can't think of presentation as just an extension of the performer

the sphere allows space for performer to do what they need to do to maintain the performance, even if their concealed behavior would break down the performance altogether if witnessed by the audience

idea of "honor" and personal integrity protect the individual in their concealment wrong to break through this sphere

control over perceptions i control over contact made!!

because even the liars and honest want to be presumes as honest, looking at how the nonhonest perform and work to legitimate their authority shows us how the already legitimate do it?

one area of concealment can make performer nervous about unrelated thing and throw whole performance off

One can maintain a status quo without rigid adherence to all demands of performance in the relationship

larger number of matters and parts of relationship = more points of secrecy

almost always there are things someone cannot openly address within performance

what is a lie and what is legitimate all highly flexible, temporally and culturally informed

A lot of ways to lie without directly lying, yet, "bare-faced" lies are the ones with all the consequences

iffy definitions of what is a valid personification of something othered or something you're not. Not all of them considered bad but the categorization of what is okay and not okay is highly flexible

line is more blurred where claims to be something have less tangible means of legitimating

have empathy for those with one hidden thing vs those who conceal everything

not usually offended when someone is acting down- acting as a lower status than they actually are

we get offended by these performers who aren't who they say they are because they weaken our belief in a connection between authority to perform apart and the capability to play the part - one can act and not have the credentials, what does that mean for everyone else?

When we find out someone is a fraud, what we discover is that they did not have authority to play the part they played- they don't have the means of status by the right materials or legitimating factors

in our heads- a true or honest performer- would not mess up. Assumption that there IS an honest performer or someone who embodies something without a need for front. This view isn't analytically very applicable

as members, we assess validity or phoniness of performance

identification with a figure- not with oneself

we do not let or are expected not to allow momentary emotions to impact performance- should be homogenous

our presentations are fragile and easily destroyed by minor mishaps

we usually designate strict social expectations of presentation to other cultures but exist well within our own.

1. actor can make it seem like they care too much or care too little

three examples in slip ups of the facade

takes very little to signal this meaning- susceptible to misinterpretation

often beneficial for actor to downplay routinization of actions- act like that interact is special, personal, meaningful, etc.

Not always a demand for "authenticity" - audience benefits from seeing front appropriate for the context of THEIR relation to the individual

both med schools and patients act like they choose doctors on aptitude and training as opposed to ethnic origins

conceal motives that break idea of what a "good" clergymen should do

Can you be a good clergymen and still wish to progress socailly?

training extensive to five image that they are set apart

fulfill idealized ideas of routines and roled

many people consume things in secret to keep up an image

never a performance about bettering or reflecting what is really there, people embody identities for a certain result

while reasons for hiding lack of material wealth and making it seem like one has more, other instances requires or rewards the downplaying of material wealth. A. Serving a lot of food when guests are over even if you live off very little. B. Letting your house look bad so landlords don't think they can charge extra

even in America is material wealth is downplayed as if other moral standards are stronger forces for prosperity

Indian caste system allows for social movement in non-material gains (by adopting certain belief systems and practices) which re-centers the lower classes around the practices that allow for them to be upwardly mobile

minimal separation between the status and the front- Here, Goffman doesn't distinguish something like yearly income attaining status than the means to attain the front of a certain status

upward mobility frequently requires presenting a proper performance and the efforts to move up are expressed in maintaining the front that someone is of a higher class

performance is more focused on living up to standard societal expectations than an individuals actual behabior

some say aristocracy is injecting expressions of character into minor activities

not about doing his best in ALL routines- focus on the one his identity most invested in?

people often choose a certain routine to invest own ego into

Performing a task well takes time away from the effort required to LOOK like you're performing a task well

Creates an irony "It takes a lot of work to make this look easy" One embodies the characteristics not present in the role itself for the appearance of the role. Sometimes this characteristics contradict the role.

merchants overcharge for things that look expensive (are in which to role of money is dramatically fulfilled) so that they can pay for what costs but is not dramatic

issue- exists a nonbelief in other people that are doing fulfilling their role unless they see a dramatized representation of the task

sometimes this dramatization becomes an issue

example- umpire must act sure of call for the sake of their own authority

actors constantly confirming own role

often the front or selection of t- or title- isn't suitable for all the tasks at hand

diversification of society too vast- break up into distinct categories and provide based on those- allowing or forcing people to maintain social front

appearances provide a expectations for manner and behavior someone should expect- varying thing such as actions become standardized, ease the stress of someone like a client.

our ideal type is when the manner and appearance of presentation match

elevator operator in a tux??

appearance are status and other identifications (can show things like what you do for work) Manner tells us of oncoming action within a persons role (what the person expects to happen or how others are to respond)

apart of the "front" is expressive equipment, what is used to express oneself

Labeling- The "front" of the stage

so little distinction between performing and becoming

there is often a demand to be insincere that the cynical performers, in their hyperawareness of the gap between reality and the performers, more willingly offer up this insincerity.

not every cynical performer evil- some work to do good with such performance

MMEEEE

other extreme- individual isn't at all convinced of own performance, may not actually care if its real me asf

ethnography vibes- iffy methodology but good theories?

except this time, the audience is the performers

use the metaphor of the theater- presentation of oneself in response to presentation of others for an audience- world of make-believe

Yani, bir dişin antagonisti, o dişin karşısında yer alan, çiğneme sırasında onunla temas eden diştir.

Daniela Pajtašová z Ústavu hematologie a krevní transfuze:

[P]o užití marihuany je třeba dodržet minimálně týden odstup, než půjdete darovat krev. Užití musí být výjimečné, pravidelné užívání se s dárcovstvím neslučuje.

[Užívání marihuany] více jak 1x za půl roku [je považováno za pravidelné].

[Krev od lidí, kteří užívají marihuanu pravidelně, není žádoucí] především [proto, že] je to známka dalšího možného rizikového chování.

Darovat krev tedy mohu, pokud jsem v posledním půlroce užil marihuanu maximálně jednou a od tohoto užití uplynul nejméně týden. Častější užívání marihuany je považováno za rizikové, protože značí další možné rizikové chování.

Daniela Pajtašová z Ústavu hematologie a krevní transfuze:

[P]o užití marihuany je třeba dodržet minimálně týden odstup, než půjdete darovat krev. Užití musí být výjimečné, pravidelné užívání se s dárcovstvím neslučuje.

[Užívání marihuany] více jak 1x za půl roku [je považováno za pravidelné].

[Krev od lidí, kteří užívají marihuanu pravidelně, není žádoucí] především [proto, že] je to známka dalšího možného rizikového chování.

Darovat krev tedy mohu, pokud jsem v posledním půlroce užil marihuanu maximálně jednou a od tohoto užití uplynul nejméně týden. Častější užívání marihuany je považováno za rizikové, protože značí další možné rizikové chování.

Reviewer #3 (Public review):

The aim of the study was to map, a) whether different tissues exhibit different metabolic profiles (this is known already), what differences are found between female and male mice and how the profiles changes with age. In particular, the study recorded the activity of respirasomes, i.e. the concerted activity of mitochondrial respiratory complex chains consisting of CI+CIII2+CIV, CII+CIII2+CIV or CIV alone.

The strength is certainly the atlas of oxidative metabolism in the whole mouse body, the inclusion of the two different sexes and the comparison between young and old mice. The measurement was performed on frozen tissue, which is possible as already shown (Acin-Perez et al, EMBO J, 2020).

Weakness: The assay reveals the maximum capacity of enzyme activity, which is an artificial situation and may differ from in vivo respiration, as the authors themselves discuss. The material used was a very crude preparation of cells containing mitochondria and other cytosolic compounds and organelles. Thus, the conditions are not well defined and the respiratory chain activity was certainly uncoupled from ATP synthesis. Preparation of more pure mitochondria and testing for coupling would allow evaluation of additional parameters: P/O ratios, feedback mechanism, basal respiration, and ATP-coupled respiration, which reflect in vivo conditions much better. The discussion is rather descriptive and cautious and could lead to some speculations about what could cause the differences in respiration and also what consequences these could have, or what certain changes imply.<br /> Nevertheless, this study is an important step towards this kind of analysis.

Comments on the second revision:

I believe this is an important and interesting area of study, although I recognise that the assay which measures maximal enzyme activity under unphysiological conditions has its limitations. Nevertheless, it does seem possible to get a first glance of the respiratory situation in the respective tissue. There is a typo in the source data (Fig. xC) for skeletal muscle.

I think how you incorporate small details that preview the topics you address in this essay are really creative. This paragraph really sets the scene of your listening guide which I think mine would benefit from.

Charakterystyka procesu przełączanie się między różnymi rodzajami uwagi/trybami przetwarzanie.

Makroskopowe działanie LC NE

Związek LC z autonomicznym układem nerwowym

Wyjaśnienie jednokierunkowych projekcji z LC do nbM - mała aktywność w LC a duża w nbM (LC, wpływa na nbM i go aktywnuje)

LC reguluje również układ autonomiczny (jądroprzedmotoryczne)

poprzez projekcje do jądra pośrednio-bocznego w rdzeniu przedłużonym, między innymi [14]. To poprzez te projekcje LC ma wpływ na rozszerzenie źrenicy [27–31]

Jurisprudência em teses nº 234 (26/04/2024)

• A necessidade da demonstração de urgência para o deferimento da medida cautelar de indisponibilidade de bens em ação de improbidade administrativa reveste-se de caráter processual, de modo que a alteração legislativa do art. 16 da Lei n. 8.429/1992, dada pela Lei n.14.230/2021, tem aplicação imediata ao processo em curso.

• A partir da vigência da Lei n. 14.230/2021, exige-se a demonstração do requisito da urgência, além da plausibilidade do direito invocado, para o deferimento da indisponibilidade de bens em ação de improbidade administrativa.

Jurisprudência em teses nº 234 (26/04/2024)

• A contratação de servidores públicos temporários sem concurso público, mas baseada em legislação local, por si só, não configura ato de improbidade administrativa previsto no art. 11 da Lei n. 8.429/1992, por estar ausente o elemento subjetivo (dolo) necessário para a configuração do ato de improbidade violador dos princípios da administração pública.

• A nova redação do art. 11 da LIA, dada pela Lei n. 14.230/2021, que tipificou de forma taxativa os atos de improbidade administrativa por ofensa aos princípios da administração pública, obsta a condenação genérica com base nos revogados incisos I e II do mesmo artigo para atos praticados na vigência do texto anterior da lei e sem condenação transitada em julgado.

• Não se extingue a ação de improbidade administrativa se a exclusão da conduta anteriormente disposta no art. 11 da LIA - violação genérica aos princípios administrativos - aboliu a tipicidade, mas a nova previsão legal especifica em seus incisos a conduta descrita, em razão do princípio da continuidade típico-normativa.

• Não caracteriza ato de improbidade administrativa praticado por prefeito a ausência de prestação ou de repasse de informações solicitadas pelo Poder Legislativo ou por municípes, quando inexistente o intuito malicioso, desonesto ou corrupto.

REsp 1.929.685-TO , Rel. Ministro Gurgel de Faria, Primeira Turma, por unanimidade, julgado em 27/8/2024. (Informativo 823)

Destaque

A exigência do efetivo prejuízo, em relação ao ato de improbidade administrativa que causa lesão ao erário, prevista no art. 10, caput , da Lei n. 8.429/92 (com redação dada pela Lei 14.320/2021) se aplica aos processos ainda em curso.

Informações do Inteiro Teor

Após alterações operadas pela Lei n. 14.230/21, a norma do art. 10, caput , da Lei de Improbidade passou a prever expressamente que "constitui ato de improbidade administrativa que causa lesão ao erário qualquer ação ou omissão dolosa, que enseje, efetiva e comprovadamente, perda patrimonial, desvio, apropriação, malbaratamento ou dilapidação dos bens ou haveres das entidades referidas no art. 1º desta Lei, e notadamente (...)".

Em razão disso, o dano presumido, para qualquer figura típica do art. 10 da LIA (inclusive os incisos VIII e XI do caso) não pode mais dar suporte à condenação pela prática de ato ímprobo.

Diante desse novo cenário, os casos anteriores à alteração legal, ainda em trâmite, e que apresentem a supracitada controvérsia devem ser solucionados com a posição externada na nova lei, que reclama dano efetivo. Sem este (o dano efetivo), não há como reconhecer o ato ímprobo.

Não se trata exatamente de discussão sobre a aplicação retroativa de alteração normativa benéfica, já que, anteriormente, não havia norma expressa prevendo a possibilidade do dano presumido. Na realidade, o dano presumido só foi admitido após construção pretoriana, a partir da jurisprudência que se consolidara no Superior Tribunal de Justiça até então, e que vinha sendo prolongadamente aplicada.

Ocorre que esse entendimento (fruto de construção jurisprudencial, e não decorrente de texto legal) não pode continuar balizando as decisões do STJ, se o próprio legislador deixou expresso não ser cabível a condenação por ato ímprobo mediante a presunção da ocorrência de um dano. Assim, cabe ao Judiciário prestar a devida deferência à opção que foi a escolhida pelo legislador ordinário para dirimir a questão.

Agint no REsp 1.991.470-MG , Rel. Ministro Herman Benjamin, Segunda Turma, por unanimidade, julgado em 11/6/2024. (Informativo 816)

Destaque

• A absolvição criminal com fundamento na atipicidade da conduta não faz coisa julgada no juízo cível, considerando a independência das instâncias.

Informações do Inteiro Teor

Conforme entendimento sufragado no Superior Tribunal de Justiça, a absolvição operada no Juízo criminal somente se comunica com a esfera administrativa quando negada a existência do fato ou da autoria (AREsp n. 1.358.883/RS, relator Ministro Francisco Falcão, Segunda Turma, julgado em 3/9/2019).

A absolvição criminal com fundamento na atipicidade da conduta, não faz coisa julgada no cível, considerando a independência das instâncias que, ademais, consta do próprio art. 37, § 4º, da CF: "Os atos de improbidade administrativa importarão a suspensão dos direitos políticos, a perda da função pública, a indisponibilidade dos bens e o ressarcimento ao erário, na forma e gradação previstas em lei, sem prejuízo da ação penal cabível".

Nesse mesmo sentido da independência das instâncias, diversos são os precedentes do STJ: RMS n. 32.319/GO, Rel. Min. Napoleão Nunes Maia Filho, Rel. para acórdão Min. Sérgio Kukina, Primeira Turma, DJe de 22/9/2016 e REsp n. 1.364.075/DF, Rel. Min. Mauro Campbell Marques, Segunda Turma, DJe de 2/12/2015.

O referido entendimento jurisprudencial encontra-se em consonância com o disposto no art. 21, § 3º, da Lei n. 8.249/1992 (na redação da Lei n. 14.230/2021), no sentido de que as "sentenças civis e penais produzirão efeitos em relação à ação de improbidade quando concluírem pela inexistência da conduta ou pela negativa da autoria".

Apesar de o disposto no art. 21, § 4º, da Lei n. 8.429/1992, na redação da Lei n. 14.230/2021, apontar que a "absolvição criminal em ação que discuta os mesmos fatos, confirmada por decisão colegiada, impede o trâmite da ação da qual trata esta Lei, havendo comunicação com todos os fundamentos de absolvição previstos no art. 386 do Decreto-Lei 3.689/1941 (Código de Processo Penal)", tal disposição está suspensa por liminar deferida na ADI/STF 7.236.

Ademais, nem sempre há correspondência exata entre o dolo que autoriza a improcedência da ação penal por atipicidade da conduta com o dolo exigido no crime de apropriação.

Súmula Vinculante 13 do STF: A nomeação de cônjuge, companheiro ou parente em linha reta, colateral ou por afinidade, até o terceiro grau, inclusive, da autoridade nomeante ou de servidor da mesma pessoa jurídica investido em cargo de direção, chefia ou assessoramento, para o exercício de cargo em comissão ou de confiança ou, ainda, de função gratificada na administração pública direta e indireta em qualquer dos Poderes da União, dos Estados, do Distrito Federal e dos Municípios, compreendido o ajuste mediante designações recíprocas, viola a Constituição Federal.

O §21 do mesmo artigo fala a mesma coisa:

Art. 17 § 21. Das decisões interlocutórias caberá agravo de instrumento, inclusive da decisão que rejeitar questões preliminares suscitadas pelo réu em sua contestação.

O art. 17-C também fala que não haverá remessa necessária:

Art. 17-C. § 3º Não haverá remessa necessária nas sentenças de que trata esta Lei.

O MP não tem legitimidade exclusiva!

Nas ADIs 7042 e 7043, o STF decidiu que os entes públicos lesados também tem competência para propor a ação de improbidade e para a celebração de acordos de não persecução civil.

STF decide que entes públicos interessados podem propor ação de improbidade administrativa

lizeth Duarte ID 684503

Según el análisis e identificación de la autora ella no considera suficiente mente completa la explicación de los fenómenos sociales que no tienen en cuenta la perspectiva subjetiva de los actores. Esta Hipótesis se enmarca en la tradición fenomenológica y sociológica que plantea que las ciencias sociales deben diferenciarse de las ciencias naturales al estudiar y analizar al ser humano, que no solo actúa si o que también interpreta y da sentido a sus acciones. La autora presenta y resalta diferentes argumentos como la experiencia subjetiva en el que el punto de partida de acción social , ya que los individuos actúan basándose en su percepción y significados de la realidad el análisis sociológico debe incorporar el contexto de interpretación individual para captar las motivaciones detrás de las acciones. La suposición planteada parece adecuada en la medida en que reconoce la diferencia entre ciencias sociales y ciencias naturales. La subjetividad es en efecto un componente esencial en el análisis de los fenómenos sociales. No obstante, se debe reflexionar sobre la necesidad de un equilibrio: mientras que la inclusión de la subjetividad es valiosa, la investigación también debe buscar cierta objetividad y consistencia para generar teorías aplicables en diferentes contextos.

Bryan Segura - ID 977365

El sentido de las experiencias cotidianas son mucho mejor comprendidas en este fragmento del texto.

La autora puntualiza que las experiencias cotidianas son las que construyen la realidad social de los individuos. Su actitud se adapta al entorno social y es el factor fundamental para entender su actuar en el mundo. La subjetividad no sólo es relevante, sino que es base importante en la construcción social.

Según Schutz, la crítica que hace a Parsons es que al ver los valores morales solo desde un punto de vista subjetivo, se está perdiendo el actor que siempre enfrenta un dilema cuando toma decisiones. Por un lado, tiene una meta o fin que quiere lograr, pero, por otro lado, tiene que elegir los medios o formas de llegar a esa meta, y cada elección viene con consecuencias que pueden ser buenas o malas.

Argumenterar han för någon slags subjektiv rättviseteori? Har för mig att han sa att den inte var subjektiv - men på vilket sätt är den objektiv förrutom att det är viktigt att allt sker offentligt?

Welcome back, this is part two of this lesson.

We're going to continue immediately from the end of part one, so let's get started.

Okay, so all three of these mount targets are now in an available state and that means we can connect into this EFS file system from any of the availability zones within the Animals for Life VPC.

So what we need to do is test out this process and we're going to interact with this file system from our EC2 instances.

