre-Operative Physiotherapy Improve Outcomes in Primary Total Knee Arthroplasty? - A Systematic Review. J Arthroplasty. 2015;30:1657-63. Kwok IH, 2015

2) Jordan RW, Smith NA, Chahal GS, Casson C, Reed MR, Sprowson AP. Enhanced education and physiotherapy before knee replacement; is it worth it? A systematic review. Physiotherapy. 2014;100:305-12. Jordan RW, 2014

3) Simmons L, Smith T. Effectiveness of pre-operative physiotherapy-based programmes on outcomes following total knee arthroplasty: a systematic review and meta-analysis. Phys Ther Rev. 2013;18:1-10. http://www.tandfonline.com/doi/abs/10.1179/1743288X12Y.0000000035?journalCode=yptr20

4) NSW Agency for Clinical Innovation (NSW ACI). Musculoskeletal Network: NSW Evidence Review: preoperative, perioperative and postoperative care of elective primary total hip and knee replacement. Chatswood, Australia: Agency for Clinical Innovation, 2012. Available at: http://www.aci.health.nsw.gov.au/__data/assets/pdf_file/0020/172091/EJR-Evidence-Review.PDF. Accessed 22 March 2016.

5) Wang L, Lee M, Zhang Z, Moodie J, Cheng D, Martin J. Does preoperative rehabilitation for patients planning to undergo joint replacement surgery improve outcomes? A systematic review and meta-analysis of randomised controlled trials. BMJ Open 2016;6:e009857. Wang L, 2016

On 2016 Mar 15, Tom Kindlon commented:

Depression scores in this follow-up study are very different to scores in original study (looking solely at the Reeves et al. (2005) operationalization)

Leonard Jason and colleagues previously raised concerns about the Reeves et al. (2005) chronic fatigue syndrome (CFS) criteria [which have also been described as an operationalisation of the Fukuda et al (1994) criteria] (1-4). In particular, Jason and colleagues were concerned that some people who did not have CFS might get diagnosed with CFS using this new set of criteria. They found some evidence to support this concern in a study of those with major depressive disorder who did not have CFS: 38% were found to satisfy these new criteria for CFS(4).

Looking solely at the current study, it would look like there might have been little basis for these concerns. Of 71 people classified with CFS in the current study, only one (1.4%) had a Zung self-rating depression scale (SDS) (5) score of >=60. The mean SDS score for the 71 CFS participants was 44.78 (calculated from the data in Table 4) (6).

However, it should be noted that the SDS (depression) scores in the follow-up study are very different from the scores in the original Georgia cohort(7). Of the 113 people diagnosed with CFS in the original Georgia cohort, data for 112 (99.1%) was published(7). The average SDS score was considerably higher at 56.2. Possibly more revealingly, 40.2% had a SDS score of >=60. As described in the paper, the SDS scale provides an index score and categories reflecting no (<50), mild (50-59), moderate (60-69), and severe (>=70) depression.

I am not sure why there should be such a large difference in a cohort between the initial and follow-up studies in the rate of those with moderate or severe depression (40.2% vs 1.4%). But it does mean that caution should be used in terms of interpreting the findings reported in the current paper and their significance regarding the Reeves et al. (2005) criteria (1,6).

References:

[1]. Reeves WC, Wagner D, Nisenbaum R, Jones JF, Gurbaxani B, Solomon L, Papanicolaou DA, Unger ER, Vernon SD, Heim C. Chronic fatigue syndrome--a clinically empirical approach to its definition and study. BMC Med. 2005 Dec 15;3:19.

[2]. Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, Komaroff A. The chronic fatigue syndrome; a comprehensive approach to its definition and study. Ann Int Med 1994, 121:953-959.

[3]. Jason LA, & Richman JA. How science can stigmatize: The case of chronic fatigue syndrome. Journal of CFS 2007;14:85-103.

[4]. Jason LA, Najar N, Porter N, Reh C. Evaluating the Centers for Disease Control's empirical chronic fatigue syndrome case definition. Journal of Disability Policy Studies 2009;20;93.

[5]. Zung WW, Richards CB, Short MJ. Self-rating depression scale in an outpatient clinic: further validation of the SDS. Arch Gen Psychiatry.1965;13(6):508-515.

[6]. Unger ER, Lin JM, Tian H, Gurbaxani BM, Boneva RS, Jones JF. Methods of applying the 1994 case definition of chronic fatigue syndrome - impact on classification and observed illness characteristics. Popul Health Metr. 2016 Mar 12;14:5.

[7]. Heim C1, Nater UM, Maloney E, Boneva R, Jones JF, Reeves WC. Childhood trauma and risk for chronic fatigue syndrome: association with neuroendocrine dysfunction. Arch Gen Psychiatry. 2009 Jan;66(1):72-80.

On 2016 Feb 29, Gary Goldman commented:

Marin et al report a vaccine effectiveness (VE) of 81% (95% C.I.: 78-84) for the one-dose varicella vaccination protocol. [1] This figure is biased high and declines rapidly as the vaccine is widely used and exogenous boosting becomes rare. Many of the clinical trials and studies that reported VE were conducted within the first few years of the start of varicella vaccination—during a time period when vaccinees were additionally boosted by exogenous exposures to those shedding wild-type (or natural) varicella-zoster virus during annual outbreaks. Annual VE, derived from secondary family attack rate (SFAR) data among contacts aged <20 years reporting to the Antelope Valley Varicella Active Surveillance Project (VASP), demonstrated an annual increase from 87% in 1997 to 96% in 1999 (the last year that varicella displayed its characteristic seasonality), then declined to 85% and 74% in 2000 and 2001, respectively, when exogenous boosting substantially decreased. [2]

The conclusion given in the Meta-analysis by Marin et al states that several studies reported a lower risk for herpes zoster among varicella vaccinated children “and a decline in herpes zoster incidence among cohorts targeted for varicella vaccination.” [1] The later part of that statement is patently false. There are two confounders in the cited studies that contribute to this erroneous conclusion and a consideration of these confounders helps to explain why the VASP study [3] (referenced in the Meta-analysis [1]) reports that (a) the 2000-2006 HZ incidence increased by 63% among 10- to 19-year olds and (b) HZ incidence decreased by 32% from 98.3/100,000 person-years (p-y) in 2006 to 66.7/100,000 p-y in 2010, with “substantial fluctuation in annual HZ rates.” [3]

The authors of both the Meta-analysis [1] and supporting reference [3] have erroneously assumed that the HZ cases reported to the VASP represent 100% reporting completeness. However, using capture-recapture with two ascertainment sources (schools and health cares), it was demonstrated that varicella cases among 2- to 18-year-olds were under-reported by approximately 45%. [4] Likewise, it can be shown that VASP also experienced approximately 50% under-reporting of HZ cases [2], leading to the Marin et al study [3] reporting incidence rates that are one-half the actual rates. HZ incidence rates that have not been ascertainment corrected simply reflect the incidence of reported HZ cases to the VASP and not the HZ incidence rate in the community. It is invalid to compare the uncorrected VASP-reported HZ rates to those rates reported by other studies that possess much higher case ascertainment. [5]

Additionally, the >10- to 19-year-old age category consists of three different cohorts with widely differing HZ incidence rates. Marin et al [3] only considers the mean HZ incidence rate for each age category instead of stratifying by (1) those still susceptible to varicella and never vaccinated (0 cases/100,000 p-y); (2) those that have had a prior history of wild-type varicella who exhibit increasing HZ incidence rates from approximately 120 cases/100,000 p-y to 500 cases/100,000 p-y (in the absence of exogenous boosting); and (3) those vaccinated who exhibit an HZ incidence rate less than 120 cases/100,000 p-y.

In summary, unless HZ incidence rates are ascertainment corrected [5], such rates will erroneously be reported as “lower” than other studies. [1] Also, reporting the mean HZ incidence of a bimodal distribution masks the widely differing incidence rates among those vaccinated and those with a prior history of varicella. Further, this invalid mean masks the significant effects of exogenous boosting. [7] Varicella vaccination innoculates children with the Oka-strain VZV. When these children are exposed to natural varicella or herpes zoster in adults, they may additionally harbor the natural VZV strain. Both strains are subject to reactivation as HZ. This is another confounder in the reporting of HZ incidence rates. Health officials initially believed that only a single dose of varicella vaccine would provide long-term protection and have negligible impact on the incidence of HZ. These assumptions are incorrect and have led to a continual cycle of treatment and disease. The shingles (herpes zoster) vaccine now provides the boosting to postpone or suppress the reactivation of HZ in adults aged 60 years and older—a substitute for the exogenous boosting that was prevalent in the pre-varicella vaccination era at no cost. [6]

References:

[1] Marin M, Marti M, Kambhampati A, Jeram SM, Seward JF. Global varicella vaccine effectiveness: A meta-analysis. Pediatrics Feb. 16, 2016; DOI: 10.1542/peds.2015-3741. Marin M, 2016

[2] Goldman GS. Universal Varicella Vaccination: Efficacy Trends and Effect on Herpes Zoster. Int J Toxicol 2006 Sep-Oct; 25(5):313-317. Goldman GS, 2005

[3] Marin M. Civen R, Zhang J, et al. Update on incidence of herpes zoster among children and adolescents following implementation of varicella vaccination, Antelope Valley, CA. 2000-2010. Presented at IDweek 2015, October 7-11, 2015; San Diego, CA.

[4] Seward JF, Watson BM, Peterson CL, Mascola L, Pelosi JW, Zhang JX, et al. Varicella disease after introduction of varicella vaccine in the United States, 1995-2000. JAMA 2002; 287(5):606-611. Seward JF, 2002

[5] Hook EB, Regal RR. The value of capture-recapture methods even for apparent exhaustive surveys: the need for adjustment for source of ascertainment intersection in attempted complete prevalence studies. Am J Epidemiol 1992; 135:1060-1067. Hook EB, 1992

[6] Goldman GS, King PG. Review of the United States universal varicella vaccination program: Herpes-zoster incidence rates, cost effectiveness, and vaccine efficacy based primarily on the Antelope Valley Varicella Active Surveillance Project data. Vaccine 2013; 31(13):1680-1694. Goldman GS, 2013

[7] Guzzetta G, Poletti P, Del Vava E, et al. Hope-Simpson’s progressive immunity hypothesis as a possible explanation for herpes –zoster incidence data. Am J Epidemiol 2013; 77(10):1134-1142. Guzzetta G, 2013

On 2016 Mar 15, Lionel Christiaen commented:

In this article, the author presents an extensive account of the extreme diversity of adult anatomies and life histories encountered across the thousands of tunicate species that roam the oceans worldwide, and occupy multitudes of ecological niches. The author then emphasizes that tunicate genomes are markedly more compact and evolve faster than the genomes of their chordate relatives, the cephalochordates and vertebrates. Several recent studies support this notion, and the argument that rapid genome diversification may have fostered tunicate evolution is reasonable. Since the early development of tunicates, in particular ascidians, has been considerably simplified and streamlined in a manner analogous to what is observed in nematodes, the author argues that tunicates must have lost most ancestral genomic, developmental and anatomical features that could inform reconstruction of the evolutionary history of vertebrate traits. We wish to provide alternative interpretations and propose a more inclusive approach to the problems posed by tunicates in building models for the evolution of vertebrates. First, the argument about faster evolutionary rates implies that every part of the genome evolves at similarly faster rates; yet, phylogenomic analyses of concatenated coding sequences unequivocally revealed that tunicates and vertebrates form a monophyletic group referred to as olfactores [1, 2]. Moreover, conserved anatomical features including the notochord, the dorsal neural tube and the pharyngeal gill slits depend upon ancestral regulatory inputs from conserved transcription factors, as noted by the author. These simple examples argue against a complete relaxation of evolutionary constraints on ancestral features in tunicates, especially in ascidians. In other words, high average rates of sequence evolution and profound morphological changes are not incompatible with deep conservation of cellular and molecular mechanisms for embryonic patterning and cell fate specification. Instead, the apparent incompatibility between high rates of genome divergence and the maintenance of ancestral olfactores features over long evolutionary distances hints at the notion of developmental system drift (DSD), whereby mechanistically connected developmental features may be conserved between distantly related species exhibiting extensive divergence of the intervening processes [3]. Ascidians provide an attractive test-bed to study DSD since their early embryos have barely changed in almost half a billion years, despite considerable genomic divergence [4]. This is a lively area of research as illustrated by the 11 tunicate genomes recently made openly available to the worldwide research community [4-6]. We argue that comparative developmental studies are poised to identify additional features conserved between tunicates and vertebrates, such as those recently reported for the neural crest, the cranial placodes and the cardiopharyngeal mesoderm [7-10]. These "islands of conservation" will continue to shed light on the mechanisms of tunicate diversification and the deep evolutionary origins of the vertebrate body plan.

REFERENCES 1. Delsuc, F., Brinkmann, H., Chourrout, D., and Philippe, H. (2006). Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439, 965-968. 2. Putnam, N.H., Butts, T., Ferrier, D.E., Furlong, R.F., Hellsten, U., Kawashima, T., Robinson-Rechavi, M., Shoguchi, E., Terry, A., Yu, J.K., et al. (2008). The amphioxus genome and the evolution of the chordate karyotype. Nature 453, 1064-1071. 3. True, J.R., and Haag, E.S. (2001). Developmental system drift and flexibility in evolutionary trajectories. Evolution & development 3, 109-119. 4. Stolfi, A., Lowe, E.K., Racioppi, C., Ristoratore, F., Brown, C.T., Swalla, B.J., and Christiaen, L. (2014). Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians. eLife 3, e03728. 5. Voskoboynik, A., Neff, N.F., Sahoo, D., Newman, A.M., Pushkarev, D., Koh, W., Passarelli, B., Fan, H.C., Mantalas, G.L., Palmeri, K.J., et al. (2013). The genome sequence of the colonial chordate, Botryllus schlosseri. eLife 2, e00569. 6. Brozovic, M., Martin, C., Dantec, C., Dauga, D., Mendez, M., Simion, P., Percher, M., Laporte, B., Scornavacca, C., Di Gregorio, A., et al. (2016). ANISEED 2015: a digital framework for the comparative developmental biology of ascidians. Nucleic acids research 44, D808-818. 7. Abitua, P.B., Gainous, T.B., Kaczmarczyk, A.N., Winchell, C.J., Hudson, C., Kamata, K., Nakagawa, M., Tsuda, M., Kusakabe, T.G., and Levine, M. (2015). The pre-vertebrate origins of neurogenic placodes. Nature 524, 462-465. 8. Abitua, P.B., Wagner, E., Navarrete, I.A., and Levine, M. (2012). Identification of a rudimentary neural crest in a non-vertebrate chordate. Nature 492, 104-107. 9. Diogo, R., Kelly, R.G., Christiaen, L., Levine, M., Ziermann, J.M., Molnar, J.L., Noden, D.M., and Tzahor, E. (2015). A new heart for a new head in vertebrate cardiopharyngeal evolution. Nature 520, 466-473. 10. Stolfi, A., Ryan, K., Meinertzhagen, I.A., and Christiaen, L. (2015). Migratory neuronal progenitors arise from the neural plate borders in tunicates. Nature 527, 371-374.

On 2017 Feb 08, Tony Gardner-Medwin commented:

This paper (Gomes et al., 2016) raises a dilemma for readers. If well founded, it clearly merits much work to understand it and its implications. But the conclusions conflict so strongly with conventional wisdom that it is tempting to dismiss it as probably somehow incorrect. All credit therefore to Barbour (2016) for critiquing it and pinpointing questions that clearly need answering. Hopefully, both the authors and others with their own perspectives may contribute to clarify the situation.

Broadly I concur with the points Barbour raises. I would add however what seems possibly a key oversight in the papers from Gomes' group. This is in the argument that observed filter characteristics and cell input impedances that fall off with the square root of frequency at high frequencies are indicative of diffusion processes, rather than R-C elements as in conventional modelling. Cable equations for the input impedance of even the simplest dendritic model (with uniform characteristics and a length of many space constants: V'' = Λ^-2 (1+ jωτ) V, I= -V'/R, Z = RΛ / √(1+jωτ), |Z| = RΛ (1+ω² τ² )^-0.25 ) predict just such a relation (Rall & Rinzel, 1973: see equations A13, A15 for the more general solution with dendrites of any length). So the argument of Gomes et al. that the data implicate diffusion processes (which can also lead to square root relationships) seems to collapse.

Though the external pathway for current generated by neurons is usually regarded as largely within interstitial space, it is not exclusively so, even at low frequencies or DC. Around 6% of DC current passed through rat cortex is accounted for by K⁺ flux (Gardner-Medwin, 1983). Since current in interstitial space would only account for a K⁺ flux of 1.2%, the difference is presumably due to trans-cellular passage of at least 5% of long distance current flow, probably largely through the astrocytic syncytium. This is a small adjustment to the notion that low frequency currents are largely extracellular, but it does represent a 5-fold enhancement of K⁺ flux driven by an electro-chemical gradient, which when applied to chemical (concentration) gradients implies greatly enhanced K⁺ dispersal around regions of build up in interstitial space, compared with diffusion alone - the so-called 'spatial buffer' mechanism for K⁺ .

An additional, larger, component of macroscopic cortical conductance appears to arise from extracellular but not interstitial pathways, possibly via perivascular tissue. This may not have been studied in detail, but is indicated by the fact that measured cortical impedance is in at least some circumstances only around half what would be expected on the basis of measurements of the volume and tortuosity of local interstitial space around a microelectrode (Gardner-Medwin, 1980; Nicholson & Phillips, 1981). Barbour (2016) points out that these two ways of approaching impedance both give an order of magnitude hugely below that of Gomes et al. (2016). Taking account of interstitial tortuosity shows, however, that they do differ by a factor of about 2.

Barbour B. (2016) Analysis of claims that the brain extracellular impedance is high and non-resistive. https://arxiv.org/abs/1612.08457

Gardner-Medwin A.R. (1980) Membrane transport and solute migration affecting the brain cell microenvironment. Neurosci. Res. Progr. Bull. 18:208-226

Gardner-Medwin A.R. (1983) A study of the mechanisms by which potassium moves through brain tissue in the rat. J Physiol 335:353-374

Gomes J.-M., C. Bédard, S. Valtcheva, M. Nelson, V. Khokhlova, P. Pouget, L. Venance, T. Bal, and A. Destexhe (2016) Intracellular impedance measurements reveal non-ohmic properties of the extracellular medium around neurons. Biophysical Journal, 110(1):234-246

Nicholson C. & Phillips J.M. (1981) Ion diffusion modified by tortuosity and volume fraction in the extracellular microenvironment of the rat cerebellum. J. Physiol. 321:225-257

Rall W. & Rinzel J. (1973) Branch input resistance and steady attenuation for input to one branch of a dendritic neuron model. Biophysical Journal 13(7):648-688

On 2016 Aug 17, Kirk O'Reilly commented:

McIntyre et al. have published a series of papers evaluating the use of soil bioretention systems to reduce the toxicity of stormwater and highway runoff (McIntyre et al. 2014, 2016, 2016b). Their recent paper (McIntyre et al. 2016) investigates the use of soil bioretention to treat artificial runoff from a test plot treated with a refined tar sealant (RTS). What readers may not know is that there is an on-going controversy concerning the environmental implications of RTS use (LeHuray 2015; USGS 2016). According to McIntyre’s acknowledgement, U.S. Geological Survey personnel responsible for the agency’s effort to ban RTS assisted in project planning. The goal of this comment is not to criticize McIntyre’s research on the use of soil bioretention systems but to discuss the results in the context of other studies so that the paper is not misused by those advocating for RTS product bans. The technical basis for these comments are summarized in Exponent (2016).

Key Points:

The title overstates the toxicity of RTS runoff.

The title suggests that sealant runoff causes “severe” toxicity. But severe is neither defined nor used in the text of the article. Severe toxicity is not a commonly used term in aquatic toxicology. Use of “acute toxicity” or just “toxicity” in the title would be less inflammatory and more consistent with the study results.

As noted by the authors in a prior publication (McIntyre et al. 2014), “Developing fish embryos are particularly vulnerable to the harmful effects of chemical contaminants and have long been a focus for toxicity screening. More recently, model species such as the zebrafish have provided an increasingly sophisticated experimental context for evaluating the developmental toxicity of individual chemical constituents in stormwater.” McIntyre et al. (2016b) says that their tests measure subtle biological effects. A positive bioassay result with a particularly vulnerable fish embryo test system is insufficient to support the suggestion of severe toxicity.

While the survival of another test species, juvenile Coho salmon, was less in runoff from a sealant test plot than the control, only the runoff collected within a few hours of sealant application killed all the organisms. Mortality decreased substantially for subsequent samples and remained significantly different from controls with runoff collected two weeks but not three weeks after application.

The toxicity of sealant runoff is consistent with the toxicity of runoff from unsealed surfaces.

McIntyre et al. (2014, 2016b) describe tests conducted on highway runoff collected during six storm events. Two of the six stormwater samples killed all Zebrafish embryos, and a third sample resulted in a significant reduction in survival. All six of the stormwater samples caused sublethal effects similar to those discussed in the RTS paper.

Greenstein et al (2004) tested the toxicity of artificial runoff from an operating asphalt parking lot in Southern California. There was no evidence that RTS was ever applied on the lot. Using a sensitive marine aquatic bioassay, sea urchin egg fertilization, toxicity was noted in all runoff samples.

Soil Bioretention treatment of RTS and highway runoff can eliminate toxicity and significantly reduced the response of sensitive molecular indicators.

Soil bioretention is a sustainable approach in which runoff is filtered by soil. It mimics natural processes that can reduce the toxicity of runoff from urban surfaces including sealed parking lots.

Acknowledgment

The author of this comment has conducted research funded by the Pavement Coating Technology Council.

References

McIntyre JK, Edmunds RC, Anulacion BF, Davis JW, Incardona JP, Stark JD, Scholz NL. 2016. Severe coal tar sealcoat runoff toxicity to fish and reversal by bioretention filtration. Environmental Science & Technology 50(3): 1570–1578.

LeHuray, A. 2015. In response to Bales (2014). Integrated Environmental Assessment and Management, 11(2), 185–187.

USGS. 2016. Information Quality - Information Correction Request. At: https://www2.usgs.gov/info_qual/archives/coal_tar_sealants.html

Exponent 2016. Summary of McIntyre et al. 2016. “Severe Coal Tar Sealcoat Runoff Toxicity to Fish Is Prevented by Bioretention Filtration” Environ. Sci. Technol. 50:1570−1578. Pavement Coatings Technology Council. 2016-08-14. URL:http://www.pavementcouncil.org/wp-content/uploads/2016/08/Tech-Memo-McIntyre-2016-review-Final.pdf. Accessed: 2016-08-14. (Archived by WebCite® at http://www.webcitation.org/6jlOpIS8t)

McIntyre JK, Davis J, Incardona J, Stark J, Anulacion B, Scholz N. 2014. Zebrafish and clean water technology: Assessing soil bioretention as a protective treatment for toxic urban runoff. Science of the Total Environment 500:173–178. Open Access: http://www.sciencedirect.com/science/article/pii/S0048969714012455

McIntyre JK, Edmunds RC, Redig MG, Mudrock EM, Davis JW, Incardona JP, Stark JD, Scholz NL. 2016b. Confirmation of stormwater bioretention treatment effectiveness using molecular indicators of cardiovascular toxicity in developing fish. Environmental Science & Technology 50(3): 1561–1569.

Greenstein D, Tiefenthaler L, and Bay S. 2004. Toxicity of parking lot runoff after simulated rainfall Arch Environ Contam Toxicol. 47:199–206.

On 2015 Nov 28, Friedrich Thinnes commented:

Arguments for plasmalemmal VDAC-1 to form the channel part of VRAC

The inclusion of VDAC-1 = voltage dependent anion channel of isotype-1 into the plasma membrane of mammalian cells was first demonstrated in 1989, this by its immuno-topochemical flagging on human B lymphocyte, and those data were meanwhile corroborated by several laboratories using manifold approaches world wide (1-4).

Concerning the function of plasmalemmal VDAC-1 (5-9) it has been shown that the channel is involved in cell volume regulation. Cell outside applied monoclonal mouse anti-human type-1 porin antibodies blocked the RVD of HeLa cells, proving that VDAC-1 is involved in the process. HeLa cells pre-incubated with the antibodies dramatically increased their volume within about 1 min after a stimulus by hypotonic Ringer solution, but did not move backward towards their starting volume, thus indicating abolished RVD.

To notice, corresponding blocking effects were induced by the established anion channel inhibitor DIDS or BH4BClXL peptides, respectively. Video camera monitoring of cell size over time was used in this direct and noninvasive approach (9; www.futhin.de Supplement 1). Corroboration of these data came from the laboratory of Dr. R. Boucher (10) using VDAC knock out mice, this study, furthermore, pointing to the channel as an ATP pathway.

First data concerning the involvement of plasmalemmal VDAC-1 in the apoptotic process came from Dr. F. Elinder´s laboratory, demonstrating that opening of plasma membrane voltage-dependent anion channels (VDAC) precedes caspase activation in neuronal apoptosis induced by toxic stimuli (11). In line, the laboratory of Dr. Raquel Marin demonstrated that voltage dependent anion channel (VDAC-1) participates in amyloid Aß-induced toxicity and also interacts with the plasma membrane estrogen receptor alpha (mERa) in septal and hippocampal neurons (12). Noteworthy: Alzheimer Disease disproportionally affects women.

To notice, plasmalemmal VDAC and amyloid Aß, too, carry GxxxG peptide interaction motifs (2-4).

Concerning VDAC-1 agonists there are many data on low molecular weight agonists working on VDAC in varying settings, which may be helpful in studies on VRAC: DIDS, cholesterol, ATP, König's polyanion, dextran sulfate, Ga3+, Al3+, Zn2+, polyamines, compound 48/80, ruthenium red, fluoxetine, cisplatin, curcumin. Further studies looked for corresponding effect of peptides e.g. BH4-BClXL peptides, peptides including the free N-terminal part of VDAC-1 and amyloid Aβ peptides (4,9-16).

There is increasing evidence on interactions of VDAC-1 and proteineous modulators: e.g. α-synuclein shows high affinity interaction with voltage dependent anion channel, suggesting mechanisms of regulation and toxicity in Parkinson Disease (17). It has, furthermore, been shown that interaction of human plasminogen kringle 5 and plasmalemmal VDAC-1 links the channel to the extrinsic apoptotic pathway (18). Finally, an early study pointed to cancer cell cycle modulation by functional coupling between sigma-1 receptors and Cl- channels, here GxxxG motifs putatively playing a role (19,20).

Noteworthy, a SwissProt alignment of the LRC8A-D sequences shows two GxxxG motifs in a critical loop of LRC8E (Thinnes, unpublished).

Conclusion

While the expression of VDAC-1 in in the plasma membranes is beyond reasonable doubt (1-4) its function in this compartment is still in debate (5-20, 21-23).

VDAC-1 shows ubiquitous multi-toplogical expression, standing in outer mitochondrial membranes, the endoplasmic reticulum, as well as in the plasmalemma. To fulfill putatively varying functions in differing compartments, from the beginning on, my laboratory postulated proteineous channel modulators, which in varying heteromer complexes may adjust membrane-standing VDAC-1 to local needs.

Meanwhile, several of those come to the fore. VRAC/VSOAC candidates appear to be amongst them.

Finally, concerning medical relevance VDAC-1 complexes are involved in the pathogenesis of e.g. Cystic Fibrosis (13), Alzheimer Disease (3,4,12) and cancer (4).

References

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3) Thinnes FP. Front Aging Neurosci. 2015 Sep 30;7:188. doi: 10.3389/fnagi.2015.00188. eCollection 2015. No abstract available. PMID: 26483684 Free PMC Article

4) Smilansky A, Dangoor L, Nakdimon I, Ben-Hail D, Mizrachi D, Shoshan-Barmatz V. J Biol Chem. 2015 Nov 5. pii: jbc.M115.691493. [Epub ahead of print] PMID: 26542804 Free Article

5) Morris AP, Frizzell RA. Am J Physiol. 1993 Apr;264(4 Pt 1):C977-85. PMID: 7682780

6) Blatz AL, Magleby KL. Biophys J. 1983 Aug;43(2):237-41. PMID: 6311302 Free PMC Article

7) Dermietzel R, Hwang TK, Buettner R, Hofer A, Dotzler E, Kremer M, Deutzmann R, Thinnes FP, Fishman GI, Spray DC, et al. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):499-503. PMID: 7507248 Free PMC Article

8) Schwiebert EM, Egan ME, Hwang TH, Fulmer SB, Allen SS, Cutting GR, Guggino WB. Cell. 1995 Jun 30;81(7):1063-73. PMID: 7541313 Free Article

9) Thinnes FP, Hellmann KP, Hellmann T, Merker R, Brockhaus-Pruchniewicz U, Schwarzer C, Walter G, Götz H, Hilschmann N. Mol Genet Metab. 2000 Apr;69(4):331-7. PMID: 10870851 10) Okada SF, O'Neal WK, Huang P, Nicholas RA, Ostrowski LE, Craigen WJ, Lazarowski ER, Boucher RC. J Gen Physiol. 2004 Nov;124(5):513-26. Epub 2004 Oct 11. PMID: 15477379 Free PMC Article

11a) Elinder F, Akanda N, Tofighi R, Shimizu S, Tsujimoto Y, Orrenius S, Ceccatelli S. Cell Death Differ. 2005 Aug;12(8):1134-40. PMID: 15861186 Free Article

11b) Akanda N, Tofighi R, Brask J, Tamm C, Elinder F, Ceccatelli S. Cell Cycle. 2008 Oct; 7(20):3225-34. Epub 2008 Oct 20. PMID: 18927501

12a) Marin R, Ramírez CM, González M, González-Muñoz E, Zorzano A, Camps M, Alonso R, Díaz M. Mol Membr Biol. 2007 Mar-Apr;24(2):148-60. PMID: 17453421

12b) Herrera JL, Diaz M, Hernández-Fernaud JR, Salido E, Alonso R, Fernández C, Morales A, Marin R. J Neurochem. 2011 Mar;116(5):820-7. doi: 10.1111/j.1471-4159.2010.06987.x. Epub 2011 Jan 7. Review. PMID: 21214547 Free Article

13) Thinnes FP. Mol Genet Metab. 2014 Apr;111(4):439-44. doi: 10.1016/j.ymgme.2014.02.001. Epub 2014 Feb 13. Review. PMID: 24613483

14 Thinnes FP. PMID: 15781203 [PubMed - indexed for MEDLINE] Mol Genet Metab. 2005 Apr;84(4):378.

15) Thinnes FP. Mol Genet Metab. 2009 Jun;97(2):163. doi: 10.1016/j.ymgme.2009.01.014. Epub 2009 Feb 3. No abstract available. PMID: 19251445

16) Thinnes FP. Am J Physiol Cell Physiol. 2010 May;298(5):C1276. doi: 10.1152/ajpcell.00032.2010. No abstract available. PMID: 20413797 Free Article

17) Rostovtseva TK, Gurnev PA, Protchenko O, Hoogerheide DP, Yap TL, Philpott CC, Lee JC, Bezrukov SM. J Biol Chem. 2015 Jul 24;290(30):18467-77. doi: 10.1074/jbc.M115.641746. Epub 2015 Jun 8. PMID: 26055708

18) Li L, Yao YC, Gu XQ, Che D, Ma CQ, Dai ZY, Li C, Zhou T, Cai WB, Yang ZH, Yang X, Gao GQ. J Biol Chem. 2014 Nov 21;289(47):32628-38. doi: 10.1074/jbc.M114.567792. Epub 2014 Oct 8. PMID: 25296756 Free PMC Article

19) Renaudo A, L'Hoste S, Guizouarn H, Borgèse F, Soriani O. J Biol Chem. 2007 Jan 26;282(4):2259-67. Epub 2006 Nov 22. PMID: 17121836 Free Article

20) Chu U, Ruoho AE. Mol Pharmacol. 2015 Nov 11. pii: mol.115.101170. [Epub ahead of print] PMID: 26560551 Free Article

21) Liu HT, Tashmukhamedov BA, Inoue H, Okada Y, Sabirov RZ. Glia. 2006 Oct;54(5):343-57. Erratum in: Glia. 2006 Dec;54(8):891.