So move back to the tab where we have the EC2 console open.

And at this point I want you to either, and this depends on your browser, I'll either want you to right click and duplicate this tab to open another identical copy.

If you can't do this in your browser then just open a new tab and copy and paste this URL into that tab.

You'll end up with two separate tabs open to the same EC2 screen.

So on the first tab we're going to connect to A4L-EFS instance A.

So right click and then select connect.

We're going to use instance connect.

So make sure the username is EC2-user and then click on connect.

Now right now this instance is not connected to this EFS file system and we can verify that by running a DF space-k and press enter.

You'll see that nowhere here is listed this EFS file system.

These are all volumes directly attached to the EC2 instance and of course the boot volume is provided by EBS.

Now within Linux all devices or all file systems are mounted into a folder.

So the first thing that we need to do to interact with EFS is to create a folder for the EFS file system to be mounted into.

And we can do that using this command so shudu space mkdir space-p space/efs/wp-content.

Now the hyphen p option just means that everything in this path will be created if it's not already.

So this will create forward/EFS if it doesn't already exist.

So press enter to create that folder.

So I'm going to clear the screen to keep this easy to see.

And the next thing I need to do is to install a package of tools which allows this instance or specifically the operating system to interact with the EFS product.

Now the command I'm going to use to install these tools is shudu to give us admin permissions and then DNF which is the package manager for this operating system.

And then a space hyphen y to automatically acknowledge any prompts and then a space and then install because I want to install a package and then a space.

And then the name of the tools that I want to install is amazon hyphen EFS hyphen utils.

So this is a set of tools which allows this operating system to interact with EFS.

So go ahead and press enter and that will install these tools and then we can configure the interaction between this operating system and EFS.

Again I'm going to clear the screen to keep this easy to see and I want to mount this EFS file system in that folder that we've just created.

But specifically I want it to mount every time the instance is restarted.

So of course that means we need to add it to the FSTAB file.

Now if you remember this file from elsewhere in the course it's contained within the forward/ETC folder.

So we need to move into that folder cd///ETC and then the file is called FSTAB.

So we need to run shudu to give us admin permissions and then nano which is a text editor and then the name of the file which is FSTAB.

So press enter and the file will likely have only one or two lines which is the root and/or boot volume of this instance.

So let's just move to the end because we're going to add a new line and this is contained within the lesson commands document but we're going to paste in this line.

So this line tells us that we want to mount this file system ID so file system ID colon forward/.

We want to mount that into this folder so forward/efs forward/wp-content.

We tell it that the file system type is EFS.

Remember EFS is actually based on NFS which is the network file system but this is one provided by AWS as a service and so we use a specific AWS file system which is EFS.

And the support for this has been installed by that tools package which we just installed.

Now the exact functionality of this is beyond the scope of this course but if you do want to research further then go ahead and investigate exactly what these options do.

What we need to do though is to point it at our specific EFS file system.

So this is this component of the line all the way from the start here to this forward/.

So to get the file system ID we need to go back to the EFS console and we need to copy down this full file system ID and yours will be different so make sure you copy your own file system ID into the clipboard.

Then go back here and select the colon and then delete all the way through to the start of this line.

And once you've done that paste in your file system ID what it should look like is the file system ID then a colon and then a forward/.

So at this point we need to save this file so control O to save and then enter and control X to exit.

Again I'm going to clear the screen to make it easier to see.

Then I'll run a DF space -K and this is what the file systems currently attached to this instance look like.

Then we're going to mount the EFS file system into the folder that we've created and the way that we do this is with this command.

So shudu mount and then we specify the name of the folder that we want to mount.

Now the way that this works is that this uses what we've just defined in the FSTAB file.

So we're going to mount into this folder whatever file system is defined in that file.

So that's the EFS file system and if we press enter after a few moments it should return back to the prompt and that's mounted that file system.

There we go we back at the prompt and if we do a DF space -K again we'll see that now we've got this extra line at the bottom.

So this is the EFS file system mounted into this folder.

Now to show you that this is in fact a network file system let's go ahead and move into that folder using this command.

And now that we're in that folder we're going to create a file.

So we're going to use shudu so that you have admin privileges and then we're going to use the command touch which if you remember from earlier in the course just creates an empty file.

And we're going to call this file amazing test file dot txt.

Go ahead and press enter and then do an LS space -LA and you'll see that we now have this file created within this folder.

And while we're creating it on this EC2 instance it's actually put this file on a network file system.

Now to verify that let's move back to the other tab that we have open to the EC2 console the one that's still on this running instances screen.

And now let's go ahead and connect to instance B.

So right click on instance B select connect again instance connect verify the username is as it should be and click on connect.

So now we're on instance B.

Let's do a DF space -K to verify that we don't currently have any EFS file system mounted.

Next we need to install the EFS tools package so that we can mount this file system.

So let's go ahead and install that package clear the screen to make it easier to see then we need to create the folder that we're going to be mounting this file system into.

We'll use the same command as on instance A.

Then we need to edit the FSTAB file to add this file system configuration.

So we'll do that using this command so shudu space nano space forward slash ETC forward slash FSTAB press enter.

Remember this is instance B so it won't have the line that we added on instance A.

So we need to go down to the bottom paste in this placeholder and then we need to replace the file system ID at the start with the actual file system ID.

So delete this leaving the colon and forward slash go back to the EFS console copy the file system ID into your clipboard.

Move back to this instance paste that in everything looks good.

Save that file with control O press enter exit with control X then we back at the prompt clear the screen.

We'll use the shudu mount forward slash EFS forward slash WP hyphen content command again to mount the EFS file system onto this instance and again it's using the configuration that we've just defined in the FSTAB file press enter.

After a few moments you'll be placed back at the prompt we can verify whether this is mounted with DF space hyphen K.

It has mounted by the looks of things it's at the bottom.

So now if we move into that folder so CD forward slash EFS forward slash WP hyphen content forward slash and press enter.

We now in that folder and if we do a listing so LS space hyphen LA what we'll see is the amazing test file dot txt which was created on instance A.

So this proves that this is a shared network file system where any files added on one instance are visible to all other instances.

So EFS is a multi user network based file system that can be mounted on both EC2 Linux instances as well as on premises physical or virtual servers running Linux.

Now this is a simple example of how to use EFS for now we've done everything that we need to do in this demo lesson so we just need to clean up all of the infrastructure that we've used to do that.

Go back to the EFS console we're going to go ahead and delete this file system so we should already have it selected just select delete you'll need to confirm that process by pasting in the file system ID.

So go ahead and put your file system ID and then select confirm.

Now that can take some time to delete and you'll need to wait for this process to complete.

Once it has completed we're going to go ahead and move across to the cloud formation console.

You should still have this open in a tab if you don't just type cloud formation in the search box at the top and then move to the cloud formation console.

You should still have the stack name of implementing EFS which is the stack you created at the start with the one click deployment.

Go ahead and select this stack then click on delete and confirm that deletion and once that finishes deleting that's all of the infrastructure gone that we've created in this demo lesson.

So I hope this has been a fun and enjoyable demo lesson where you've gained some practical experience of working with EFS at this point though that is everything that you need to do in this demo lesson.

So go ahead and complete the video and when you're ready I'll look forward to you joining me in the next.

Welcome back and in this demo lesson I want to give you some abstract practical experience of using the Elastic File System or EFS.

Now we're going to need some infrastructure.

Before we apply that as always make sure that you're logged into the general AWS account, so the management account of the organization and you'll need the Northern Virginia region selected.

Now attached to this lesson is a one-click deployment link so go ahead and click that.

This is going to provision some infrastructure.

It's going to take you to the quick create stack screen and everything should be pre-populated.

You'll just need to scroll to the bottom, check the box beneath capabilities and then click on create stack.

You're also going to be typing some commands within this demo lesson so also attached to this lesson is a lesson commands document.

Go ahead and open that in a new tab.

So this is just a list of the commands that we're going to be using during the demo lesson and there are some placeholders such as file system ID that you'll need to replace as we go but make sure you've got this open for reference.

Now we're going to need this stack to be in a create complete state before we continue with the demo lesson so go ahead pause the video and resume it once your stack moves into a create complete state.

Okay so the stacks now moved into a create complete status and what this has actually done is create the animals for life base VPC as well as a number of EC2 instances.

So if we go to the EC2 console and click on instances running you'll note that we've created a for L - EFS instance A and a for L - EFS instance B and we're going to be creating an EFS file system and mount points and then mounting that on both of these instances and interacting with the data stored on that file system.

We're going to get you the experience of working with a network shared file system so let's go ahead and do that.

So to get started we need to move to the EFS console so in the search box at the top just type EFS and then open that in a brand new tab.

We're going to leave this tab open to the instances part of the EC2 console because we're going to come back to this very shortly.

So let's move across to the EFS console that we have open in a separate tab and the first step is to create a file system so a file system is the base entity of the elastic file system product and that's what we're going to create.

Now you've got two options for setting up an EFS file system you can use this simple dialogue or you can click on customize to customize it further.

So if we're using the simple dialogue we'd start by naming the file system so let's say we use A4L - EFS and then you'd need to pick a VPC for this file system to be provisioned into and of course we'd want to select the animals for life VPC.

Now we want to customize this further we don't want to just accept these high-level defaults so we need to click on customize.

This is going to move us to this user interface which has many more options so we've still got the A4L - EFS name for this file system.

Now for the storage class we're going to pick standard which means the data is replicated across multiple availability zones.

If you're doing this in a test or development environment or you're storing data which is not important then you can choose to use one zone which stores data redundantly but only within a single AZ.

Now again in this demonstration we are going to be using multiple availability zones so make sure that you pick standard for storage class.

You're able to configure automatic backups of this file system using AWS backup and if you're taking an appropriate certification course this is something which I'll be covering in much more detail.

You can either enable this or disable it obviously for a production usage you'd want to enable it but for this demonstration we're going to disable it.

Now EFS as I mentioned in the theory lesson comes with different classes of storage and you can configure lifecycle management to move files between those different storage classes so if you want to configure lifecycle management to move any files not accessed for 30 days you can move those into the infrequent access storage class and you can also transition out of infrequent access when anything is accessed so go ahead and select on first access for transition out of IA.

So in many ways this is like S3 with the different classes of storage for different use cases.

When you're creating a file system you're able to set different performance and throughput modes.

For throughput mode you can choose between bursting and enhanced.

If you pick enhanced you're able to select between elastic and provisioned.

I've talked more about these in the theory lesson.

We're going to pick bursting.

Now for performance you can choose between general purpose and max I/O.

General purpose is the default and rightfully so and you should use this for almost all situations.

Only use max I/O if you want to scale to really high levels of aggregate throughput and input output operations per second so only select it if you absolutely know that you need this option.

You've also got the ability to encrypt the data on the file system and if you do encrypt it it uses KMS and you need to pick a KMS key to use.

Of course this means that in order to interact with objects on this file system permissions are needed both on the EFS service itself as well as the KMS key that's used for the encryption operation.

Now this is something that you will absolutely need to use for production usage but for this demonstration we're going to switch it off.

We won't be setting any tags for this file system so let's go ahead and click on next.

You need to configure the network settings for this file system so specifically the mount targets that will be created to access this file system.

Now best practice is that any availability zones within a VPC where you're consuming the services provided by EFS you should be creating a mount target so in our case that's US - East - 1A, 1B and 1C.

So we're going to go through and configure this so first let's delete all of these default security group assignments.

Every mount target that you create will have an associated security group so we'll be setting these specifically.

For now though we need to choose the application subnet in each of these availability zones so in the top drop-down which is US - East - 1A I'm looking for app A so go ahead and do the same.

In US - East - 1B I want to select the app B subnet and then in US - East - 1C logically I'll be selecting the app C subnet so that's app A, app B and app C.

Now for security groups the CloudFormation 1 click deployment has provisioned this instance security group and by default this security group allows all connections from any entities which have this attached so this is a really easy way that we can allow our instances to connect to these mount targets so for each of these lines go ahead and select the instance security group you'll need to do that for each of the mount targets so we'll do the second one and then we'll do the third one and that's all of the network configuration options that we need to worry about so click on next it's here where you can define any policies on the file system so you can prevent root access by default you can enforce read only access by default you can prevent anonymous access or you can enforce encryption in transit for all clients connected to this EFS file system so any clients that connect to the mount targets to access the file system you can ensure that that uses encryption in transit and if you're using this in production you might want to select at least this last option to improve security for this demo lesson we're not going to use any of these policy options nor are we going to define a custom policy in the policy editor instead we'll just click on next at this point we just need to review everything's to our satisfaction everything looks good so we're going to scroll down to the bottom and just click on create now in order to continue with this demo lesson we're going to need both the file system and all of its mount targets so go into the file system click on network and you'll see three mount targets being created all three of these need to be ready before we can continue the demo lesson so this seems like a great time to end part one of this demo lesson go ahead and finish this video and then when all of these mount targets are ready to go you can start part two.

A arbitragem pode ser de direito ou de equidade.

Arbitragem de direito

• Os árbitro seguem as regras do ordenamento jurídico para solucionar os litígios.

Arbitragem por equidade

• Os árbitros se afastam das regras de direito para buscar uma solução que considerem mais justa.

Fonte: Entenda o conceito de arbitragem

• 1. O edital da licitação deve deixar explícito se o critério de aceitabilidade previsto no art. 59, inciso III, da Lei 14.133/2021 aplica-se somente ao preço global da proposta ou se, também, ao preço unitário dos itens. (Acórdão 2190/2024 Plenário, Representação, Relator Ministro Augusto Nardes.)

I think this is the problem of believing that in schools they should not be learning certain things. It is a 50/50 situations where certain parents don't want them to learn things because of how they're taught to believe theres only two genders. I feel like now it is something that should be more open and taught because the new generation of school kids will encounter and is a norm for parents to be part of the LGBTQ.

Rewrite for clarity

Keep an eye out for places in your writing where you can consider cutting redundant words to improve concision and clarity - here, for example, "may" and "possible" are serving more or less the same purpose

No need for this kind of equivocal language - just state your claim outright :-)

the "from" here doesn't have a clear referent

Les bonnes pratiques individuelles Recherche des faits sur HabiloMédias, o Vérification des faits AFP.

Les bonnes pratiques individuelles Recherche des faits sur HabiloMédias, o Vérification des faits AFP.

for - BEing journey - The Buzz - sensory substitution - for detecting somatic pressure and angle from artificial leg - Neosensory - David Eagleman

Não confundir com o relatório sintético, que é feito após a conclusão dos trabalhos de revisão.

Art. 122. Concluídos os trabalhos de revisão, o juiz ou a juíza juntará aos autos relatório sintético das operações de RAE realizadas...

Português equiparado

CF Art. 12 § 1º Aos portugueses com residência permanente no País, se houver reciprocidade em favor de brasileiros, serão atribuídos os direitos inerentes ao brasileiro, salvo os casos previstos nesta Constituição.

Divided loyalties between grandma and the rest of family

Author response:

eLife Assessment

This valuable study uses consensus-independent component analysis to highlight transcriptional components (TC) in high-grade serous ovarian cancers (HGSOC). The study presents a convincing preliminary finding by identifying a TC linked to synaptic signaling that is associated with shorter overall survival in HGSOC patients, highlighting the potential role of neuronal interactions in the tumor microenvironment. This finding is corroborated by comparing spatially resolved transcriptomics in a small-scale study; a weakness is in being descriptive, non-mechanistic, and requiring experimental validation.

We sincerely thank the editors for the valuable and constructive feedback. We appreciate the recognition of our findings and the significance of identifying transcriptional components in high-grade serous ovarian cancers. We acknowledge the insightful point on our study's descriptive nature and limited mechanistic depth. While further experimental validation would indeed enhance our conclusions, such work extends beyond the current scope of this manuscript. However, we would like to highlight that mechanistic studies demonstrating the impact of tumor-infiltrating nerves on disease progression are emerging (Zahalka et al., 2017; Allen et al., 2018; Balood et al., 2022; Jin et al., 2022; Globig et al., 2023; Restaino et al., 2023; Darragh et al., 2024). Importantly, members of our group have contributed to these findings. These studies, including in vitro and in vivo work in head and neck squamous cell carcinoma as well as high-grade serous ovarian carcinoma, demonstrate that substance P released from tumor-infiltrating nociceptors potentiates MAP kinase signaling in cancer cells, thereby influencing disease progression. This effect can be mitigated in vivo by blocking the substance P receptor (Restaino et al., 2023). Our present work identifies a transcriptional component that aligns with the presence of functional nerves within malignancies. These published mechanistic studies support our findings and suggest that this transcriptional component could serve as a potential screening tool to identify innervated tumors. Such information is clinically relevant, as patients with innervated tumors may benefit from more aggressive therapy.

Reviewer #1 (Public review):

This manuscript explores the transcriptional landscape of high-grade serous ovarian cancer (HGSOC) using consensus-independent component analysis (c-ICA) to identify transcriptional components (TCs) associated with patient outcomes. The study analyzes 678 HGSOC transcriptomes, supplemented with 447 transcriptomes from other ovarian cancer types and noncancerous tissues. By identifying 374 TCs, the authors aim to uncover subtle transcriptional patterns that could serve as novel drug targets. Notably, a transcriptional component linked to synaptic signaling was associated with shorter overall survival (OS) in patients, suggesting a potential role for neuronal interactions in the tumor microenvironment. Given notable weaknesses like lack of validation cohort or validation using another platform (other than the 11 samples with ST), the data is considered highly descriptive and preliminary.

Strengths:

(1) Innovative Methodology:

The use of c-ICA to dissect bulk transcriptomes into independent components is a novel approach that allows for the identification of subtle transcriptional patterns that may be overshadowed in traditional analyses.

We sincerely thank the reviewer for recognizing the strengths and novelty of our study. We appreciate the positive feedback on our use of consensus-independent component analysis (c-ICA) to decompose bulk transcriptomes, which we believe allowed us to detect subtle transcriptional signals often overlooked in traditional analyses.

(2) Comprehensive Data Integration:

The study integrates a large dataset from multiple public repositories, enhancing the robustness of the findings. The inclusion of spatially resolved transcriptomes adds a valuable dimension to the analysis.

Thank you for recognizing the robustness of our study through comprehensive data integration. We appreciate the acknowledgment of our efforts to leverage a large, multi-source dataset, as well as the additional insights gained from spatially resolved transcriptomes. We believe this integrative approach enhances the depth of our analysis and contributes to a more nuanced understanding of the tumor microenvironment.

(3) Clinical Relevance:

The identification of a synaptic signaling-related TC associated with poor prognosis highlights a potential new avenue for therapeutic intervention, emphasizing the role of the tumor microenvironment in cancer progression.

We appreciate the reviewer’s recognition of the clinical implications of our findings. The identification of a synaptic signaling-related transcriptional component associated with poor prognosis underscores the potential for novel therapeutic targets within the tumor microenvironment. We agree that this insight could open new avenues for intervention and further highlights the role of neuronal interactions in cancer progression.

Weaknesses:

(1) Mechanistic Insights:

While the study identifies TCs associated with survival, it provides limited mechanistic insights into how these components influence cancer progression. Further experimental validation is necessary to elucidate the underlying biological processes.