22) Sabirov RZ, Merzlyak PG. Biochim Biophys Acta. 2012 Jun;1818(6):1570-80. doi: 10.1016/j.bbamem.2011.09.024. Epub 2011 Oct 1. Review. PMID: 21986486 Free Article

23) Pedersen SF, Klausen TK, Nilius B. Acta Physiol (Oxf). 2015 Apr;213(4):868-81. doi: 10.1111/apha.12450. Epub 2015 Jan 28.

On 2018 Feb 01, Andrew Willetts commented:

The poor quality of the modelling studies of 3,6-diketocamphane monooxygenase (3,6-DKCMO) presented by Isupov et alresults from a combination of a number of well characterised deficiencies (1) in relevant structural and biochemical features that were used to develop their proposals for this Type II Baeyer-Villiger monooxygenase (2) from camphor-grown Pseudomonas putida NCIMB 10007.

An important contributory factor that stymies their studies is Isupov et al’s mistaken understanding of the nature and significance of the facial diastereoselectivity of the hydride transfer that occurs between the donated FNR cofactor and the enzyme in the active site of this flavin-dependent two-component monooxygenase (fd-TCMO [3]). Both Isupov et al’s structural and modelling studies place a significant dependence on comparative data for the luciferase from Vibrio harveyi, the first fd-TCMO to be recognised and well-characterised at both the structural and functional levels (4). However, while it has been conclusively shown that 3,6-DKCMO and the two enantiocomplementary 2,5-diketocamphane monooxygenases (2,5-DKCMOs) from camphor-grown P.putida NCIMB 10007 deploy requisite FNR exclusively in (si)-facial hydride transfers (5-7), the luciferase from V.harveyi (8,9) and all other bacterial luciferases thus characterised to date (10), deploy FNR exclusively in (re)-facial hydride transfers. This fundamental dichotomy between 3,6-DKCMO and the luciferase is not taken into account by Isupov et al who incorrectly state that ‘all enzymes of the bacterial luciferase superfamily catalyse their reaction on the si side of the ring’.

Consequential errors that result from this significant misunderstanding occur principally, but not exclusively, in Sections 3.7 (Comparison with other bacterial luciferase-family proteins) and 3.8 (The reaction mechanism) of Isupov et al’s paper. Typically, if this important biochemical difference had been appreciated, then Figure 5 of their paper might have been interpreted differently. Also, because 3,6- and the isoenzymic 2,5-DKCMOs have been shown to exhibit the same extremely high (si)-facial diastereoselectivity (5-7) with respect to the hydride transfers that characterise key biochemical events in their active sites, the outcome of which is the successive formation and stabilisation of their respective Criegee intermediates (11), Isupov et al’s prediction that these enantiocomplementary enzymes will exhibit ‘a different mode of cofactor binding’ seems highly unlikely.

The established differences in facial diastereoselectivity between these particular fd-TCMOs may help to explain why the commercially available (Sigma Aldrich) flavin reductase component of the luciferase from Vibrio harveyi failed to promote any significant electrophilic biooxidation of a small number of tested organosulfides by purified preparations of 3,6-DKCMO (12). Similar low levels of product(s) were detected in both experimental and equivalent control reactions (eg, <0.01% sulfoxide and <0.002% sulfone formed after 30h incubation with methyl phenyl sulphide +/- 3,6-DKCMO). It was concluded that the negligible levels of oxidation observed were principally, if not exclusively, the result of abiotic autooxidation, and consequently this particular research initiative was abandoned in mid-1996. Also, because they were outside the remit of the PhD programme of Jean Beecher (supervisor Dr Andrew Willetts, degree awarded 1997, University of Exeter), no equivalent studies of potential nucleophilic biooxidation of ketone substrates were considered or undertaken (13,14). Consequently, Dr Beecher’s thesis is notable for the total absence of any relevant content relating to either electrophilic or nucleophilic biooxidations with a hybrid P.putida-V.harveyi multienzyme complex. The claims in Isupov et al that ‘the commercially available Vibrio harveyi flavin reductase (Sigma) was able to demonstrate activity with purified 36DKCMO oxygenating enzyme in biotransformation reactions (McGhie, 1998)’, and that ‘commercially available NADH-FMN oxidoreductase from Vibrio harveyi has successfully been used for reduction of cofactor in activity measurements (McGhie, 1998)’ are incompatible with the above pre-1998 outcomes. McGhie is an accredited co-author of Isupov et al’s paper, and McGhie (1998) is in reference to her PhD awarded by the University of Exeter (supervisor Dr Littlechild). Most significantly, there is a total absence of either supporting data, or corresponding experimental protocols, or discussion relevant to both electrophilic and nucleophilic biooxidations by a hybrid P.putida-V.harveyimultienzyme complex in McGhie’s 1998 thesis, the sole relevant entry being a single sentence on p74 which claims that the hybrid complex can support ‘lactonising activity’ (= nucleophilic biooxidation), citing the source as ‘Beecher, personal communication’, which is clearly inconsistent with Jean Beecher’s pre-1998 studies (vide infra). The incorrect claim included in McGhie’s PhD thesis and the equivalent two incorrect claims included in Isupov et al are clearly interrelated. References. 1. Willetts, A. & Kelly, D.P. (2016). Microorganisms, 4, 38: 2. Willetts, A. (1997). Trends Biotechnol., 15, 55-62: 3. Ellis, H.R. (2010). Arch. Biochem. Biophys. , 497, 1-12: 4. Campbell, Z.T., Weichsel, A., Montfort, W.R. & Baldwin, T.O. (2008). Biochem. 48, 6085-6094: 5. Grogan, G. (1995). PhD Thesis, University of Exeter: 6. Beecher, J.E., Grogan, G., Roberts, S. & Willetts, A. (1996). Biotechnol. Lett., 18, 571-576: 7. Kelly, D.R., Knowles, C.J., Mahdi, J.G., Wright, M.A., Taylor, I.N., Roberts, S., Wan, P., Grogan, G., Pedragosa-Moreau, S. & Willetts, A.(1996). Chem. Commun., 20, 2333-2334: 8. Yamazaki, S., Tsai, L. & Stadyman, T.C. (1980). J. Biol. Chem., 255, 9025-9027: 9. Yamazaki, S., Tsai, L. & Stadyman, T.C., Teshima, T., Nakaji, A. & Shiba, T. (1985). Proc. Nat. Acad. Sci. USA, 82, 1364-1366: 10. Villa, R. & Willetts, A. (1997). J. Mol. Catal. B: Enzym., 2, 193-197:11. Yachnin, B.J., Sprules, T., McEvoy, M.B., Lau, P.C.K. & Berghuis, A.M. (2012). J. Amer. Chem. Soc., 134, 7788-7795: 12. Willetts, A. & Beecher, J.E. (1995; 1996). Laboratory records, unpublished data: 13. Willetts, A. (1996). Laboratory records, unpublished data: 14. Beecher, J.E. (1997). PhD Thesis, University of Exeter.

On 2016 Mar 01, Ram Dessau commented:

Residual symptoms after Lyme neuroborreliosis. The prevalence is unknown? Comment by Ram B. Dessau

Department clinical microbiology Slagelse Hospital, Region Zealand Ingemannsvej 46, DK4200, Slagelse, Denmark ramd@regionsjaelland.dk

Dersch et al. have performed a systematic review and meta-analysis on the prevalence and spectrum of residual symptoms in patients after treatment for Lyme neuroborreliosis (LNB)[1]. 5579 bibliographic records were screened and 38 studies were included. An overview of treatment studies reporting residual symptoms is given. The follow up time is about one year concerning most of the studies. The conclusion is that 28% of LNB patients may experience symptoms after treatment. This conclusion may be misinterpreted as associated to LNB or caused by LNB. The 38 treatment studies were not designed to elucidate the association of LNB with residual symptoms, as control groups without LNB were not included.

Many of the cited symptoms like sensory disturbances, cognitive disturbances, pain and headache are non-specific and even common in the population at large. Thus, it would be of paramount importance to compare the prevalence of events to a control group using the same methods. Only a higher frequency of residual symptoms in the LNB group may indicate association with LNB. Due to the design of the included studies the residual symptoms could as well be due to other causes such as adverse effects of treatment [2].

Reporting the gross proportion of events may be misleading for other reasons. The load of residual symptoms will depend on not only the number of events for each symptom, but also the number of recorded items and the length of time, where symptoms may occur. The more questions you ask and the higher the gross rate of symptoms will become and eventually the rate of symptoms may approach 100%. Especially if there is also a long time span for the observations.

Along this line of reasoning Dersch et al. also misinterpreted the studies including a control group[1]. The results of these studies do not “remain inconclusive”, but show comparable results, even if there are differences. These differences are not very large and a substantial proportion of the controls also have a similar level of problems. For example the mean physical component FS36 score in LNB was 44(9) compared to 51(6) in controls[3]. A significant difference in mean score was found, but the size of the standard deviation show that the distribution of scores in the two groups have a large overlap. Given that the 95% interval is about 2 SD then FS36 score in LNB is 26-62 and in controls 39-63. Even if LNB patients have “significantly” lower FS36 score than controls, this is not a large difference. Thus a weak or absent association alone could explain that some studies do not report statistical differences and some do due to random variation. Thus, residual symptoms associated with LNB are probably quite rare, if at all.

Grouping together any symptoms without considering a variable relevance to LNB is problematic. For example, it was found in Swedish children that residual symptoms were persistent nerve defects such as facial palsy, but not nonspecific subjective symptoms [4]. Thus, it would be important to distinguish different types of symptoms and their severity. The study by Dersch et al. adds to a large volume of uncontrolled case reports and case series overstating the possible risk of symptoms associated with Lyme borreliosis[5]. This may contribute to unnecessary anxiety about Lyme borreliosis.

It is acknowledged that Dersch et al [1] do state important reservations about especially the "possible" case definitions, as patients with other conditions could contribute to the high load of reported symptomps.

Conclusion The prevalence of residual symptoms associated with LNB after treatment may not be concluded from the study by Dersch et al [1] because control groups are missing. A meaningfull interpretation is not possible. The authors should have focused on the available controlled studies instead.

Conflict of interests: None to declare.

References

1.Dersch R, Sommer H, Rauer S, Meerpohl JJ. Prevalence and spectrum of residual symptoms in Lyme neuroborreliosis after pharmacological treatment: a systematic review. J Neurol 2015 Oct 12.

2.Dersch R, Freitag MH, Schmidt S, Sommer H, Rauer S, Meerpohl JJ. Efficacy and safety of pharmacological treatments for acute Lyme neuroborreliosis - a systematic review. Eur J Neurol 2015 Sep;22:1249-59.

3.Eikeland R, Mygland A, Herlofson K, Ljostad U. European neuroborreliosis: quality of life 30 months after treatment. Acta Neurol Scand 2011 Nov;124:349-54.

4.Skogman BH, Glimaker K, Nordwall M, Vrethem M, Odkvist L, Forsberg P. Long-term clinical outcome after Lyme neuroborreliosis in childhood. Pediatrics 2012 Aug;130:262-9.

5.Baker CJ, et al. Final report of the Lyme disease review panel of the infectious diseases society of America (http://www.idsociety.org).2010.

On 2015 Oct 01, Friedrich Thinnes commented:

Plasmalemmal VDAC-1 discussed as amyloid Aß-receptor

In 2010 I referred to a cell outside located GxxxG motif on the N-terminal helical stretch of plasmalemmal VDAC-1 (voltage dependent anion channel) and to a series of those inside the amyloid Aß peptide. The motifs are known to work as membrane perturbation motifs.

The hint included a first rational on an interaction of the molecules, VDAC-1 suddenly appearing to figure as a receptor of toxic Aß molecules. Another link to Alzheimer´s Dementia research arose from data pointing to amyloid Aß as an apoptotic opener of cell membrane-integrated VDAC-1 and to counteracting polyphenol preparations from several plants. Together, a revised version of the amyloid cascade hypothesis came up (F.P. Thinnes, 2015; www.futhin.de).

Accordingly, Alzheimer's disease – downstream of APP processing – can bee seen as resting on some form of extrinsic induced cell death, this via opening cell membrane-standing VDAC-1 (= receptor) and accumulating over time. The process is boosted by excessive amyloid Aß (= agonist) production via increased processing of the amyloid precursor protein (APP) of weakening cells of critical and redundant brain regions.

Recent data on the slow down of progress of Alzheimer Disease of proven Alzheimer patients in early states by monoclonal anti-amyloid antibodies that neutralize amyloid mono- or oligomers, from my point of view, corroborate plasmalemmal VDAC-1 as a receptor of those (E.R. Siemers et al., 2015)

However, a study presented by Y.H. Liu et al. (2015) reports on three monoclonal antibody preparations elaborated against different epitopes inside the amyloid Aß peptide. One of those called 6E10 a) in vitro disaggregates artificial amyloid fibrils and thus increases the number of Aß oligomers while b) injection of co-incubates into the lateral ventricle of 6-month-old C57 mice increased the neurotoxicity in vivo. Anyway, to raise amyloid Aß oligomers increases the risk of their docking to plasmalemmal VDAC-1 finally resulting in the induction of accumulating neuronal cell deaths. From here: To raise amyloid Aß oligomers accelerates AD progress.

The authors call the phenomenon dust-raising. It is tempting to think Alzheimer plaques formation as a salutary form of wipe-the-dust procedure that may even protect from Alzheimer Dementia. In other words: does plaque formation work as a buckler?

References

Thinnes, F.P. (2015) Phosphorylation, nitrosation and plasminogen K3 modulation make VDAC-1 lucid as part of the extrinsic apoptotic pathway-Resulting thesis: Native VDAC-1 indispensible for finalisation of its 3D structure. Biochim Biophys Acta. 1848:1410-1416. doi: 10.1016/j.bbamem.2015.02.031. Epub 2015 Mar 11. Review. PMID: 25771449

Siemers, E. R., Sundella, K. L., Carlson, C., Case, M., Sethuraman, G., Liu-Seifert, H., Dowsett, S. A., Pontecorvo, M. J., Dean, R.A., Demattos, R. (2015) Phase 3 solanezumab trials: Secondary outcomes in mild Alzheimer’s disease patients. Alzheimer’s & Dement. Aug 1. pii: S1552-5260(15)02148-2. doi: 10.1016/j.jalz.2015.06.1893. [Epub ahead of print].

Liu, Y. H., Bu, X. L., Liang, C. R., Wang, Y. R., Zhang, T., Jiao, S. S., Zeng, F., Yao, X. Q., Zhou, H. D., Deng, J., Wang, Y. J. (2015) An N-terminal antibody promotes the transformation of amyloid fibrils into oligomers and enhances the neurotoxicity of amyloid-beta: the dust-raising effect. J Neuroinflammation. 12:153. doi: 10.1186/s12974-015-0379-4.

On 2017 May 03, Andrea Messori commented:

Please refer to the Letters' section of the Journal.

Presenting the results of a pharmacoeconomic study: incremental cost-effectiveness ratio vs net monetary benefit

Andrea Messori and Sabrina Trippoli

HTA Unit, ESTAR Regional Health Service, 50135 Firenze (Italy)

The article by Wouters and colleagues (1) presents an exhaustive overview on how QALYs can be used in cost-effectiveness analysis. In this framework, the authors also mention the incremental cost-effectiveness ratio (ICER), which is the parameter typically employed to express the results of a cost-effectiveness study. The article, however, does not discuss the net monetary benefit (NMB), which is another parameter employed to express the results of a cost-effectiveness study.

The incremental cost (deltaC) and the incremental effectiveness (deltaE) are the two main parameters of pharmacoeconomics and cost-effectiveness analysis, along with the willingness-to-pay threshold (lambda). The decision rule (e.g. in the case of a favourable pharmacoeconomic result) is (deltaC/deltaE)<lambda (Equation 1), if based on the ICER, or (deltaE x lambda - deltaC) > 0 (Equation 2), if based on the NMB. Likewise, an unfavourable pharmacoeconomic result is when (deltaC/deltaE)>lambda or when (deltaE x lambda - deltaC) < 0; NMB is defined as deltaE x lambda - deltaC, while ICER is defined as deltaC/deltaE. Despite its apparent complexity, most part of pharmacoeconomic methodology is described by the two simple equations reported above (i.e. Equations 1 and 2), but whether the ICER or the NMB is the best parameter for the purposes of pharmacoeconomic decision-making remains on open question.

The study by Cowper et al evaluating new versus old oral anticoagulants in patients with atrial fibrillation (2) is a typical ICER-based cost-effectiveness analysis in which the ICER of apixaban versus warfarin is compared against a willingness-to-pay threshold. This analysis can be taken as an example for comparing ICER vs NMB.

In one of the base-case analyses of the study by Cowper et al, QALYs per patient were 7.94 for apixaban and 7.54 for warfarin, while pharmacological costs per patient were $22,934 and $4,392, respectively. These data yielded, for apixaban versus warfarin, an ICER of $46,355 per QALY gained, a value that remains within the willingness-to-pay threshold of $50,000 per QALY gained and is therefore considered favourable (or “high value care”). As pointed our by Hlatky (3), in interpreting a specific ICER value, more than a single willingness-to-pay threshold is frequently considered (e.g. the threshold between $50,000 and $150,000 or the threshold above $150,000), and this allows us to better understand a pharmacoeconomic result expressed on the basis of an ICER.

In the methodology of pharmacoeconomics, the net monetary benefit (NMB) plays a role similar to that of ICER, but some differences are important.

Firstly, the ICER –by definition- has always an incremental nature and consequently the absolute cost-effectiveness ratio (calculated for a single treatment in the absence of any comparison) makes little sense and, for this reason, is rarely employed. In contrast, the NMB can be calculated for a single treatment in the absence of any comparison (absolute NMB) or can conversely be calculated as an incremental parameter [according to the equation: (incremental NMB) = (incremental QALYs per patient) x (willingness-to-pay threshold) – (incremental cost per patient)]. Another feature of NMB is that the incremental NMB for the comparison of A vs B can be estimated as the absolute NMB calculated for A minus the absolute NMB calculated for B. In this sequence of calculations, calculating the absolute NMB makes sense because the absolute NMB (separately calculated for the experimental treatment and for the control treatment) represents an intermediate step in the calculation of the incremental NMB (Table 1)

The values of absolute NMB for apixaban and warfarin (Table 1) are, respectively, 374,066 and $372,608 per patient (calculated according to Equation 2). Hence, the incremental NMB for apixaban vs warfarin is simply the difference of the above two values, i.e. $1,458 per patient.

References

[1] Wouters OJ, Naci H, Samani NJ. QALYs in cost-effectiveness analysis: an overview for cardiologists. Heart. 2015 Dec;101(23):1868-73.

[2] Cowper PA, Sheng S, Lopes RD, Anstrom KJ, Stafford JA, Davidson-Ray L, Al-Khatib SM, Ansell J, Dorian P, Husted S, McMurray JJ, Steg PG, Alexander JH, Wallentin L, Granger CB, Mark DB. Economic Analysis of Apixaban Therapy for Patients With Atrial Fibrillation From a US Perspective: Results From the ARISTOTLE Randomized Clinical Trial. JAMA Cardiol. 2017 Mar 29. doi:10.1001/jamacardio.2017.0065. [Epub ahead of print]

[3] Hlatky MA. Are Novel Anticoagulants Worth Their Cost? JAMA Cardiol. 2017 Mar 29. doi: 10.1001/jamacardio.2017.0126. [Epub ahead of print]

Table 1. Cost-effectiveness of apixaban vs warfarin in atrial fibrillation: basecase analysis reported by Cowper et al.(2)

a) STARTING VALUES

apixaban: QALYs per patient = 7.94, cost per patient = $22,934

warfarin: QALYs per patient = 7.54, cost per patient = $4,392

willingness-to-pay threshold = $50,000/QALY

b) PHARMACOECONOMIC PARAMETERS

ICER = ($22,934 - $ 4,392) / (7.94 – 7.54) = 18,542 / 0.40 = $46,355/QALY

absolute NMB for apixaban = 7.94 x $50,000 – $22,934 = $374,066

absolute NMB for warfarin = 7.54 x $50,000 – $4,392 = $372,608

incremental NMB = absolute NMB for apixaban - absolute NMB for warfarin = $374,066 - $372,608 = $1,458

c) INTERPRETATION

ICER (equal to $46,355/QALY): favourable because <$50,000/QALY

incremental NMB (equal to $1,458 per patient): favourable because >0

On 2015 Sep 15, Tom Kindlon commented:

(Contd.)

References:

1 Torjesen I. Tackling fears about exercise is important for ME treatment, analysis indicates. BMJ 2015;350:h227 http://www.bmj.com/content/350/bmj.h227

2 Chalder T, Goldsmith KA, White PD, Sharpe M, Pickles AR. Rehabilitative therapies for chronic fatigue syndrome: a secondary mediation analysis of the PACE trial. Lancet Psychiatry 14 Jan 2015, doi:10.1016/S2215-0366(14)00069-8.

3 Burgess M, Chalder T. Manual for Participants. Cognitive behaviour therapy for CFS/ME.http://www.pacetrial.org/docs/cbt-participant-manual.pdf (accessed: January 17, 2015)

4 Bavinton J, Darbishire L, White PD -on behalf of the PACE trial management group. Graded Exercise Therapy for CFS/ME. Information for Participants http://www.pacetrial.org/docs/get-participant-manual.pdf (accessed: January 17, 2015)

5 Wechsler ME, Kelley JM, Boyd IO, Dutile S, Marigowda G, Kirsch I, Israel E, Kaptchuk TJ. Active albuterol or placebo, sham acupuncture, or no intervention in asthma. N Engl J Med. 2011;365(2):119-26.

6 Whiting P, Bagnall AM, Sowden AJ, Cornell JE, Mulrow CD, Ramírez G. Interventions for the treatment and management of chronic fatigue syndrome: a systematic review. JAMA. 2001 Sep 19;286(11):1360-8.

7 Burgess M, Chalder T. PACE manual for therapists. Cognitive behaviour therapy for CFS/ME.http://www.pacetrial.org/docs/cbt-therapist-manual.pdf (accessed: January 17, 2015)

8 White PD, Goldsmith KA, Johnson AL, Potts L, Walwyn R, DeCesare JC, et al, for the PACE trial management group. Comparison of adaptive pacing therapy, cognitive behaviour therapy, graded exercise therapy, and specialist medical care for chronic fatigue syndrome (PACE): a randomised trial. Lancet 2011;377:823-36.

9 McCrone P, Sharpe M, Chalder T, Knapp M, Johnson AL, Goldsmith KA, White PD. Adaptive pacing, cognitive behaviour therapy, graded exercise, and specialist medical care for chronic fatigue syndrome: a cost-effectiveness analysis. PLoS One. 2012;7(8):e40808. doi: 10.1371/journal.pone.0040808

10 Wiborg JF, Knoop H, Stulemeijer M, Prins JB, Bleijenberg G. How does cognitive behaviour therapy reduce fatigue in patients with chronic fatigue syndrome? The role of physical activity. Psychol Med. 2010 Aug;40(8):1281-7. doi: 10.1017/S0033291709992212. Epub 2010 Jan 5.

11 Heins MJ, Knoop H, Burk WJ, Bleijenberg G. The process of cognitive behaviour therapy for chronic fatigue syndrome: which changes in perpetuating cognitions and behaviour are related to a reduction in fatigue? J Psychosom Res. 2013 Sep;75(3):235-41. doi: 10.1016/j.jpsychores.2013.06.034. Epub 2013 Jul 19.

12 Friedberg F, Sohl S. Cognitive-behavior therapy in chronic fatigue syndrome: is improvement related to increased physical activity? J Clin Psychol. 2009 Apr;65(4):423-42. doi: 10.1002/jclp.20551.

13 Knoop H, Prins JB, Stulemeijer M, van der Meer JW, Bleijenberg G. The effect of cognitive behaviour therapy for chronic fatigue syndrome on self-reported cognitive impairments and neuropsychological test performance. Journal of Neurology and Neurosurgery Psychiatry. 2007 Apr;78(4):434-6.

14 Bavinton J, Darbishire L, White PD -on behalf of the PACE trial management group. Graded Exercise Therapy for CFS/ME (Therapist manual): http://www.pacetrial.org/docs/get-therapist-manual.pdf (accessed: January 17, 2015)

15 O'Dowd H, Gladwell P, Rogers CA, Hollinghurst S, Gregory A. Cognitive behavioural therapy in chronic fatigue syndrome: a randomised controlled trial of an outpatient group programme. Health Technology Assessment, 2006, 10, 37, 1-140.

16 Knoop H, Wiborg JF. What makes a difference in chronic fatigue syndrome? Lancet Psychiatry 13 Jan 2015 DOI: http://dx.doi.org/10.1016/S2215-0366(14)00145-X

17 Kindlon T. Reporting of Harms Associated with Graded Exercise Therapy and Cognitive Behavioural Therapy in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Bulletin of the IACFS/ME. 2011;19(2):59-111http://iacfsme.org/BULLETINFALL2011/Fall2011KindlonHarmsPaperABSTRACT/ta...

18 Lipkin DP, Scriven AJ, Crake T, Poole-Wilson PA. Six minute walking test for assessing exercise capacity in chronic heart failure. Br Med J (Clin Res Ed) 1986. 292:653–655. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1339640/pdf/bmjcred00224-001...

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20 Goldman MD, Marrie RA, Cohen JA. Evaluation of the six-minute walk in multiple sclerosis subjects and healthy controls. Multiple Sclerosis 2008. 14(3):383-390. http://pocketknowledge.tc.columbia.edu/home.php/viewfile/download/65399/The six-minute walk test.pdf

21 Ross RM, Murthy JN, Wollak ID, Jackson AS. The six minute walk test accurately estimates mean peak oxygen uptake. BMC Pulm Med. 2010 May 26;10:31. PMID 20504351.http://www.biomedcentral.com/1471-2466/10/31

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23 Troosters T, Gosselink R, Decramer M. Six minute walking distance in healthy elderly subjects. Eur Respir J. 1999. 14:270-4. http://www.ersj.org.uk/content/14/2/270.full.pdf

24 Rapport d'évaluation (2002-2004) portant sur l'exécution des conventions de rééducation entre le Comité de l'assurance soins de santé (institué auprès de l'Institut national d'assurance maladie invalidité) et les Centres de référence pour le Syndrome de fatigue chronique (SFC), Bruxelles, juillet 2006. (French language edition)

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On 2015 Dec 09, Donald Forsdyke commented:

PURINE LOADING AS A THERMAL ADAPTATION The proteins of thermophiles are generally more heat-stable than the corresponding proteins from mesophiles. This must be reflected in either, or both, of two major amino acid variables – composition and order. In the past the notion that amino acid composition might be reflective of the pressure in thermophiles to retain purine-rich codons (3) has been disparaged by Zeldovich et al. (6). In this elegant new paper (5), Venev and Zeldovich (2015) agree that the “multiple factors” not accounted for in their modelling “include the influence of the genetic code and guanine-cytosine (GC) content of the genomes on amino acid frequencies.” However, there is puzzlement that the “theory and simulations predict a strong increase of leucine content in the thermostable proteins, whereas it is only minimally increased in experimental data.” Perhaps it is of relevance that leucine is on the top left quadrant of the standard presentation of the genic code, its codons being extremely poor in purines.

My response to Zeldovich et al. (6) in 2007, and my follow-up references in 2012 (1, 4), are set out below. One of the coauthors of the 2007 paper has recently further contributed to this topic (2).

2007 Response

This paper draws conclusions tending to oppose those of myself and coworkers (cited). A "key question" is held to be: "Which factor - amino acid or nucleotide composition - is primary in thermal adaptation and which is derivative?" Previous evidence is considered "anecdotal." Now there is evidence for "an exact and conclusive" relationship, based on an "exhaustive study" that provides a "complete picture." A set of amino acids - IVYWREL - correlates well with growth temperature. It is noted:

"Signatures of thermal adaptation in protein sequences can be due to the specific biases in nucleotide sequences and vice versa. ... One has to explore whether a specific composition of nucleotide (amino acid) sequences shapes the content of amino acid (nucleotide) ones, or thermal adaptation of proteins and DNA (at the level of sequence compositions) are independent processes."

In other words, are primary adaptations at the nucleic acid level driving changes at the protein level, or vice- versa? To what extent are the two processes independent? Their conclusion:

"Resolving the old-standing controversy, we determined that the variation in nucleotide composition (increase of purine-load, or A + G content with temperature) is largely a consequence of thermal adaptation of proteins."