We appreciate the reviewer’s point regarding the limited mechanistic insights provided in our study. We agree that further experimental validation would enhance our understanding of how the biology captured by these transcriptional components influence cancer progression. However, we respectfully note that such validation is beyond the current scope of this article.   Our current analyses are done on publicly available expression array and spatial transcriptomic array datasets. For future studies, we therefore intend to combine spatial transcriptomic data with immunohistochemical analysis of the same tumors for validation purposes. We have started with setting up in vitro cocultures of neurons and ovarian cancer cells to obtain mechanistic insight in how genes with a large weight in TC121 regulate synaptic signaling and how that affects ovarian cancer cells.

(2) Generalizability:

The findings are primarily based on transcriptomic data from HGSOC. It remains unclear how these results apply to other subtypes of ovarian cancer or different cancer types.

In Figure 5, we present the activity of TC121 across various cancer types, demonstrating broader applicability. However, due to limited treatment response data, we were unable to assess associations between TC activity scores and patient response. Additionally, transcriptomic and survival data specific to other ovarian cancer subtypes beyond HGSOC are currently not available, limiting our ability to generalize these findings to those groups. We intend to leverage survival data from TCGA to explore associations between TC activity scores and overall survival of patients with other cancer types. Nonetheless, we recognize limitations with TCGA survival data, as outlined in this article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8726696/.

(3) Innovative Methodology:

Requires more validation using different platforms (IHC) to validate the performance of this bulk-derived data. Also, the lack of control over data quality is a concern.

We acknowledge the reviewer’s suggestion to validate our results with alternative platforms, such as IHC; however, we regret that such validation is beyond the scope of this article. Regarding data quality control, we implemented a series of checks:

• Bulk Transcriptional Profiles: We applied principal component analysis (PCA) on the sample Pearson product-moment correlation matrix, focusing on the first principal component (PCqc), which accounted for approximately 80-90% of the variance, primarily reflecting technical rather than biological variability  (Bhattacharya et al., 2020). Samples with a correlation below 0.8 with PCqc were removed as outliers. Additionally, we generated unique MD5 hashes for each CEL file to identify and exclude duplicate samples. Per gene, expression values were standardized to a mean of zero and a variance of one across the GEO, CCLE, GDSC, and TCGA datasets to minimize probeset- or gene-specific variability.

• Spatial Transcriptional Profiles: We used PCA for quality control here as well, retained samples only if their loading factors for the first principal component showed consistent signs across all profiles (i.e., all profiles had either positive or negative loading factors for the first PC) from that individual spatial transcriptomic sample. Samples that did not meet this criterion were excluded from analyses.

(4) Clinical Application:

Although the study suggests potential drug targets, the translation of these findings into clinical practice is not addressed. Probably given the lack of some QA/QC procedures it'll be hard to translate these results. Future studies should focus on validating these targets in clinical settings.

While this study is exploratory in nature, we agree that future studies should focus on validating these potential drug targets in clinical settings. As suggested, QA/QC procedures were integral to our analyses. We applied rigorous quality control, including PCA-based checks and duplicate removal across datasets, to ensure data integrity (detailed in our previous response).

In terms of clinical application, which we partially discussed in the manuscript, we will discuss additional strategies to prevent synaptic signaling and neurotransmitter release in the tumor microenvironment (TME). Drugs such as ifenprodil and lamotrigine are used in treating neuronal disorders to block glutamate release responsible for subsequent synaptic signaling, whereas the vesicular monoamine transporter (VMAT) inhibitor reserpine can block the formation of synaptic vesicles (Reid et al., 2013; Williams et al., 2001). Previous in vitro studies with HGSOC cell lines showed a significant effect of ifenprodil alone on cancer cell proliferation, whereas reserpine seemed to trigger apoptosis in cancer cells (North et al., 2015; Ramamoorthy et al., 2019). Such strategies could potentially be used to inhibit synaptic neurotransmission in the TME.

Reviewer #2 (Public review):

Summary:

Consensus-independent component analysis and closely related methods have previously been used to reveal components of transcriptomic data that are not captured by principal component or gene-gene coexpression analyses.

Here, the authors asked whether applying consensus-independent component analysis (c-ICA) to published high-grade serous ovarian cancer (HGSOC) microarray-based transcriptomes would reveal subtle transcriptional patterns that are not captured by existing molecular omics classifications of HGSOC.

Statistical associations of these (hitherto masked) transcriptional components with prognostic outcomes in HGSOC could lead to additional insights into underlying mechanisms and, coupled with corroborating evidence from spatial transcriptomics, are proposed for further investigation.

This approach is complementary to existing transcriptomics classifications of HGSOC.

The authors have previously applied the same approach in colorectal carcinoma (Knapen et al. (2024) Commun. Med).

Strengths:

(1) Overall, this study describes a solid data-driven description of c-ICA-derived transcriptional components that the authors identified in HGSOC microarray transcriptomics data, supported by detailed methods and supplementary documentation.

We thank the reviewer for acknowledging the strength of our data-driven approach and the use of consensus-independent component analysis (c-ICA) to identify transcriptional components within HGSOC microarray data. We aimed to provide comprehensive methodological detail and supplementary documentation to support the reproducibility and robustness of our findings. We believe this approach allows for the identification of subtle transcriptional signals that might be overlooked by traditional analysis methods.

(2) The biological interpretation of transcriptional components is convincing based on (data-driven) permutation analysis and a suite of analyses of association with copy-number, gene sets, and prognostic outcomes.

We appreciate the reviewer’s positive feedback on the biological interpretation of our transcriptional components. We are pleased that our approach, which includes data-driven permutation testing and analyses of associations with copy-number alterations, gene sets, and prognostic outcomes, was found convincing. These analyses were integral to enhancing the robustness and biological relevance of our findings.

(3) The resulting annotated transcriptional components have been made available in a searchable online format.

Thank you for acknowledging the availability of our annotated transcriptional components in a searchable online format.

(4) For the highlighted transcriptional component which has been annotated as related to synaptic signalling, the detection of the transcriptional component among 11 published spatial transcriptomics samples from ovarian cancers appears to support this preliminary finding and requires further mechanistic follow-up.

Thank you for acknowledging the accessibility of our annotated transcriptional components. We prioritized making these data available in a searchable online format to facilitate further research and enable the community to explore and validate our findings.

Weaknesses:

(1) This study has not explicitly compared the c-ICA transcriptional components to the existing reported transcriptional landscape and classifications for ovarian cancers (e.g. Smith et al Nat Comms 2023; TCGA Nature 2011; Engqvist et al Sci Rep 2020) which would enable a further assessment of the additional contribution of c-ICA - whether the c-ICA approach captured entirely complementary components, or whether some components are correlated with the existing reported ovarian transcriptomic classifications.

We appreciate the reviewer’s insightful suggestion to compare our c-ICA-derived transcriptional components with previously reported ovarian cancer classifications, such as those from Smith et al. (2023), TCGA (2011), and Engqvist et al. (2020). To address this, we will incorporate analyses comparing the activity scores of our transcriptional components with these published landscapes and classifications, particularly focusing on any associations with overall survival. Additionally, we plan to evaluate correlations between gene signatures from these studies and our identified TCs, enhancing our understanding of the unique contributions of the c-ICA approach.

(2) Here, the authors primarily interpret the c-ICA transcriptional components as a deconvolution of bulk transcriptomics due to the presence of cells from tumour cells and the tumour microenvironment. However, c-ICA is not explicitly a deconvolution method with respect to cell types: the transcriptional components do not necessarily correspond to distinct cell types, and may reflect differential dysregulation within a cell type. This application of c-ICA for the purpose of data-driven deconvolution of cell populations is distinct from other deconvolution methods that explicitly use a prior cell signature matrix.

Thank you for highlighting this nuanced aspect of c-ICA interpretation. We acknowledge that c-ICA, unlike traditional deconvolution methods, is not specifically designed for cell-type deconvolution and does not rely on a predefined cell signature matrix. While we explored the transcriptional components in the context of tumor and microenvironmental interactions, we agree that these components may not correspond directly to distinct cell types but rather reflect complex patterns of dysregulation, potentially within individual cell populations.

Our goal with c-ICA was to uncover hidden transcriptional patterns possibly influenced by cellular heterogeneity. However, we recognize these patterns may also arise from regulatory processes within a single cell type. To investigate further, we plan to use single-cell transcriptional data (~60,000 cell-types annotated profiles from GSE158722) and project our transcriptional components onto these profiles to obtain activity scores, allowing us to assess each TC’s behavior across diverse cellular contexts after removing the first principal component to minimize background effects.

References

Allen JK, Armaiz-Pena GN, Nagaraja AS, Sadaoui NC, Ortiz T, Dood R, Ozcan M, Herder DM, Haemerrle M, Gharpure KM, Rupaimoole R, Previs R, Wu SY, Pradeep S, Xu X, Han HD, Zand B, Dalton HJ, Taylor M, Hu W, Bottsford-Miller J, Moreno-Smith M, Kang Y, Mangala LS, Rodriguez-Aguayo C, Sehgal V, Spaeth EL, Ram PT, Wong ST, Marini FC, Lopez-Berestein G, Cole SW, Lutgendorf SK, diBiasi M, Sood AK. 2018. Sustained adrenergic signaling promotes intratumoral innervation through BDNF induction. Cancer Res 78:canres.1701.2016.

Balood M, Ahmadi M, Eichwald T, Ahmadi A, Majdoubi A, Roversi Karine, Roversi Katiane, Lucido CT, Restaino AC, Huang S, Ji L, Huang K-C, Semerena E, Thomas SC, Trevino AE, Merrison H, Parrin A, Doyle B, Vermeer DW, Spanos WC, Williamson CS, Seehus CR, Foster SL, Dai H, Shu CJ, Rangachari M, Thibodeau J, Rincon SVD, Drapkin R, Rafei M, Ghasemlou N, Vermeer PD, Woolf CJ, Talbot S. 2022. Nociceptor neurons affect cancer immunosurveillance. Nature 611:405–412.

Bhattacharya A, Bense RD, Urzúa-Traslaviña CG, Vries EGE de, Vugt MATM van, Fehrmann RSN. 2020. Transcriptional effects of copy number alterations in a large set of human cancers. Nat Commun 11:715.

Darragh LB, Nguyen A, Pham TT, Idlett-Ali S, Knitz MW, Gadwa J, Bukkapatnam S, Corbo S, Olimpo NA, Nguyen D, Court BV, Neupert B, Yu J, Ross RB, Corbisiero M, Abdelazeem KNM, Maroney SP, Galindo DC, Mukdad L, Saviola A, Joshi M, White R, Alhiyari Y, Samedi V, Bokhoven AV, John MSt, Karam SD. 2024. Sensory nerve release of CGRP increases tumor growth in HNSCC by suppressing TILs. Med 5:254-270.e8.

Globig A-M, Zhao S, Roginsky J, Maltez VI, Guiza J, Avina-Ochoa N, Heeg M, Hoffmann FA, Chaudhary O, Wang J, Senturk G, Chen D, O’Connor C, Pfaff S, Germain RN, Schalper KA, Emu B, Kaech SM. 2023. The β1-adrenergic receptor links sympathetic nerves to T cell exhaustion. Nature 622:383–392.

Jin M, Wang Y, Zhou T, Li W, Wen Q. 2022. Norepinephrine/β2-adrenergic receptor pathway promotes the cell proliferation and nerve growth factor production in triple-negative breast cancer. J Breast Cancer 26:268–285.

denial as a tool to understand our moral imperative

!!!

climate challenge requires full deconstruction and reinvention of our current worldviews and societal rules

no reward for big companies to combat the climate crisis --> even less motivation to act

successful movements were not made possible by just nonviolent approaches, but also a combination of violent tactics

compares demands of the New Deal following the Great Depression to the demands of the current climate crisis

this kind of economic shift HAS happened before, as a result of the efforts of ordinary citizens

Esta es una idea muy buena porque muchos otros programas sociales de otros países excluyen las personas sin residencias en el proceso del programa. Entonces, me parece este enfoque es muy interesante e impactante

Bu metinde, diş restorasyonlarıyla ilgili önemli bir durumdan bahsedilmektedir. Kutular (box form) genellikle dişin iç kısmındaki çürükleri veya hasarları onarmak için açılır. Ancak, iki kutu açıldığında, dişin yapısal bütünlüğü tehlikeye girebilir. Şu şekilde açıklanabilir:

İki kutu hazırlanırsa: Dişin mesial ve distal (ön ve arka) yüzeylerinde iki ayrı kutu şekli oluşturulursa, dişin o bölgedeki yapısal sağlamlığı azalabilir.

Lingual duvarın bozulma olasılığı yüksektir: Dişin lingual yüzeyi, yani dil tarafındaki duvar, bu durumda zayıflayabilir ve bozulma riski artar. Dişin iç kısmındaki kutular, lingual duvarın dayanıklılığını azaltabilir çünkü bu alan daha ince kalır ve basınca daha duyarlı hale gelir.

I don't think this applies to me, so I can feel free to busy myself more

remove capitalization

I love using a question at the beginning of the paragraph. It might be interesting to carry it through the rest of the introduction.

Welcome back.

This is part two of this lesson.

We're going to continue immediately from the end of part one.

So let's get started.

Now the next thing that I want to demonstrate is how we can restore RDS if we have data corruption.

The way that we're going to simulate this is to go back to our WordPress blog and we're going to corrupt part of this data.

So we're going to change the title of this blog post from the best cats ever to not the best cats ever, which is clearly untrue.

But we're going to change this and this is going to be our simulation of data corruption of this application.

So go ahead and click on update to update the blog post with this new obviously incorrect data.

Now let's assume that we need to restore this database from an earlier snapshot.

Now let's ignore the automatic backup feature of RDS and just look at manual snapshots.

Well, let's move back to the RDS console and click on snapshots and we'll be able to see the snapshot that we created at the start of this demo lesson.

Remember, this does have the blog post contained within it in its original correct form.

Now to do a restore, we need to select this snapshot, click on actions and then restore snapshot.

Now I mentioned this in the theory lesson about backups and restores within RDS.

Restoring a snapshot actually creates a brand new database instance.

It doesn't restore to the existing one using normal RDS.

So we have to restore a snapshot.

Obviously the engine set to MySQL community and we're provided with an entry box for a brand new database identifier.

And we're going to use a4lwordpress-restore.

So this allows us to more easily distinguish between this and the original database instance.

We also need to decide on the deployment option.

So go ahead and select single DB instance.

This is only a demo, so we don't need to select multi-AZ DB instance.

We need to pick the type of instance that we're going to restore to.

And again, because this is a new instance, we're not limited to the previous free tier restrictions.

So we're able to select from any of the available instance types.

So go ahead and select burstable classes and then pick either t2 or t3.micro.

We'll leave storage as default.

We'll need to provide the VPC to provision this new database instance into.

So we'll make sure that a4l-vpc1 is selected and we'll use the same subnet group that was created by the one-click deployment, which you used at the start of this demo.

You're allowed to choose between public access yes or no.

We'll choose no.

You'll have to pick a VPC security group to use for this RDS instance.

Now the one-click deployment did create one, so click in the drop-down and select the RDS multi-AZ snap RDS security group.

So not the instance security group, but the RDS security group.

Once you've selected that, then delete default, scroll down.

You can specify database authentication and encryption settings.

And again, if applicable in the course that you're studying, I'll be covering these in a separate lesson.

We'll leave all of that as default and click on restore DB instance.

Now this is going to begin the process of restoring a brand new database instance from that snapshot.

Now the important thing that you need to understand is this is a brand new instance.

We're not restoring the snapshot to the same database instance.

Instead, it's creating a brand new one.

Now when this finishes restoring, when it's available for use, if we want our application to make use of it, and the restored non-corrupted data, then we're going to need to change the application to point at this newly restored database.

So at this point, go ahead and pause the video because for the next step, which is to adjust the WordPress configuration, we need this database to be in an available state.

So pause the video, wait for the status to change from creating all the way through to available, and then we're good to continue.

Okay, so the snapshot restore is now completed and we have a brand new database instance, A4LWordPress-Restore.

And in my case, it took about 10 minutes to perform that restoration.

Now just to reiterate this concept, because it's really important, it features all the time in the exams, and you'll need this if you operate in the real world using AWS.

If we go into the original RDS instance, just pay attention to this endpoint DNS name.

So we have a standard part, which is the region, and then .rds, and then .amazonaws.com.

Before this, though, we have this random part.

Now this represents the name of the database instance as well as some random.

If we go back to the databases list and then go into the restored version, now we can see that we have A4LWordPress-Restore.

And this is different than that original database endpoint name for the original database.

So the really important, the critical thing to understand is that a restore with a normal RDS will create a brand new database instance.

It will have a brand new database endpoint DNS name, the CNAME, and you will need to update any application configuration to use this brand new database.

So go ahead and just leave this open in this tab because we'll be needing it very shortly.

Click on Services, find EC2, and open that in a new tab.

So as a reminder, if we go back to the WordPress tab and just hit Refresh, we can see that we still have the corrupt data.

Now what we want to do is point WordPress at the restored correct database.

So to do that, go to the EC2 tab that you just opened, right click on the A4LWordPress instance, select Connect.

We're going to use Instance Connect, so choose that to make sure the username is EC2-user and then connect to the instance.

This process should be familiar by now because we're going to edit the WordPress configuration file.

So cd/var/www/html, then we'll do a listing ls-la, and we want to edit the configuration file which is wp-config.php, so shudu, space, nano which is the text editor, space, wp-config.php.

Once we're in this file, just scroll down and again we're looking for the dbhost configuration which is here.

Now this DNS name you'll recognize is pointing at the existing database with the corrupt data.

So we need to delete all of this just to leave the two single quotes.

Make sure your cursor's over the second quote.

Go back to the RDS console and we need to locate the DNS name for the A4LWordPress-Restore instance.

Remember this is the one with the correct data.

So copy that into your clipboard, go back to EC2 and paste that in, and then Ctrl+O and Enter to save, and Ctrl+X to exit.

That's all of the configuration changes that we need.

If we go back to the WordPress application and hit refresh, we'll see that it's now showing the correct post, the best cats ever, because we're now pointing at this restored database instance.

So the key part about this demo lesson really is to understand that when you're restoring a normal RDS snapshot, you're restoring it to a brand new database instance, its own instance with its own data and its own DNS endpoint name.

So you have to update your application configuration to point at this new database instance.

With normal RDS, it's not possible to restore in place.

You have to restore to a brand new database instance.

Now this is different with a feature of Aurora which I'll be covering later in this section, but for normal RDS, you have to restore to a brand new instance.

So those are the features which I wanted to demonstrate in this demo lesson.

I wanted to give you a practical understanding of the types of recovery options and resilience options that you have available using the normal RDS version, so MySQL.

Now different versions of RDS such as Microsoft SQL, PostgreSQL, Oracle, and even AWS specific versions such as Aurora and Aurora Serverless, they all have their own collections of features.

For the exam and for most production usage, you just need to be familiar with a small subset of those.

Generally, you'll either be using Oracle, MSSQL, or one of the open source or community versions, so you'll only have to know the feature set of a small subset of the wider RDS product.