Thus, the superficial reader of the paper, while noting the purine-richness of some of the codons corresponding to the IVYWREL amino acids, will conclude that the "independent processes" alternative has been excluded. Reading the paper (e.g. Figure 7) one can question the validity of this conclusion. Many of the IVYWREL amino acids have purine-poor alternative codons (especially IYLV, which at best can only change one purine unit in their codons). One of the IVYWREL amino acids has relatively purine-rich alternative codons (R, which at best can change two purine units). Two (EW) are always purine-rich, and there are no alternatives.

Displaying more EW's as the temperature got hotter would satisfy a need both for more purines and for more tryptophan and glutamate, so here there is no discrimination as to whether one "shapes" the organism’s content of the other. Displaying more IYLVs gives only minimal flexibility in accommodating a purine-need. Most flexibility is provided by R codons.

The authors do not give statistics for the differences between the slopes of Figs. 7a (unshuffled codons) and 7b (shuffled codons), but they appear real, presumably reflecting the choice biologically of purine-rich codons, a choice the organisms might not have to make if there were no independent purine-loading pressure. Thus, the authors note, but only in parenthesis, that the slopes "are somewhat different suggesting that codon bias may be partly responsible for the overall purine composition of DNA."

2012 Response

As a follow up, it can be noted that Dehouck et al. (2008) report that relationship between a protein's thermostability and the optimum growth temperature of the organism containing it, is not so close as previously thought (1). Furthermore, Liu et al. (2012) now conclude from a study of xylanase purine-rich coding sequences that "The codons relating to enzyme thermal property are selected by thermophilic force at [the] nucleotide level," not at the protein level (4).

1.Dehouck Y, Folch B, Rooman M (2008) Revisiting the correlation between proteins' thermoresistance and organisms' thermophilicity. Protein Engineering, Design and Selection 21:275-278.Dehouck Y, 2008

2.Goncearenco A, Berezofsky IN (2014) The fundamental tradeoff in genomes and proteomes of prokaryotes established by the genetic code, codon entropy, and the physics of nucleic acids and proteins. Biology Direct 9:29 Goncearenco A, 2014

3.Lambros RJ, Mortimer JR, Forsdyke DR (2003) Optimum growth temperature and the base composition of open reading frames in prokaryotes. Extremophiles 7:443–450.Lambros RJ, 2003

4.Liu L, Wang L, Zhang Z, Wang S, Chen H (2012) Effect of codon message on xylanase thermal activity. J. Biol. Chem. 287:27183-27188 Liu L, 2012

5.Venev SV, Zeldovich KB (2015) Massive parallel sampling of lattice proteins reveals foundations of thermal adaptation. J. Chem. Phys. 143: 055101Venev SV, 2015

6.Zeldovich KB, Berezofsky IN, Shakhnovich EI (2007) Protein and DNA sequence determinants of thermophilic adaptation. PLOS Comput. Biol. 3(1), e5.Zeldovich KB, 2007

On 2016 Aug 18, David C. Norris commented:

This JAMA Viewpoint hinges on a categorical claim: “probability is not meaningful in an individual context.” In a subsequent exchange with Van Calster, Steyerberg and Harrell¹ , the authors have backed away slightly from this precipice, stating that it was rather the verifiability (and not meaningfulness) of ‘individual probability’ that was at issue—and indeed that only a frequentist probability notion was targeted by this statement.² The authors also explain that their original citation of Cohen³ in the context of this statement was meant “to direct the reader to the excellent discussion by Cohen of the limitations of the frequentist notion of probability”² . Cohen’s discussion is indeed excellent, and the reader who follows up this citation cannot fail to find a forceful rebuke of this Viewpoint's entire treatment of ‘probability’, delivered no less with particular reference to the very context under consideration—medical decision making:

Nor is it open to a frequency theorist to claim that all important probabilities are indeed general, not singular. It often seems very important to be able to calculate the probability of success for your own child’s appendectomy... ^3(p49)

Cohen proceeds from this observation to advance a Bayesian perspective; why Sniderman, D’Agostino and Pencina don’t do likewise would be a mystery if they did not reveal some peculiar methodological preoccupations in the ensuing development of their argument.

Eschewing a (meaningful|verifiable) notion of ‘individual probability’, the authors substitute the petitio principii of ‘individual risk’—the continuous, probabilistic character of which they conceal through the conceit of “clinically meaningful risk categories” [emphasis mine]. Tellingly, they label these categories “clinically meaningful” because they have forfeited the philosophic basis for making them so. Ultimately, what makes any concept clinically meaningful is its openness to connection with the values and circumstances of individual patients. Classification schemes that prematurely close patients’ decision problems have precisely the opposite character. Without such artificial categories, however, the characteristically incoherent⁴ frequentist approach to decision-making under uncertainty would lack even a semblance of that singular uncertainty which confronts the patient-physician dyad.

The authors conclude by calling on physicians to mop up this shambles. They utter the shibboleth, “models cannot replace the physician,” then incant some vague magic by which physicians should restore the individual patient to a scheme that has excluded the individual from its very epistemology. Mathematically, the requisite magic translates to conditioning on individuals after frequentist methods have already averaged individuals out.^4(pp61,509)

The ‘art of medicine’ has long enough been defined by quixotic attacks upon mathematical impossibilities. Physicians of the future will gladly relinquish the merely computational tasks of medicine to predictive models and other forms of automation. They will rather find a purposive role in the creative, irreplaceably human endeavor of helping patients to formulate their medical decision problems in alignment with their values and circumstances,⁵ and to decide these problems in accordance with appropriate evidence drawn from ever-improving⁶ predictive models.

1] Van Calster B, 2015

2] Sniderman AD, 2015

3] Cohen, L. Jonathan. An Introduction to the Philosophy of Induction and Probability. Oxford : New York: Clarendon Press; Oxford University Press, 1989.

4] Robert, Christian P. The Bayesian Choice: From Decision-Theoretic Foundations to Computational Implementation. 2nd ed. Springer Texts in Statistics. New York: Springer, 2007.

5] Moroff SV, 1983

6] Mazzola E, 2015

On 2015 Jul 23, Neil Davies commented:

Østergaard and colleagues report that fewer people with a high genetic risk of high blood pressure develop Alzheimer disease compared to those with a lower genetic risk of high blood pressure (1). This is consistent with evidence that use of anti-hypertensive medication is associated with incidence and progression of Alzheimer disease (2–4). We investigated these associations in an observational longitudinal cohort study using data from the Clinical Practice Research Datalink, a database of anonymous UK National Health Service primary and secondary health care data. We found that patients prescribed angiotensin-receptor blockers were less likely to be diagnosed with Alzheimer disease (OR 0.47, 95% CI: 0.37 to 0.58) than those prescribed other anti-hypertensives. Our results were based on observational data and may suffer from residual confounding, but they provide suggestive evidence that blood pressure or blood pressure medication is implicated in the aetiology of Alzheimer disease. A meta-analysis of randomised controlled trials has also suggested that treatment with blood pressure lowering medication may help slow cognitive decline (5).

However, there are alternative explanations for these associations. Although Mendelian randomization analyses may remove biases due to confounding and reverse causality, survival bias is still likely to be an issue. Individuals with a greater burden of high blood pressure SNPs may have higher mortality, which could cause them to die before developing Alzheimer’s disease and reduce the frequency of blood pressure increasing SNPs in cases compared to controls. Survival bias may also explain the apparent protective effect on Alzheimer’s disease risk of variants that are associated with heaviness of smoking. The smoking heaviness increasing allele located in the CHRNA5-A3-B4 gene cluster has been shown to be associated with increased mortality risk amongst ever smokers (6), which is likely to explain the reduced frequency of this allele amongst older populations of ever compared to never smokers.(7) This highlights the difficulties in using Mendelian randomisation for diseases of old age.

The authors discuss survival bias, and conclude it is unlikely to be a problem. However, in our opinion more research is needed to quantify the extent and impact of survival bias in Mendelian randomisation studies. It may be possible to assess the extent of survival bias by comparing risk allele frequencies to known allele frequencies in younger populations, or to track their change in a population as it ages. Survival bias is likely to only occur if there are fewer risk alleles in older populations. Simulations may allow us to estimate the size of survival bias and aid the interpretation of Mendelian randomisation studies.

Neil M. Davies, Amy E. Taylor, Marcus R. Munafò

[1] S. D. Østergaard et al., Associations between Potentially Modifiable Risk Factors and Alzheimer Disease: A Mendelian Randomization Study. PLOS Med. 12, e1001841 (2015).

[2] N. Li et al., Use of angiotensin receptor blockers and risk of dementia in a predominantly male population: prospective cohort analysis. Br. Med. J. 340, b5465 (2010).

[3] N. M. Davies, P. G. Kehoe, Y. Ben-Shlomo, R. M. Martin, Associations of anti-hypertensive treatments with Alzheimer’s disease, vascular dementia, and other dementias. J. Alzheimers Dis. JAD. 26, 699–708 (2011).

[4] P. G. Kehoe, N. M. Davies, R. M. Martin, Y. Ben-Shlomo, Associations of Angiotensin Targeting Antihypertensive Drugs with Mortality and Hospitalization in Primary Care Patients with Dementia. J. Alzheimers Dis. JAD. 33, 999–1008 (2013).

[5] N. Levi Marpillat, I. Macquin-Mavier, A.-I. Tropeano, A.-C. Bachoud-Levi, P. Maison, Antihypertensive classes, cognitive decline and incidence of dementia: a network meta-analysis. J. Hypertens. 31, 1073–1082 (2013).

[6] L. Rode, S. E. Bojesen, M. Weischer, B. G. Nordestgaard, High tobacco consumption is causally associated with increased all-cause mortality in a general population sample of 55,568 individuals, but not with short telomeres: a Mendelian randomization study. Int. J. Epidemiol. 43, 1473–1483 (2014).

[7] A. E. Taylor, M. R. Munafò, CARTA consortium, Commentary: Does mortality from smoking have implications for future Mendelian randomization studies? Int. J. Epidemiol. 43, 1483–1486 (2014).

On 2015 Jun 06, A Martinez-Arias commented:

This manuscript claims the discovery of a new kind of Embryonic Stem (ES) cell, so called ‘region specific Epiblast stem cells (rsEpiSC). A close look at the protocols, genetics and cell behaviours indicates that what is reported is likely to be an improved method for the maintenance of EpiSCs and the closely related human ES cells; it would be misleading to describe a more stable population as a new entity.

The main finding reported here is that inhibition of Wnt signalling in cultures of EpiSCs, and also human ES cells, leads to a stable pluripotent population. As briefly indicated below, this has been reported a number of times before, as has the ability of EpiSCs to integrate in postimplantation embryos, the other finding presented here as new. The observation that stabilized human ES cells can undergo a similar integration is, however, novel, though the degree to which this happens and its value will await further experiments.

It is well known that ES and EpiS cells are, for the most part, heterogeneous populations in dynamic equilibria. In the case of ES cells this can be biased towards a stable state, called ‘ground state’, by application of two inhibitors, one for MEK and another for GSK3 [1]. It is also well known that in contrast with ES cells, when EpiSCs and human ES cells are presented with high levels of Wnt signalling they differentiate [2, 3] and over the last few years a number of reports have shown that inhibition of Wnt secretion or Wnt/ß-catenin signalling increases the self renewal of EpiSCs populations [4-6]. Thus a cocktail of Activin, FGF2 together with Wnt/ß-catenin inhibitors leads to efficient derivation of EpiSCs as well as their stable culture. This is confirmed in this report without acknowledging the earlier studies –interestingly some of these studies are referred to but only as discussing Wnt signalling in pluripotency. Having repeated this observation, the authors now notice that removal of Activin from the cocktail –but not inhibition of Activin signalling, which is not tested- improved the stability of the culture; this tweak is novel and mechanistically intriguing, though it is not pursued further. Thus the main message of the work is that EpiSCs grown or derived in the presence of FGF2 and Wnt inhibitors are stable (this is no new kind of ES cell).

With regard to the ability of the cells to integrate in the posterior part of postimplantation embryos, there are also precedents showing that EpiSCs, which indeed are not able to integrate in preimplantation embryos under normal conditions, can and will integrate in postimplantation embryos [7, 8]. The point made here that ‘stable EpiSCs integrate preferentially in posterior regions of the embryo is not surprising as integration is likely to be easier in the region undergoing gastrulation, which is ongoing in the posterior region at the time of the injection. Furthermore, the conclusion that ‘rsEpiSCs’ are related to the posterior proximal epiblast of stage E6.5 is similar to that obtained by Kojima et al. that although it is possible to obtain EpiSCs from a range of stages during gastrulation, EpiSC lines tend to correspond to anterior primitive streak i.e. posterior proximal E6.5 [8]. The results presented here are compatible with the observation that EpiSCs derived from embryos at different stages of gastrulation can be different [8] i.e there might be many rsEpiSCs, which is probably not a helpful notion.

One of the arguments used by the authors to claim the identification of a new type of stem cell is their transcriptional profile. However, EpiSC lines derived from different stages around gastrulation have different properties and transcriptional profiled and much of these differences are likely to be down to signalling [8]. Thus it is not that surprising that a population of EpiSCs severely deprived of Wnt and Activin signalling exhibits a special transcriptional profile and maps, in a PCA plot, away from other cells in different states and notably from EpiSCs grown in Activin and FGF2, a very different cocktail. It is likely that under appropriate experimental conditions, rsEpiSCs will be shown to correspond to one of the EpiSCs derived by Kojima et al. [8], though the changes and adaptations associated with growth in specific signalling environments would make the comparison challenging.

In summary, this report is a protocol to obtain a state which is to EpiSCs what the ground state (2i) is to the ES cells. This is certainly useful but not enough to talk about a new kind of ES cells. Of course, this is an opinion. Nevertheless, the work provides further support to the importance of Wnt signalling in the control of the dynamics of stem cell populations.

References [1] Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, et al. The ground state of embryonic stem cell self-renewal. Nature 2008;453:519-23. [2] Singh AM, Reynolds D, Cliff T, Ohtsuka S, Mattheyses AL, Sun Y, et al. Signaling network crosstalk in human pluripotent cells: a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation. Cell Stem Cell 2012;10:312-26. [3] Davidson KC, Adams AM, Goodson JM, McDonald CE, Potter JC, Berndt JD, et al. Wnt/beta-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4. Proc Natl Acad Sci U S A 2012;109:4485-90. [4] Kurek D, Neagu A, Tastemel M, Tuysuz N, Lehmann J, van de Werken HJ, et al. Endogenous WNT signals mediate BMP-induced and spontaneous differentiation of epiblast stem cells and human embryonic stem cells. Stem cell reports 2015;4:114-28. [5] Kim H, Wu J, Ye S, Tai CI, Zhou X, Yan H, et al. Modulation of beta-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal. Nat Commun 2013;4:2403. [6] Sumi T, Oki S, Kitajima K, Meno C. Epiblast ground state is controlled by canonical Wnt/beta-catenin signaling in the postimplantation mouse embryo and epiblast stem cells. PLoS One 2013;8:e63378. [7] Huang Y, Osorno R, Tsakiridis A, Wilson V. In Vivo differentiation potential of epiblast stem cells revealed by chimeric embryo formation. Cell Rep 2012;2:1571-8. [8] Kojima Y, Kaufman-Francis K, Studdert JB, Steiner KA, Power MD, Loebel DA, et al. The transcriptional and functional properties of mouse epiblast stem cells resemble the anterior primitive streak. Cell Stem Cell 2014;14:107-20.

On 2015 May 28, Peter Good commented:

Cochran et al. measured brain glutamate, glutamine, GABA, and other metabolites by MRS in the anterior cingulate cortex of ASD adolescents to test whether autistic behavior arises from imbalance of excitatory vs. inhibitory neurotransmitters. They found normal concentrations of excitatory transmitter glutamate, but significantly high non-transmitter glutamine and significantly low inhibitory GABA. They concluded high brain glutamine and/or low GABA might explain imbalance of excitatory vs. inhibitory transmission – i.e. autistic behavior.

True, low brain GABA could be responsible – but implicating high brain glutamine in autistic behavior is risky at best, and potentially dangerous. Not only do ASD children consistently have low plasma glutamine, high brain glutamine appears to protect against ASD. This is most obvious in children with high brain glutamine from inborn urea cycle disorders (UCD) or propionic acidemia (PA) [Good 2014]. In UCD, failure of the liver urea cycle to detoxify blood ammonia at first pass allows high levels into the brain, which astrocytes detoxify by combining with glutamate to form glutamine. Krivitzky et al.: “Children in this cohort [UCD] show other behavioral/emotional strengths, including a minimal percentage with previous diagnoses of Autism spectrum disorders, mood disorders, and other psychiatric disorders.”[Krivitzky L, 2009] Saul Brusilow (MD) never mentioned autism or autistic behavior in any paper on UCD or hepatic encephalopathy (HE) [personal communication 2013]. Gropman et al., however, noted patients with partial deficiencies of urea cycle enzymes and late-onset presentations may show signs of autism [Gropman AL, 2007]. Cochran et al. also found the osmolyte myoinositol normal in ASD brains; when astrocyte glutamine is high in urea cycle disorders or HE, myoinositol is consistently low. Gabis et al., however, found myoinositol high in ASD astrocytes [Gabis L, 2008].

Propionic acidemia is another inborn metabolic disorder where high blood ammonia becomes high brain glutamine. Al Owain et al.: “In the consensus conference about diagnosis and management of PA hosted in Washington, D.C. in January 2011, there was no reported association among the neurological sequelae of the disease between PA and autism.”[Al-Owain M, 2013] Other physicians who treat PA and UCD children also report they rarely show autistic behavior. Sabine Scholl-Bürgi (MD): “In our PA patient group none has an ASD.”[personal communication 2014]. Professor of pediatrics (MD): “I see lots of kids with PA and UCD but few (perhaps none) have ASD.”[personal communication 2013]

Moreover, plasma concentrations of glutamine and GABA in ASD children are consistently opposite brain concentrations detected by Cochran et al. Plasma glutamine is consistently low [e.g. Moreno-Fuenmayor H, 1996; Aldred S, 2003; Shimmura C, 2011] and plasma GABA high [Cohen 2000; Dhossche D, 2002]. Ghanizadeh concluded: “The low level of plasma glutamine . . . is suggested as a screening test for detecting autism in children especially those with normal IQ. The decreased level has been reported before in all children with autism.”[Ghanizadeh A, 2013] Dhossche et al. thought high plasma GABA explained mood disorders and stupor. Burrus concluded a reaction between ammonia and propionic acid could produce a molecule structurally very similar to GABA [Burrus CJ, 2012].

Glutamine is normally the most abundant amino acid in blood [Souba WW, 1991], a primary brain osmolyte, alternative fuel for brain neurons and astrocytes (especially during hypoglycemia) [Stelmashook EV, 2011], and primary fuel in rapidly replicating cells (e.g. blood vessel endothelial cells, intestinal enterocytes, liver cells, and lymphocytes) [Souba WW, 1987; Souba WW, 1991; Deutz NE, 2008]. Glutamine released from skeletal muscles for anabolic responses to infection may explain the dramatic ability of fever to relieve autistic behavior [Good P, 2013]. A new clue to this phenomenon was recently published at <www.autismstudies.net>.

Autism Research Institute practitioners commonly give ASD patients oral glutamine to heal their intestines, from 250mg–8g/day, with few side effects (some hyperactivity) [Good P, 2013] – although one neurologist reported seizures. Only two practitioners, however, reported improved behavior from glutamine. Franco Verzella (MD) in Bologna, Italy gives ASD children 5–7g/day of oral glutamine after cleansing their intestines of pathogens like bacteria and Candida: “Multifactorial and multisystemic is the condition, so that the improvement has different aspects in different children. Most common: sedation, less stereotypes, better sleep, more concentration.”[personal communication 2013]

Cochran and colleagues need to think twice about reducing brain glutamine in ASD children – whose plasma and brain glutamine are more likely TOO LOW than too high. Pangborn (2013) recommended the free amino acid taurine as a natural way to increase conversion of ammonia + glutamate to glutamine.

Peter Good Autism Studies La Pine OR www.autismstudies.net autismstudies1@gmail.com

Aldred S, Moore KM, Fitzgerald M, Waring RH. Plasma amino acid levels in children with autism and their families. J Autism Dev Disord 2003;33:93–97.

Al-Owain M, Kaya N, Al-Shamrani H, et al. Autism spectrum disorder in a child with propionic acidemia. JIMD Rep 2013;7:63–66.

Burrus CJ. A biochemical rationale for the interaction between gastrointestinal yeast and autism. Med Hypotheses 2012;79:784–785.

Cohen BI. Infantile autism and the liver: a possible connection. Autism 2000;4:441–442.

Deutz NEP. The 2007 ESPEN Sir David Cuthbertson Lecture: amino acids between and within organs. The glutamate-glutamine-citrulline-arginine pathway. Clin Nutr 2008;27(3):321–327.

Dhossche D, Applegate H, Abraham A, et al. Elevated plasma gamma-aminobutyric acid (GABA) levels in autistic youngsters: stimulus for a GABA hypothesis of autism. Med Sci Monit 2002;8:PR1–PR6.

Gabis L, Wei Huang, Azizian A, et al. 1H-magnetic resonance spectroscopy markers of cognitive and language ability in clinical subtypes of autism spectrum disorders. J Child Neurol 2008;23(7):766–774.

Ghanizadeh A. Increased glutamate and homocysteine and decreased glutamine levels in autism: a review and strategies for future studies of amino acids in autism. Dis Markers 2013;35:281–286.

Good P. Does infectious fever relieve autistic behavior by releasing glutamine from skeletal muscles as provisional fuel? Med Hypotheses 2013;80:1–12.

Good P. Why do children with propionic acidemia or urea cycle disorders rarely show autistic behavior? Autism–Open Access 2014;4:3.

Gropman AL, Summar M, Leonard JV. Neurological implications of urea cycle disorders. J Inherit Metab Dis 2007;30:865–879.

Krivitzky L, Babikian T, Lee HS, et al. Intellectual, adaptive, and behavioral functioning in children with urea cycle disorders. Pediatr Res 2009;66:96–101.

Moreno-Fuenmayor H, Borjas L, Arrieta A, et al. Plasma excitatory amino acids in autism. Invest Clin 1996;37:113–128.

Pangborn JB. Nutritional Supplement Use for Autism Spectrum Disorder. San Diego: Autism Research Institute; 2013.

Shimmura C, Suda S, Tsuchiya KJ, et al. Alteration of plasma glutamate and glutamine levels in children with high functioning autism. PLoS One 2011;6:e25340–e25346.

Souba WW. Interorgan ammonia metabolism in health and disease: a surgeon’s view. J Parenter Enteral Nutr 1987;11:569–579.

Souba WW. Glutamine: a key substrate for the splanchnic bed. Ann Rev Nutr 1991;11:285–308.

Stelmashook EV, Isaev NK, Lozier ER, et al. Role of glutamine in neuronal survival and death during brain ischemia and hypoglycemia. Int J Neurosci 2011;121:415–422.

On 2015 Aug 24, Eiko Fried commented:

We have published a commentary on this article in Frontiers of Psychiatry, in which we introduce Symptomics as a novel research paradigm for psychiatry and clinical psychology. We provide a brief summary of the commentary below.

Summary:

"Research has now shown that distinct depression symptoms differ in the risk factors that predispose them, their underlying biology, their response to specific life events, and their impact on impairment of psychosocial functioning. The recently published work by Hieronymus et al. adds the differential reactivity of depression symptoms to antidepressant medication to this prior body of work. The authors argue that the findings stress the importance of analyzing individual depression symptoms in future studies. We would like to extend their claim: these results mandate the examination of symptom-specific effects throughout the realm of psychopathology. Symptomics invites the application of new modeling efforts to the level of individual symptoms as fundamental building blocks of mental disorders. Focusing (A) on the level of symptoms and (B) analyzing the causal relations among them—as an alternative approach to the dominant focus on diagnoses—is likely to extend our understanding of psychopathology directly and significantly. As such, symptomics may herald a time of renewed research energy that could, finally, provide an inroad to achieve real understanding of the mechanisms underlying psychopathology."

Reference:

Fried EI, Boschloo L, van Borkulo CD, Schoevers RA, Romeijn J-W, Wichers MC, de Jonge P, Nesse RM, Tuerlinckx F, & Borsboom D (2015). Commentary: "Consistent superiority of selective serotonin reuptake inhibitors over placebo in reducing depressed mood in patients with major depression", Frontiers in Psychiatry 6, 1–3. DOI: 10.3389/fpsyt.2015.00117.

URL: http://journal.frontiersin.org/article/10.3389/fpsyt.2015.00117/full

On 2015 Apr 24, CHARLES KING commented:

I feel there is a serious methodological flaw in the recently published Cochrane Review “Circulating antigen tests and urine reagent strips for diagnosis of active schistosomiasis in endemic areas” by Ochodo, et el., 2015. The inaccurate analysis used in the HSROC estimation skews the reported summary results on the diagnostic performance of both antigen tests and dipsticks, and a correct analysis would actually invalidate several of the conclusions and recommendations found in the current version of the review: My objection is to the use of egg count diagnostics as a reference standard for the diagnosis of Schistosoma infection. The stool egg count for S. mansoni and S. japonicum and the urine filtration egg count for S. haematobium have long been known to be poorly sensitive for low intensity infections. When subjects are repeatedly tested for 7-15 days in a row, single day egg count testing has a sensitivity of 40-60%. Although these imperfect tests remain in widespread use in field studies and control campaigns because they accurately detect persons with heavy infections, they cannot be relied upon to establish infection in all subjects. The failings of stool counting for S. mansoni were documented in the work of de Vlas and colleagues cited below [1,2]. The limitations of stool counting for S. japonicum have been established by Carabin, et al.,[3] and Hubbard, et al.,[4] and the limitations of egg counting for S. haematobium can be found in Savioli et al.,[5] and Warren, et al.[6]

In all cases, egg counting cannot be considered a ‘gold standard’ diagnostic. Such a test, with ~50% sensitivity, is so inaccurate to be useless on a per-individual diagnostic basis. I feel that the appropriate comparison for the Ochodo, et al., review would have been Latent Class Analysis, in which two imperfect tests are compared in their attempt to classify an unmeasured ‘true’ infection status. Using egg count results as a reference standard in the HSROC was a serious error--reporting a new test’s performance benchmarked against an already flawed test is meaningless, and in fact, the reported comparisons are misleading to the practitioner or public health officer.

Secondly, I feel that the exclusion of results from populations or areas without significant Schistosoma risk was also a tactical error. If we are concerned about the specificity of new tests, there is great value in measuring results among persons who have a very low prior probability of infection.

I would strongly recommend that the Cochrane review be revised and re-issued after the authors revisit the data using the approach of Dendukuri, et al., 2012 [7] for situations where there is no gold standard. Their SAS code is available online, and the reanalysis could be done in a matter of a day. The Bayesian LCA should be informed by prior observations on the estimated specificity of single (or treble stool) examinations, and the known specificity estimates of dipsticks and antigen testing among non-endemic populations.

The concern is that while the authors discuss and reiterate the lack of a gold standard and the insensitivity of the egg count procedures they go right ahead and use them as their comparator and they present strong conclusions that are biased by their approach. This will only add to policymakers’ confusion about the utility of these alternative test approaches.

By way of disclosure, I have no relation to the manufacturers of these tests, and I have no conflict of interest in this matter.

1. de Vlas SJ, Engels D, Rabello AL, Oostburg BF, Van Lieshout L, Polderman AM, Van Oortmarssen GJ, Habbema JD, Gryseels B, 1997. Validation of a chart to estimate true Schistosoma mansoni prevalences from simple egg counts. Parasitology 114 ( Pt 2): 113-21.
2. de Vlas SJ, Gryseels B, 1992. Underestimation of Schistosoma mansoni prevalences. Parasitol Today 8: 274-277.
3. Carabin H, Marshall CM, Joseph L, Riley S, Olveda R, McGarvey ST, 2005. Estimating the intensity of infection with Schistosoma japonicum in villagers of Leyte, Philippines. Part I: A Bayesian cumulative logit model. The Schistosomiasis Transmission & Ecology Project (STEP). Am J Trop Med Hyg 72: 745-753.
4. Hubbard A, Liang S, Maszle D, Qiu D, Gu X, Spear RC, 2002. Estimating the distribution of worm burden and egg excretion of Schistosoma japonicum by risk group in Sichuan Province, China. Parasitology 125: 221-31.
5. Savioli L, Hatz C, Dixon H, Kisumku UM, Mott KE, 1990. Control of morbidity due to Schistosoma haematobium on Pemba Island: egg excretion and hematuria as indicators of infection. Am J Trop Med Hyg 43: 289-295.
6. Warren KS, Arap Siongok TK, Hauser HB, Ouma JH, Peters PAS, 1978. Quantification of infection with Schistosoma haematobium in relation to epidemiology and selective population chemotherapy. I. Minimal number of daily egg counts in urine necessary to establish intensity of infection. Journal of Infectious Diseases 138: 849-55.
7. Dendukuri N, Schiller I, Joseph L, Pai M, 2012. Bayesian meta-analysis of the accuracy of a test for tuberculous pleuritis in the absence of a gold standard reference. Biometrics 68: 1285-1293.

On 2016 Jan 16, Lewis G Halsey commented:

Fay worries that in the real world of data collection, the power of a study is not known in advance. If true, this argument would only compound our own, which is that unless power is very high (>90%) P has surprisingly low repeatability (Halsey et al., 2015), and the power of most studies calculated after the data analysis is far lower than this (Button et al., 2013, Maxwell, 2004). Therefore, researchers would not be able to design their experiment to ensure it has a very high power, and they could not rely on good fortune instead for their experiment to turn out this way.

But anyway, an integral step in testing the null hypothesis generates an estimate of the variance of the pooled population. Using this estimated parameter, obtained as the data are analysed, the researcher can immediately gauge the study’s power. The exact parameters of the population are never known. These parameters are hypothesised and then estimated, with varying certainty, according to the sample that we have. With a limited sample, these estimates can vary substantially each time an experiment is repeated. If our samples, and the estimates they generate, suggest that power is poor, then any P value that we obtain, low or not, is untrustworthy. A small P value is of little import: a repetition of the same study would give another result (our study, figure 4). This is like looking at the world through a pinhole. When the theoretical power is 0.48, P values less than 0.05 are no more likely than P values greater than 0.05. Why get excited if P is <0.01, when the next replicate experiment could give a P of 0.6?