So I do recommend experimenting with all of the different features and depending on the course that you're taking, I will be going into much more depth on those specific features elsewhere in this section.

For now though, that is everything that I wanted to talk about, so all that remains is for us to tidy up the infrastructure that we've used in this demo lesson.

So go to databases.

I want you to select the A4L WordPress -Restore instance because we're going to delete this fully.

We're not going to be using this anymore in this section of the course, so select it, click on the Actions drop down, and then select Delete.

Don't create a final snapshot.

We don't need that.

Don't retain automated backups and because we don't choose either of these, we need to acknowledge our understanding of this and type Delete Me into this box.

So do that and then click on Delete.

Now that's going to delete that instance as well as any snapshots created as part of that instance.

So if we go to Snapshots, we only have the one manual snapshot.

If we go to System Snapshots, we can see that we have one snapshot for this Restore database, and if you're deleting a database instance, then any system created snapshots for that database instance will also be deleted either immediately or after the retention period expires.

So those will be automatically cleared up as part of this deletion process.

We're not going to delete the manual snapshot that we created at the very start of this lesson with the catpost in because we're going to be using this elsewhere in the course.

So leave this in place.

Click on Databases again.

We're going to need to wait for this Restored Database instance to finish deleting before we can continue.

So go ahead and pause the video, wait for this to disappear from the list, and then we can continue.

Okay, so that Restored Database instance has completed deleting.

So now all that remains is to move back to the CloudFormation console.

You should still have a tab open.

Select the stack deployed as part of the one-click deployment.

It should be called RDS Multi-AZ Snap.

Select Delete and then confirm that deletion, and that will clear up all of the infrastructure that we've used in this demo lesson.

It will return the account into the same state as it was at the start of this demo with one exception.

And that one exception is the snapshot that we created of the RDS instance as part of this deployment.

So that's everything you need to do in this demo lesson.

I hope you've enjoyed it.

I know it's been a fairly long one where you've been waiting a lot of the time in the demo for things to happen, but it's important for the exam and real-world usage that you get the practical experience of working with all of these different features.

So you should leave this demo lesson with some good experience of the resilience and recovery features available as part of the normal RDS product.

Now at this point, that's everything you need to do, so go ahead and complete this video, and when you're ready, I look forward to you joining me in the next.

Welcome back.

This is part two of this lesson.

We're going to continue immediately from the end of part one.

So let's get started.

Okay, so the instance is now in an available state.

Let's just close down this informational dialogue at the top.

And let's just minimize this menu on the left.

Let's maximize the amount of screen space that we have for this specific purpose.

So I just want us to go inside this database instance and explore together the information that we have available.

So I talked in the theory lesson how every RDS instance is given an endpoint name and an endpoint port.

So this is the information that we'll use to connect to this RDS instance.

Networking wise, this instance has been provisioned in US-EAST-1A.

It's in the Animals for Life VPC and it's used our A4L subnet group that we created at the start of this demo.

And that means that it's currently utilizing all three database subnets in the Animals for Life VPC.

But it's chosen because we only deployed one instance to use US-EAST-1A.

Now this is the VPC security group that we're going to need to configure.

So right click on this and open it in a new tab and move to that tab.

This is the security group which controls access to this RDS instance.

So let's expand this at the bottom.

So currently it has my IP address being the only source allowed to connect into this RDS instance.

So the only inbound rule on the security group protecting this RDS instance is allowing my IP address.

So we're going to click on Edit and then click on Add Rule.

And we're going to add a rule which allows our other instances to connect to this RDS instance.

So first in the type drop down click and then type mySQL to get the same option as the line above and then click to select.

Next go ahead and type instance into the source box and then select the migrate to RDS-instance security group.

Now this is the security group that's used by any instances deployed by our one click deployment.

And this allows those instances to connect to our RDS instance and that's what we want.

So go ahead and select that and then click on Save Rules.

And this means now that our WordPress instance can communicate with RDS.

So now let's move back to the RDS tab and then make sure we're inside the A4L WordPress database instance.

So that's the connectivity and the security tab.

We also have the monitoring tab and it's here where you can see various CloudWatch provided metrics about the database instance.

You also have logs and events related to this instance.

So if we go and have a look at recent events we can see all of the events such as when the database instance was created, when its first backup was created.

And you can explore those because they might be different in your environment.

You can click on the Configuration tab and see the current configuration of the RDS instance.

The Maintenance and Backups tab is where you can configure the maintenance and backup processes and then of course you can tag the RDS instance.

Now in other lessons in this section of the course and depending on what course you're taking I will be talking about many of these options, what you can modify and which actions you can perform on RDS instances.

But for now we're just going to move on with this demo.

So the next step is that we need to migrate our existing data into this RDS instance.

So what we're going to do is to click on the Connectivity and Security tab and we're going to leave this open.

We're going to need this endpoint name and port very shortly.

You should still have a tab open to the EC2 console.

If you don't you can reach that by going on Services and then opening EC2 in a new tab.

But I want you to select the A4L-WordPress instance and then right click and connect to it using Instance Connect.

So go ahead and do that.

Once you've done that we're going to start referring to the lesson commands document.

So make sure you've got that open.

We're going to use this command to take a backup of the existing MariaDB database.

So we need to replace a placeholder.

What we need to do is delete this and replace it with the private IP address of the MariaDB EC2 instance.

So go back to the EC2 console, select the DB-WordPress instance and copy the private IP version 4 address into your clipboard.

And then let's move back to the WordPress instance and paste that in.

Go ahead and press Enter and it will prompt you for the password.

And the password is the same Animals for Life strong password that we've been using everywhere.

Copy that into your clipboard.

So this is the password for the A4L WordPress user on the MariaDB EC2 instance.

So paste that in and press Enter and then LS-LA to confirm that we now have this A4L WordPress.SQL database backup file.

And we do, so that's good.

So as we did in the previous demo lesson, we're going to take this backup file and we're going to import it into the new destination database, which is going to be the RDS instance.

To do that, we'll use this command, but we're going to need to replace the placeholder hostname with the CNAME of the RDS instance.

So go ahead and delete this placeholder, then go back to the RDS console and I'll want you to copy the endpoint name into your clipboard.

So select it, right click and then copy.

We won't need the port number because this is the standard MySQL port and if you don't specify it, most applications will assume this default.

So just make sure that you have the endpoint DNS name or endpoint CNAME in your clipboard.

And then back on the WordPress EC2 instance, go ahead and paste this database name into this command and press Enter.

And again, you'll be asked for the password and that's the same Animals for Life strong password.

So copy that into your clipboard, paste that in and press Enter.

And that's imported this A4LWordPress.SQL file into the RDS instance.

So now we need to follow the same process and change WordPress so that it points at the RDS instance.

And we do that by moving to where the WordPress configuration file is.

So cd space forward slash var forward slash ww w forward slash html and press Enter.

And then shudu.

So we have admin privileges, nano, which is a text editor and then wp-config.php and press Enter.

Then we need to scroll down and we're looking for where it says DB host and currently it has a host name here.

Now if you go back to the EC2 console and you look at the A4L-DB-WordPress instance, you'll see that its private IP version for DNS name is what's listed inside this configuration item.

So it's currently pointing at this dedicated database instance.

What we need to do is replace that and we're going to replace it with the RDS database DNS name or the CNAME of this RDS instance.

So copy that into your clipboard and then go ahead and delete this private DNS name for the MariaDB EC2 instance and then paste in the RDS endpoint name, also known as the RDS CNAME.

Once you've done that, control O and Enter to save and control X to exit.

And now our WordPress instance is pointing at the RDS instance for its database.

Now we can verify that by checking WordPress, move back to instances, select the WordPress instance, copy the public IP version for addressing to your clipboard.

Don't use this open address link.

Open that in a new tab.

Go ahead and just click on the best cats ever to verify the functionality and it does look as though it's working.

And to verify that, if we go back to the EC2 console, select the A4L-DB-WordPress instance and right click and then stop that instance.

Now the original database that was providing database services to WordPress is going to move into a stopped state.

And if our WordPress blog continues functioning, we know that it's using the RDS instance.

So let's keep refreshing and wait for this to change into a stopped state.

There we go.

It's stopped.

And if we go back to our WordPress page and refresh, it still loads.

And so we know that it's now using RDS for its database services.

So at this point, that's everything that I wanted you to do in this demo lesson.

You've stepped through the process of provisioning an RDS instance.

So you've created a subnet group, provisioned the instance itself, explored the functionality of the instance, including how to provide access to it by selecting a security group.

And then editing that security group to allow access.

You've performed a database migration and you've explored how the RDS instance is presented in the console.

So that's everything that you need to do within this demo lesson.

And don't worry, we're going to be exploring much more of the advanced functionality of RDS as we move through this section of the course.

For now, though, I want us to clear up the infrastructure that we've created as part of this demo lesson.

Now, because we've provisioned RDS manually outside of CloudFormation, unfortunately, there is a little bit more manual work involved in the cleanup.

So I want you to go to the RDS console, move to databases, select this database, click on actions, and then select delete.

Now it will prompt you to create a final snapshot and we're not going to do that.

We're not going to retain automated backups and so you'll need to acknowledge that upon instance deletion, automated backups including any system snapshots and pointing time recoveries will no longer be available.

And don't worry, I'll be talking about backups and recovery in another lesson in this section of the course.

For now, just acknowledge that and then type delete me into this box and confirm the deletion.

Now this deletion is going to take a few minutes.

It's not an immediate process.

It will start in a deleting state and we need to wait for this process to be completed before we continue the cleanup.

So go ahead and pause this video and wait for this instance to fully delete before continuing.

Now that the instance has been deleted, it vanishes from this list.

Next, we need to delete the subnet group that we created earlier.

So click on subnet groups, select the subnet group and then delete it.

You'll need to confirm that deletion.

Once done, it too should vanish from that list.

Next, go to the tab you've got open to the VPC console, scroll down and select security groups.

Now look through this list and locate the security group that you created as part of provisioning the RDS instance.

It should be called a4LVPC-RDS-SG.

Select that, click on actions and then delete security group and you'll need to confirm that process as well.

Once that's deleted, the final step is to go to the cloud formation console and then you'll need to delete the cloud formation stack that was created using the one-click deployment at the start of the demo.

It should be called migrate to RDS.

Select it, click on delete and confirm that deletion.

And once deleted, the account will be returned into the same state as it was at the start of the demo lesson.

So all of the infrastructure that we've used will be removed from the account and the account will be in the same state as at the start of the demo.

Now I hope you've enjoyed this demo and that we're repeating the same WordPress installation and then the creation of the blog post over and over again.

But I want you to get used to different parts of this process over and over again.

You need to know why not to use a database on EC2.

You need to know why not to perform a lot of these processes manually.

From this point onward in the course, we're going to be using RDS to evolve our WordPress design into something that is truly elastic.

And so all of these processes, the things I'm having you repeat are really useful to aid in your understanding of all of these different components.

So from this point onward, we're going to be automating the creation of RDS and focusing on the specific pieces of functionality that you need to understand.

But at this point, that's everything that you need to do in this demo.

So go ahead, complete the video and when you're ready, I look forward to you joining me in the next.

Welcome to this demo lesson where you're going to migrate from the monolithic architecture on the left of your screen towards a tiered architecture on the right.

Essentially you're going to split the WordPress application architecture, you're going to move the database from being on the same server as the application to being on a different server and this will form step one of moving this architecture from being a monolith through to being a fully elastic architecture.

Now this is the first stage of many but it is a necessary one.

Now in order to perform this demonstration you're going to need some infrastructure.

Before we apply the infrastructure just make sure that you're logged in to the general AWS account, so the management account of the organization and as always you need to have the Northern Virginia region selected.

Now once you've got both of those set there's a one-click deployment link attached to this lesson so go ahead and click on that link.

What this is going to do is deploy the Animals for Life base infrastructure, it's going to deploy the monolithic WordPress application instance and it's also going to deploy a separate MariaDB database instance that you're going to use as part of the migration.

Now everything set, the stack name should be set to a suitable default, all you need to do is to scroll all the way down to the bottom, check this capabilities box and click on create stack.

Now also attached to this lesson is a lesson commands document which contains all the commands you'll be using throughout this demo.

So go ahead and open that in a new tab, you'll be referencing it constantly as you're making the adjustments to the WordPress architecture.

Now we're going to need this CloudFormation stack to be fully complete before we can continue so go ahead and pause the video and resume once the CloudFormation stack moves into a create complete state.

So now the stacks moved into a create complete state, we're good to continue.

Now this has created the base Animals for Life infrastructure which includes a number of EC2 instances so let's take a look at those, let's click on services and then locate and open EC2 in a brand new tab.

Once you're at the EC2 console if you do see any dialogues around user interface updates then just go ahead and close those down and then click on instances running.

Once you're here you'll see two EC2 instances, one will be called A4L-WordPress and this is the monolith so this is the EC2 instance which contains the WordPress application and the built-in database.

So this is the WordPress installation that we're going to migrate from and then this instance A4L-DB-WordPress this contains a standalone MariaDB installation so we're going to migrate the database for WordPress from this instance onto the DB instance and this will create a tiered application architecture rather than the monolith which we currently have.

So step number one is to perform the WordPress installation so to do that I want you to go ahead and copy the public IP version for address of the WordPress EC2 instance into your clipboard and then open it in a new tab.

Now be careful not to use the open address link that will use HTTPS which we're not currently using so copy the IP address into your clipboard and open that in a new tab.

Now when you do that you'll see a familiar WordPress installation dialog we're going to create a simple blog for site title go ahead and call it the best cats for username pick admin and then for the password instead of using the randomly selected one go ahead and use this same complex password that we've used for the CloudFormation template so this is animals for life but with number substitution.

So if you go back to your CloudFormation tab and go to the parameters tab this is the same password that we use for the DB password and the DB root password.

Now of course in production this is incredibly bad practice we're just doing it in this demo to keep things simple and avoid any mistakes.

So back to the WordPress installation screen site title the best cats username admin this for the password and then just go ahead and type a fake email so I don't want to use my real email for this I'm going to type test at test.com you can do the same and then go ahead and click on install WordPress so this is installed the WordPress application and it's using the Maria DB server that's on the same EC2 instance so part of the same monolith.

So we're going to log in we'll need to type admin and then use the animals for life strong password and click on login and once we logged in we're going to create a simple blog post so click on posts we're going to select the existing hello world post select trash this time then click on add new then we're going to add a new post we can close down this introduction dialogue and for title go ahead and type the best cats ever and then some exclamation points next click on this plus sign and we're going to add a gallery now at this point you're going to need some images to upload to this blog post I've attached an images link to this lesson so if you go ahead and click that link it will download a zip file if you extract that zip file it's going to contain four image files all four of my cats so at this point once you've downloaded and extracted that file go ahead and click on upload locate those images there should be four select them all and click on open that will add these images to this blog post and once you've added them all you can go ahead and click on publish and then publish again and this will publish this blog post so it will add data to the database that's running on the monolithic application instance as well as store these images on the local instance file system now making a point of mentioning that these images are stored on the file system because as you'll see later in the course this is one of the things that we need to migrate when we're moving to a fully elastic architecture we can't have images stored on the instances themselves we need to move that to a shared file system for now though we're focusing on the database so at this point we have the working blog the images for this blog are stored on the local file system of a4l-wordpress and the data for that blog post is stored on the MariaDB database that's also running on this EC2 instance so the next step of this demo lesson is that you're going to migrate the data from a4l-wordpress onto a4l-db-wordpress and this is an isolated MariaDB instance this is dedicated for the database so to do this migration select a4l-wordpress right click we're going to connect to this instance we'll be using EC2 instance connect so just make sure that the username is set to EC2-user and then click on connect now this is where you're going to be using the commands that are stored within the lesson commands document so you need to make sure that you have this ready to reference because it's far easier to copy and paste these commands and then adjust any placeholders rather than type them out manually because that's prone to errors the first step is to get the data from the database that's running on this monolithic application instance and store it in a file on disk so that's the first thing we need to do we need to do a backup of the database into a .sql file now to do that we use this command so it's a utility called my sql-dump it uses the -u to specify the user that we're going to be using to connect to the database then we use -p to specify that we want to provide a password and we could either provide the password on the command line or we could have it prompt us now if we supply the password with no spaces next to this -p then it will accept it as input on this command if we don't specify anything so there's a space here then it's going to ask us for the password the next thing we specify is the database name that we want to do the dump of in this case it's a4l WordPress which is the database for the animals for life WordPress instance now if we just run this command on its own it would output the dump so all of the data in the database to standard output which in this case is our screen we don't want it to do that we want it to store the results in a file called a4l WordPress dot sql and so we use this symbol which means that it's going to take the output of this component of the command and it's going to redirect it into this file so let's go ahead and run this command and it's going to prompt us for the password for this database now to get that go back to cloud for information make sure parameters are selected and it's this password that we need which is the DB password so copy that into your clipboard go back to the instance paste that in press enter and that will output all the data in the database to this file now you won't see any indication of success or failure but if you do an LS space -la and press enter one of the files that you'll see is a4l WordPress dot sql so now we have a copy of the WordPress database containing our blog post the next thing that we need to do is to take this file this backup of the database and inject it into the new database that we want to use so the dedicated Maria DB EC2 instance and to do that we're going to use this command so this command has two components the first component is this which connects to the Maria DB database instance the second component is this which takes the backup that we've just made and it feeds it into this command so this backup contains all the necessary definitions to create a new database and inject the data required this component of the command just allows us to connect to this new dedicated Maria DB instance now there are some place holders that we need to change the database name that we're going to use is the same so a4l WordPress we're still going to want to be prompted for a password so -p is what we use this time though we're going to connect using a user called a4l WordPress so we're not using the root user we're going to connect to this separate Maria DB database instance using a user a4l WordPress the only thing that we need to change is that we need to connect to a non-local host so when we used the mysql dump command we didn't specify a host to connect to and this defaulted to local host so the current machine in the case of this command we're operating with a separate server this dedicated EC2 instance which is running the Maria DB database server so a4l -db -wordpress we need to connect to this so what we'll need to connect to this is the private IP version for address of this separate database instance so select it look for private IP version for addresses and then click on the icon next to this to copy the private IP version for address of this separate database server into your clipboard then return to the application instance and we need to replace the placeholder here with that value so make sure that you're one space after the end of this placeholder and just delete this leave a space between -h and where the cursor is and then paste in that IP address so this is going to connect to this separate EC2 instance using its private IP it's going to use the a4l WordPress user it will prompt us for a password it will perform the operation on the a4l WordPress database and it's going to use the contents of this backup file to perform those tasks so go ahead and press enter and you'll be prompted for a password now again it's the same password this has all been set up as part of the cloud formation one-click deployment this lesson is about the migration process not setting up a database server so I've automated this component of the infrastructure so copy the DB password into your clipboard go back to the instance paste it in and press enter so now we've uploaded our WordPress application database into this separate MariaDB database server the next step is to configure WordPress to point at this new database server so to do that cd space forward slash var forward slash www forward slash html and press enter and then we're going to run a shudu space nano which is a text editor space wp-config.php and this is the WordPress configuration file so press enter now what we're looking for if we scroll down is we're looking for the line which says define and then a space and then DB host so this is the database host that WordPress attempts to connect to and currently it's set to local host which means it will use the database on the same EC2 instance as the application we're going to delete this local host so delete until we have two single quotes and then make sure that you still have the private IP version for address of this separate database instance in your clipboard if you don't just go ahead and copy it again from the EC2 console and then paste that in place of local host so now you should see DB underscore host and this now represents this private IP address and now the private IP address that you should use here will be different you need to use your private IP address of your a4l - DB - WordPress EC2 instance so now that you've updated this configuration file press control o and enter to save and then control x to exit out of editing this file now this now means that the WordPress instance is going to be communicating with the separate MariaDB database instance let's verify that let's go back to the tab that we have to our WordPress application and let's just go ahead and do a refresh if everything's working as expected we should see that the blog reloads successfully now this means that this blog is now pointing at this separate MariaDB database instance to be doubly sure of this though let's go back to the WordPress instance and let's shut down the MariaDB database server and we do that using this command so shudu space service space MariaDB space and then stop so type or copy and paste that command in and press enter and that's going to stop the MariaDB database service which is running on a4l WordPress so now the only MariaDB database that we have running is on the a4l - DB - WordPress EC2 instance now we can go back to the WordPress tab and hit refresh and assuming it loads in as it does in my case this now confirms that WordPress is communicating with this dedicated MariaDB EC2 instance now the reason why I wanted to step you through all these tasks in this demo lesson is the time a firm believer that in order to understand best practice architecture you need to understand bad architecture and as I mentioned in the theory lesson there is almost no justification for running your own self-managed database server on an EC2 instance in almost all situations it's preferable to use the RDS service but I need you to understand exactly how the architecture works when you're self managing a database and how to migrate from a monolithic all-in-one architecture through to having a separate self managed database in the demo lesson that's coming up next in the course you're going to migrate from this through to an RDS instance so that's step two but at this point you've done everything that I wanted you to do in this demo lesson you've implemented the architecture that's on screen now on the right all we need to do is to tidy up all of the infrastructure that we've used within this lesson so to do that it's nice and easy just go back to the cloud formation console make sure that you have the monolith to EC2 DB stack selected click on the delete button and then confirm that deletion and that stack deleting will clean up all of the infrastructure that we've used throughout this demo lesson and it will return the account into the same state as it was at the start of the lesson at this point you've completed all of the tasks that I want you to do so I hope you've enjoyed this demo lesson go ahead and complete this video and when you're ready I'll look forward to you joining me in the next.

Mookie's whole character is so well written and the way that the characters are describes as "Statues" earlier in the essay which is a piece of public art allow Mookie to be interpreted in many different ways

This is what I believe makes Do the right thing so well regarded as a piece of public art as it makes you think whether Mookie's actions by the end of the movie were justified and right and I believe that the comparison of MLK and Malcom fits perfectly with it as you can make an argument that Mookie's action was or wasn't justified using their beliefs

The use of public art in the film "Do The right thing" is amazing and the public art being Sal's wall and how it origin point of all of the violence in the movie which helps with what Mitchell asks earlier in the paper which is "Is public art inherently violent".

This I believe is what Mitchell was talking about in the first half which was how art (in this case music) can cause violence or inherently has violence in it as the song talks about fighting against the "system" which would be the government and all of the racial discrimination they face.

Do the right thing talks about many of the different spheres in the public that were talked about in film or at least not in the way Do the right thing did

Public art cant outright monumentalize violence as it goes against the public sphere

Artists are creating Art for the purpose to get a reaction of the public whether it be violent or slander the piece of work

Most of the public Art we see is being created just for the sake of aesthetically pleasing and have no real meaning or symbolism behind them

The use of Violence and the symbolism of it has been used for as long as we can remember.

Even if something is labeled as public art there is always some form of legal and political control over it thus never making it truly public art and most art that enters the public can cause some sort of violence to happen.

Violence is something that has been embedded in the history of public arts and the use of violence is to fight back against the something, in this case its Chinese government vs. The students of China.

Mechanizm działania neuromodulacyjnego cholinergiczny i adenergiczny

tady už se pracuje s procenty a ne body zmiňovanými výše, tak možná vysvětlit, co přesně ta procenta říkají

tomu nerozumím

zvážit používání pojmu "osoba samostatně pečující o dítě"

vystoupalo Česko o 5 p.b. nad evropský průměr.

Son rengi onaylamak için, seçilen kompozitten küçük bir miktar restorasyon yapılacak bölgenin yanına yerleştirilir ve ardından uyum sağlamak için ışıkla kürlenir.

Posterior kompozit restorasyonlar için, renk seçimi anterior restorasyonlara göre o kadar kritik değildir.

Sobre a Guerra

Sobre o Interesse Nacional

vynechat - opakuje se

staati website o=populate the page

Csendes éj (dal)

Overvej hvor relevant det er at opsummere disse

DOI: 10.1038/s41586-024-07144-2

Resource: (IMSR Cat# JAX_026220,RRID:IMSR_JAX:026220)

Curator: @AleksanderDrozdz

SciCrunch record: RRID:IMSR_JAX:026220

What is this?

DOI: 10.1523/JNEUROSCI.0381-24.2024

Resource: NWB Inspector (RRID:SCR_025465)

Curator: @scibot

SciCrunch record: RRID:SCR_025465

What is this?

1. The configuration factor (C-factor) is the ratio of bonded surface of the restoration to the unbonded surfaces. Konfigürasyon faktörü (C-faktörü), restorasyonun bağlı yüzeyinin bağlı olmayan yüzeylere oranıdır.

2. The higher the value of ‘C’-factor, the greater is the polymerization shrinkage. C-faktör değeri ne kadar yüksekse, polimerizasyon büzülmesi o kadar fazla olur.

Reçine matrisinin içeriği ne kadar fazla ise, deformasyon da o kadar fazla olur.

daha sert

La entrada de una célula a fase M depende entonces, del factor promotor de maduración, el cual tiene 2 subunidades: 1. cinasa: transfiere grupos P del ATP a residuos de serina y treonina 2. ciclina: su concentración activa o inhibe a la cinasa. A mayor concentración de esta, mayor actividad de la cinasa. Las bajas concentraciones de ciclina provocan la inhibición de las cinasas, provocando que la célula NO entre a fase M

giải thuật decode: sử dụng thuật toán tham lam theo đoạn, sẽ chọn các đoạn phù hợp dựa trên các điểm. Các đoạn có điểm > 0.5 sẽ được coi là đúng. Có 3 giải thuật decode:

• Flat NER: giải thuật này chọn đoạn không chồng chéo có điểm cao nhất và tiếp tục như vaatyj đến khi nào tất cả các đoạn đều được chọn.
• Nested NER: tương tự NestNER, nhưng cho phép chọn các đoạn được đóng gói toàn bộ trong các thực thể khác mà vẫn tránh được bị chồng chéo 1 phần.
• Hiệu quả thuật toán: Chiến lược decode được cài đặt sử dụng hàng đợi ưu tiên cho các đoạn, đảm bảo độ phức tạp là O(n log n) với n là số lượng đoạn ứng viên.

Mục tiêu của quá trình huấn luyện là cải thiện điểm phù hợp của các cặp đoạn-nhãn đúng và giảm thiểu điểm của các cặp sai. Hàm mất mát được sử dụng ở mỗi mẫu dữ liệu, bao gồm 1 các đoạn S và các nhãn T, được định nghĩa như sau:....

biến s tượng trưng cho cặp đoạn-nhãn

Mô hình gồm 3 thành phần chính: - Một bộ encode văn bản (BiLM như BERT) - một module thể hiện đoạn văn bản, có chức năng tính toán các embedding văn bản từ embedding của các token. - Một module thể hiện thực thể, có chức nằng tính các embedding của thực thể mà mô hình cần trích xuất ra.

Mục tiêu là có được thể hiện thực thể va thể hiện văn bản ở trong cùng 1 không gian ẩn để đạt được tính phù hợp trong việc bắt cặp.

các mô hình llm mạnh yêu cầu tài nguyên tính toán lớn. Mặc dù có thể truy cập vào các LLM này thông qua API, ví dụ như openAI, việc sử dụng chúng ở một quy mô nhất định sẽ dấn đến phát sinh chi phí lớn.

¿Como sería la solución tecnológica o innovación tecnológica que para cuantificar y valorar las relaciones de la biodiversidad con el negocio o la empresa?

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:  

Reviewer #1 (Public Review): 

Summary: 

The fungal cell wall is a very important structure for the physiology of a fungus but also for the interaction of pathogenic fungi with the host. Although a lot of knowledge on the fungal cell wall has been gained, there is a lack of understanding of the meaning of ß-1,6-glucan in the cell wall. In the current manuscript, the authors studied in particular this carbohydrate in the important humanpathogenic fungus Candida albicans. The authors provide a comprehensive characterization of cell wall constituents under different environmental and physiological conditions, in particular of ß-1,6glucan. Also, β-1,6-glucan biosynthesis was found to be likely a compensatory reaction when mannan elongation was defective. The absence of β-1,6-glucan resulted in a significantly sick growth phenotype and complete cell wall reorganization. The manuscript contains a detailed analysis of the genetic and biochemical basis of ß-1,6-glucan biosynthesis which is apparently in many aspects similar to yeast. Finally, the authors provide some initial studies on the immune modulatory effects of ß-1,6-glucan. 

Strengths: 

The findings are very well documented, and the data are clear and obtained by sophisticated biochemical methods. It is impressive that the authors successfully optimized methods for the analyses and quantification of ß-1-6-glucan under different environmental conditions and in different mutant strains. 

Weaknesses: 

However, although already very interesting, at this stage there are some loose ends that need to be combined to strengthen the manuscript. For example, the immunological studies are rather preliminary and need at least some substantiation. Also, at this stage, the manuscript in some places remains a bit too descriptive and needs the elucidation of potential causalities.

Reviewer #2 (Public Review): 

Summary: 

The authors provide the first (to my knowledge) detailed characterization of cell wall b-1,6 glucan in the pathogen Candida albicans. The approaches range from biochemistry to genetics to immunology. The study provides fundamental information and will be a resource of exceptional value to the field going forward. Highlights include the construction of a mutant that lacks all b-1,6 glucan and the characterization of its cell wall composition and structure. Figure 5a is a feast for the eyes, showing that b-1,6 glucan is vital for the outer fibrillar layer of the cell wall. Also much appreciated was the summary figure, Figure 7, which presents the main findings in digestible form.

Strengths: 

The work is highly significant for the fungal pathogen field especially, and more broadly for anyone studying fungi, antifungal drugs, or antifungal immune responses.

The manuscript is very readable, which is important because most readers will be cell wall nonspecialists.

The authors construct a key quadruple mutant, which is not trivial even with CRISPR methods, and validate it with a complemented strain. This aspect of the study sets the bar high. The authors develop new and transferable methods for b-1,6 glucan analysis. 

Weaknesses: 

The one "famous" cell type that would have been interesting to include is the opaque cell. This could be included in a future paper.

Reviewer #3 (Public Review): 

Summary: 

The cell wall of human fungal pathogens, such as Candida albicans, is crucial for structural support and modulating the host immune response. Although extensively studied in yeasts and molds, the structural composition has largely focused on the structural glucan b,1,3-glucan and the surface exposed mannans, while the fibrillar component β-1,6-glucan, a significant component of the well wall, has been largely overlooked. This comprehensive biochemical and immunological study by a highly experienced cell wall group provides a strong case for the importance of β-1,6-glucan contributing critically to cell wall integrity, filamentous growth, and cell wall stability resulting from defects in mannan elongation. Additionally, β-1,6-glucan responds to environmental stimuli and stresses, playing a key role in wall remodeling and immune response modulation, making it a potential critical factor for host-pathogen interactions.

Strengths: 

Overall, this study is well-designed and executed. It provides the first comprehensive assessment of β-1,6-glucan as a dynamic, albeit underappreciated, molecule. The role of β-1,6-glucan genetics and biochemistry has been explored in molds like Aspergillus fumigatus, but this work shines an important light on its role in Candida albicans. This is important work that is of value to Medical Mycology, since β-1,6-glucan plays more than just a structural role in the wall. It may serve as a PAMP and a potential modulator of host-pathogen interactions. In keeping with this important role, the manuscript rigor would benefit from a more physiological evaluation ex vivo and preferably in vivo, assessment on stimulating the immune system within in the cell wall and not just as a purified component. This is a critical outcome measure for this study and gets squarely at its importance for host-pathogen interactions, especially in response to environmental stimuli and drug exposure.

Response to reviewers (Public reviews):

We thank all the three reviewers for their opinion on our work on Candida albicans β-1,6-glucan, which highlights the importance of this cell wall component in the biology of fungi. Here are our responses to their comments for public reviews:

(1) Indeed, the data presented for immunological studies is preliminary. It has been acknowledged by the reviewers that our analysis providing insights into the biosynthetic pathways involved in comprehensive in dealing with organization and dynamics of the β-1,6-glucan polymer in relation with other cell wall components and environmental conditions (temperature, stress, nutrient availability, etc.). However, we anticipated that there would be immediate curiosity as to what the immunological contribution of β-1,6 glucan and we therefore felt we needed to initiative these studies and include them. We therefore performed immunological studies to assess whether β-1,6-glucans act as a pathogen-associated molecular pattern (PAMP), and if so, what its immunostimulatory potential is. Our data clearly suggest that β-1,6-glucan is a PAMP, and consequently lead to several questions: (a) what are the host immune receptors involved in the recognition of this polysaccharide, and thereby the downstream signaling pathways, (b) how is β-1,6-glucan differentially recognized by the host when C. albicans switches from a commensal to an opportunistic pathogen, and (c) how does the host environment impact the exposure of this polysaccharide on the fungal surface. We believe addressing these questions is beyond the scope of the present manuscript and aim to present new data in future manuscript. Nonetheless, in the revised manuscript, suggest approaches that we can take to identify the receptor that could be involved in the recognition of β-1,6-glucan. Moreover, we have modified the discussion presenting it based on the data rather than being descriptive.  

(2) It will be interesting to assess the organization of β-1,6-glucan and other cell wall components in the opaque cells. It is documented that the opaque cells are induced at acidic pH and in the presence of N-acetylglucosamine and CO2. Our data shows that pH has an impact on β-1,6-glucan, which suggests that there will be differential organization of this polysaccharide in the cell wall of opaque cells. As suggested by the reviewer, we will include analysis of opaque cells (and other C. albicans cell types) in future studies. 

With the exception of these major new avenues for this research, our revision can address each of the comments provided by the reviewers.

Recommendations for the authors

Reviewer #1 (Recommendations For The Authors):

Although the study is very interesting, there are some loose ends that need to be combined to strengthen the manuscript. For example, the immunological studies are rather preliminary and need at least some substantiation. Also, at this stage, the manuscript in some places remains a bit too descriptive and needs the elucidation of potential causalities.

Specifically: 

(1) As you showed, defects in chitin content led to a decrease in the cross-linking of β-glucans in the inner wall that corresponded to the effect of nikkomycin-treated C. albicans phenotype; conversely, an increase in chitin content led to more cross-linking of β-glucans as observed in the FKS1 mutant or in the presence of caspofungin. What is the mechanistic reason for these observations? 

On one hand, yeast cell wall chitin occurs in three forms: free and covalently linked to β-1,3-glucan or β-1,6-glucan; crosslinked β-glucan-chitin forms core fibrillar structure resistant to alkali. A decrease in the chitin content, therefore, affect β-glucan-chitin crosslinking thereby making β-glucan alkali-soluble. On the other hand, a decrease in the β-glucan content, as in FKS1 mutant or upon caspofungin treatment, results in increased cell wall chitin and β-glucan-chitin contents. A decrease in the β-1,3-glucan biosynthesis is associated with upregulation of CRH1 involved in the β-glucan-chitin crosslinking, which explains an increased β-glucan-chitin content in the FKS1 mutant or upon caspofungin treatment. We have included in this discussion in the revised manuscript (p14, lines 2-10).     

(2) The β-1,6-glucan biosynthesis is stimulated via a compensatory pathway when there is a defect in O- and N-linked cell wall mannan biosynthesis. Why? causality? Hypothesis?  

Two phenomena were observed related to β-1,6-glucan and mannan biosynthesis: 1) a defect in the elongation of N-mannan led to an increase in the β-1,6-glucan content; 2) a defect of O-mannan elongation resulted in the reduce size of β-1,6-glucan chains, however, increased their branching. These observations of our study suggest a global rescue program of the cell wall damage that could occur due to defect in one of the cell wall contents. We have discussed this in the revised manuscript (p14, last paragraph, p15 first paragraph). Moreover, β-1,3-glucan and chitin are synthesized by respective membrane bound synthases, and a defect in of their synthesis is compensated by the other. In line, although need to be validated for β-1,6-glucan, biosynthesis of mannan and β-1,6-glucan seem to initiate intracellularly. Therefore, possibility is that the defective mannan biosynthesis could be compensated by β-1,6-glucan biosynthesis, but need to be further validated experimentally. 

(3) You showed that the removal of β-1,6-glucan by periodate oxidation (AI-OxP) led to a significant decrease in the IL-8, IL-6, IL-1β, TNF-α, C5a, and IL-10 released, suggesting that their stimulation was in part β-1,6-glucan dependent. What is the consequence of the stimulation, e.g. better phagocytosis, etc.? This needs some more experiments, otherwise the data is purely descriptive, as the conclusion. Also, what do you want to show with the activation of the complement system? Is ß1,6-glucan detected by complement receptors? I think this is really a loose end. I think it is necessary to provide more data on this observation, which I think lacks control with serum lacking complement, this should then be moved to the main manuscript. 

In this study, our aim was to assess whether β-1,6-glucan acts as a pathogen-associated molecular pattern (PAMP) of C. albicans, and if yes, what is its immunostimulatory capacity/potential. Our data confirms that, indeed, β-1,6-glucan acts as a PAMP, and its removal significantly reduces the immunostimulatory capacity of the fibrillar core structure of the C. albicans cell wall. On the other hand, data provided in the revised manuscript (see updated Figure S14, discussion p13 lines 16-21) indicate that the human serum factors significantly enhance the immunostimulatory capacity of β1,6-glucan and that β-1,6-glucan interacts with the complement component C3b. However, addressing the role of β-1,6-glucan in phagocytosis using β-1,6-glucan deletion mutant will not be possible as the cell wall of this mutant is modified, and β-1,6-glucan is not the only cell wall component interacting with C3b. Alternate is to coat β-1,6-glucan on beads and use to study phagocytosis and identify immune receptors; however, these are beyond the scope of our present study/focus.      

(4) Also, you suggested that β-1,6-glucan and β-1,3-glucan stimulate innate immune cells in distinct ways. Please provide more data on this interesting suggestion. You can block the dectin-1 receptor for example or use dectin-1 deficient macrophages from mice. The part on the immune stimulation needs to be optimized. 

Stimulation of immune cells by pustulan (insoluble linear β-1,6-glucan) via a dectin-1independent pathway has been described previously (PMIDs: 18005717, 16371356) as discussed in the manuscript. Our preliminary data indicate that dectin-1 blocking on immune cells (using antidectin-1 antibodies) has no effect on the immunostimulatory potential of β-1,6-glucan, unlike AI and AI-OxP that showed significantly reduced cytokine secretion by the immune cells upon dectin-1 blocking. Deciphering the β-1,6-glucan recognition and its immunomodulatory pathways are underway, and will be the subject of our future study/manuscript.   

(5) β-1,6-glucan and mannan productions are coupled. What is the hypothesis? Is it due to the necessity of mannan residues in ß-1,6-glucan biosynthesis enzymes from the ER? Can that be experimentally proven? 

β-1,6-glucan and mannan synthesis should be coupled in two ways. First, as mentioned above (Response 2), defects in mannan elongation led to an alteration of β-1,6-glucan production. Second, early steps of N-glycosylation led to a strong reduction of β-1,6-glucan size and its cell wall content. However, we do not believe that the synthesis of N-glycan is required for the synthesis of an acceptor essential to β-1,6-glucan synthesis. Defect in N-mannan elongation led to a global cell wall remodeling as described above. Kre5, Rot2 and Cwh41 are part of the calnexin cycle involved in the control of N-glycoprotein folding in the ER, suggesting that some protein directly involved in the β-1,6-glucan synthesis required a folding quality control to be active. We modified our discussion, accordingly, highlighting these points (p14, last paragraph, p15 second paragraph).

(6) As PHR1 and PHR2 genes are strongly regulated by external pH, the compensatory differences described may be explained by pH-dependent regulation of β-1,6-glucan synthesis.' Please check. Also, could the pH regulation form the basis of e.g. differences you found for ß-1,6-glucan under different environmental conditions, i.e., growth on different carbon sources leads to different external pH values, as shown for many fungi?  

We agree that environmental pH is dependent on carbon source and pH varies during growth curve. To test the effect of pH we buffered the medium with 100 mM MOPS or MES. Clearly, Fig. 2 and S1 show that the pH has an effect on the cell wall composition and polymer exposure as previously described (PMID: 28542528). Here, we show that pH has an impact on the β-1,6-glucan size as well as its branching. However, in buffered medium, addition of organic acid (such as acetate, propionate, butyrate or lactate) had an impact on cell wall composition, showing that not only pH has an effect on cell wall composition. About _phr1_Δ/Δ and _phr2_Δ/Δ mutants, we believe that the difference in the cell wall composition observed between mutants is mainly due to the pH-dependent regulation, which we indicated in the discussion (p14, end of first paragraph).

Minor: 

(1) In Figure 7B: dynamism should be replaced by dynamic and in term is rather in terms.  

Modified as suggested.

(2) Replace molecular size with molecular mass when you give daltons. 

Molecular size has been replaced by molecular weight, when presented as daltons.

(3) Page 7: for explanation, please add that nikkomycin is a chitin biosynthesis inhibitor.   

As suggested, explained that nikkomycin is a chitin biosynthesis inhibitor.

Reviewer #2 (Recommendations For The Authors):

(1) I wondered if the increased chitin content of hyphae might reflect growth on the precursor GlcNAc. Have you tested hyphae that are induced in other ways? (2) Related to point 1, did you look at the relative abundance of yeast vs hyphae in the preparation? I wonder if yeast contamination might have reduced the extent of the composition changes observed. 

We used GlcNAc as hyphae inducer as: 1) in presence of GlcNAc, hyphae are produced without any yeast contamination; in this condition, we observed an increase in the chitin content, as described, in hyphae (PMID: 16423067); 2) we excluded using of serum, another condition inducing hyphal formation, as we could not control serum factors that may impact cell wall composition. We now indicate in the methods section that hyphae induced by GlcNAc were not contaminated by yeast (p17, line 3). 

(3) I recommend rephrasing the first sentence of the Figure 2 legend: "Cells were grown in liquid SD medium at 37oC at exponential phase under different growth conditions." The conditions varied extensively - stationary is not exponential; biofilm is probably not exponential. Also, the "D" in "SD" stands for dextrose, and the carbon source varied a good deal. Perhaps you could say: "Cells were grown in liquid synthetic medium at 37oC under different growth conditions, as specified in Methods." 

Sentences have been rephrased.  

(4) Figure 7b has a typo: "dependant" for "dependent".

Typo-error has been corrected.

Reviewer #3 (Recommendations For The Authors):

To explore the biochemical composition of the cell wall, the authors fractionated the wall component into three categories based on polymer properties and reticulations: sodium-dodecyl-sulphate-βmercaptoethanol (SDS-β-ME) extract, alkali-insoluble (AI), and alkali-soluble (AS) fractions, and they developed several independent methods to distinguish between β-1,3-glucans and β-1,6-glucans. The composition and surface exposure of fungal cell wall polymers is known to depend on environmental growth conditions. It was shown that the cell wall of C. albicans hyphae increased chitin content (10% vs. 3%) and decreased β-1,6-glucan (18% vs. 23%) and mannan (13% vs. 20%) compared to the yeast form, and the reduced β-1,6-glucan content was associated with a smaller β1,6-glucan size (43 vs. 58 kDa), suggesting that both the content and structure of β-1,6-glucan are regulated during growth and cellular morphogenesis. Similar behavior was observed when exposing cells to acid and neutral medium pH. The most significant cell wall alteration occurred in a lactatecontaining medium, which led to a sharp reduction in structural core polysaccharides: chitin (-43%), β-1,3-glucan (-48%), and β-1,6-glucan (-72%). This reduction aligns with the previously observed decreases in inner cell wall layer thickness. As expected, the authors found that modulating chitin content genetically (chs3Δ/Δ knockout mutant) led to an increase of both β-1,3-glucan and β-1,6glucan. An increase in chitin content following genetic alteration of FKS genes impacting glucan synthase or after exposure to the echinocandin caspofungin led to enhanced cross-linking of βglucans. A slight increase in the β-1,3-glucan branching was also observed in the mnt1/mnt2Δ/Δ double mutant, suggesting that β-1,6-glucan and mannan synthesis may be coupled.

- This effect is not that pronounced, and the relationship appears somewhat overstated and may reflect an indirect interaction. The authors should address accordingly. 

We agree that this sentence was overstated. To make it clearer and less pronounced, we divided this sentence into to two with less pronounced statements (p8, line 34).

The genetics of β-1,6-glucan biosynthesis appear complex and a figure describing putative roles for specific genes would be beneficial. For example, KRE6 is a glucosyl hydrolase required for beta1,6-glucan biosynthesis.

- It would be valuable to better understand the overall biosynthetic process. Please elaborate more in a figure. 

Although proteins/enzymatic activities directly involved in the β-1,6-glucan biosynthesis have not yet been identified, as suggested by this reviewer, we included a schematic representation of this process based on our hypothesis (Figure S15, and p15 lines 17-22 in revised manuscript), indicating the possible involvement of Kre6p.  

The deletion of KRE6 homologs, essential for β-1,6-glucan biosynthesis, resulted in the absence of β-1,6-glucan production, and significant structural alterations of the cell wall. This result nicely confirms the important role of β-1,6-glucan in regulating cell wall homeostasis. The absence of β1,6-glucan was associated with increased (mutant v. WT) chitin content (9.5% vs. 2.5%) and highly branched β- β-1,6-glucan 1,3-glucan (48% vs. 20%). TEM ultrastructure studies nicely showed the change in cell wall overall architecture. From a drug discovery perspective, since the blockade of β1,6-glucan did not block growth, it may have more value as a potential virulence target. This would be valuable but needs to be assessed in animal model challenge competition experiments.

- The authors may want to elaborate more. 

We agree and modified “antifungal target” as “potential virulence target”.

It is well known that β-1,3-glucan, mannan, and chitin function serve as PAMPs, which induce immune responses. The role of β-1,6-glucan as a PAMP is not well understood, and the authors provide evidence that different cell wall extracted fractions with enriched constituents induce immune responses invoking cytokines, chemokines, and acute phase proteins, as well as the complement system. While this data clearly shows that β-1,6-glucan is immunologically active and potentially important for host-pathogen interactions, the analysis is preliminary and falls short of making this case. 

- This is a critical point in getting at the potential host signaling of β-1,6-glucan contained in the cell wall or shed by the cell (is this known?)

- This analysis would be bolstered significantly by examining stimulation relative to other cell wall components, and most importantly, whole cell modulation of β-1,6-glucan exposure for immune presentation, and not just unnatural concentrated extracts. This can be readily accomplished with the various mutants in hand, as well as after exposure to various antifungal agents echinocandins and nikkomycins) (see Hohl et al. 2008 JID). Additional validation would benefit from animal model studies to examine in vivo immune modulation.

We agree with the reviewer. However, the main focus of our present work was to study the organization and dynamics of C. albicans cell wall β-1,6-glucan, and to explore its possible role as pathogen-associated molecular pattern (PAMP). Our study indicates that, indeed, β-1,6-glucan acts as a PAMP with immunostimulatory potential. As pointed by this reviewer, and similar to β-1,3glucans, the exposure of β-1,6-glucan is probably a key point in immune response. However, this investigation beyond the scope of this study, underway and will be presented in our future work.

- The Discussion would also benefit from an analysis of how β-1,6-glucan in Aspergillus fumigatus, which was largely elucidated by the same primary authors. 

To our knowledge, β-1,6-glucan has never been identified, either by chemical analysis (PMID: 10869365; PMID: 36836270) or solid-state NMR (PMID: 34732740), in the cell wall of A. fumigatus, although a homolog of KRE6 is present in A. fumigatus but with unknown function.

o reduce or be reduced in size or importance 減少，減小，降低

• I don't want to diminish her achievements, but she did have a lot of help. 我不想貶低她的成就，但她確實得到了很多幫助。

Narrative citation with the inclusion of a URL in parenthesis that links to the source article.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This work sets out to elucidate mechanistic intricacies in inflammatory responses in pneumonia in the context of the aging process (Terc deficiency - telomerase functionality).

Strengths:

Very interesting, conceptually speaking, approach that is by all means worth pursuing. An overall proper approach to the posited aim.

We want to thank the reviewer for taking the time to review our manuscript and for providing positive feedback regarding our research question.  

Weaknesses:

The work is heavily underpowered and may have statistical deficits. This precludes it in its current state from drawing unequivocal conclusions.

Thank you for this essential and valuable comment. We fully accept that the small sample size of the Tercko/ko mice is a major limitation of our study and transparently discuss this in our manuscript.  However, due to Animal Welfare regulations, only a reduced number of mice were approved because of the strong burden of disease. Consequently, only three non-infected and five infected mice were available to us. This reduced number of mice presents a clear limitation to our study. However, due to ethical considerations related to animal welfare and sustainability, as well as compliance with German animal welfare regulations, it is not possible to obtain additional Tercko/ko mice to increase the dataset.

The animal studies are an important aspect of our study; however, our hypothesis was also investigated at multiple levels, including in an in vitro co-culture model (Figure 5), to ensure comprehensive analysis. Thus, we clearly demonstrated that S. aureus pneumonia in Tercko/ko mice leads to a more severe phenotype, orchestrated by the dysregulation of both innate and adaptive immune response.

Reviewer #2 (Public Review):

Summary:

The authors demonstrate heightened susceptibility of Terc-KO mice to S. aureus-induced pneumonia, perform gene expression analysis from the infected lungs, find an elevated inflammatory (NLRP3) signature in some Terc-KO but not control mice, and some reduction in T cell signatures. Based on that, They conclude that disregulated inflammation and T-cell dysfunction play a major role in these phenomena.

Strengths:

The strengths of the work include a problem not previously addressed (the role of the Terc component of the telomerase complex) in certain aspects of resistance to bacterial infection and innate (and maybe adaptive) immune function.

We would like to thank the reviewer for the positive feedback regarding our aim to investigate the impact of Terc deletion on the pulmonary immune response to S. aureus.  

Weaknesses:

The weaknesses outweigh the strengths, dominantly because conclusions are plagued by flaws in experimental design, by lack of rigorous controls, and by incomplete and inadequate approaches to testing immune function. These weaknesses are as follows

(1) Terc-KO mice are a genomic knockout model, and therefore the authors need to carefully consider the impact of this KO on a wide range of tissues. This, however, is not the case. There are no attempts to perform cell transfers or use irradiation chimera or crosses that would be informative.

We thank the reviewer for bringing up this important point. The aim of our study, however; was to investigate the impact of Terc deletion in the lung and on the response to bacterial pneumonia, rather than to provide a comprehensive characterization of the Tercko/ko model itself. This characterization of different tissues and cell types has already been conducted by previous studies. For instance, studies that characterize the general phenotype of the model (Herrera et al., 1999; Lee et al., 1998; Rudolph et al., 1999) but also investigations that shed light on the impact of Terc deletion on specific cell types such as microglia (Khan et al., 2015) or T cells (Matthe et al., 2022). The impact of Terc deletion on T cells is also discussed in our manuscript in lines 89 to 105. Furthermore, a section about the general phenotype of the Terc deletion model is included in the introduction in lines 126 to 138. Thus we discussed the relevant literature regarding Tercko/ko mice in our manuscript and attempted to provide a more in-depth characterization of the lung by investigating the inflammatory response to infection as well as changes in the gene expression (Figure 2-4).  

(2) Throughout the manuscript the authors invoke the role of telomere shortening in aging, and according to them, their Terc-KO mice should be one potential model for aging. Yet the authors consistently describe major differences between young Terc-KO and naturally aging old mice, with no discussion of the implications. This further confuses the biological significance of this work as presented.

Thank you for mentioning this relevant point. We want to apologize for the confusion regarding this matter. While Tercko/ko mice are a well-established model for premature aging, these effects become more apparent with increasing generations (G) and thus, G5 and 6 mice are the most affected by Terc deletion (Lee et al., 1998; Wong et al., 2008).

Thus, while Tercko/ko mice are a common model for premature aging, this accelerated aging phenotype is predominantly apparent in later-generation Tercko/ko (G5 and 6) or aged Tercko/ko mice (Lee et al., 1998; Wong et al., 2008). Since the aim of this study was to analyze the impact of Terc deletion on the lung and its immune response to bacterial infections instead of the impact of telomere shortening and telomerase dysfunction, young G3 Tercko/ko mice (8 weeks) were used in this study. This is also mentioned in the lines 131-134. In this study, Tercko/ko mice were used not as a model of aging, but rather as a model specifically for Terc deletion. The old WT mice function as a control cohort to observe possible common but also deviating effects between aging and Terc deletion. In our sequencing data, we observe that uninfected young WT mice are very similar to uninfected Tercko/ko mice. Other studies have also reported this lack of major differences between uninfected WT and Tercko/ko mice in the G3 knockout mice (Kang et al., 2018). Conversely, uninfected young WT and Tercko/ko mice exhibited great differences, for instance, regarding the numbers of differentially expressed genes (Supplemental Figure 1H). Thus, differences between naturally aged mice and young G3 Tercko/ko mice are not surprising. To clarify this aspect we reconstructed the paragraph discussing the Tercko/ko mice (lines 126-134). Additionally we added a paragraph explaining the purpose of the naturally aged mice to the lines 134 to 138:

“As control cohort age-matched young WT mice were utilized. To investigate whether Terc deletion, beyond critical telomere shortening, impacts the pulmonary immune response, we used young Tercko/ko mice. Additionally, naturally aged mice (2 years old) were infected to explore the potential link to a fully developed aging phenotype.”

(3) Related to #2, group design for comparisons lacks a clear rationale. The authors stipulate that TercKO will mimic natural aging, but in fact, the only significant differences seen between groups in susceptibility to S. aureus are, contrary to the authors' expectation, between young Terc-KO and naturally old mice (Figures 1A and B, no difference between young Terc-KO and young wt); or there are no significant differences at all between groups (Figures 1, C, D,).

We thank the reviewer for this essential comment. As mentioned above the Tercko/ko mice in this study are not selected to model natural aging. To model telomerase dysfunction and accelerated aging selection of later generation or aged Tercko/ko mice would have been more suitable. 

The lack of statistical significance in some figures is likely due to the heterogeneity of disease phenotype of S. aureus infection in mice, which is a limitation of our study that we discuss in our discussion section in lines 576-582. The phenotype of S. aureus infection can vary greatly within a mouse population, highlighting the limitations of mice as a model for S. aureus infections. To account for this heterogeneity we divided the infected Tercko/ko mice cohort into different degrees of severity based on the clinical score and the presence of bacteria in organs other than the lung (mice with systemic infection). 

Despite the heterogeneity especially within the Tercko/ko mice cohort the differences between the knockout and young as well as old WT mice were striking. Including the fatal infections, 80% of the Tercko/ko mice had a severe course of disease, while none of the WT mice displayed a severe course (Figure 1A, B and Supplemental Figure 1A, B). This hints towards a clear role of Terc in the response to S. aureus infection in mice. Thus while in some figures the differences are not significant, strong trends towards a more severe phenotype of S. aureus infection in the Tercko/ko mice regarding bacterial load, score and inflammatory response could be observed in our study. 

Another example of inadequate group design is when the authors begin dividing their Terc-KO groups by clinical score into animals with or without "systemic infection" (the condition where a bacterium spreads uncontrollably across the many organs and via blood, which should be properly called sepsis), and then compare this sepsis group to other groups (Supplementary Figures 1G; Figure 2; lines 374-376 and 389391). This gives them significant differences in several figures, but because they did not clearly indicate where they applied this stratification in the figure legends, the data are somewhat confusing. Most importantly, methodologically it is highly inappropriate to compare one mouse with sepsis to another one without. If Terc-KO mice with sepsis are a comparator group, then their controls have to be wild-type mice with sepsis, who are dealing with the same high bacterial load across the body and are presumably forced to deploy the same set of immune defenses.

We sincerely appreciate the significant time and effort you have invested in reviewing our manuscript. However, with all due respect, we must point out that the definition of sepsis you have referenced is considered outdated. According to the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3), sepsis is defined as "a life-threatening organ dysfunction caused by a dysregulated host response to infection" (Marvin Singer, 2016, JAMA). Given this fundamental misunderstanding of our findings, we find the comment regarding the inadequacy of our groups to be both dismissive and lacking in scientific merit. We would like to emphasize that the group size used in our study is consistent with accepted standards in infection research. We strongly reject any insinuations of inadequacy that have been repeatedly mentioned throughout the review.

In order to provide a nuanced investigation of disease severity in Tercko/ko mice, we added the term “systemic infection” to the figures whenever the mice were divided into groups of mice with and without systemic infection. This is the case for Figure 2A and Supplemental Figure 1C-E. The division into mice with and without systemic infection is also mentioned in the figure legend of Figure 2A in lines 932 to 935 and for Supplemental Figure 1 in lines 1052-1053. We agree that Supplemental Figure 1G is somewhat confusing as the mice with systemic infection are highlighted in this graph but not included as a separate group within our sequencing analysis. We added a sentence to the figure legend clarifying this (lines 1042-1044):

“Nevertheless, the infected Tercko/ko mice were considered one group for the expression analysis and not split into separate groups for the subsequent analysis.”

Additionally, we revised the section regarding this grouping in different degrees of severity in our Material and Methods section to clarify that this division was only performed for specific analysis (line 191):

“…for the indicated analysis.”

Furthermore, the mice which were classified as systemically infected mice were not septic mice, as mentioned above. Those mice were classified by us as systemically infected based on their clinical score and the presence of bacteria in other organs than the lung as stated in the lines 188-191 and 377-381. Bacteremia is a symptom of very severe cases of hospital-acquired pneumonia with a very high mortality (De la Calle et al., 2016).

Therefore, the systemically infected mice or rather mice with bacteremia display an especially severe pneumonia phenotype, which is distinct from sepsis. The presence of this symptom in our Tercko/ko mice further highlights the clinical relevance of our study. This aspect was added to the manuscript in the lines 568-570.

“The detection of bacteria in extra pulmonary organs is of particular interest, as bacteremia is a symptom of severe pneumonia and is associated with high mortality (De la Calle et al., 2016).”

(4) The authors conclude that disregulated inflammation and T-cell dysfunction play a major role in S. aureus susceptibility. This may or may not be an important observation, because many KO mice are abnormal for a variety of reasons, and until such reasons are mechanistically dissected, the physiological importance of the observation will remain unclear.

Two points are important here. First, there is no natural counterpart to a Terc-KO, which is a complete loss of a key non-enzymatic component of the telomerase complex starting in utero. 

Second, the authors truly did not examine the key basic features of their model, including the features of basic and induced inflammatory and immune responses. This analysis could be done either using model antigens in adjuvants, defined innate immune stimuli (e.g. TLR, RLR, or NLR agonists), or microbial challenge. The only data provided along these lines are the baseline frequencies of total T cells in the spleen of the three groups of mice examined (not statistically significant, Figure 4B). We do not know if the composition of naïve to memory T cell subsets may have been different, and more importantly, we have no data to evaluate whether recruitment of the immune response (including T cells) to the lung upon microbial challenge is similar or different. So, what are the numbers and percentages of T cells and alveolar macrophages in the lung following S. aureus challenge and are they even comparable or are there issues in mobilizing the T cell response to the site of infection? If, for example, Terc-KO mice do not mobilize enough T cells to the lung during infection, that would explain the paucity in many T-cellassociated genes in their transcriptomic set that the authors report. That in turn may not mean dysfunction of T cells but potentially a whole different set of defects in coordinating the response in Terc-KO mice.

We thank the reviewer for highlighting these important aspects. Regarding the first point, indeed there is no naturally occurring deletion of Terc in humans. However, studies reported reduced expression of Terc and Tert in the tissues of aged mice and rats (Tarry-Adkins et al., 2021; Zhang et al., 2018). Terc itself has been found to have several important immunomodulatory functions such as the activation of the NFκB or PI3-kinase pathway (Liu et al., 2019; Wu et al., 2022). As those aforementioned pathways are relevant for the immune response to S. aureus infections, the authors were interested in exploring the impact of Terc deletion on the pulmonary immune response. The potential immunomodulatory functions of Terc are discussed in lines 106-121. To further clarify our rationale we added a sentence to the introduction in lines 121-125.

“Interestingly, downregulation of Terc and Tert expression in tissues of aged mice and rats has been found (Tarry-Adkins, Aiken, Dearden, Fernandez-Twinn, & Ozanne, 2021; Zhang et al., 2018). Therefore, as a potential immunomodulatory factor reduced Terc expression could be connected to agerelated pathologies.”

Regarding the second point, as we focused on the effect of Terc deletion in the lung and its role in S. aureus infection, we investigated inflammatory and immune response parameters relevant to this setting. For instance, inflammation parameters in the lungs of all three mice cohorts were measured to investigate differences in the inflammatory response in the non-infected and infected mice (Figure 2A). Those measurements showed no baseline difference in key inflammatory parameters between young WT and Tercko/ko mice, which is consistent with previous findings (Kang et al., 2018). The inflammatory response to infection with S. aureus in the Tercko/ko mice cohort differed significantly from the other cohorts (Figure 2A), hinting towards a dysregulated inflammatory response due to Terc deletion. Furthermore, we investigated general immune cell frequencies such as dendritic cells, macrophages, and B cells in the spleen of all three mice cohorts to gather a baseline understanding of the general immune cell populations. In our manuscript only total T cell frequencies were included due to its relevance for our data regarding T cells (Figure 4B). This data could show that there was no difference of total amount of T cells in the spleen of all three mice cohorts. For a more detailed insight into our analysis we added the frequencies of the other immune cell populations analyzed in the spleen as a Supplemental Figure 3B-F. Additionally, a figure legend for the graphs was added to lines 1075-1094.

Therefore, while we did not analyze baseline frequencies of specific populations of T cells, we analyzed and characterized the inflammatory and immune response of our model in a way relevant to our research question. 

The differences observed in T cell marker and TCR gene expression was also partly present between the uninfected and infected Tercko/ko mice such as the complete absence of CD247 expression in infected Tercko/ko, which is however expressed in uninfected mice of this cohort (Figure 4A, C and D). Thus, this effect cannot be solely attributed to an inadequate mobilization of T cells to the lung after infectious challenge. However, we agree that a more detailed insight into recruited immune cells to the lung or frequencies of different T cell populations could contribute to a better understanding of the proposed mechanism and would be an interesting experiment to conduct in further studies. We accept this as a limitation of our study and included it in our discussion section in lines 719-723:

“As total CD4+ T cells were analyzed in this study, it would be useful to investigate specific T cell populations such as memory and effector T cells to elucidate the potential mechanism leading to T cell dysfunctionality in further detail. Additionally, analysis of differences in immune cell recruitment to the lungs between young WT and Tercko/ko mice would be relevant.”

(5) Related to that, immunological analysis is also inadequate. First, the authors pull signatures from the total lung tissue, which is both imprecise and potentially skewed by differences, not in gene expression but in types of cells present and/or their abundance, a feature known to be affected by aging and perhaps by Terc deficiency during infection. Second, to draw any conclusions about immune responses, the authors would have to track antigen-specific T cells, which is possible for a wide range of microbial pathogens using peptide-MHC multimers. This would allow highly precise analysis of phenomena the authors are trying to conclude about. Moreover, it would allow them to confirm their gene expression data in populations of physiological interest

We thank the reviewer for highlighting this important and relevant point. In our study, we aimed to investigate the role of Terc expression in modulating inflammation and the immune response to S. aureus infection in the lung. To address this, we examined the overall impact of age, genotype, and infection on lung inflammation and gene expression. Therefore, sequencing of total lung tissue was essential for addressing the research question posed. Our findings demonstrate that Tercko/ko mice exhibit a more severe phenotype following S. aureus infection, characterized by an increased bacterial load and heightened lung inflammation (Figures 1 and 2). Furthermore, our data suggest that Terc plays a role in regulating inflammation through activation of the NLRP3 inflammasome, along with the dysregulation of several T cell marker genes (Figures 2, 4, and 5). However, this study lacks a detailed analysis of distinct T cell populations, including antigen-specific T cells, as noted earlier. Investigating these aspects in future studies would be valuable to validate and expand upon our findings. We have incorporated these suggestions into the discussion section (lines 719-723)

“As total CD4+ T cells were analyzed in this study, it would be useful to investigate specific T cell populations such as memory and effector T cells to elucidate the potential mechanism leading to T cell dysfunctionality in further detail. Additionally, analysis of differences in immune cell recruitment to the lungs between young WT and Tercko/ko mice would be relevant.”

Nevertheless, our study provides first evidence of a potential connection between T cell functionality and Terc expression.  

Third, the authors co-incubate AM and T cells with S. aureus. There is no information here about the phenotype of T cells used. Were they naïve, and how many S. aureus-specific T cells did they contain? Or were they a mix of different cell types, which we know will change with aging (fewer naïve and many more memory cells of different flavors), and maybe even with a Terc-KO? Naïve T cells do not interact with AM; only effector and memory cells would be able to do so, once they have been primed by contact with dendritic cells bringing antigen into the lymphoid tissues, so it is unclear what the authors are modeling here. Mature primed effector T cells would go to the lung and would interact with AM, but it is almost certain that the authors did not generate these cells for their experiment (or at least nothing like that was described in the methods or the text).

Thank you for bringing up this important question. For the co-cultivation experiment of T cells and alveolar macrophages, total CD4+ T cells of both young WT and Tercko/ko were used. We did not select for a specific population of T cells. Our sequencing data indicated the complete downregulation of CD247 expression, which is an important part of the T cell receptor, in the lungs of infected Tercko/ko mice (Figure 4A, C and D). Given that this factor is downregulated under chronic inflammatory conditions, we investigated the impact of the inflammatory response in alveolar macrophages on the expression of various T cell-derived cytokines, as well as CD247 expression (Figure 5D, E) (Dexiu et al., 2022). This aspect is also highlighted in the discussion in lines 622-636. Therefore, a co-cultivation model of T cells and alveolar macrophages was established and confronted with heat-killed S. aureus to elicit an inflammatory response of the macrophages. To emphasize this purpose, we have revised our statement about the model setup in lines 516-518 of the manuscript: 

“An overactive inflammatory response could be a potential explanation for the dysregulated TCR signaling.”

The authors hope this will clarify the intent behind the model setup.

(6) Overall, the authors began to address the role of Terc in bacterial susceptibility, but to what extent that specifically involves inflammation and macrophages, T cell immunity, or aging remains unclear at present.

We thank the reviewer for the helpful and relevant comments. The authors accept the limitations of the presented study such as the reduced number of Tercko/ko mice and the limitations of murine models for S. aureus infection itself and discuss those in the discussion section in the lines 558-560; 576-582; 688-690 and 719-725. However, we hope that our responses have provided sufficient evidence to convince the reviewer that our data supports a clear role for Terc expression in regulating the immune response to bacterial infections, particularly with respect to inflammation and its potential connection to T cell functionality.  

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

The good element first:

I read this paper with genuine interest and applaud the authors for investigating the posited question. I consider it by all means scientifically relevant in the context of physiological/pathophysiological aging and reaction to a disease (here pneumonia). The Terc deletion model looks very appropriate for the question and the methodology is very advanced/in-depth. The data flow/selection of endpoints and assays is very logical to me. Moreover, I like the breakdown of pneumonia into varying levels of severity.

We thank the reviewer for their time and effort taken to revise our manuscript. Additionally, we are grateful to receive your positive feedback regarding our study design and research question.

The weaknesses:

(1) I cannot help but notice that the study is heavily underpowered. As such, it is inadmissible. The key reason is that it is the first of its kind and seminal findings must be strongly propped by the evidence. It is apparent to me that the data scatter presented in the figures tends to be abnormally distributed (e.g. obvious bimodal distribution in some groups). Therefore, the presented comparisons (even if stat. sign) can be heavily misleading in terms of: i) the true magnitude of the observed effects and ii) possible type 2 error in some cases of p value >0.05. Solution: repeat the study to ensure reasonable power/reliability. This will also make it stronger as it will immediately demonstrate its reproducibility (or lack of it).

Thank you for bringing up this extremely relevant point. We acknowledge the issue of the small sample size of Tercko/ko mice as a major limitation of our study. This limitation is also included in our discussion section in the lines 558-560. Thus we fully agree with this limitation and transparently discuss this in our manuscript. However, due to the strict German animal welfare regulations it is not possible to obtain more Tercko/ko mice, as mentioned above. Furthermore, since fatal infections occurred in the Tercko/ko mice cohort we had a reduced number of mice available. 

However, the differences between the Tercko/ko and WT mice were striking. Including the fatal infections 80% of the Tercko/ko mice had a severe course of disease, while none of the WT mice displayed a severe course. This hints towards a clear role of Terc in the response to S. aureus infection in mice.  

(2) In the stat analysis section of M&Ms, the authors feature only 1 sentence. This cannot be. A detailed stats workup needs to be included there. This is very much related to the above weakness; e.g. it is impossible to test for normality (to choose an appropriate post-hoc test) with n=3. Back to square one: study underpowered.

We thank the reviewer for highlighting this important aspect. We carefully revised the method section in lines 357-360 to include all relevant information: 

“Data are presented as mean ± SD, or as median with interquartile range for violin and box plots, with up to four levels of statistical significance indicated. P-values were calculated using Kruskal-Wallis test. Individual replicates are represented as single data points.”

(3) Pneumonia severity. While I noted that as a strength, I also note it as weakness here. It looks to me like the authors stopped halfway with this. I totally support testing a biological effect(s) such as the one investigated here across a spectrum of a given disease severity. The authors mention that they had various severity phenotypes produced in their model but this is not visible in the data figs. I strongly suggest including that as well; i.e., to study the posited question in the severe and mild pneumonia phenotype. This is a very smart path and previous preclinical research clearly demonstrated that this severe/mild distinction is very relevant in the context of the observed responses (their presence/absence, longevity, dynamics, etc). I realize this is challenging, thus, I would probably use this approach in the Terc k/o model as sort of a calibrator to see whether the exacerbation observed in the current setup (severe?) will be also present in a mild pneumonia phenotype. S. aureus can be effectively titrated to produce pneumonia of varying severity.

We thank the reviewer for bringing up this relevant point. 

In our study, we could observe heterogeneity within the infected Tercko/ko cohort. Therefore as pointed out by the reviewer we assigned different degrees of severity to those groups based on clinical scores, the fatal outcome of the disease (fatal subgroup), and the presence of bacteria in organs other than the lungs (systemic infection subgroup) as stated in our materials and methods part in the lines 188-191 (Supplemental Figure 1A and B). Moreover, we highlighted this difference in a number of our figures. For example, when categorizing the mice into groups with and without systemic infection, we noticed that the mice with systemic infection demonstrated a higher bacterial load, significant body weight loss, and increased lung weight (see Supplemental Figure 1C-E). Interestingly, the two mice with systemic infection clustered separately from the other mice, indicating that the mice with systemic infection are transcriptomically distinct from the other mice cohorts (Supplemental Figure 1G). Additionally, the inflammatory response was exclusively elevated in the lungs of mice with systemic infection (Figure 2C). Thus, we included this distinction in several figures and attempted to study the differences between those subgroups but also their similarities. For instance, we could observe that some changes in the transcriptome were present in all three infected Tercko/ko mice such as the complete absence of CD247 expression at 24 hpi (Figure 4D). This distinction therefore provided a more detailed insight into the underlying mechanisms of disease severity in Tercko/ko mice and is lacking in other studies. We agree with the reviewer, that a study investigating mild and severe pneumonia phenotypes would be clinically relevant. However, as noted above, due to ethical considerations related to animal welfare and sustainability, as well as compliance with German animal welfare regulations, it is not possible to obtain additional Tercko/ko mice to carry out the proposed experiment. 

(4) Please read ARRIVE guidelines and note the relevant info in M&Ms as ARRIVE guidelines point out.

Thank you for emphasizing this crucial aspect. We revised our materials and methods section according to the ARRIVE guidelines (lines 179-206).

“Tercko/ko mice aged 8 weeks, were used for infection studies (n = 8; non-infected = 3; infected = 5). Female young WT (age 8 weeks) and old WT (age 24 months) C57Bl/6 mice (both n = 10; non-infected = 5; infected = 5) were purchased from Janvier Labs (Le Genest-Saint-Isle, France). All infected mouse cohorts were compared to their respective non-infected controls, as well as to the infected groups from other cohorts. Additionally, comparisons were made between the non-infected cohorts across all groups.

All mice were anesthetized with 2% isoflurane before intranasal infection with S. aureus USA300 (1x108 CFU/20µl) per mouse. After 24 hours, the mice were weighed and scored as previously described (Hornung et al., 2023). Infected Tercko/ko mice were grouped into different degrees of severity based on their clinical score, fatal outcome of the disease (fatal) and the presence of bacteria in organs other than the lung (systemic infection) for the indicated analysis. Mice with fatal infections were excluded from subsequent analyses, with only their final scores being reported. The mice were sacrificed via injection of an overdose of xylazine/ketamine and bleeding of axillary artery after 24 hpi. BAL was collected by instillation and subsequent retrieval of PBS into the lungs. Serum and organs were collected. Bacterial load in the BAL, kidney and liver was determined by plating of serially diluted sample as described above. For this organs were previously homogenized in the appropriate volume of PBS. Gene expression was analyzed in the right superior lung lobe. Lobes were therefore homogenized in the appropriate amount of TriZol LS reagent (Thermo Fisher Scientific, Waltham, MA, US) prior to RNA extraction. The left lung lobe was embedded into Tissue Tek O.C.T. (science services, Munich, Germany) and stored at 80°C until further processing for histological analysis. Cytokine measurements were performed using the right inferior lung lobe. Lobes were previously homogenized in the appropriate volume of PBS. Remaining organs were stored at -80°C until further usage. Mouse studies were conducted without the use of randomization or blinding.“

(5) There are also some other descriptive deficits but they are of a much smaller caliber so I do not list them.

We thank the reviewer for their valuable and insightful suggestions for improving our manuscript. We hope that our responses and the corresponding revisions address these suggestions satisfactorily.

Concluding: the investigative idea is great/interesting and the methodological flow is adequate but the low power makes this study of low reliability in its current form. I strongly urge the authors to walk the extra mile with this work to make it comprehensive and reliable. Best of luck!

Reviewer #2 (Recommendations For The Authors):

(1) Many legends are uninformative and do not contain critical information about the experiments. For example, Figure 2A with cytokine measurements (in lung homogenates?) is likely showing data from an ELISA or Luminex test, but there is no mention of that in the legend. It stands next to Figure 2B, which is a gene expression map, again, likely from the lung (prepared how, normalized how, etc?) lacking even the most basic information. Further, Figure 2D has no information on the meaning/effect size of gene ratios on the x-axis. Figures 3 and 4 are presumably the subsets of their transcriptome data set (whole lung, harvested on d ?? post-infection), but that is just a guess on my part. Even in the main text, the timing and the controls for the transcriptomic study are not stated (ln. 398 and onwards). The authors really need to revise the figure legends and provide all the details that an average reader would need to be able to interpret the data.

We thank the reviewer for bringing up this important point. The figure legends of all figures including supplemental figures were revised to ensure they include all relevant data necessary for accurate interpretation of the graphs. Additionally, we clarified the sequenced samples in lines 427-429:

“We performed mRNA sequencing of the murine lung tissue of infected and non-infected mice at 24 hpi to elucidate potential differentially expressed genes that contribute to the more severe illness of Tercko/ko mice.”

(2) Telomere shortening affects differentially different cells and its role in aging is nuanced - different in mesenchymal cells with no telomerase induction, in non-replicating cells, and in hematopoietic cells that can readily induce telomerase. The authors should be mindful of that in setting up their introduction and discussion.

Thank you for mentioning this essential aspect. We revised our introduction and discussion to reflect the nuanced role of telomerase shortening in different tissues (lines 83-92 and 690-695):

“Telomerase activity is restricted to specific tissues and cell types, largely dependent on the expression of Tert. While Tert is highly expressed in stem cells, progenitor cells, and germline cells, its expression is minimal in most differentiated cells (Chakravarti, LaBella, & DePinho, 2021). Consequently, the impact of telomerase dysfunction on tissues varies according to their self-renewal rate. (Chakravarti et al., 2021). One important aspect of telomere dysfunction is the impact of telomere shortening on the immune system as well as the hematopoietic system. Tissues or organ systems that are highly replicative, such as the skin or the hematopoietic system, are affected first by telomere shortening (Chakravarti et al., 2021).”

“It is important to note that telomere shortening has a significant impact on the immune system. Although young Tercko/ko mice were used in this study, telomere shortening is still likely to be a contributing factor. Therefore, further experiments investigating the role of T cell senescence in this model should therefore be conducted.”

(3) Syntax and formulations need to be improved and made more scientifically precise in several spots. Specifically, in 62-63, the authors say that the aged immune system "is also discussed to be more irritable", please change to reflect the common notion that the reaction to infection is dysregulated; in many cases inflammation itself is initially blunted, misdirected, and of different type (e.g. for viruses, the key IFN-I responses are not increased but decreased). In lines 114-117, presumably, the two sentences were supposed to be connected by a comma, although some editing for clarity is probably needed regardless. Line 252, please change "unspecific" to "non-specific". Line 264, please capitalize German.

We thank the reviewer for bringing these important points to our attention. We revised our introduction regarding the aged immune response in lines 61-69:

“Age-related dysregulation of the immune response is also characterized by inflammaging, defined as the presence of elevated levels of pro-inflammatory cytokines in the absence of an obvious inflammatory trigger (Franceschi et al., 2000; Mogilenko, Shchukina, & Artyomov, 2022). Additionally, immune cells, such as macrophages, exhibit an activated state that alters their response to infection (Canan et al., 2014). In contrast, the immune response of macrophages to infectious challenges has been shown to be initially impaired in aged mice (Boe, Boule, & Kovacs, 2017). Thus aging is a relevant factor impacting the pulmonary immune response.”

Sentences were edited to provide more clarity in lines 131-134:

“Although G3 Tercko/ko mice with shortened telomeres were used in this study, they were infected at a young age (8 weeks). This approach allowed for the investigation of Terc deletion effects rather than telomere dysfunction.”

“Unspecific was changed to “non-specific” in line 282 and “German” was capitalized in line 293 and 558.

We appreciate and thank you for your time spent processing this manuscript and look forward to your response.

References

De la Calle, C., Morata, L., Cobos-Trigueros, N., Martinez, J. A., Cardozo, C., Mensa, J., & Soriano, A. (2016). Staphylococcus aureus bacteremic pneumonia. European Journal of Clinical Microbiology & Infectious Diseases, 35(3), 497-502. https://doi.org/10.1007/s10096-015-2566-8  

Dexiu, C., Xianying, L., Yingchun, H., & Jiafu, L. (2022). Advances in CD247. Scand J Immunol, 96(1), e13170. https://doi.org/10.1111/sji.13170  

Herrera, E., Samper, E., Martín-Caballero, J., Flores, J. M., Lee, H. W., & Blasco, M. A. (1999). Disease

states associated with telomerase deficiency appear earlier in mice with short telomeres. Embo j, 18(11), 2950-2960. https://doi.org/10.1093/emboj/18.11.2950  

Hornung, F., Schulz, L., Köse-Vogel, N., Häder, A., Grießhammer, J., Wittschieber, D., Autsch, A., Ehrhardt, C., Mall, G., Löffler, B., & Deinhardt-Emmer, S. (2023). Thoracic adipose tissue contributes to severe virus infection of the lung. International Journal of Obesity, 47(11), 10881099. https://doi.org/10.1038/s41366-023-01362-w  

Kang, Y., Zhang, H., Zhao, Y., Wang, Y., Wang, W., He, Y., Zhang, W., Zhang, W., Zhu, X., Zhou, Y., Zhang, L., Ju, Z., & Shi, L. (2018). Telomere Dysfunction Disturbs Macrophage Mitochondrial Metabolism and the NLRP3 Inflammasome through the PGC-1α/TNFAIP3 Axis. Cell Reports, 22(13), 3493-3506. https://doi.org/https://doi.org/10.1016/j.celrep.2018.02.071  

Khan, A. M., Babcock, A. A., Saeed, H., Myhre, C. L., Kassem, M., & Finsen, B. (2015). Telomere dysfunction reduces microglial numbers without fully inducing an aging phenotype. Neurobiology of Aging, 36(6), 2164-2175. https://doi.org/https://doi.org/10.1016/j.neurobiolaging.2015.03.008  

Lee, H.-W., Blasco, M. A., Gottlieb, G. J., Horner, J. W., Greider, C. W., & DePinho, R. A. (1998). Essential role of mouse telomerase in highly proliferative organs. Nature, 392(6676), 569-574. https://doi.org/10.1038/33345  

Liu, H., Yang, Y., Ge, Y., Liu, J., & Zhao, Y. (2019). TERC promotes cellular inflammatory response independent of telomerase. Nucleic Acids Research, 47(15), 8084-8095. https://doi.org/10.1093/nar/gkz584  

Matthe, D. M., Thoma, O. M., Sperka, T., Neurath, M. F., & Waldner, M. J. (2022). Telomerase deficiency reflects age-associated changes in CD4+ T cells. Immun Ageing, 19(1), 16. https://doi.org/10.1186/s12979-022-00273-0  

Rudolph, K. L., Chang, S., Lee, H. W., Blasco, M., Gottlieb, G. J., Greider, C., & DePinho, R. A. (1999). Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell, 96(5), 701-712. https://doi.org/10.1016/s0092-8674(00)80580-2  

Tarry-Adkins, J. L., Aiken, C. E., Dearden, L., Fernandez-Twinn, D. S., & Ozanne, S. (2021). Exploring Telomere Dynamics in Aging Male Rat Tissues: Can Tissue-Specific Differences Contribute to Age-Associated Pathologies? Gerontology, 67(2), 233-242. https://doi.org/10.1159/000511608  

Wong, L. S. M., Oeseburg, H., de Boer, R. A., van Gilst, W. H., van Veldhuisen, D. J., & van der Harst, P. (2008). Telomere biology in cardiovascular disease: the TERC−/− mouse as a model for heart failure and ageing. Cardiovascular Research, 81(2), 244-252. https://doi.org/10.1093/cvr/cvn337  

Wu, S., Ge, Y., Lin, K., Liu, Q., Zhou, H., Hu, Q., Zhao, Y., He, W., & Ju, Z. (2022). Telomerase RNA TERC and the PI3K-AKT pathway form a positive feedback loop to regulate cell proliferation independent of telomerase activity. Nucleic Acids Res, 50(7), 3764-3776. https://doi.org/10.1093/nar/gkac179  

Zhang, M. W., Zhao, P., Yung, W. H., Sheng, Y., Ke, Y., & Qian, Z. M. (2018). Tissue iron is negatively correlated with TERC or TERT mRNA expression: A heterochronic parabiosis study in mice. Aging (Albany NY), 10(12), 3834-3850. https://doi.org/10.18632/aging.101676

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Contents

• • [

    Designing a tympanum through stereographic projection

    ](https://en.wikipedia.org/wiki/Astrolabe#Designing_a_tympanum_through_stereographic_projection) - [

    The tropics and the equator define the tympanum
    
    ](https://en.wikipedia.org/wiki/Astrolabe#The_tropics_and_the_equator_define_the_tympanum)
    

    • [

      The horizon and the measurement of altitude

      ](https://en.wikipedia.org/wiki/Astrolabe#The_horizon_and_the_measurement_of_altitude) - [

      The meridians and the measurement of azimuth

      ](https://en.wikipedia.org/wiki/Astrolabe#The_meridians_and_the_measurement_of_azimuth) - [

  See also

  ](https://en.wikipedia.org/wiki/Astrolabe#See_also) - [

  References

  ](https://en.wikipedia.org/wiki/Astrolabe#References) - [

  Bibliography

  ](https://en.wikipedia.org/wiki/Astrolabe#Bibliography) - [

  External links

  ](https://en.wikipedia.org/wiki/Astrolabe#External_links)

Astrolabe

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From Wikipedia, the free encyclopedia

For other pages with a similar name, see Astrolabe (disambiguation). Not to be confused with Cosmolabe.

North African, 9th century AD, Planispheric Astrolabe. Khalili Collection.

A modern astrolabe made in Tabriz, Iran in 2013.

An astrolabe (Greek: ἀστρολάβος astrolábos, 'star-taker'; Arabic: ٱلأَسْطُرلاب al-Asṭurlāb; Persian: ستاره‌یاب Setāreyāb) is an astronomical instrument dating to ancient times. It serves as a star chart and physical model of visible heavenly bodies. Its various functions also make it an elaborate inclinometer and an analog calculation device capable of working out several kinds of problems in astronomy. In its simplest form it is a metal disc with a pattern of wires, cutouts, and perforations that allows a user to calculate astronomical positions precisely. It is able to measure the altitude above the horizon of a celestial body, day or night; it can be used to identify stars or planets, to determine local latitude given local time (and vice versa), to survey, or to triangulate. It was used in classical antiquity, the Islamic Golden Age, the European Middle Ages and the Age of Discovery for all these purposes.

The astrolabe, which is a precursor to the sextant,^[1]^ is effective for determining latitude on land or calm seas. Although it is less reliable on the heaving deck of a ship in rough seas, the mariner's astrolabe was developed to solve that problem.

Applications

16th-century woodcut of measurement of a building's height with an astrolabe

The 10th-century astronomer ʿAbd al-Raḥmān al-Ṣūfī wrote a massive text of 386 chapters on the astrolabe, which reportedly described more than 1,000 applications for the astrolabe's various functions.^[2]^ These ranged from the astrological, the astronomical and the religious, to navigation, seasonal and daily time-keeping, and tide tables. At the time of their use, astrology was widely considered as much of a serious science as astronomy, and study of the two went hand-in-hand. The astronomical interest varied between folk astronomy (of the pre-Islamic tradition in Arabia) which was concerned with celestial and seasonal observations, and mathematical astronomy, which would inform intellectual practices and precise calculations based on astronomical observations. In regard to the astrolabe's religious function, the demands of Islamic prayer times were to be astronomically determined to ensure precise daily timings, and the qibla, the direction of Mecca towards which Muslims must pray, could also be determined by this device. In addition to this, the lunar calendar that was informed by the calculations of the astrolabe was of great significance to the religion of Islam, given that it determines the dates of important religious observances such as Ramadan.^[citation needed]^

Etymology

The Oxford English Dictionary gives the translation "star-taker" for the English word astrolabe and traces it through medieval Latin to the Greek word ἀστρολάβος : astrolábos,^[3]^^[4]^ from ἄστρον : astron "star" and λαμβάνειν : lambanein "to take".^[5]^

In the medieval Islamic world the Arabic word al-Asturlāb (i.e., astrolabe) was given various etymologies. In Arabic texts, the word is translated as ākhidhu al-Nujūm (Arabic: آخِذُ ٱلنُّجُومْ, lit. 'star-taker'), a direct translation of the Greek word.^[6]^

Al-Biruni quotes and criticises medieval scientist Hamza al-Isfahani who stated:^[6]^ "asturlab is an arabisation of this Persian phrase" (sitara yab, meaning "taker of the stars").^[7]^ In medieval Islamic sources, there is also a folk etymology of the word as "lines of lab", where "Lab" refers to a certain son of Idris (Enoch). This etymology is mentioned by a 10th-century scientist named al-Qummi but rejected by al-Khwarizmi.^[8]^

History

Ancient era

An astrolabe is essentially a plane (two-dimensional) version of an armillary sphere, which had already been invented in the Hellenistic period and probably been used by Hipparchus to produce his star catalogue. Theon of Alexandria (c. 335 -- c. 405) wrote a detailed treatise on the astrolabe.^[9]^ The invention of the plane astrolabe is sometimes wrongly attributed to Theon's daughter Hypatia (born c. 350--370; died AD 415),^[10]^^[11]^^[12]^^[13]^ but it's known to have been used much earlier.^[11]^^[12]^^[13]^ The misattribution comes from a misinterpretation of a statement in a letter written by Hypatia's pupil Synesius (c. 373 -- c. 414),^[11]^^[12]^^[13]^ which mentions that Hypatia had taught him how to construct a plane astrolabe, but does not say that she invented it.^[11]^^[12]^^[13]^ Lewis argues that Ptolemy used an astrolabe to make the astronomical observations recorded in the Tetrabiblos.^[9]^ However, Emilie Savage-Smith notes "there is no convincing evidence that Ptolemy or any of his predecessors knew about the planispheric astrolabe".^[14]^ In chapter 5,1 of the Almagest, Ptolemy describes the construction of an armillary sphere, and it is usually assumed that this was the instrument he used.

Astrolabes continued to be used in the Byzantine Empire. Christian philosopher John Philoponus wrote a treatise (c. 550) on the astrolabe in Greek, which is the earliest extant treatise on the instrument.^[a]^ Mesopotamian bishop Severus Sebokht also wrote a treatise on the astrolabe in the Syriac language during the mid-7th century.^[b]^ Sebokht refers to the astrolabe as being made of brass in the introduction of his treatise, indicating that metal astrolabes were known in the Christian East well before they were developed in the Islamic world or in the Latin West.^[15]^

Medieval era

Astrolabes were further developed in the medieval Islamic world, where Muslim astronomers introduced angular scales to the design,^[16]^ adding circles indicating azimuths on the horizon.^[17]^ It was widely used throughout the Muslim world, chiefly as an aid to navigation and as a way of finding the Qibla, the direction of Mecca. Eighth-century mathematician Muhammad al-Fazari is the first person credited with building the astrolabe in the Islamic world.^[18]^

The mathematical background was established by Muslim astronomer Albatenius in his treatise Kitab az-Zij (c. AD 920), which was translated into Latin by Plato Tiburtinus (De Motu Stellarum). The earliest surviving astrolabe is dated AH 315 (AD 927--928). In the Islamic world, astrolabes were used to find the times of sunrise and the rising of fixed stars, to help schedule morning prayers (salat). In the 10th century, al-Sufi first described over 1,000 different uses of an astrolabe, in areas as diverse as astronomy, astrology, navigation, surveying, timekeeping, prayer, Salat, Qibla, etc.^[19]^^[20]^

An Arab astrolabe from 1208

The spherical astrolabe was a variation of both the astrolabe and the armillary sphere, invented during the Middle Ages by astronomers and inventors in the Islamic world.^[c]^ The earliest description of the spherical astrolabe dates to Al-Nayrizi (fl. 892--902). In the 12th century, Sharaf al-Dīn al-Tūsī invented the linear astrolabe, sometimes called the "staff of al-Tusi", which was "a simple wooden rod with graduated markings but without sights. It was furnished with a plumb line and a double chord for making angular measurements and bore a perforated pointer".^[21]^ The geared mechanical astrolabe was invented by Abi Bakr of Isfahan in 1235.^[22]^

The first known metal astrolabe in Western Europe is the Destombes astrolabe made from brass in the eleventh century in Portugal.^[23]^^[24]^ Metal astrolabes avoided the warping that large wooden ones were prone to, allowing the construction of larger and therefore more accurate instruments. Metal astrolabes were heavier than wooden instruments of the same size, making it difficult to use them in navigation.^[25]^

Spherical astrolabe

A depiction of Hermann of Reichenau with an astrolabe in a 13th-century manuscript by Matthew Paris

Herman Contractus of Reichenau Abbey, examined the use of the astrolabe in Mensura Astrolai during the 11th century.^[26]^ Peter of Maricourt wrote a treatise on the construction and use of a universal astrolabe in the last half of the 13th century entitled Nova compositio astrolabii particularis. Universal astrolabes can be found at the History of Science Museum in Oxford.^[27]^ David A. King, historian of Islamic instrumentation, describes the universal astrolobe designed by Ibn al-Sarraj of Aleppo (aka Ahmad bin Abi Bakr; fl. 1328) as "the most sophisticated astronomical instrument from the entire Medieval and Renaissance periods".^[28]^

English author Geoffrey Chaucer (c. 1343--1400) compiled A Treatise on the Astrolabe for his son, mainly based on a work by Messahalla or Ibn al-Saffar.^[29]^^[30]^ The same source was translated by French astronomer and astrologer Pélerin de Prusse and others. The first printed book on the astrolabe was Composition and Use of Astrolabe by Christian of Prachatice, also using Messahalla, but relatively original.

Front of an Indian astrolabe now kept at the Royal Museum of Scotland at Edinburgh.

In 1370, the first Indian treatise on the astrolabe was written by the Jain astronomer Mahendra Suri, titled Yantrarāja.^[31]^

A simplified astrolabe, known as a balesilha, was used by sailors to get an accurate reading of latitude while at sea. The use of the balesilha was promoted by Prince Henry (1394--1460) while navigating for Portugal.^[32]^

The astrolabe was almost certainly first brought north of the Pyrenees by Gerbert of Aurillac (future Pope Sylvester II), where it was integrated into the quadrivium at the school in Reims, France, sometime before the turn of the 11th century.^[33]^ In the 15th century, French instrument maker Jean Fusoris (c. 1365--1436) also started remaking and selling astrolabes in his shop in Paris, along with portable sundials and other popular scientific devices of the day.

Astronomical Instrument Detail by Ieremias Palladas 1612

Thirteen of his astrolabes survive to this day.^[34]^ One more special example of craftsmanship in early 15th-century Europe is the astrolabe designed by Antonius de Pacento and made by Dominicus de Lanzano, dated 1420.^[35]^

In the 16th century, Johannes Stöffler published Elucidatio fabricae ususque astrolabii, a manual of the construction and use of the astrolabe. Four identical 16th-century astrolabes made by Georg Hartmann provide some of the earliest evidence for batch production by division of labor. In 1612, Greek painter Ieremias Palladas incorporated a sophisticated astrolabe in his painting depicting Catherine of Alexandria. The painting was entitled Catherine of Alexandria and featured a device called the System of the Universe (Σύστημα τοῦ Παντός). The device featured the planets with the names in Greek: Selene (Moon), Hermes (Mercury), Aphrodite (Venus), Helios (Sun), Ares (Mars), Zeus (Jupiter), and Chronos (Saturn). The device also featured celestial spheres following the Ptolemaic model and Earth was depicted as a blue sphere with circles of geographic coordinates. A complex line representing the axis of the Earth covered the entire instrument.^[36]^

Medieval astrolabes

• 

  A treatise explaining the importance of the astrolabe by Nasir al-Din al-Tusi, Persian scientist

• 

  Astrolabe of Jean Fusoris, made in Paris, 1400

• 

  An 18th-century Persian astrolabe

• 

  Disassembled 18th-century astrolabe

• 

  Exploded view of an astrolabe

• 

  Animation showing how celestial and geographic coordinates are mapped on an astrolabe's tympan through a stereographic projection. Hypothetical tympan (40° north latitude) of a 16th-century European planispheric astrolabe.

• 

  Astrolabe manual from the Alfonso X of Castile work Libros del saber de astronomía, 1276.

• 

  A page from the 1575 book "Astrolabium" depicting an astrolabe. Masha'Allah Public Library Bruges [nl] Ms. 522