Fay asks if there is a single better measure than P to test the likelihood that the null hypothesis is untenable. First, this brings us back to the nub of the problem - P is only a good test of the null in the ideal circumstances that study power is very high. Second, there are long-held, big concerns about the value of null hypothesis significance testing as a method for analysing and interpreting data (Cohen, 1994).

Lewis G Halsey and Gordon B Drummond

BUTTON, K., IOANNIDIS, J., MOKRYSZ, C., NOSEK, B., FLINT, J., ROBINSON, E. & MUNAFO, M. (2013) Power failure: why small sample size undermines the reliability of neuroscience. Nature Reviews Neuroscience, 14, 365-376. COHEN, J. (1994) The Earth is round (p < 0.05). American Psychologist 49, 997-1003. HALSEY, L., CURRAN-EVERETT, D., VOWLER, S. & DRUMMOND, G. (2015) The fickle P value generates irreproducible results. Nature Methods, 12, 179-185. MAXWELL, S. (2004) The persistence of underpowered studies in psychological research: Causes, consequences, and remedies. Psychological Methods, 9, 147-163.

On 2015 Feb 23, William Grant commented:

Concerns over Vitamin D and clinical practice at a crossroads recommendations

In their viewpoint piece, Vitamin D and clinical practice at a crossroads, Manson and Bassuk state among other things that the Institute of Medicine (IOM) set the recommended dietary allowance for vitamin D at 600 IU/d for those living in the upper latitudes of North America aged to 70 years and 800 IU/d for those older in order to reach a 25-hydroxyvitamin D [25(OH)D] concentration of 20 ng/mL (50 nmol/L) [1]. However, it is not clear how the Dietary Reference Intakes for Calcium and Vitamin D committee arrived at that number. For example, on p. 3-20 of the vitamin D and calcium IOM report [2], Figure 3-4 from Cashman et al. [3] is given as Figure 3-4, although without the 95% confidence intervals as in the original paper. The results were based on a 22-week placebo, randomized controlled supplementation study involving men and women aged 20-40 years. Inspection of Figure 2 in Ref. 3 indicates that it would take 1155 IU/d vitamin D3 for 97.5% of the 20-40 year old population sampled to reach 50 nmol/L. In a subsequent paper based on a systematic review and meta-analysis of vitamin D intake and 25(OH)D concentrations, the same group determined that it would take 930 IU/d vitamin D3 for people living in northern Europe to reach 50 nmol/L [4]. Further complicating matters is the fact that not all of the contributions from diet are accounted for in most studies. It is becoming apparent that some food such as meat has vitamin D in the form of 25(OH)D. Thus, vegans in the UK have 25(OH)D concentrations 20 nmol/L lower than omnivores [5], so require higher vitamin D intake from non-dietary sources or solar UVB exposure.

Their comment that "while awaiting the results of the large trials now in progress, physicians would be well advised to follow current USPSTF and IOM recommendations and avoid overscreening and overprescribing supplemental vitamin D" is also not well grounded. The IOM restricted its assessment of the benefits of vitamin D to vitamin D randomized controlled trials [RCTs] with substantial benefits by 2010. A number of trials since then demonstrated health benefits, e.g., for biomarkers of inflammation, where it was found that RCTs with baseline 25(OH)D concentrations below 48 nmol/L had a 50% chance of findings benefits from vitamin D supplementation compared to 25% with baseline 25(OH)D concentrations above 50 nmol/L [6]. In addition, ecological, observational, clinical, and laboratory studies have found many health benefits of solar UVB exposure and/or vitamin D. Since the IOM report was published (29 November, 2010), 13,535 publications with vitamin D in the title or abstract have been published at PubMed.gov as of 23 February, 2015, compared with 27,775 published before that date. Many of these publications strengthen the case for vitamin D supplementation and UVB exposure.

In terms of confounding factors related to observational studies, one not mentioned in Ref. 1 is the possibility that solar UV exposure may have health benefits in addition to vitamin D production. As a result, 25(OH)D concentrations may be an index of UVB exposure. Beneficial effects of UV exposure in addition to vitamin D production have been reported for intestinal cancer [7], multiple sclerosis [8], and blood pressure [9].

As for concern about adverse effects of higher 25(OH)D concentrations based on observational studies, it should be noted that most such studies do not obtain any information from participants about vitamin D supplementation prior to having 25(OH)D concentrations measured. Thus, those with adverse health outcomes and high 25(OH)D concentrations may have started taking vitamin D supplements shortly before blood draw. For example, studies of frailty vs. 25(OH)D concentration found a U-shaded relation for elderly women [10] but a linear inverse relation for elderly men [11]. Elderly women in the U.S. are much more likely to be advised to take vitamin D supplements than men, and starting to take vitamin D late in life cannot erase the adverse effects of years of low 25(OH)D concentrations. In addition, meta-analyses of observational studies of health outcomes with respect to 25(OH)D concentrations do not show U-shaped relations for cardiovascular disease [12] or all-cause mortality rates [13].

As to their comment regarding how interest in vitamin D could jeopardize ongoing vitamin D RCTs, that should not be the case if trial participants are screened by measuring 25(OH)D concentration prior to acceptance and including only those with 25(OH)D concentrations below 50 nmol/L then dropping any who are subsequently prescribed supplements in excess of the IOM recommendations. Over 99% of the population is not enrolled in vitamin D RCTs and should not be held hostage to ongoing or planned trials since there appear to be many health benefits and very few risks of vitamin D supplementation below 4000 IU/d [14] even by the IOM's admission [2].

References 1. Manson JE, Bassuk SS. Vitamin D research and clinical practice: at a crossroads. JAMA. 2015 Feb 19. doi: 10.1001/jama.2015.1353. [Epub ahead of print] 2. Ross AC, Taylor CL, Yaktine AL, Del Valle HB, eds. ; Committee to Review Dietary Reference Intakes for Vitamin D and Calcium; Dietary Reference Intakes for Calcium and Vitamin D. Institute of Medicine. ISBN: 0-309-16395-1, 482 pages, (2010) Available from National Academies Press at: http://www.nap.edu/catalog/13050.html 3. Cashman KD, Hill TR, Lucey AJ, et al. Estimation of the dietary requirement for vitamin D in healthy adults. Am J Clin Nutr. 2008;88(6):1535-42. 4. Cashman KD, Fitzgerald AP, Kiely M, Seamans KM. A systematic review and meta-regression analysis of the vitamin D intake-serum 25-hydroxyvitamin D relationship to inform European recommendations. Br J Nutr. 2011;106(11):1638-48. 5. Crowe FL, Steur M, Allen NE, et al. Plasma concentrations of 25-hydroxyvitamin D in meat eaters, fish eaters, vegetarians and vegans: results from the EPIC-Oxford study. Public Health Nutr. 2011;14(2):340-6. 6. Cannell JJ, Grant WB, Holick MF. Vitamin D and inflammation. Dermato-Endocrinology. 2014;6(1):e983401-1-10.<br> 7. Rebel H, der Spek CD, Salvatori D, et al.UV exposure inhibits intestinal tumour growth and progression to malignancy in intestine-specific Apc mutant mice kept on low vitamin D diet. Int J Cancer. 2015;136(2):271-7. 8. Zivadinov R, Treu CN, Weinstock-Guttman B, et al. Interdependence and contributions of sun exposure and vitamin D to MRI measures in multiple sclerosis. J Neurol Neurosurg Psychiatry. 2013;84(10):1075-81. 9. Opländer C, Volkmar CM, Paunel-Görgülü A, et al. Whole body UVA irradiation lowers systemic blood pressure by release of nitric oxide from intracutaneous photolabile nitric oxide derivates. Circ Res. 2009;105(10):1031-40. 10. Ensrud KE, Ewing SK, Fredman L, et al. Circulating 25-hydroxyvitamin D levels and frailty status in older women. J Clin Endocrinol Metab. 2010;95(12):5266-73. 11. Ensrud KE, Blackwell TL, Cauley JA, et al. Circulating 25-hydroxyvitamin D levels and frailty in older men: the osteoporotic fractures in men study. J Am Geriatr Soc. 2011;59(1):101-6. 12. Wang L, Song Y, Manson JE, et al. Circulating 25-hydroxy-vitamin D and risk of cardiovascular disease: A meta-analysis of prospective studies. Circ Cardiovasc Qual Outcomes. 2012;5(6):819-29. 13. Garland CF, Kim JJ, Mohr SB, et al. Meta-analysis of all-cause mortality according to serum 25-hydroxyvitamin D. Am J Pub Health. 2014;104(8):e43-50. 14. Vieth R. Implications for 25-hydroxyvitamin D testing of public health policies about the benefits and risks of vitamin D fortification and supplementation. Scand J Clin Lab Invest Suppl. 2012;243:144-53.

Disclosure I receive funding from Bio-Tech Pharmacal, Inc. (Fayetteville, AR), MediSun Technology (Highland Park, IL), and the Vitamin D Council (San Luis Obispo, CA).

On 2015 Jun 10, Paul Golding commented:

Baggott and Tamura imply that my experiment was flawed because: “The study design by Golding (2014) should have included a balanced supplementation of micronutrients at the level of generally recommended dietary intakes.” and advise that “Therefore, the data presented by Golding (2014) must be cautiously interpreted”.

My full response is available here.

Was the 500 mg/day vitamin C supplement excessive, and could it have abnormally extended the time taken to deplete the liver folate store?

Baggott and Tamura claim that my 500 mg/day vitamin C supplement was excessive, and that my saturated vitamin C status was abnormal. They propose that by abnormally protecting the reduced (unstable) form of folate from oxidation, which they claim would not have been preserved by recommended vitamin C intakes, the “excessive” vitamin C preserved the liver folate store. I strongly disagree with these assertions.

Carr et al (2012), Levine et al. (1996) and Lykkesfeldt and Poulsen (2010) recommend that vitamin C intake should be sufficient to ensure plasma saturation. If the plasma vitamin C concentration is insufficient to prevent oxidation of fully reduced folate, although sufficient to prevent scurvy, there would be sub-optimal vitamin C status.

There is no evidence that, once there is sufficient vitamin C present to ensure plasma saturation, and thereby protect the fully reduced folates, additional amounts will affect the time taken to develop folate deficiency.

Levine et al. (1996) recommend 200 mg vitamin C daily, very significantly higher than the 70 mg taken by Herbert (Herbert 1962), and a level that ensures plasma saturation. They plotted plasma vitamin C concentration against the daily vitamin C dose (Levine et al. 1996 Figure 1C); this produced a sigmoid curve with 200 mg the lowest dose to reach a plateau.

I used data from Levine et al. (1996 Table 1) to produce a chart to illustrate the relationship between vitamin C dose and the plasma concentration, at relevant levels. At Herbert’s dose of 70 mg/day, plasma concentration is 32 µmol/L; at 200 mg/day recommended by Levine et al. (1996), plasma concentration is 66 µmol/L; at my dose of 500 mg/day, plasma concentration is 71 µmol/L. A 500 mg dose of vitamin C will not produce a significantly higher plasma concentration than the 200 mg dose recommended by Levine et al. (1996), but a 70 mg dose will produce a very significantly lower plasma concentration. The Excel file, high-resolution PDF and Microsoft PowerPoint slide for my chart are available at the link above.

The predicted plasma vitamin C concentration of 71 µmol/L, produced by my intake of 500 mg/day, is very close to the value considered by Carr et al. (2012) to be necessary for good health.

The vitamin C supplement used by me was integrated with the iron tablets taken to prevent the iron deficiency reported by Herbert. As stated in Golding (2014), the iron tablets (Abbott Australia Ferro-Grad C) comprised 325 mg ferrous sulphate (equivalent to 105 mg elemental iron), with 562 mg sodium ascorbate (equivalent to 500 mg vitamin C). Sodium ascorbate is necessary to ensure adequate absorption of the iron (Hurrell 2002, McCurdy 1968).

Was the 1000 µg/day vitamin B12 supplement excessive, and could it have abnormally extended the time taken for me to deplete my liver folate store?

Although Baggott and Tamura raise the possibility of such a confounding effect, they have not suggested any mechanism for it, or cited any published reports of such findings.

I found no published reports of vitamin B12 used to overcome a dietary folate deficiency, or any findings of interference by vitamin B12 supplements with folate metabolism where there was no initial vitamin B12 deficiency. An initially untreated vitamin B12 deficiency can cause a secondary functional folate deficiency; the “methylfolate trap” (Tisman and Herbert 1973). Such a secondary folate deficiency might be corrected by vitamin B12 supplements, depending on the cause of the vitamin B12 deficiency, but there is no published evidence that increasing the vitamin B12 intake beyond that will have any effect on folate metabolism.

As explained in Golding (2014), the 1000 µg/day vitamin B12 supplement was necessary to prevent vitamin B12 deficiency. Extensive testing during my previous vitamin B12 investigation showed that my vitamin B12 is absorbed and transported to cells normally but is not utilised adequately within the cells, presumably because of a deficiency of one of the B12 cofactors. At least 250 µg was needed to avoid deficiency but 1000 µg is the most readily available, allows a reasonable margin for error, and is within the recommended range for the oral vitamin B12 dose (Kuzminski et al. 1998).

Did the experiment include appropriate supplementation of micronutrients and was the title adequate?

Baggott and Tamura have not provided any evidence that taking either of these supplements could have interfered with the development of folate deficiency in my experiment, and have not offered any plausible mechanism for such confounding effects. In addition, my supplementation with vitamin C and vitamin B12 was reasonable, in the doses used, to prevent deficiencies.

I therefore disagree with their advice that “Therefore, the data presented by Golding (2014) must be cautiously interpreted”, and with what they imply in their conclusion; that my experiment was flawed because the study design failed to include “a balanced supplementation of micronutrients at the level of generally recommended dietary intakes.”

I also disagree with their assertion that “The title … should have contained words clearly indicating that the subject had a high initial folate status and was saturated with ascorbate.” My replete folate status was clearly stated in Objective, Experiment Design and The Subject. According to Levine et al. (1996), Carr et al. (2012) and Lykkesfeldt and Poulsen (2010), plasma saturation is the normal optimal vitamin C status.

References

Baggott JE, Tamura T (2014) The second study of experimental human folate deficiency. SpringerPlus 3:719

Carr AC, Pullar JM, Moran S, Vissers MC (2012) Bioavailability of vitamin C from kiwifruit in non-smoking males: determination of 'healthy' and 'optimal' intakes. J Nutr Sci 1:e14

Golding PH (2014) Severe experimental folate deficiency in a human subject – a longitudinal study of biochemical and haematological responses as megaloblastic anaemia develops. SpringerPlus 3:442

Herbert V (1962) Experimental nutritional folate deficiency in man. Trans Assoc Am Physicians 75:307–320

Herbert V (1987) Recommended dietary intakes (RDI) of folate in humans. Am J Clin Nutr 45(4):661–670

Hurrell RF (2002) Fortification: overcoming technical and practical barriers. J Nutr 132(4 Suppl):806S–812S

Kuzminski AM, Del Giacco EJ, Allen RH, Stabler SP, Lindenbaum J (1998) Effective treatment of cobalamin deficiency with oral cobalamin. Blood 92(4):1191-1198

Levine M, Conry-Cantilena C, Wang Y, Welch RW, Washko PW, Dhariwal KR, Park JB, Lazarev A, Graumlich JF, King J, Cantilena LR (1996) Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. Proc Natl Acad Sci 93(8):3704–3709

Lykkesfeldt J, Poulsen HE (2010) Is vitamin C supplementation beneficial? Lessons learned from randomised controlled trials. Br J Nutr 103(9):1251-1259

McCurdy PR, Dern RJ (1968) Some therapeutic implications of ferrous sulfate-ascorbic acid mixtures. Am J Clin Nutr 21(4):284–288

Tisman G, Herbert V (1973) B12 dependence of cell uptake of serum folate: an explanation for high serum folate and cell folate depletion in B 12 deficiency. Blood 41(3):465–469

On 2015 Jul 13, Graham Coop commented:

After our paper appeared we were made aware of a great potential example of paternal control of female meiotic transmission (Herrera et al. 1996). In many species dispensable supernumerary B-chromosomes are preferentially transmitted to offspring, with this preferential segregation (drive) often occurring through female meiosis (Jones 1991, Burt and Trivers 2006). Herrera et al. studied a widespread B chromosome polymorphism in the grasshopper, Eyprepocnemis plorans. In crosses between 1B females and males from the same focal population in which B-chromosomes were present, B-chromosomes were transmitted following Mendelian ratios. However, when the same females were crossed to males from a population where the B chromosome was not present, they transmitted their B-chromosome to much more than half of their progeny. There was no obvious reduction in fertility, suggesting that this was not due to lethality and potentially due to meiotic drive. Herrera et al suggested that the male control of female meiosis is exerted during the first meiotic division, perhaps due to an effect of substances in the male ejaculate. These results are consistent with our hypothesis that sperm-based suppressors of drive may arise and spread in response to the spread of female meiotic drive elements (such as B-chromosomes), such that female meiotic drive can re-emerge when eggs are exposed to specific sperm from a population where drive suppression had not evolved. We thank Juan Pedro M. Camacho (Universidad de Granada) for kindly bringing this example to our attention and for feedback on this note.

Yaniv Brandvain and Graham Coop

Cited References:

Jones, R.N. 1991. B-Chromosome Drive. American Naturalist 137: 430-442.

Burt, A. and R. Trivers, 2006. Genes in conflict. Belknap Press, Cambridge.

Herrera, J. A., M. D. López-León, J. Cabrero, M. W. Shaw and J. P. M. Camacho. 1996. Evidence for B chromosome drive suppression in the grasshopper Eyprepocnemis plorans. Heredity 76: 633–639.

On 2015 Feb 01, William Grant commented:

The paper by Jorde and Grimes outlines the case for conducting more vitamin D randomized controlled trials (RCTs) [1]. They also point out that RCTs conducted on those with low baseline 25-hydroxyvitamin D [25(OH)D] concentration are more likely to find beneficial effects of vitamin D supplementation than those with higher baselines. In agreement with this point, we just published a review of vitamin D RCTs and biomarkers of inflammation. Among the 22 trials with baseline 25(OH)D concentration below 47 nmol/L, 49% found reduced biomarkers of inflammation while for the 12 trials with baseline 25(OH)D concentration above 48 nmol/L, only 27% did [2]. In addition, achieved 25(OH)D concentration was relatively unimportant.

Robert Heaney recently presented guidelines for designing nutrient RCTs. The key steps for vitamin D RCTs include starting with an understanding of the 25(OH)D concentration-health outcome relation, generally from observational studies, measure 25(OH)D concentrations of prospective participants, include only those with 25(OH)D concentrations near the low end of the relation, supplement with enough vitamin D3 to raise 25(OH)D concentrations to near the upper end of the relation, remeasure 25(OH)D concentrations, and optimize conutrient status [3]. Few vitamin D RCTs have been designed in accordance with these guidelines, including the major ones currently underway.

As to the concern about J- an U-shaped 25(OH)D concentration-health outcome relations, one of the reasons for such findings appears to be that those with the highest 25(OH)D concentrations started taking vitamin D supplements late in life, possibly after developing some vitamin D deficiency conditions such as osteoporosis. In support of this hypothesis, two similar observational studies of 25(OH)D concentration and frailty conducted in the United States found different results: for men, there was a nearly linear inverse relation between 25(OH)D and frailty [4] while for women, there was a U-shaped relation [5]. In the United States, postmenopausal women are often advised to take vitamin D but older men are not.

One of the emerging topics of research is whether the observational studies finding inverse correlations between health outcomes and 25(OH)D concentrations might be due to non-vitamin D effects of solar UV exposure. There is mounting evidence that this may be the case for at least three types of health outcomes.

For many types of cancer, geographical ecological studies find inverse correlations between solar UVB doses and cancer incidence and/or mortality rates [6]. A mouse model study recently found that UVB exposure was more effective in slowing the progression of intestinal tumors than was oral vitamin D intake when both raised 25(OH)D concentrations by similar amounts [7].

Several recent studies have found that there are non-vitamin D effects associated with solar UVB exposure for multiple sclerosis [8-11].

Long wave UV (UVA) has been found to lower blood pressure [12], a risk factor for cardiovascular disease.

The mechanisms whereby UVB reduces the risk of disease independent of vitamin D are not well known and determining what they are remains an active field of research.

Based on the knowledge to date, what seems to be a prudent policy is spending time in the sun daily when it is possible to make vitamin D, generally when the solar elevation angle is greater than 45 deg. [13] and taking vitamin D supplements when not.

References 1. Jorde R, Grimnes G. Vitamin D and health: The need for more randomized controlled trials. J Steroid Biochem Mol Biol. 2015 Jan 27. pii: S0960-0760(15)00033-3. doi: 10.1016/j.jsbmb.2015.01.021. [Epub ahead of print] Review. 2. Cannell JJ, Grant WB, Holick MF. Vitamin D and inflammation. Dermato-Endocrinology. 2015;6(1): e983401-1-10. DOI:10.4161/19381980.2014.983401 3. Heaney RP. Guidelines for optimizing design and analysis of clinical studies of nutrient effects. Nutr Rev. 2014;72(1):48-54. 4. Ensrud KE, Blackwell TL, Cauley JA, Cummings SR, Barrett-Connor E, Dam TT, Hoffman AR, Shikany JM, Lane NE, Stefanick ML, Orwoll ES, Cawthon PM; Osteoporotic Fractures in Men Study Group. Circulating 25-hydroxyvitamin D levels and frailty in older men: the osteoporotic fractures in men study. J Am Geriatr Soc. 2011;59(1):101-6. 5. Ensrud KE, Ewing SK, Fredman L, Hochberg MC, Cauley JA, Hillier TA, Cummings SR, Yaffe K, Cawthon PM; Study of Osteoporotic Fractures Research Group. Circulating 25-hydroxyvitamin D levels and frailty status in older women. J Clin Endocrinol Metab. 2010;95(12):5266-73. 6. Moukayed M, Grant WB. Molecular link between vitamin D and cancer prevention. Nutrients. 2013;5(10):3993-4023. 7. Rebel H, der Spek CD, Salvatori D, van Leeuwen JP, Robanus-Maandag EC, de Gruijl FR.UV exposure inhibits intestinal tumour growth and progression to malignancy in intestine-specific Apc mutant mice kept on low vitamin D diet. Int J Cancer. 2015;136(2):271-7. 8. Lucas RM, Ponsonby AL, Dear K, Valery PC, Pender MP, Taylor BV, Kilpatrick TJ, Dwyer T, Coulthard A, Chapman C, van der Mei I, Williams D, McMichael AJ. Sun exposure and vitamin D are independent risk factors for CNS demyelination. Neurology. 2011;76(6):540-8. 9. Zivadinov R, Treu CN, Weinstock-Guttman B, Turner C, Bergsland N, O'Connor K, Dwyer MG, Carl E, Ramasamy DP, Qu J, Ramanathan M. Interdependence and contributions of sun exposure and vitamin D to MRI measures in multiple sclerosis. J Neurol Neurosurg Psychiatry. 2013;84(10):1075-81. 10. Bjørnevik K, Riise T, Casetta I, Drulovic J, Granieri E, Holmøy T, Kampman MT, Landtblom AM, Lauer K, Lossius A, Magalhaes S, Myhr KM, Pekmezovic T, Wesnes K, Wolfson C, Pugliatti M. Sun exposure and multiple sclerosis risk in Norway and Italy: The EnvIMS study. Mult Scler. 2014;20(8):1042-1049. 11. Knippenberg S, Damoiseaux J, Bol Y, Hupperts R, Taylor BV, Ponsonby AL, Dwyer T, Simpson S, van der Mei IA. Higher levels of reported sun exposure, and not vitamin D status, are associated with less depressive symptoms and fatigue in multiple sclerosis. Acta Neurol Scand. 2014;129(2):123-31. 12. Liu D, Fernandez BO, Hamilton A, Lang NN, Gallagher JM, Newby DE, Feelisch M, Weller RB. UVA irradiation of human skin vasodilates arterial vasculature and lowers blood pressure independently of nitric oxide synthase. J Invest Dermatol. 2014;134(7):1839-46. 13. Engelsen O. The relationship between ultraviolet radiation exposure and vitamin D status. Nutrients. 2010;2(5):482-95.

On 2015 Jan 28, William Grant commented:

Vitamin D does reduce risk of upper respiratory infections!

The paper by Yawn and colleagues reported that the evidence for vitamin D for the treatment of respiratory diseases was weak since random controlled trials (RCTs) have not supported the observational studies finding inverse correlations between 25-hydroxyvitamin D [25(OH)D] concentrations and incidence of respiratory diseases [1]. However, the analysis of the RCTs and the literature search were not conducted well.

There have been two vitamin D RCTs that demonstrated a beneficial effect in reducing risk of influenza. The first was conducted on black post-menopausal women living on Long Island. Those taking 800 or 2000 IU/d vitamin D3 had significantly lower incidence of colds and influenza than those taking the placebo [2]. However, the same research group conducted a second vitamin D RCT on a similar group without finding a beneficial effect as noted in Ref. 1 [3]. The primary difference between the two trials was the baseline 25(OH)D concentration. As reported in Ref. 3, in the first one, it was 46.9±20.6 nmol/l (95% CI 43.9–50.39) while in the second one it was 64.3±25.4 nmol/L. The second was a vitamin D RCT conducted on school children in Japan. For those not taking vitamin D prior to entering the trial, there was a 64% reduction in incidence of type A influenza for those taking 1200 IU/d vitamin D3 compared to the placebo [4].

Another vitamin D RCT overlooked was conducted in Mongolia. In this study, school children with mean baseline 25(OH)D concentration of 7.0 (5.0–9.9) ng/mL (to convert ng/mL to nmol/L, divide by 2.5). were given 300 IU/d vitamin D3 which raised 25(OH)D concentration to 18.9 (15.5–22.9) ng/mL [5]. A significant reduction in acute respiratory infections was found.

Two negative studies were reviewed in Ref. 1. A study from New Zealand started with a baseline 25(OH)D concentration of 29 (SD, 9) ng/mL, achieved a 25(OH)D concentration of 48 ng/mL, and found no beneficial effect [6]. Another vitamin D RCT followed two groups, each with mean baseline 25(OH)D concentration near 25 ng/mL and achieved 25(OH)D concentration near 32 ng/mL [7]. Again, no significant effect of vitamin D supplementation was found.

Summarizing the findings from vitamin D trials, those trials that had baseline 25(OH)D concentrations below 50 nmol/L (20 ng/mL) found reduced risk of respiratory tract infections from taking vitamin D3 while those with higher baseline concentrations did not.

Robert Heaney recently outlined guidelines for optimizing designs of nutrients such as vitamin D [8]. The important points for vitamin D are to start with an understanding of the 25(OH)D concentration-health outcome relation, measure 25(OH)D concentrations, enroll only those with low 25(OH)D concentrations, supplement with enough vitamin D3 to raise concentrations high enough to see an effect, and remeasure 25(OH)D concentrations. Very few vitamin D trials satisfied these steps. Many 25(OH)D concentration-health outcome relations show rapid changes below 15 ng/mL with very little change after 30 ng/mL [9, 10]. Thus, vitamin D RCTs should seek to enroll people with baseline 25(OH)D concentrations below 20 ng/mL.

Additional evidence that vitamin D reduces risk of respiratory tract infections comes from an ecological study of influenza case-fatality rates in 12 communities in the United States from the 1918–1919 influenza pandemic. Case fatalities were generally due to pneumonia and occurred about 10 days after the influenza infection. In a comparison with solar UVB doses for summer or winter, rates were significantly lower in communities with higher solar UVB doses [11]. The mechanisms proposed were that vitamin D induces cathelicidin, which can kill bacteria, and reduction in the cytokine storm that often accompanies influenza. The cytokine storm can damage the lining of the lungs, thereby permitting the ever present bacteria to give rise to pneumonia. The influenza in that pandemic was type A H1N1.

Thus, there is plenty of evidence that vitamin D can prevent and treat upper respiratory tract infections.

References 1. Yawn J, Lawrence LA, Carroll WW, Mulligan JK. Vitamin D for the treatment of respiratory diseases: Is it the end or just the beginning? J Steroid Biochem Mol Biol. 2015 Jan 24. pii: S0960-0760(15)00029-1. doi: 10.1016/j.jsbmb.2015.01.017. [Epub ahead of print] 2. Aloia JF, Li-Ng M. Re: epidemic influenza and vitamin D. Epidemiol Infect. 2007;135(7):1095-6; author reply 1097-8. 3. Li-Ng M, Aloia JF, Pollack S, et al. A randomized controlled trial of vitamin D3 supplementation for the prevention of symptomatic upper respiratory tract infections. Epidemiol Infect. 2009;137(10):1396-404. 4. Urashima M, Segawa T, Okazaki M, Kurihara M, Wada Y, Ida H. Randomized trial of vitamin D supplementation to prevent seasonal influenza A in schoolchildren. Am J Clin Nutr. 2010;91(5):1255-60. 5. Camargo CA Jr, Ganmaa D, Frazier AL, et al. Randomized trial of vitamin D supplementation and risk of acute respiratory infection in Mongolia. Pediatrics. 2012;130(3):e561-7. 6. Murdoch DR, Slow S, Chambers ST, et al. Effect of vitamin D3 supplementation on upper respiratory tract infections in healthy adults: the VIDARIS randomized controlled trial. JAMA. 2012;308(13):1333-9. 7. Rees JR, Hendricks K, Barry EL, et al. Vitamin d3 supplementation and upper respiratory tract infections in a randomized, controlled trial. Clin Infect Dis. 2013;57(10):1384-92. 8. Heaney RP. Guidelines for optimizing design and analysis of clinical studies of nutrient effects. Nutr Rev. 2014;72(1):48-54. 9. Grant WB. Relation between prediagnostic serum 25-hydroxyvitamin D level and incidence of breast, colorectal, and other cancers. J Photochem Photobiol B, 2010;101(2):130–6. 10. Garland CF, Kim JJ, Mohr SB, et al. Meta-analysis of all-cause mortality according to serum 25-hydroxyvitamin D. Am J Pub Health. 2014;104(8):e43-50. 11. Grant WB, Giovannucci E. The possible roles of solar ultraviolet-B radiation and vitamin D in reducing case-fatality rates from the 1918–1919 influenza pandemic in the United States. Dermatoendocrinol. 2009;1(4): 215-9.

Disclosure I receive funding from Bio Tech Pharmacal (Fayetteville, AR), MediSun Technology (Highland Park, IL), and the Vitamin D Council (San Luis Obispo, CA).

On 2016 Feb 18, Abdulla A-B Badawy commented: