1,078 Matching Annotations
  1. Jan 2019
    1. we tested 17 single mutants with both targeting and nontargeting guides, under the assumption that background luciferase restoration in the nontargeting condition would be indicative of broader off-target activity.

      It would be too expensive to test all the mutants by RNA sequencing to find whole-transcriptome off-targets. Instead, the authors looked at a single transcript for Cluc luciferase.

      The authors assumed that if luciferase activity was restored even non-targeting guide RNAs, it would mean that ADAR2 increased off-target activity.

    2. To reduce the size, we tested a variety of N-terminal and C-terminal truncations of dCas13

      AAVs can accommodate up to 4.7kb, meaning dCas13b and the extra regulatory sequences are too big to fit in one virus. To solve this problem, the authors truncated (shortened) dCas13b by removing sections of the protein with unnecessary functions.

      The dCas13b nuclease is composed of different domains (parts), some of which are used for RNA binding and some which are used for cleavage. The RNA cleavage is mediated by two well-defined HEPN domains which are located close to the N- and C-terminus of the protein. We are still unsure which domains mediate binding.

      However, the REPAIRv1 system needs only the RNA binding ability of dCas13b. Therefore, the researchers were able to remove sections of dCas13b containing HEPN while retaining its binding function.

    3. AAV vectors have a packaging limit of 4.7 kb

      Almost the entire AAV genome can be replaced with a desired construct of max length of 4.7kb. This limits how much genetic material the virus can carry, so some essential viral genes are carried in an additional construct or constructs (the "helper plasmid" in the picture below).

      Learn more about AAV at Addgene.

    4. We then tested the ability of REPAIRv1 to correct 34 different disease-relevant G→A mutations

      The researchers chose 34 disease causing mutations to test. Each mutation was a G to A substitution that changed the amino acid sequence of the protein and disrupted normal function.

    5. Using guide RNAs containing 50-nt spacers

      The authors chose three 50-nt spacers to target two genes carrying disease-relevant mutations. As was determined at the previous step, the spacers of 50-nt length showed higher rates of editing but more off-target effects than 30-nt spacers.

    6. To demonstrate the broad applicability of the REPAIRv1 system for RNA editing in mammalian cells, we designed REPAIRv1 guides against two disease-relevant mutations

      After the authors had generated successful REPAIRv1-mediated editing of mRNA for an exogenous gene (i.e., a gene introduced from outside the organism), they chose two endogenous genes (i.e., native to the organism) to further explore the power of REPAIRv1.

      They tested Cas13b nuclease activity in the same way as they did with the exogenous gene.

    7. we modified the linker between dCas13b and ADAR2DD(E488Q)

      The dCas13b and ADAR2 protein parts are joined together via a stretch of amino acids called a linker. There are more than a thousand linker variants in different multi-domain proteins. The sequence of a linker can influence protein folding and stability, as well as functional properties of individual domains. Therefore, the choice of linker is an important step in protein design.

      The authors tested linkers of different length and flexibility and found that shorter and more flexible variants produced the best results.

    8. To validate that restoration of luciferase activity was due to bona fide editing events, we directly measured REPAIRv1-mediated editing of Cluc transcripts via reverse transcription and targeted next-generation sequencing.

      The ADAR deaminase can introduce changes not only into the target adenosine but also in the surrounding adenosine bases. To check how specific the dCas13-ADAR2 protein was in targeting the correct adenosine base, the researchers sequenced edited Cluc transcripts and determined all positions with A to I substitutions.

      Further, they used tiling gRNA molecules in order to determine the influence of the spacer (PFS) length and the mismatch distance on the off-target editing.

    9. position-matched guides

      The authors directly compared the RNA knockdown efficiency of two technologies, Cas13 cleavage and RNA interference (RNAi). Both technologies require guide RNA (gRNA) molecules for targeted recognition. Cas13 is directed by a gRNA, while RNAi complex uses a molecule termed shRNA.

      Different parts of an RNA molecule can be more or less accessible to gRNAs. Therefore, gRNA and shRNA target sequences have to be selected such that they are close to each other, i.e. position-matched, so that a more fair comparison between Cas13 and RNAi can be made.

    10. Cas13b from Prevotella sp. P5-125 (PspCas13b) and Cas13b from Porphyromonas gulae (PguCas13b) C-terminally fused to the HIV Rev nuclear export sequence (NES), and Cas13b from Riemerella anatipestifer (RanCas13b) C-terminally fused to the mitogen-activated protein kinase NES

      Why did the authors test several NESs from different proteins?

      Over 200 NESs from different proteins have been described. Each NES is around 10 amino acids long and has a unique structure. The variation between NESs means that they have different effects on export efficiency and protein stability in different environments.

    11. To engineer a PspCas13b lacking nuclease activity (dPspCas13b, referred to as dCas13b hereafter), we mutated conserved catalytic residues in the HEPN domains and observed loss of luciferase RNA knockdown

      The authors hypothesized that even without nuclease activity, Cas13b would still be able to recognize target molecules directed by a gRNA.

      Mutations of the key part of the protein responsible for RNA cleavage eliminated Cas13b's catalytic ability. The next step was to test whether the mutated Cas13b was still able to find the target sequences, even if it couldn't cleave them.

    12. We found that LwaCas13a and PspCas13b had a central region that was relatively intolerant to single mismatches

      The sequences with substitutions in the middle part (between 12 and 26 nucleotides) had the lowest depletion scores. A lower depletion score means that the nuclease was not as successful at cleaving the RNA sequence (i.e. depleting expression).

    13. To characterize the interference specificities of PspCas13b and LwaCas13a, we designed a plasmid library of luciferase targets containing single mismatches and double mismatches throughout the target sequence and the three flanking 5′ and 3′ base pairs

      To figure out how specifically the Cas13 orthologs would target RNA, they created a "library" (collection) of sequences with one or two mutations in the gRNA target site for the Gluc gene. The idea was to see how different a sequence could be and still be recognized by the nuclease.

      Remember that the goal is to find a highly specific nuclease. This means that ideally the nuclease would not recognize sequences with mutations.

    14. without msfGFP

      The authors ran the assay without msfGFP to determine if any of the orthologs do not require stabilization domains. This is important because orthologs that do not require these domains could be used when an experimental construct needs to be small.

    15. For each Cas13 ortholog, we designed PFS-compatible guide RNAs, using the Cas13b PFS motifs derived from an ampicillin interference assay

      Cas13a prefers to recognize target sequences when a spacer (target sequence) is surrounded by specific PFS motifs.

      To determine which PFS sequences Cas13b orthologs prefer, the researchers used an ampicillin interference assay. They transformed bacterial cells with two plasmids: One contained a Cas13b ortholog while the second plasmid included an ampicillin resistance gene and a gRNA against this gene with randomly generated 5' and 3' PFS around the target sequence. If the PFS led to the Cas13b ortholog targeting the correct gene, bacterial cells would die because they would lose ampicillin resistance when the gene was cleaved.

      The authors collected and analyzed cells that survived to determine which PFS sequences were irrelevant for targeting. They then subtracted these PFSs from the starting pool to determine which PFSs are preferred by Cas13b orthologs.

    16. To assay interference in mammalian cells, we designed a dual-reporter construct expressing the independent Gaussia (Gluc) and Cypridina (Cluc) luciferases under separate promoters, allowing one luciferase to function as a measure of Cas13 interference activity and the other to serve as an internal control.

      To monitor the effect of Cas13 nuclease activity, the researchers constructed a vector that contained genes for two different luciferases. These luciferases use different substrates (source materials) to generate light.

      One luciferase was used to measure Cas13 interference, and the other was used as a control. In cells where Cas13 did not interfere, both luciferases would emit light. In cells with interference, however, activity from one of the luciferases would decrease.

    1. A genome-wide PCA analysis

      Analyzing multiple whole genomes at the same time is so complex that scientists needed to simplify the data to make it easier to see patterns and differences between the genomes. The mathematical tool that they used was PCA analysis, which reduces the complexity of the data and retains its variance.

      PCA analysis works by setting principal components (e.g. PC1, PC2, PC3), which are essentially directions in which the data has the largest spread.

      In this case, PC1 and PC2 are studied.

    2. sequenced and analyzed 59 hypervariable mtDNA fragments from ancient dogs spread across Europe, and we combined those with 167 modern sequences

      The d-loop sequences of mitochondrial DNA are considered to be mutational hotspots (places where mutations appear to happen more frequently than others), and therefore looking at this region can provide important information about how evolution occurred.

      Modern dog sequences and seven of the 59 ancient d-loop sequences were already available on a public database. The rest of the sequences were generated from ancient DNA samples by polymerase chain reaction (PCR). The sequences were then compared to each other using DomeTree, a program that creates haplogroup phylogenetic trees based on mitochondrial DNA.

    3. we used the radiocarbon age of the Newgrange dog to calibrate the mutation rate for dogs

      The team of scientists used radiocarbon dating techniques to calculate the dog as 4700-4900 years old.

      Since they knew how old the Newgrange dog was, they were able to estimate the time at which the Newgrange dog and the Portuguese village dogs last had a common ancestor, and figure out when they diverged from each other.

      Based on the assumption that a new generation of dogs were born every 3 years, the scientists were able to calculate the mutation rate.

    4. we defined Western Eurasian and East Asian “core” groups (Fig. 1A), supported by the strength of the node leading to each cluster (12).

      The information represented in Figure 1A was used to define the two core groups. The dogs needed to have very high bootstrap values (>90)—high-quality genetic data—to support their placement in the groups.

      The Western Eurasian core group consisted of all modern breeds and Portugal village dogs. The East Asian core group consisted of Sharpei, Village dogs from China, Tibet, and Vietnam, and Tibetan Mastiffs.

    5. We used principal components analysis (PCA), D statistics, and the program TreeMix (12) to further test this pattern.

      Three tools for mining the data for ancestry information.

      Principal components analysis (PCA) simplifies the data. The number of variables is reduced but the trends and patterns are retained.

      D-statistics detects admixture events; it is used to detect gene flow between closely-related species.

      TreeMix is a genetics computer program used to estimate ancestral relationships. It can detect population splits and migration events.

    6. radiocarbon dated

      This technique allows scientists to estimate the age of a plant or animal based on the amount of carbon-14 that is present at the time of measurement. Carbon-14, also known as radiocarbon, is a weakly radioactive type of carbon molecule that decays over time. The age of samples up to 60,000 years old can be estimated.

      To learn more about radiocarbon dating, check out this video from Scientific American.

    7. CanineHD 170,000 (170 K)

      A type of genetic test that covers 170,000 single-position variations in the genome of a dog. It is essentially a chip, upon which the genetic material of the dog of interest is placed. It contains thousands (170,000 in this case) of probes—short DNA sequences that can stick to the complementary sequence in the sample, if that matching DNA variant is present. Each interaction can be recorded to easily measure the presence of a large number of genes at the same time.

    8. (28x) of an ancient dog dated to ~4800 calendar years before the present (12) from the Neolithic passage grave complex of Newgrange (Sí an Bhrú) in Ireland.

      The scientists isolated DNA from a portion of the temporal bone in the dog's skull.

      They made a library of single-stranded DNA sequences; smaller pieces of DNA that together represent the entire genome of the dog. The DNA library is sequenced on a machine that can read the order of the bases (As, Ts, Gs, and Cs) that make up the genome of the particular dog being studied.

      Check out this video from TED-Ed on how to sequence the human genome (it also applies to the dog genome).

    9. sequences from European dogs (from 14,000 to 3000 years ago)

      They sequenced the d-loop of mitochondrial DNA, an area where mutations happen more often than other parts of mitochondrial DNA.

      Seven of the d-loop sequences were already available on a public database, and the others were generated from DNA in bone samples. A very small amount of bone was ground to a fine powder. The scientists were very careful to make sure contamination of the samples did not occur. Once the cells in the bone sample were broken open, and the DNA was isolated from other parts of the cell, the DNA could be sequenced by polymerase chain reaction (PCR).

      Check out this video from Khan Academy to learn how DNA is sequenced by PCR.

    10. ancient

      In this case, the DNA specimens are ~14,000 to 3000 years old.

  2. Dec 2018
    1. Ecdysozoa (molting animals) is a major protostome clade (Figure 1) proposed by Aguinaldo et al. (1997) that includes the large phyla Arthropoda (Figure 3) and Nematoda (both of tremendous ecological, economic, and biomedical importance) and their close relatives (Tardigrada [water bears, 1150 species], Nematomorpha [351 species], Onychophora [velvet worms; 182 species], Kinorhyncha [179 species], Loricifera [30 species], and Priapulida [19 species]). Ecdysozoans are characterized by their ability to molt the cuticle during their life cycle, and for having reduced epithelial ciliation, which requires locomotion via muscular action. They include segmented or unsegmented, acoelomate, pseudocoelomate, or coelomate animals; many have annulated cuticles and a mouth located at the end of a protrusible or telescopic proboscis, and some lack circular musculature (i.e., Nematoda, Nematomorpha, Tardigrada). Here we restrict the proposed sampling to the noninsect and nonnematode ecdysozoans. The clade includes the only animals (loriciferans) thought to complete their life cycles in anoxic environments (Danovaro et al. 2010). This group is also relevant for studies of extreme cell size reduction. Other than the mentioned arthropod and nematode genomes, no genome is available for any member of the Ecdysozoa.

      This is one of the limitations that will be placed on researchers that plan on conducting research to contribute to this database. They restrict a certain proposed sampling to the non-insect and non-nematode ecdysozoans.

    2. Many of the roughly 70 invertebrate species whose genomes have been sequenced belong to the Arthropoda or Nematoda, although the number of other invertebrate genomes continues to grow (e.g., Olson et al. 2012; Takeuchi et al. 2012; Zhang et al. 2012; Simakov et al. 2013; Tsai et al. 2013; Flot et al. 2013). We propose to focus on noninsect/nonnematode phyla, and specifically on an important group of currently neglected arthropods, the crustaceans.

      They propose a certain focus for researchers that wish to dedicate time to contributing to the database by stating that they should focus more on neglected groups of arthropods, specifically crustaceans.

    3. GIGA has adopted a set of standards and best practices to help ensure that genomic resources, data, and associated metadata are acquired, documented, disseminated, and stored in ways that are directly comparable among projects and laboratories. These data should be easily and equitably shared among GIGA members and the broader scientific community, and GIGA will obey appropriate laws and regulations governing the protection of natural biodiversity. Briefly, all genome projects will report on a set of parameters that will allow assessment of genome assembly, annotation, and completeness (e.g., NG50, N50 of contigs and scaffolds, number of genes, assembled vs. estimated genome size) (Jeffery et al. 2013). Detailed descriptions of these standards and compliant protocols will be posted on the GIGA Web site. These will be revised periodically to facilitate the establishment and maintenance of current best practices common to many invertebrate genome and transcriptome sequencing projects and to help guide the researcher in selecting and assessing genomes for further analyses. The following recommendations summarize minimal project-wide standards designed to accommodate the large diversity of invertebrates, including extremely small and rare organisms, as well as those that live in close association with other organisms. Permissions: GIGA participants must comply with treaties, laws, and regulations regarding acquisition of specimens or samples, publication of sequence data, and distribution or commercialization of data or materials derived from biological resources. Participants must acquire all necessary permits required for collection and transport of biological materials prior to the onset of the work. The CBD recognizes the sovereignty of each nation over its biological resources, and under the auspices of the CBD, many nations and jurisdictions rigorously regulate the use and distribution of bioIogical materials and data. GIGA participants must be aware of these regulations and respect the established rights of potential stakeholders, including nations, states, municipalities, commercial concerns, indigenous populations, and individual citizens, with respect to any materials being collected, to all derivatives and progeny of those materials, and to all intellectual property derived from them. GIGA participants must also familiarize themselves with the conservation status of organisms to be sampled and any special permits that may be required (e.g., CITES). Moreover, GIGA participants should collect in ways that minimize impacts to the sampled species and their associated environments. Field collection and shipping: Methods for field collection and preservation of specimens and tissues should be compatible with recovery of high-quality (e.g., high molecular weight, minimally degraded) genomic DNA and RNA (Dawson et al. 1998; Riesgo et al. 2012; Wong et al. 2012). Many reagents commonly used for tissue and nucleic acid preservation (e.g., ethanol, dry ice) are regulated as hazardous and/or flammable materials. These reagents may be restricted from checked and carry-on luggage and may require special precautions for shipping or transport. GIGA participants should contact the appropriate airline carriers or shippers for information regarding safe and legal shipment of preserved biological materials. When possible, multiple samples will be collected so that extractions can be optimized and samples resequenced as technologies improve. Specimens of known origin (i.e., field-collected material) will be favored over specimens of unknown origin (e.g., material purchased from the aquarium trade). Collection data will include location (ideally, with GPS coordinates) and date, and also other data such as site photographs and environmental measurements (e.g., salinity) when relevant. Selection and preparation of tissues: It is often advisable to avoid tissues that may contain high concentration of nucleases, foreign nucleic acids, large amounts of mucus, lipid, fat, wax, or glycogen or that are insoluble, chitinous, or mineralized. To obtain the highest quality material for sequencing or library construction, it may be preferable to extract nucleic acids from living or rapidly preserved tissue from freshly sacrificed animals, from gametes or embryos, or from cell lines cultivated from the target organism (Ryder 2005; Rinkevich 2011; Pomponi et al. 2013). When appropriate, select tissues or life history stages that will avoid contamination by symbionts, parasites, commensal organisms, gut contents, and incidentally associated biological and nonbiological material. Whenever possible, DNA or RNA will be sequenced from a single individual because many taxa display sufficient polymorphism among individuals to complicate assembly. Similarly, heterozygosity can also hinder assembly: inbreeding may be used to reduced heterozygosity (Zhang et al. 2012) or, when crossings are impossible (for instance in asexual species), haplotypes may have to be assembled separately (Flot et al. 2013). Quantity and Quality: The quantity of DNA or RNA required for sequencing varies widely depending on the sequencing platform and library construction methods to be used and should be carefully considered. Recent consensus from the G10KCOS group of scientists suggests that at least 200 – 800 µg of high-quality genomic DNA is required to begin any project because of the requirement for large insert mate-pair libraries (Wong et al. 2012). However, these minimum quantities are expected to decline with improving technology. DNA quality can be assessed by size visualizatons and 260/280nm ratios. Quality of RNA will be checked using RNA integrity number (RIN > 7 is preferred); however, these values have been shown to appear degraded in arthropods due to artifacts during quantification (Schroeder et al. 2006; Winnebeck et al. 2010). Taxonomic identity: The taxonomic identity of source organisms must be verified. Whenever possible, consensus should be sought from expert systematists, supportive literature, and sequence analysis of diagnostic genes (see next section). Voucher specimens: As a prerequisite for inclusion as a GIGA sample, both morphological and nucleic acid voucher specimens must be preserved and deposited in public collections, and the associated accession numbers must be supplied to the GIGA database. Photographs should be taken of each specimen and cataloged along with other metadata. The GIGA Web site lists cooperating institutions willing to house voucher specimens for GIGA projects, such as the Smithsonian Institution or the Ocean Genome Legacy (http://www.oglf.org). Documentation of projects, specimens, and samples: Unique alphanumeric identification numbers (GIGA accession numbers) will be assigned to each GIGA project and to each associated specimen or sample used as a source of genome or transcriptome material for analysis. A single database with a web interface will be established to accommodate metadata for all specimens and samples. Metadata recording will also aim to coordinate and comply with previously established standards in the community, such as those recommended by Genomics Standards Consortium (http://gensc.org/; Field et al. 2011). Sequencing Standards: Standards for sequencing are platform and taxon specific, and sensitive to the requirements of individual sequencing facilities. For these reasons, best practices and standards will be established for individual applications. Coverage with high-quality raw sequence data is a minimal requirement to obtain reliable assemblies. An initial sequencing run and assembly will be used to estimate repeat structure and heterozygosity. These preliminary analyses will make it possible to evaluate the need for supplemental sequencing, with alternative technologies aimed at addressing specific challenges (e.g., mate-pair sequencing to resolve contig linkage). Moreover, all raw sequence reads generated as part of a GIGA project will be submitted to the NCBI Sequence Read Archive. Sequence Assembly, Annotation, and Analyses: Because assemblies vary widely in quality and completeness, each assembly should be described using a minimum set of common metrics that may include: (1) N50 (or NG50) length of scaffolds and contigs (see explanation of N50 in Bradnam et al. 2013), (2) percent gaps, (3) percent detection of conserved eukaryotic genes (e.g., Core Eukaryotic Genes Mapping Approach (Parra et al. 2007), (4) statistical assessment of assembly (Howison et al, 2013), (5) alignment to any available syntenic or physical maps (Lewin et al. 2009), and (6) mapping statistics of any available transcript data (Ryan 2013). The current paucity of whole invertebrate genome sequence projects can pose problems for gene calling, gene annotation, and identification of orthologous genes. In cases where the genome is difficult to assemble, we recommend that genome maps be developed for selected taxa via traditional methods or new methods (e.g., optical mapping of restriction sites) to aid and improve the quality of genome assembly (Lewin et al. 2009) and that GIGA genome projects be accompanied by transcriptome sequencing and analysis when possible. Such transcriptome data will assist open reading frame and gene annotation and are valuable in their own right.

      GIGA aims to set standards for data acquisition and the processing of that data as well as the input of that data into their system. These standards will be progressively revised as better methods are discovered. These standards are to be used for a large variety of invertebrates. This standardization will allow for a more easily accessible and usable bank of information.

    4. We also recognize the existence and formation of other recent genome science initiatives and coordination networks and will synchronize efforts with such groups through future projects. Because GIGA is an international consortium of scientists, agencies, and institutions, we will also abide by the rules of global funding agencies for data release (e.g., those proposed by the Global Research Council; http://www.globalresearchcouncil.org). We are aware that different nations have different constraints and regulations on the use of biological samples. Given the international nature of GIGA, we will work to ensure that national genomic legacies are protected and will consult with the pertinent governmental agencies in the countries from which samples originate. We will deposit sequence data in public databases (e.g., GenBank), as well as deposit DNA vouchers in publically accessible repositories (e.g. Global Genome Biodiversity Network, Smithsonian). GIGA is an inclusive enterprise that invites all interested parties to join the effort of invertebrate genomics. We will attempt to capture the impact of the effort in the wider scientific and public arenas by following relevant publications and other products that result from GIGA initiatives.

      The project coordinators will also join with other networks to work together on future projects of common interest. They also understand that different countries have limits on the use of biological samples and thus their governments will be consulted with so that the international program is protected from any government agencies in the countries where the samples come from.

    5. GIGA embraces a transparent process of project coordination, collaboration, and data sharing that is designed to be fair to all involved parties. The ENCODE project may be emulated in this regard (Birney 2012). We are committed to the rapid release of genomic data, minimizing the period of knowledge latency prior to public release while protecting the rights of data product developers (Contreras 2010). The data accepted as part of GIGA resources will undergo quality control steps that will follow preestablished and evolving standards (see Standards section) prior to data release. Efforts such as those of Albertin et al. (2012) have addressed data sharing issues relevant to GIGA and other large-scale genomics consortia.

      Once the data has been submitted by researchers, it will go through a quality control process to make sure that it reaches the quality and accuracy which the project requires. As well as to avoid duplicate data being published.

    6. but there are currently no published genomes for the other 21 invertebrate phyla. We examined current phylogenetic hypotheses and selected key invertebrate species that span the phylogenetic diversity and morphological disparity on the animal tree of life (see Supplementary Material). New invertebrate genome data can reveal novel sequences with sufficient phylogenetic signal to resolve longstanding questions.

      The currently available genome sequences at the time were reviewed and the missing phyla were taken note of. These missing areas in the so called "tree of life" were added to the list of species to be researched so that the data could be used to further other research.

    7. Therefore, the geographic scope of the project in terms of participation, taxa collected, stored, and sequenced, data analysis and sharing, and derived benefits, requires global partnerships beyond the individuals and institutions represented at the inaugural workshop. Because international and interinstitutional cooperation is essential for long-term success, the new GIGA Web site will be used to foster cooperative research projects. For now, the GIGA Web site can serve as a community nexus to link projects and collaborators, but it could also eventually expand to host multiple shared data sets or interactive genome browsers. The broad scope of GIGA also necessitates growth in the genomics-enabled community overall. Sequencing and analyzing the large amount of resulting data pose significant bioinformatic and computational challenges and will require the identification and creation of shared bioinformatics infrastructure resources.

      The information gathered from experimentation will be unified by being submitted to the GIGA website, thus allowing researchers from all over the world to have access to the information at a moments notice. The website will also be used to host cooperative research projects that scientists from around the world could participate in and contribute data towards.

    8. Selection and prioritization of taxa to be sequenced will occur through future discussion and coordination within GIGA. Thus, the target number is a somewhat arbitrary compromise between the desire to encompass as much phylogenetic, morphological, and ecological diversity as possible and the practical limitations of the initiative. Given the large population sizes of many invertebrate species, collection of a sufficient number of individuals may be relatively easy for the first set of targets. Collection of invertebrates usually involves fewer difficulties with regard to permits than collection of vertebrate tissues. However, some invertebrate taxa pose various logistic and technological challenges for whole-genome sequencing: many species live in relatively inaccessible habitats (e.g., as parasites, in the deep sea, or geographically remote) or are too small to yield sufficient amounts of DNA from single individuals. These challenges will be considered with other criteria as sequencing projects are developed and prioritized.

      Selecting which organisms genetic information is to be gathered and recorded first will be determined at a meeting among researchers in the field. The priority of these organisms will also be determined by their accessibility to researchers as well as their ease of retrieving genetic information.

    9. We propose to sequence, assemble, and annotate whole genomes and/or transcriptomes of 7000 invertebrate species, complementing ongoing efforts to sequence vertebrates, insects, and nematodes (Genome 10K Community of Scientists [G10KCOS], 2009; Robinson et al. 2011; Kumar et al. 2012) (Table 1).

      The purpose of the experiment is to create a genetic database of 7000 invertebrate species.

    10. A main goal of GIGA is to build an international multidisciplinary community to pursue comparative invertebrate genomic studies. We seek to develop tools to enable and facilitate genomic research and encourage collaboration. We will develop standards that ensure data quality, comparability, and integration. By coordinating sample collecting and sequencing efforts among invertebrate biologists, we aim to avoid duplication of effort and leverage resources more efficiently. We envision a scientific commons where shared resources, data, data standards, and innovations move the generation and analysis of invertebrate genomic data to a level that likely could not be achieved with the traditional piecemeal single-investigator–driven approach.

      The main goal of the project is to build an international community whose goal is to perform invertebrate research. This community will be used to avoid duplicate research and wasting time on studies that have already been conducted. They will also set in place standards by which the data must be gathered and presented so that is can be universally understood by everyone around the world and so that the data is accurate enough.

    1. Datta, A., Rane, A., 2013. Trop. Conserv. Sci. 6, 674–689.

      Referencing Box 1, this study was reviewed by the author classified this study as looking at population/species and as qualitative or quantitative estimate of flower or fruit production. The big conclusion of this study is that a rare species is seed-limited while dispersal limitation may play a secondary role in determining its abundance.

    2. Peres, C.A., 1994a. Biotropica 285-294.

      Referencing Box 1; The author of this paper looks at how plant phenology changes across succession in a community. Peres is studying keystone resources over many different seasons and how phenology changes over time. This aids in answering this papers research question of how phenology can be used in conservation to help keystone species.

    3. Schmidt, I.B., Figueiredo, I.B., Scariot, A., 2007. Econ. Bot. 61, 73–85.

      Referencing Box 1; Schmidt and his colleagues focused on sustainability in harvesting non-timber forest products. They did this by analyzing flowering and fruiting time, as well as, the abundance and size of the fruits produced. This conservation effort was done using a large scale population control effort.

    4. Rossi, S., Morin, H., Deslauriers, A., 2012. J. Exp. Bot. 63, 2117–2126.

      Referencing Box 1; Rossi and his colleagues focused their studies and conservations efforts on estimating carbon stocks and developing growth models in order to track and plan for future phenological changes and their effects on different tree species. Their models were developed by looking at tree growth rings within the trunks of various trees in order to look at the relative amounts of carbon dioxide intake and atmospheric carbon stocks at the time of the ring growth.

    5. Ali, N.S., Trivedi, C., 2011. Biodivers. Conserv. 20, 295–307.

      Referencing Box 1; Ali and his colleagues established a calendar for the collection of seeds and other plant resources in order to properly manage the conservation of genetic resources in natural plant populations. This was done throughout population and species scale conservation. They made direct ground observations of plant phenophases (leafing, flowering, fruiting) and the environmental factors that effected these actions. Advancements in this conservation can be seen in efforts such as seed banks.

    6. We focus on shifts in plant phenology induced by global change, their impacts on species diversity and plant–animal interactions in the tropics, and how conservation efforts could be enhanced in relation to plant resource organization

      Research question:

      How can phenology be used to further our knowledge and success in conservation biology? What are the influences of climate change on phenology and what are the implications of those influences within a tropical environment?

    1. Treatments applied to caterpillars were: (1) no barriers (no tanglefoot/no cage), (2) tanglefoot present/no cage, (3) no tanglefoot/cage with holes present, (4) tanglefoot present/cage with holes present, (5) no tanglefoot/cage present, and (6) tanglefoot present/cage present.

      These are the parameters made to make sure that the experiment itself is not biased and there are different variables being tested.

    1. The role of histone variants was further investigated by studying H2A.X, H2A.Z and macroH2A protein expression levels (Fig. 3)

      The scientists further studied the relationship between histone proteins and stress levels after acknowledging that a possible cause and effect exists.

      By discovering that antibodies connected to the histones were valid, it also proved the oyster to be the best model to test on.

    2. Genomic DNA was purified from gill tissue as described elsewhere (Fernandez-Tajes et al., 2007)

      The authors recorded and purified DNA from the gill tissue of the oysters in order to measure DNA methylation.

      Later, the DNA sequences were amplified.

      By using PCR methods, the samples were digested in order to asses the patterns of methylation and used to identify loci using Gowers Coefficient of Similarity.

    3. Histone protein isolation was performed as described elsewhere (Ausio and Moore, 1998), adapting the protocol to oyster gill tissues in the present work

      The purpose in the experiment was conducted in the extraction of histone proteins. By using classic centrifuging and electrophoresis techniques, the histone proteins were separated in order to retrieve the different types of anti-proteins. The antibodies were then analyzed with the Western Blot using the antibodies.

    4. Dosage was adjusted to maintain homogeneous microalgae concentrations across replicates (Fig. 1B). Specimens (n = 2 oysters per biological replicate) were collected at 4 different time points: T0, before exposure begins; T1, after 3 h exposure; T2, after 5 h exposure; T3, after 24 h exposure.

      The collection of the the oysters were at different time intervals in order to monitor and view different relationships between the algae concentrations and oyster infection number.The gills were the main focus of the time intervals to indicate how much of the algae was found in the oysters system.

    5. Experimental HAB simulation

      A 24h simulation using a culture of K. brevis and the eastern oyster specimens.

      They were fed less prior so that during the simulation they would actively feed.

      There were 4 time points where data was collected: 0hr, 3hr, 5hr, and 24hr.

      The oysters were opened and the gills were flash frozen for later examination. This is because the brevetoxins contact the gills first and thus would experience DNA damage first.

    6. Specimen collection and laboratory acclimatization

      Explains how oysters were collected and kept.

      Collected: Rookery Bay National Estuarine Research Reserve, Naples FL

      Kept: In lab tanks with controlled conditions. Fed twice a day.

    7. The study of the epigenetic mechanisms mediating exposure-response relationships constitutes the basis for environmental epigenetic analyses (Baccarelli and Bollati, 2009; Bollati and Baccarelli, 2010), providing information about how different environmental factors influence phenotypic variation (Cortessis et al., 2012; Suarez-Ulloa et al., 2015; Etchegaray and Mostoslavsky, 2016)

      In one experiment, one of the authors (Suarez-Ulloa) had previously studied the cause-effect relationship between environmental factors and subsequent epigenetic modifications triggering adaptive responses in marine invertebrates.

    8. Consequently, bivalve molluscs are generally used as sentinel organisms to study HAB pollution

      In previous experiments conducted on Eastern Oysters, the author has successfully used bivalve mollusks as sentinel organisms.

    1. We compared absolute and qualitative differences in leaf defense. First, we compared the dry mass of leaf defense chemicals in a linear model with soil type and study region as independent variables. Second, we tested whether habitat type, sampling site, or the interaction of the two, was a significant determinant of the relative allocation among flavan, flavone, quinic acid derivatives, and oxidized terpenes in each plant using a factorial analysis of variance.

      When doing comparison, it helps to create a table to have a better understanding on the differences in leaf defense.

    2. Second, we used a factorial analysis of variance (McArdle and Anderson 2001) to assess whether habitat type, sampling site, or the interaction of the two was a significant determinant of herbivore species composition.

      This was done to determine the different variables that affect insect populations and which ones were more important than others. With this information they set the parameters to create an observable experiment with a limited amount of discrepancies.

    3. But little is known about the mechanisms underlying the evolution of habitat specialization and the extent to which herbivores contribute to phenotypic divergence during the speciation process,

      The authors have a clear goal in their experiment. Through this observation that is lacking a response they build an experiment to identify natures processes.

    1. We therefore developed protocols for fluorescent in situ hybridization (FISH) targeting specific ascomycete and cystobasidiomycete rRNA sequences.

      A molecular probe is created against a specific DNA sequence, in this case rRNA from either ascomycetes or basidiomycetes. The probe is designed with a recognizable tag on the end. Then a second probe is used to match the first probe, and finally a third probe to match the second one with a fluorescent tag on it. By using multiple probes we can amplify even the smallest signals to our first probe. These probes are then mixed with a biological sample that is fixed in place. If fluorescence is observed the probe has found a target.

      In some experiments, as in this example, researchers use multiple probes in a single experiment. This provides information about how close two organisms of interest are in a given sample.

    2. incorporated into a broader sample of published cystobasidiomycete rDNA sequence data

      Often researchers only publish a single gene for a newly described taxon, as it is sometimes too expensive to generate sequence information for the entire genome. When research first began working with DNA sequence data to determine evolutionary relationships it was decided that the ribosomal DNA (rDNA), shared by every living thing, was the best single gene to determine evolutionary relationships between taxa. Currently this is hotly debated, however, the use of rDNA is still very common in eukaryotic taxonomy.

    3. using gene sequences inferred from our transcriptome data set and other available genomes (table S1).

      Phylogenies are often used to determine the taxonomy of a lineage, that is the evolutionary context of an organism within the greater tree of life. The researchers could recognize that the new symbiont was a basidiomycete from the sequence information, but only by comparing the sequence information of the new organism to the previously known organisms can we determine when and from which ancestor the new species evolved. To account for the millions of years that have passed after all the basidiomycetes diverged from one another, the researchers incorporate as much sequence data as possible. Here, they use 349 loci to determine the placement of the new lineage.

    4. amplicon sequencing

      Refers to sequencing of target genes obtained by a procedure called polymerase chain reaction (PCR) using gene-specific primers. Amplicon sequencing provides a snapshot of the microbial populations in an environment.

    5. attempts to culture the basidiomycete from fresh thalli were unsuccessful

      Researchers attempted to grow and isolate basidiomycete cells independent from their lichen partners, the algae and the ascomycete fungus. Often times it is very difficult to culture new organisms because we do not know enough about the growth requirements. This is frequently the case with organisms that form symbiotic relationships, as they often lose the ability to grow independent of their symbiotic partners.

    6. we designed specific primers for ribosomal DNA [rDNA; 18S, internal transcribed spacer (ITS), and D1D2 domains of 28S) to screen lichens growing physically adjacent to Bryoria in Montana forests.

      Primers are small oligonucleotide sequences that are used as probes to determine if something is present in an unknown mixed sample. In this case, the primer is designed to match a piece of ribosomal DNA specifically in basidiomycete genomes found in the previous experiments. If the primer finds a target in a biological sample it is highly likely that a basidiomycete is present in the sample. In these experiments, the biological samples are other wild lichens found next to Bryoria lichens in Montana forests. Primers operate on the molecular level, and so very little biological material is needed to determine if basidiomycetes are present.

    7. expanding the taxonomic range to consider all Fungi, we found 506 contigs with significantly higher abundances in vulpinic acid–rich B. tortuosa thalli

      The author initially looked only at the expressed gene copies (contiguous sections of messenger RNA, or contigs) that corresponded either to known ascomycete or known algal DNA. They limited their search to these two because until then, a lichen was thought to only consist of those two partners. When they could not find any difference that would explain the phenotype differences between the two lichens, they expanded their search to see what other kinds of DNA was found in every single lichen, and perhaps had not been accounted for yet. They found 506 contiguous snippets of DNA that were found in every lichen that appeared to come from a second fungus, a basidiomycete, that wasn’t supposed to occur in lichens.

    8. transcriptome-wide analysis

      A transcriptome is a way for researchers to count the number of messenger RNA copies that has been produced from each gene in an organism, which is a measure of how much this gene is expressed. By comparing the level of gene expression between two different species, researchers can get an idea of which genes might be involved in producing different phenotypes in those species.

    1. the complexity of the evolutionary history of dogs

      For a recent study on the history of dogs in the Americas (published by some of the same authors of this paper), check out the 2018 research article in the "Related content" tab.

    1. To test if mating is rewarding, males were exposed sequentially for 30 min to two odorants [ethyl acetate (EA) or isoamyl alcohol (IAA)], one in the absence and the other in the presence of virgin females, and tested for odor preference 24 hours later in the absence of females

      This sentence describes how the researchers used the conditioning assay to test whether mating is rewarding. The two odors they used came from the chemicals ethyl acetate (EA) and isoamyl alcohol (IAA). The specifics of these two chemicals are not important—any two clearly distinct smelling chemicals could have been used. Male flies were exposed to one of the two odors in the presence of sexually receptive females (who the males presumably mated with), and were exposed to the remaining odor in the absence of females. They then tested to see whether males preferred the females/mating-associated odor over the other neutral odor.

    2. To test these predictions, we used a series of conditioning assays in which male flies were trained to associate the proposed rewarding experiences (mating, ethanol exposure, or NPF circuit activation) with one of two neutral odor cues.

      Here, the researchers are aiming to verify their theory about how mating, ethanol, and activity of the NPF circuit are connected (for more on this, check out the previous paragraph) by proving that each of these three things are inherently rewarding.

      An example of how they could do this would be to repeatedly expose the flies to alcohol while simultaneously also exposing them to a particular odor cue—let's call it odor A. After repeated exposure to alcohol and odor A in conjunction, the flies will learn that if they smell odor A, alcohol must be near.

      Once this association has formed, the researchers could put the flies in a Y maze, with one prong containing odor A, and the other prong containing a random odor—say odor B. Since odor A predicts alcohol, if the flies spend the majority of their time in the prong containing odor A, it would indicate that they are seeking out alcohol, presumably because it is rewarding.

      This type of manipulation is exactly what the researchers did here, except that they also investigated mating and manual activation of the NPF circuit (in addition to alcohol) as possible rewarding elements.

    3. Several experiments were designed to determine which of these was the predominant contributor to the enhanced ethanol preference seen in rejected-isolated males

      Since the initial experiment does not allow us to clearly conclude why the virgin males preferred the alcohol (the list of possible reasons is given in the prior sentence), the researchers designed a series of follow-up experiments, each designed to examine a slightly different possibility.

    4. rejected-isolated

      The group of male flies that were exposed only to females who had already mated and were thus no longer sexually receptive. These male flies experience sexual rejection.

    5. mated-grouped

      The group of male flies that experienced repeated, lengthy mating sessions with multiple virgin female flies.

  3. Nov 2018
    1. D statistics

      Can detect admixture events; it is used to detect gene flow between closely related species.

    2. Mitochondrial sequences as well as genotype files (in plink format) were deposited on Dryad (doi:10.5061/dryad. 8gp06).

      Genetic sequences of the modern and ancient dogs used in this study are publicly available. Check out their data on Dryad!

    3. cross coalescence rate (CCR)

      A method of estimating the time at which populations had a common ancestor, based on their genetic differences and similarities.

    4. multiple sequentially Markovian coalescent (MSMC)

      A technique that looks at the pattern of DNA sequence changes (mutations) in multiple individuals, focusing on the most recent common ancestor of any two sequences. It can provide information about the timing of shared ancestry, population sizes, population splits, and migration rates.

    5. complete (28x) genome

      Certain bases that are always present in dogs were covered by sequencing 28 times, so the whole genome is said to have been covered 28 times.

    1. To perform a fair comparison, we fixed the computational complexity of both algorithms to be the same

      The fly algorithm is so much more efficient that it can perform more computations for the same cost as the LSH algorithm. This is the because the cost per projection is so different—for each projection in LSH, it requires 2d operations. For each projection in the fly algorithm, it only requires 0.1d operations. To even it out so that the total operational cost is the same means that the fly algorithm is able to generate 20 times the number of projections as the LSH algorithm.

    2. mean average precision

      The mean average precision (MAP) is a way of measuring how well an algorithm is doing at a task.

      Here, the authors took each algorithm (fly and LSH) and ran it against each data set (SIFT, GLOVE, and MNIST) for 1,000 different query inputs. This produced a list—also called a rank—of all the images or words that were similar to the query. Each item in the rank comes with a certain precision value that represents its accuracy.

      Next, the authors added up all the precision values in the ranking (with the top-ranked values carrying the most weight) and divided the sum by the total number of items in the ranking to get the average precision (AP). They then took the average of all 1,000 APs (one for each query) to get the MAP.

      Calculating a MAP for each algorithm and data set allowed the authors to compare the effectiveness of the algorithms.

    3. We compared the two algorithms for finding nearest neighbors in three benchmark data sets: SIFT (d = 128), GLOVE (d = 300), and MNIST (d = 784)

      SIFT, GLOVE, and MNIST are three well-known data sets that researchers and computer scientists often use to test similarity search algorithms.

      MNIST is the Modified National Standards of Instruments and Technology data set. It consists of handwritten digits, and each row of the matrix corresponds to an image.

      SIFT stands for Scale-Invariant Feature Transform. It consists of reference images and can be used to extract specific features of those images (e.g. the corners of a door frame).

      GLOVE, or Global Vectors for Word Representation, consists of word vectors. It is useful for measuring the linguistic or semantic similarity between two words.

    4. provides a conceptually new algorithm for solving a fundamental computational problem

      This "fundamental computing problem" is the problem of being able to quickly and efficiently pick out objects that are similar to one another from very large data sets.

      For example, Google needs to be able to pick out search results that are similar to what you typed—and it needs to do this very quickly. Likewise, Netflix needs to compare your watch history to that of all its other users so that it can make recommendations for what else you might like.

      These and other similar problems pop up all over the place in your everyday life. The process of solving them is called a "similarity search" and is of intense interest to computer scientists who want to make these searches faster, more efficient, and better quality.

    1. We injected Yob mRNA into nonsexed preblastoderm embryos of A. gambiaeand its sibling species A. arabiensis to assess whether ectopic Yob transcripts affect mosquito sex ratios. To control for successful injection, we coinjected a plasmid with a green fluorescent protein (GFP) expression cassette (embryos that receive sufficient nucleic acids develop into larvae transiently expressing GFP; fig. S9). Surviving individuals were sorted at the larval stage into a GFP-positive and a GFP-negative group (Fig. 3A), and at the pupal stage mosquitoes were sexed.

      In this experiment, the authors injected normal Yob transcripts into normal embryos, along with a fluorescent marker to let them know that injection was successful, and allowed the embryos to grow. Once at the larval stage, they could see the fluorescent injection marker and sorted the larvae based on expression of the marker. At the pupal state, the authors were able to sex the mosquitoes.

    2. We transfected the Sua5.1 cells with two modified Yobtranscripts containing putative nonsynonymous point mutations r.1A>C and [r.5U>A; 6U>G] and investigated the effects of the deduced amino acid changes Met1→Leu and Phe2→Stop, respectively, on dsx splicing.

      Based on what the authors found by analyzing other species, they hypothesize that Yob actually codes for a protein. They made new lab-generated transcripts of Yob with sequence changes that would result in no protein product (either removing the start codon, or adding in a premature stop codon) and transfected those transcripts in to cells to monitor dsx splicing.

    3. we evaluated the protein-coding potential of Yob by comparing its sequence to PCR-isolated orthologous sequences from Anopheles arabiensis and Anopheles quadriannulatus, two members of the Anopheles gambiae complex.

      The authors isolated sequences from closely related mosquito species and compared similarities and differences in those sequences computationally.

    4. We investigated the effect of in vitro–synthesized mRNA corresponding to the shortest, presumably mature A. gambiae Yob transcript isoform on dsxsplicing

      The authors made mRNA in the lab. The mRNA has the same sequence of what they think is the shortest, but functional Yob transcript. They are testing whether Yob has a direct effect on dsx.

    5. ectopic embryonic delivery of Yob transcripts

      The scientists artificially inserted the gene-reading Yob into the embryos of Anopheles gambiae.

    6. transcription of Yob is limited to males. Transcription begins in embryos between 2 and 2.5 hours after oviposition,

      As seen in Figure 1, they are analyzing Yob mRNA through use of a gel, not sequencing, in this experiment. The authors find that transcription of this male factor begins very early after fertilization, at the same time as other genetic markers of zygotic expression.

    7. we analyzed transcriptomes of male and female embryos (18), whose sexual identity was determined by polymerase chain reaction (PCR) (fig. S1). Separate pools of mRNA were sequenced, yielding ~500,000 Roche 454 reads from each sex.

      Because mosquito embryos look identical whether male or female, the authors had to use molecular techniques to find out the sex of each embryo. The embryos were separated based on their sexual identity (male or female) and mRNA was extracted and sequenced from the collective male or female embryos.

    1. We are sometimes asked what the result would be if we put four +'s in one gene. To answer this my colleagues have recently put together not merely four but six +'s. Such a combination is active, as expected on the basis of our theory, although sets of four or five of them are not.

      This experiment is an extension of the frameshift experiment. If the bases are read in triplets, then you'd expect that four or five extra bases would ruin the code. However, six extra bases would still encode the amino acids for the native protein (with two extra amino acid residues).

    1. FGFP data set provided a unique opportunity to perform an informed power analysis

      The authors performed a power analysis study, which allows researchers to determine the sample size required to detect an effect, due to the large number of samples with broad characteristics. They first calculated the number of samples needed to determine a difference in microbiota diversity when the cause is unknown. To do so, they need to determine the effect size (the minimum deviation that is considered significant), the significance level (the probability of determining that a condition is true given that it is false), and the power (the probability of determining that a condition is false given that it is true).

      Read more: http://www.biostathandbook.com/power.html

    1. SEM

      Acronym for standard error of the mean, a measure that represents how far the mean of a sample is from the estimated true population mean. It is a good estimate of how accurately your mean reflects the true population.

      To learn more about SEM: https://www.khanacademy.org/math/ap-statistics/sampling-distribution-ap/sampling-distribution-mean/v/standard-error-of-the-mean

    2. Analysis of covariance

      An analysis of covariance is used to see how two independent variables change together.

      Here, the authors used it to determine whether the response to a reward or devaluated stimuli was due to habitual learning, rather than external factors such as outcome-action knowledge, working-memory/inhibition, or the ability to learn how to discriminate during the training phase of the test.

      To learn more about analysis of covariance: http://www.lehigh.edu/~wh02/ancova.html

  4. Oct 2018
    1. To test whether activation of the NPF-NPFR pathway is rewarding per se, we trained virgin males to associate artificial activation of NPF neurons with either EA or IAA. Males expressing dTRPA1 in NPF neurons (NPF-GAL4 + UAS-dTRPA1) and the genetic controls each carrying only one of the two transgenes were trained for three 1-hour sessions at 29°C, with dTRPA1 active, interspersed with three 1-hour rest periods at 18°C, with dTRPA1 inactive (Fig. 4B).

      The final thing the researchers wanted to test was whether artificial activation of the NPF circuit was inherently rewarding. To do this, they once again relied on the dTRPA1 proteins, which can activate neurons at high temperatures. The researchers inserted this protein into neurons that were part of the NPF circuit. They then exposed the flies to one smell while keeping a high temperature (thus activating the NPF neurons). This smell became associated with a state of high NPF circuit activity. Conversely, they exposed the flies to the second smell while keeping a low temperature. This smell became associated with a state of normal/low NPF circuit activity. Finally, they placed the flies in a Y maze (as before) and checked to see if the flies would prefer the prong containing the NPF-activity paired odor, or the unpaired odor.

    2. A conditioned odor preference index (CPI) for mating was calculated by averaging preference indices for reciprocally trained groups of flies. Positive CPI values indicate conditioned preference, negative values indicate aversion.

      The researchers used time spent in the two prongs of the Y maze to develop a preference score. Per this score, a positive value would indicate that the flies preferred the odor that was associated with one of the three activities (mating, alcohol exposure, or activation of the NPF circuit). Conversely, a negative score would indicate that the flies preferred the odor that was not associated with these activities. A score of zero would indicate no obvious preference between the two.

    3. To determine whether the inverse correlation between NPF levels and ethanol preference reflects a cause-and-effect relationship, we manipulated the NPF-NPFR system genetically.

      So far, the researchers have shown that changes in NPF levels correlate with both sexual experience and alcohol preference. Specifically, a more active sexual history is correlated with an increase in NPF levels, which is correlated with a decrease in preference for alcohol (and vice versa). However, one of the most important rules to remember in science is that correlation does not equal causation. The researchers thus decided to follow up with a direct genetic manipulation of NPF in order to establish a causal relationship.

    4. To further test the strength of this conclusion, we divided a cohort of rejected-isolated males into two subgroups, one of which was left undisturbed, and the other of which was allowed to mate with virgin females for 2.5 hours immediately before testing.

      As a final follow-up experiment, the researchers took a group of sexually rejected males and split them up into two. One subgroup was left alone, and then tested for alcohol preference. The other subgroup was exposed to virgin females just before testing. As such, these males did in fact get an opportunity to mate before undergoing the alcohol preference test. If the first subgroup continued to prefer alcohol, but this preference were to disappear for the second group (who initially experienced rejection, but then were able to mate just before testing), it would indicate that the main cause for the alcohol preference was indeed sexual deprivation.

    5. We next investigated ethanol preference in males that were sexually deprived (blocked from copulating) but not exposed to the social experience of rejection.

      For the second follow-up experiment, the researchers wanted to investigate the possibility that it was not actually the lack of sex, but rather the experience of rejection by a female that was driving the virgin males to prefer alcohol.

    1. To identify nonredundant covariates of microbiome variations from our shortlist of 69 correlating factors

      A redundant covariate is a factor that either has the same value for every sample or multiple factors that are highly correlated and can be used as substitutes for one another. The researchers removed redundant covariates from their study because they can hamper interpretability of the results.

    2. forward stepwise redundancy analysis (RDA)

      This is a statistical technique used to describe linear relationships between components of dependent variables that can be explained by a set of independent variables. Here, the independent variables were the 69 covariates and the dependent variable was the microbiota variation in each sample.

    3. PCoA

      Stands for Principal Coordinate Analysis, a statistical technique used to visualize similarities between variables by representing the variance in as few axes/dimensions as possible. The arrows show the direction of the original variables and the angle between arrows gives a rough estimate of the correlation between features.

    4. 16S ribosomal RNA (rRNA) gene amplicon sequencing

      Almost all bacteria have a copy of the 16S rRNA gene. Comparing their sequences is a common tool to identify and differentiate bacteria at the species level.

      Learn more about how bacteria are identified with this resource from iBiology: https://www.ibiology.org/microbiology/genome-sequencing/

    1. We then found the top 2% of predicted nearest neighbors in m-dimensional hash space

      For the fly, the data is organized into a higher dimensional space. For LSH, it is organized in a lower dimensional space. But if the algorithms work effectively, the data should still be arranged so that similar features are near one another.

      Instead of feature vectors, the algorithms arrange items in hashes or "tags" (just like the fly brain uses a tag to represent an odor, the algorithms use a tag to represent a specific image or word). Finding the hashes that are closest to each other in this new m-dimensional space should reveal the images/words that are most similar to each other (again, if the algorithm works correctly).

      These nearby hashes are called "predicted nearest neighbors" because they predict which items in the data set are the most similar.

    2. we show analytically that sparse, binary random projections of the type in the fly olfactory circuit generate tags that preserve the neighborhood structure of input points.

      In a mathematical proof, the authors took a matrix of input odors and multiplied it by a vector of projection values to create a new matrix of Kenyon Cell values. In this new matrix, the relative positions of the input odors were approximately the same as they were in the original matrix.

      This shows that the fly algorithm preserves the distance between input points when sending information from PNs to KCs.

    1. scanning electron microscopy

      In bright field microscopy, an image is produced by bouncing light off of an object and the image is magnified by lenses. In scanning electron microscopy, an image is produced by bouncing electrons off of an object and the image is interpreted and produced by a computer. By using electrons instead of light, researchers can observe incredibly fine detail in the image created.

    2. initial microscopic imaging

      Researchers look at a sample through a bright field microscope to look for evidence of cell morphology that could be basidiomycetes.

    3. When assaying for the basidiomycete

      To take a measurement of a sample, usually meaning a biochemical or chemical measurement of a biological sample; in this case, basidiomycete yeasts.

    4. we estimated transcript abundances by mapping raw reads back to a single, pooled metatranscriptome assembly and binning

      In a biological sample that contains more than one organism, before gene expression can be evaluated, the researchers must identify which genes belong to which organism. To do this, gene expression data is mapped onto the genomes of the organisms that are known in the mixture, and genes are sorted, or binned, into groups by the genomes they match. This way the gene expression data for each known organism can be determined.

    5. We hypothesized that differential gene expression might account for the increased production of vulpinic acid in B. tortuosa.

      The authors knew that the difference between these two lichen species was poorly understood. One species produces vulpinic acid, and the other does not. Phylogenetic analysis could not identify differences in the symbionts of the two species. The authors decided to test if differential gene expression of a gene shared between the species might be the cause of the phenotypic difference.

    1. We purified the corresponding recombinant I. sakaiensis proteins (fig. S5) and incubated them with PET film at 30°C for 18 hours.

      Using genetic engineering, the authors isolated the protein strand the two organisms had in common and exposed it to a PET film without any microbes present.

    2. We collected 250 PET debris–contaminated environmental samples including sediment, soil, wastewater, and activated sludge from a PET bottle recycling site (7). Using these samples, we screened for microorganisms that could use low-crystallinity (1.9%)

      The authors collected samples from different environments where PET plastic waste was likely to be found. In this case they chose to sample near a PET recycling plant. The plant specifically recycled high-crystallinity PET. They took some dirt, water, and sludge and put the samples in an environment including low-crystallinity PET. Low crystallinity PET is more disorganized and easier to break down than polymers with higher crystallinity.

      It was ultimately one of the samples of sediment that grew bacteria. When looking at the bacteria under a microscope, the researchers were able to see that it was growing on a thin layer of PET plastic.

    3. We compared the activity of the ISF6_4831 protein with that of three evolutionarily divergent PET-hydrolytic enzymes identified from a phylogenetic tree that we constructed using published enzymes (Fig. 2C and table S2). We purified TfH from a thermophilic actinomycete (10), cutinase homolog from leaf-branch compost metagenome (LC cutinase, or LCC) (11), and F. solanicutinase (FsC) from a fungus (fig. S5) (12), and we measured their activities

      The authors used published data of proteins having similar activity as the ISF6_4831 enzyme and analyzed it using statistical tests to select three enzymes that divergently evolved—meaning that the enzymes evolved from a common ancestor and accumulated enough differences to result in the formation of a new species.

    1. Monte Carlo analysis

      This technique is used in calculations for complicated systems where changes in one variable influences many parts of the calculation. In this technique many calculations are run with each calculation having different beginning conditions. The end results are then analyzed to look at the range of possible outcomes. The error bars in parentheses in Figure 4 are determined by a Monte Carlo simulation.

      See more about Monte Carlo simulations from the Massachusetts Institute of Technology: http://news.mit.edu/2010/exp-monte-carlo-0517

    2. R2

      R-squared (\(R^2\)) values represent how close the data points are to the predictions from a model. An \(R^2\) value of 1 means that all of the data points lie on the prediction from the model. In this case, the researchers are using the solid line that represents perfect agreement between the box model calculation and the experimental data. An \(R^2\) value of 1 in this case would mean that the box model calculations and the experimental data are in perfect agreement. In general, the closer the \(R^2\) value is to 1, the closer the model and data match.

      Without the inclusion of VCPs in the model calculation the \(R^2\) value was 0.59 (Panel A of Figure 3). With the inclusion of VCPs into the model the \(R^2\) value improved to 0.94 (Panel B of Figure 3).

    3. Here, we assess the importance of VCP emissions to ambient air pollution, again using Los Angeles as a test case (Fig. 4). Los Angeles currently violates the U.S. 8-hour O3 standard,

      The air in Los Angeles, California has ozone (\(O_3\)) levels that are higher than those recommended by the Environmental Protection Agency. The amounts and types of VOCs present influences ozone levels.

    4. We therefore conclude that large underpredictions are due to missing emission sources.

      Comparisons are made between experimental data and model calculations. The model calculations are influenced by what molecules are emitted, how those emissions chemically react, and how wind and other variables cause emissions to move from one place to another. The calculations use techniques from other researchers (including this paper's co-authors Joost de Gouw and Si-Wan Kim) to take into account chemical reactions and movement of the emissions from one place to another.

      The authors then make different predictions with their model assuming different types of chemical emissions (fossil fuels and/or VCPs).

    5. we found that fossil fuel VOCs [from mobile sources and from local oil and natural gas production and distribution (36)] can only account for 61% of the mass of freshly emitted VOCs measured, and 59% of their variability

      Box model calculations were carried out under various conditions. The authors can make various assumptions about what sources are emitting VOCs into the air (mobile sources or VCPs). The results of the calculations under these various conditions are then compared to experimental data. This allows the researchers to examine what sources are important contributors to VOCs in air.

    1. boundary conditions and idealizations in the simulation

      The authors define a series of constraints for their simulation. The scientific model assumes facts about the phenomenon that make the problem easier to solve. In this case, the authors assume among others that the pore structure is homogeneous, that the vapor pressure is the same for the MOF and the condenser, and that the diffusivity of the crystal does not vary.

    2. For visualization purposes, we used a condenser with a temperature controller to maintain the temperature slightly below ambient levels but above the dew point, in order to prevent vapor condensation on the inner walls of the enclosure.

      To simplify the experiments, the author maintain the condenser at a temperature cooler than the surrounding temperature. This results in the condenser being cooler than the walls of the box, so that all the water condenses on the condenser and can be easily collected. In practice, ambient temperature is enough to harvest water without the need for additional cooling of the condenser.

    3. A solar flux (1 kW m–2, air mass 1.5 spectrum) was introduced to the graphite-coated substrate layer

      The authors simulate natural sunlight in the laboratory. The air mass is the ratio of the path length that sunlight has to travel through the atmosphere to the vertical path length (when sun is at the highest point in the sky, which corresponds to the shortest path length). An air mass of 1.5 is the condition most commonly use to quantify the efficiency of solar devices.

    4. The environmental temperature above standard ambient temperature was necessary to perform the experiments at >1 kPa

      In order to efficiently harvest water, a large difference of temperature between the MOF and the condenser is needed. In their experiment, the authors set the temperature at 35°C in order to be able to condense the vapor with a condenser at 10°C.

    5. In this simulation, MOF-801 was initially equilibrated at 20% RH, and the vapor content in the air-vapor mixture that surrounds the layer during desorption increased rapidly from 20 to 100% RH at 25°C

      The authors performed a simulation in conditions reflecting an accurate, real-life use of the MOF. The results show an increase in RH upon water desorption, followed by return to the initial 20% value upon water adsorption.

    6. the crystal diameter of MOF-801 is only ~0.6 μm

      The diameter of MOF-801 was determined by a method called scanning electron microscopy. The surface of the MOF is scanned with a beam of electrons which gives information about the surface.

      See figure S5 in the Supplementary Materials.

    7. A theoretical model was developed to optimize the design of the water-harvesting process with MOF-801, which was further validated with the experimental data.

      The authors developed a computational model to simulate the behavior of the MOF material. Through this approach, they can study the influence of the material parameters on the water harvesting properties and then confirm their result experimentally. Their goal is to screen a large number of parameters computationally to optimize the system.

    8. We carried out the adsorption-desorption experiments for water harvesting with MOF-801 at 20% RH

      The author first quantified the water harvesting capacity of the MOF. For this purpose, they use 20% relative humidity for their measurements, as it is representative of the low humidity in dry regions of the world where an efficient water-harvesting method is most needed.

    9. Therefore, to predict the prototype’s water-harvesting potential under equilibrium conditions, we extended the desorption time for the simulation

      The design of the prototype results in a slower desorption process than was considered in the initial calculations. The authors modified the parameters of their simulation to take this into account and have a model reflecting the experimental conditions.

    10. Last, a proof-of-concept MOF-801 water-harvesting prototype was built to demonstrate the viability of this approach outdoors

      To demonstrate the utility of their material in real conditions, the authors built a small device containing the MOF and a condenser and tested it outdoors.

    11. The powder was infiltrated into a porous copper foam with a thickness of 0.41 cm and porosity of ~0.95, which was brazed on a copper substrate to create an adsorbent layer

      The MOF is pressed onto a substrate made of copper metal in order to have a material that conducts heat efficiently and that can cover a large surface.

    1. Ordinary least squares (OLS) regression summary statistics (adjusted R2)

      Like PGLS, this method examines the relationship between two or more variables. However, this method does not account for phylogeny. OLS regression was used to compare with PGLS results to see if it would produce similar results.

    2. We measured differences in species’ northern and southern range limits, the warmest or coolest temperatures occupied, and their mean elevations in three periods (1975 to 1986, 1987 to 1998, and 1999 to 2010) (figs. S2 to S4) relative to a baseline period (1901 to 1974)

      To test whether climate change has impacted species ranges, the authors had to measure, for each species, the latitudes of the northern and southern range limits (and the distance between these), the warmest and coolest temperature the species were observed, and the mean elevation of the species.

      These measurements are based on the average of the five most relevant species observations in each case. For example, to find the northern range limit, the authors took the average of the five northernmost observations of the species, while to find the coolest temperatures within the species' range, they took the average of the five coolest points that the species was observed at. This helps avoid skewed results from a signal sighting.

      Check out the Supplemental Materials for more information on how the authors took all their measurements.

    1. glial fibrillary acidic protein (GFAP) promoter to target ChR2 to local astroglia

      Glial fibrillary acidic protein (GFAP) is an intermediate filament (cytoskeletal component) protein expressed by glial cells including astrocytes. Glial cells are non-neuronal cells within the central and peripheral nervous systems whose roles include providing support and protection for neurons. When a gene like ChR2 is under the control of a GFAP promoter, only those cells that express the transcription factor for GFAP will express ChR2.

    2. Optical circuit interventions were tested in rats that had been made hemiparkinsonian by injection of 6-hydroxydopamine (6-OHDA) unilaterally into the right medial forebrain bundle (MFB)

      To test whether optogenetics can be used to dissect neural circuits, the authors utilized an established model of induced Parkinson's disease. 6-OHDA is a synthetic neurotoxin that selectively destroys dopaminergic and noradrenergic neurons in the brain. When injected on one side, it can cause side-biased motor deficits. The right medial forebrain bundle is a neural pathway that passes through the hypothalamus and basal forebrain and contains a high number of dopaminergic neurons.

  5. Sep 2018
    1. Johnson-Lindenstrauss

      This is a mathematical statement that shows that you can take a small number of points in a high-dimensional space and transfer them to a lower-dimensional space while (very nearly) maintaining the distance between all the points.

      You could think of this like taking all the points on a sculpture and projecting them onto a piece of paper while keeping every point in its proper position relative to all the other points on the sculpture.

    2. Illustrative odor responses.

      Here, the authors use chemicals (ethanol, methanol, and dimethyl sulfide) to demonstrate similarity tagging/hashing.

    3. we used 20k random projections for the fly to equate the number of mathematical operations used by the fly and LSH

      As stated earlier in the article, the authors could only fairly compare the LSH and fly algorithms if each used the same number of mathematical operations.

      They determined that if they used 20k random projections in the fly algorithm (where k is the length of the output tag, or hash) then the total number of operations for the fly and LSH algorithms would be equal.

      For more detail, see the second paragraph under "Materials and Methods" in the Supplementary Materials document.

    4. we selected 1000 random query inputs from the 10,000 and compared true versus predicted nearest neighbors

      From the 10,000 vectors, the authors randomly chose 1,000 of them to be query inputs (images or words that they would test against the rest of the feature vectors to find which ones represent similar images or words).

    5. We used a subset of each data set with 10,000 inputs each, in which each input was represented as a feature vector in d-dimensional space

      They chose 10,000 vectors of length d from each data set. Each vector represented an image (SIFT, MNIST), or word (GLOVE).

    6. computed the overlap between the ranked lists of true and predicted nearest neighbors

      This is a method of measuring how well an algorithm performed. The more overlap between the two lists, the more similar they are. If they are exactly the same, this means that the algorithm performed perfectly.

      For example, it might mean that it found the closest match to all query images, or that it correctly found all the correct features that match part of an image (see picture below).

    7. determined on the basis of Euclidean distance between feature vectors

      Each feature vector contains a row of values representing features of an image or word (depending on the data set). Because the data is arranged so that similar features are near each other, measuring the straight-line (Euclidean) distance between two vectors allows the authors to determine the similarity between them.

      That is, the closer the vectors, the more similar their features. The vectors closest to one another are the "true nearest neighbors," or the images/words in the data set most similar to one another.

    1. AIC

      The Akaike information criterion (AIC) is a statistic that measures whether some models are more or less informative than others. This means that a model with a lower score for AIC is more informative than a competing statistical model and may tell us something that is meaningful in a biological sense.

      Like many other aspects of statistics, we have to be careful about blindly believing test results, so we apply our experience as scientists to make sure that numbers like the AIC score actually make biological sense!

      Click here for a more in-depth view of what AIC is, how it is calculated, and what it can (and can't) do.

    2. PGLS models

      Phylogenetic Generalized Least Squares (PGLS) is a method that accounts for phylogenetic relationships within a group of species (phylogeny reveals how closely related species are to each other). This method will determine if variables of interest are closely related.

      In this table, models of trait evolution indicated whether traits are ancestral and kept over evolutionary time. Species’ upper thermal limit (one of the traits studies here) was found to be shared by close relatives. The authors pointed out in this paper that niche conservatism (i.e. the tendency for species to keep their ancestral traits) could explain such findings.

    3. applications accounted for changes in bumblebee species’ range or thermal limits (table S3)

      Without also testing competing theories (land-use and pesticides), the authors would not be able to say how important the effects of climate change were in influencing range limits. Remember that the scientific method does not allow us to definitively prove a theory, only add to the evidence that one hypothesis is more likely than the alternatives.

    4. We investigated whether land use affected these results. Finally, we used high-resolution pesticide application data available in the United States after 1991 to investigate whether total pesticide or

      Because climate change is not the only possible explanation for changes in species' latitudinal and thermal range limits, the authors also tested other competing theories, like land-use changes and pesticides.

    1. hybrid optical stimulation–electrical recording device (optrode)

      An optrode is a combined optical fiber with a metal electrode that can both stimulate via light and record via a tungsten wire. The tungsten wire was tightly attached to the optical fiber with the electrode tip being slightly deeper to ensure proper recording from light stimulated neurons.

    2. opsin vector introduction

      To accurately target the subthalamic nucleus (STN), the authors used extracellular recordings to determine accurate position within the deep brain structure since the STN has a characteristic firing pattern. Guided by this method, accurate injection of opsin vectors to the region was accomplished.

    1. Cyclone intensities around the world are estimated

      The authors used satellite imagery and characteristic wind and moisture patterns in order to gauge the intensity of storms and hurricanes.

      This method of gauging hurricane intensity has been tested against aircraft sensor measurements and does a solid job of accurately recognizing hurricane characteristics.

    2. we conducted a comprehensive analysis of global tropical cyclone statistics for the satellite era (1970–2004)

      These authors used satellite data from 1970 to 2004 in order to examine cyclones and hurricanes in every tropical ocean basin.

      They examined: 1) number of storms and hurricanes 2) number of storm days 3) hurricane intensities

      Most of the data examined came in the form of best track data archived in hurricane warning centers.

    1. instrumental learning

      A method used to reinforce an association with a certain stimulus. Unlike classical conditioning, instrumental learning (otherwise known as operant conditioning) is active and involves a person performing behaviors that are positively or negatively reinforced. Reinforcement is also known as learning.

      The two instrumental tasks used in this study were reward and avoidance learning. Participants learned that using a pedal would allow them to avoid being shocked when presented with pictures that they had learned to associate with a shock.

    2. slip-of-action test

      A test for habitual behavior. Certain stimuli that were previously associated with a reward are devalued, meaning the reward is removed.

      Once the reward is removed, responding to a stimulus no longer makes sense. If people continue to respond, their response is considered habitual.

    3. skin conductance

      Skin conductance measures the activity of certain sweat glands, which become more active during the avoidance response (resulting in a higher skin conductance).

      It was measured 0.5 to 5 seconds after each stimulus was presented. Conductance allowed the authors to measure how the participants respond to seeing the stimulus before reacting to it and to make sure they had learned to fear the stimuli associated with a shock. This way, they knew that if participants performed poorly in the task, it was not because they did not learn to fear the stimuli.

    1. Early hominin stature reconstructions are notoriously difficult to assess: the limited number of intact long bones available in the fossil record often requires reconstruction of the long bone length from fragmentary remains, before different methods can be used to estimate the stature; the eventual results can differ according to the method employed.

      The authors based early hominin stature estimates on the length of intact fossil femurs, reconstructed femurs, and femur head diameters.

    2. For both of the described methods, mean estimates of stature and body mass for S1 were computed by averaging the estimates obtained from individual tracks (Tables 2 and 3). The average footprint length values were considered more reliable than minimum values (which from a theoretical point of view could be regarded as more representative of the foot length) for the following reasons.

      The authors used average footprint length when estimating stature and body mass. They did so to minimize overestimates and underestimates of footprint size and to compensate for poor preservation.

    3. Similarly, we estimated the body mass of the Laetoli track-makers using the 'walk only' regression equation that relates footprint area (i.e., footprint length x max. width) to body mass

      The authors used a regression equation based on walking pace as it relates to the size of the footprint and body mass. Australopithecus afarensis proportions were used rather than those of modern humans.

    4. we also computed some estimates using the foot:stature ratio known for Au. afarensis (Dingwall et al., 2013). This ratio is 0.155–0.162 (Dingwall et al., 2013), so we obtained stature estimates (Tables 2–3) predictably close to or slightly lower than the lower limit of the estimates given by the Tuttle (1987) method.

      The authors also estimated the stature of the Laetoli trackmakers using Australopithecus afarensis foot:stature ratios.

    5. Given that the foot length in H. sapiens is generally about 14% to 16% of stature (Tuttle [1987], and references therein), we computed two estimates for the Laetoli hominins assuming that their feet were, respectively, 14% and 16% of their body height (Tables 2–3). This method, however, is not fully reliable because it is based on the body proportions of modern humans, and because it does not take into account that the footprint length does not accurately reflect the foot length.

      The authors used two different methods to estimate the stature of the hominin trackmakers. One used modern Homo sapiens body proportions. This was considered to be less accurate since the body proportions of the hominids were likely different from those of modern humans.

    6. The following morphometric measures were taken on the contour maps: footprint length – maximum distance between the anterior tip of the hallux and the posterior tip of the heel; footprint max width – width across the distal metatarsal region; footprint heel width; angle of gait – angle between the midline of the trackway and the longitudinal axis of the foot; step length – distance between the posterior tip of the heel in two successive tracks; stride length – distance between the posterior tip of the heel in two successive tracks on the same side.

      The authors gathered the following data on tracks from both G and S.

    7. Data acquisition and processing (Supplementary file 4) were performed following the workflow described above for the Site S test-pits. We positioned four perimeter control points and 11 inner targets. The latter were used to model in detail six selected tracks (G2/G3–29, G1–35, G1–34, G2/3–26, G2/3–25 and G2/3–18, listed in the direction of walking)

      The authors used the same data gathering and visualization techniques on the fiberglass casts as they had used on the tracks at S.

    8. Data processing started by checking measurements in plan and height. This step is preliminary to the definition of the control point coordinates

      The authors checked their measurements and used STAR*NET software to combine their conventional observations with GPS vectors. They used the leveling observations the software provided to make 3D adjustments in the footprint images.

    9. The photographic survey was carried out by three shooting modes:

      Each test-pit was surveyed and photographed in order to determine its relationship to the other test-pits and to Site G.

    10. n the second step, the perimeter target positions were measured. We placed two rods equipped with a spherical level on successive pairs of targets and we marked points at the same height on the rods for each pair by using the water level device

      The authors carefully measured distances between targets so that the location and depth of each of the modeled footprints could be precisely determined.

    11. Each test-pit was entirely surveyed at lower resolution and then detailed 3D models of some inner portions (single prints or groups of close prints) were acquired (Figures 4–6).

      The authors made 3D models of selected footprints and sets of footprints. Models of some of the footprints were not made due to their poor state of preservation.

    1. We explored a low, moderate, and high scenario for atmospheric CO2 concentration (550, 850, and 950 ppm, respectively: EPA and IPCC 2007) and each climate driver: mean annual air temperature (+1, +2.5, and +4.2°C; IPCC 2013) and precipitation (−2, +7, and +14%; IPCC 2013) (Fig. 4).

      Here, the authors explains how they ran different scenarios in the simulation system with the data they had collected in the field, making for more realistic results.

    2. Fig. 2. Long-term daily weather data from the NCDC Royal Palm Ranger Station from 1963 to 2012. In climate change simulations weather data variability during 2000 to 2100 was based on variability at the Royal Palm weather station during 1963 to 2011 and weather data from TS and SRS in 2012.

      These data collected from 1963-2012 wereinputted into the DAYCENT system to simulate conditions during the next 100 years (from 2000-2100).

    3. Table 1. DAYCENT site characteristics for Taylor Slough (TS) and Shark River Slough (SRS). Site data was obtained from the Florida coastal Everglades Long-term Ecological Research (FCE LTER sites TS-1 and SRS-2), AmeriFlux and the literature.

      This table shows the two different sites, Taylor Slough (TS) and Shark River Slough (SRS) along with their exact location using the coordinate system, characteristics of each site, such as root:shoot ratio, soil composition, depth of roots, Nitrogen deposition along with the amount of Carbon found at each site.

    1. All islands were sampled at the completion of the growing season (August) between 2001 and 2003. We established a 30 m by 30 m plot at each of the grid crosspoints (12 to 32 per island, depending on island size) (12), within which we sampled plant species presence and cover; aboveground plant biomass; total soil N, P, and δ15N; and %N and δ15N from a common grass (in most cases Leymus mollis but in some instances Calamagrostis nutkanensis) and forb (Achillea borealis) (12).

      In this experiment, grids were designated over the island and 30 x 30 meter plots were established to sample within. In these plots the total soil nitrogen, phosphorous, amount of stable nitrogen isotope, and percentage nitrogen were sampled across grasses, forbs, dipterans, arachnids, passerines, and mollusks to determine how much of these nutrients were derived from the ocean. Organisms and soil that derive their nitrogen from a local source will have fewer amounts of nitrogen isotopes than those that get their nitrogen from higher trophic levels as is shown in Figure 3.

    2. Experimental nutrient additions to a community representative of fox-infested islands over 3 years caused a 24-fold increase in grass biomass (24.33 ± 6.05 g m–2) compared with control plots (0.51 ± 0.38 g m–2 increase; two-factor analysis of variance, F1,20 = 23.96, P < 0.001) and a rapid shift in the plant community to a grass-dominated state. In fertilized plots, grass increased from 22 (±2.7%) to 96 (±17.3%) of total plant biomass, whereas grass biomass in control plots was relatively unchanged (11.4 ± 3.0% and 12.1 ± 1.2% of total biomass at the start and end of the experiment, respectively) (12). In a parallel experiment (18), we disturbed and fertilized plots to mimic the effects of both seabird burrowing and guano addition. Here we found that disturbance negatively rather than positively affected grass biomass; the effects of fertilization alone were far greater than the joint effects of disturbance and fertilization. These results confirm the importance of nutrient limitation in these ecosystems and establish that nutrient delivery in the form of seabird guano is sufficient to explain observed differences in terrestrial plant communities between islands with and without foxes.

      In this experiment, nutrients were added to a community that represented the fox-infested islands which increased the grass biomass and also created a more grass dominated environment overall.

      The nutrient additions were meant to represent the spread of guano by the seabirds. In the fox-infested islands the seabird populations were scarce, which decreased guano accumulation. Therefore, the added nutrients represented the guano production by seabirds in the absence of seabird on the island that is fox-infested.

      These nutrient additions are similar to the nutrient states of fox-free islands and show what the changes in ecosystem could have been like during the introduction of foxes.

      In another experiment, they tried to mimic the disturbance in soil of seabirds burrowing, this negatively impacted the grass biomass and it was shown that fertilization by itself had the greatest positive effect on grass biomass and distribution. This helps explain the higher incidence of grasses in the fox-free islands.

    3. Fig. 1. The Aleutian archipelago with sample islands indicated in red (fox-infested) and blue (fox-free). Adak Island, the location of fertilization experiments, is indicated with a yellow dot.

      This figure shows the islands that were studied and their relative position on Earth. There are 9 fox-infested areas shown in red and 9 fox-free areas shown in blue, the sample size of islands is the same so the results cannot be attributed to the sample size difference. The yellow dot is the location of fertilization experiments.

    4. We use this experiment to show how differing seabird densities on islands with and without foxes affect soil and plant nutrients; plant abundance, composition, and productivity; and nutrient flow to higher trophic levels

      This is very broadly what the experiment seeks to discover through looking at fox-infested and fox-free islands. This experiment is designed to assess effects of sea bird densities on soil nutrients and composition, plant abundance, primary productivity, and the flow of nutrients from lower organisms to higher ones (i.e. from plants to herbivores to carnivores.)

    1. Physics had to be modified

      It would be more accurate to say that the models used to describe physics had to be modified.

      As physics progressed, physicists revised their theories in light of new evidence and knowledge (and they continue to do so today). Here, Einstein describes how the special theory of relativity came about, in part, to solve the problem of classical mechanics' compatibility with electromagnetic theory.

      Modern physicists consider classical mechanics an approximate theory that is useful for the study of non-quantum mechanical, low-energy particles in weak gravitational fields. It is usually the starting point for students learning physics.

  6. Aug 2018
    1. Fig. 1 High-resolution melting profiles showing the resolved Symbiodinium strain genotypes.

      Different genetic sequences melt at slightly different rates. Therefore, they can be analyzed using these melting point curves, where fluorescence is plotted on the y-axis and temperature on the x-axis.

      If the DNA strand is composed of many guanine and cytosine bases, it will take a higher temperature to break the hydrogen bonds apart as opposed to adenine and thymine bases. This is why the E2 clade fluoresced at a higher temperature than the other clades.

    2. denaturing gradient gel electrophoresis (DGGE)

      Denaturing Gradient Gel Electrophoresis (DGGE) has been used, along with yeast cultures, to analyze saliva samples of 24 adults to study the bacteria present. This study showed ample variation of bacteria among individuals. The conclusion obtained from this experiment was that there is a lack of association between yeasts and bacterial DGGE fingerprint clusters in saliva, implying a significant ecological specificity.

    3. In this study, we evaluated the effectiveness of HRMas a tool to rapidly and precisely genotype monotypic Symbiodinium populations using the internal transcribed spacer, region 2, ribosomal DNA (ITS2 rDNA).

      This study (experiment) wanted to identify the effectiveness of high-resolution melting for the genotype (genetic makeup) of the Symbiodinium. Symbiodinium are single-celled algae that can be found in the endoderm (innermost layer of cells or tissue of an embryo in early development) of tropical cnidarians such as corals, sea anemones, and jellyfish.

    4. High-resolution melting

      A technique that detects mutations, and differences in DNA samples. HRM has been used to target various microbial communities on tadpole intestines and feces. In this study, HRS targeted a short amplicon (piece of DNA or RNA that is the source of amplification or replication events) of the 16S rRNA gene, which was the sequence being tested. Along with the HRM, gel electrophoreses and DNA sequencing were also used in order to study the results more closely. All three methods revealed several types of bacteria living in a tadpole's intestines and feces.

    1. We were unable to accurately calculate pollinator importance confidence intervals as we only estimated the relative frequency data (see above).

      Since much of the calculations were done using estimations, the authors could not provide a range of values that's likely to encompass the true value. For example, when scientists say this value falls within the 95% confidence interval it means that the intervals contain the true value 95 percent of the time and fail to contain the true value the other 5 percent of the time.

    2. Foraging behaviour was categorized by following visitor movements after they visited A. berteroi flowers. Visitation frequency of the floral visitors was estimated by counting the number of visits of each of the visitor groups to A. berteroi flowers during the observation periods where at least one visitor was seen, and calculating the corresponding percentage of the total visits observed in those periods.

      In order to determine the pollination efficiency of the insects, the authors tracked how often a specific visitor groups arrived and how long each individual stayed. Then the authors calculated an “efficiency” by estimating pollen on the visitors’ mouthparts. This was calculated as the average number of pollen grains per individual visitor of each group.

    3. Pollen grains were collected from the insect bodies to see whether visitors carried A. berteroi and/or other pollen.

      The reason behind this technique is to determine what species the carriers favored.

    4. We conducted pollinator watches weekly, for 3 h per week per site (12 intervals of 15 min per day), from 9:00 am to 12:00 pm (the hours with the highest visitation rates, B. B. Roque, personal observations) during the flowering period (April–June)

      The author monitored when the pollinators came around and interacted with the plants. Reason behind this is to determine how frequently the flower will have visitors. Another reason why the author monitored the plants, was to determine the period of time when the flower had the most visitors. With both of these factors being monitored, the author can determine more or less how frequently these plants can have their pollen spread throughout an area.

    1. Total genomic DNA of fresh leaves of a single individual of C. argentata was also isolated with the DNeasy Mini Plant kit. This DNA sample was subsequently used to obtain microsatellite loci that were developed by the Georgia Genomics Facility at the University of Georgia (Athens, Georgia, USA).

      This experiment isolated the genome of an individual C. argentata sample using the same method described in the previous experiment (DNeasy). The DNA sample obtained from this group was then taken to the Georgia Genomics Facility at the University of Georgia to develop microsatellite loci which are single sequence repeats that allows researchers to see any variability in the genome of this specific plant compared to the others. The researchers then used an Illumina HiSeq 2000 to see the microsatellite markers. The process through which they obtained these microsatellite loci is not described in the paper; however, the significance of this step when comparing different samples of DNA is described in the video below: https://www.youtube.com/watch?v=9bEAJYnVVBA

    1. Finally, knowing object distance is a prerequisite (or corequisite) in the model for deconfounding size, impedance and shape, so these features would first appear in the torus and higher areas. Although this proposal is not yet based on quantitative simulation or modeling, we believe it may be a useful working hypothesis for interpreting and further exploring parts of the electrosensory nervous system.

      Here, the authors are hypothesizing that the EOD pattern incorporated into electrosensory nervous system of the electric fish uses the information of size, shape, and distance of the objects in an algorithm to process and relay information to the electric fish brain.

    2. we have focused on reconstructing quantitatively the entire pattern of currents resulting from the fish’s discharge and environment.

      The authors here talk about the premise of their experiment and how they want to take all the data that the team has collected visually and through pattern tracking and turn it into data that has numbers. That is why in the chart that follows there is system mapping for EOD of both "wave" and "pulse" fish.

    1. Table 1. Evolution of spread-relevant traits as a function of landscape patchiness.

      Table 1 shows data indicating linear relationships between variables that changed in an evolving population based on the variability of the environment. The P column explains whether the data is statistically significant, meaning it can be concluded as a relationship between data and not just due to random chance. The two values observed for each variable are Y intercept (which indicates whether the trait changed from the original population) and the slope (which indicates how much the trait changed). These values are calculated as P values to determine if they are significant.

    2. Fig. 3. Genotypes and traits at the invasion fronts.

      Figure 3 shows the initial and final genotype compositions as well as trait changes among the different conditions (A. continuous, B. gap size of four times the mean dispersal rate, C. gap size of eight times the dispersal rate, and D. gap size of 12 times the dispersal rate). The central pie chart found in each of the four graphs shows the equal frequency of genotypes in the founding population, while the other pie charts are used to represent the genotypic make-up of the top 10 leading individuals after 6 generations of spreading within the different conditions. The placement of the pie charts is based on rankings of three traits: the competitive ability of the plant (dominance of the plant in ways that do not directly affect it's ability to spread offspring), dispersal (the average distance of the farthest dispersed seed from the plant it was dispersed from), and the height of the plant.

      These values show that even as the gap size increases, the trait values hardly increased at all. Similarly, as can be seen by the color of the pinwheels, the genotype composition change between the different gap sizes was rather consistent. The only outlier is the continuous condition that has random trait values and genotype compositions.

    3. We initiated each replicate invasion in the leftmost pot of the array by sowing equal fractions of 14

      In the first experiment, the authors simulated invasion by planting 14 genotypes on one side of the pot array. The plants were allowed to proceed for several generations to follow evolution in A. thaliana. Habitat patchiness was tested by putting space between the pots at varying distances.

    1. Error bars reflect the 95% confidence interval of the mean or expert judgment

      Notice that each bar in the figure has small capped lines that extend above and below the bar. These lines represent error bars for the various VOC emission factors. Error bars reflect the experimental uncertainty in determining VOC emission factors. The error bars in this figure are 95% confidence intervals, meaning that we are 95% certain that the true value lies somewhere in between the top and bottom of the error bar. Any measurement made has uncertainty associated with it.

      In scientific communication it is important to give the value that was found and the uncertainty in that value. We typically talk about uncertainty as being the precision of the measurement, where a smaller uncertainty is a more precise measurement and a larger uncertainty is a less precise measurement.

    2. We used energy and chemical production statistics, together with near-roadway and laboratory measurements, to construct the mass balance shown in Fig. 1 (17).

      Economic data was used to estimate the mass of various chemical products used in the United States. You can see how the estimates were derived in Tables S2 and S3 of the supporting information.

      Tables S2 and S3 can be found on pages 23 and 24 of the Supplementary Materials document

    3. California has an extensive regulatory reporting program for consumer products (34), including residential and commercial uses, which we used to speciate emissions. These speciation profiles provided us with target compounds to characterize in both outdoor and indoor environments. We also accounted for industrial emissions from VCPs (e.g., degreasing, adhesives, and coatings). The reporting data are in agreement with a U.S. database of chemicals (35) used as key constituents in chemical products (table S7). The VOC speciation profiles of VCPs (table S8) are distinguishable from those of fossil fuels (table S9), although there is some overlap in species present.

      Every emission source of VOCs emits different compounds. Many databases of the different emission profiles exist. The emission profiles allow the researchers to determine what activities (driving a car, painting a house, spraying perfume, etc.) lead to the presence of different VOCs in the air. The researchers use these profiles to differentiate between sources that come from fossil fuels (e.g. driving a car) and those that come from VCPs (e.g. painting or perfume).

    4. If chemical products are an important source of urban air pollution, then their chemical fingerprint (fig. S3) should be consistent with ambient and indoor air quality measurements.

      Previous studies have measured volatile organic compounds in outdoor and indoor air around Los Angeles, California. This study is using that data to see how it agrees with model calculations when accounting for volatile organic compounds from different sources. The video below gives an overview of how atmospheric modeling helps us understand what is occurring.


      The study looks at two different environments (indoor air and outdoor air) that can influence each other.

      See Figure S4 on page 18 from the Supplementary Materials document

    5. The fraction that can be emitted to the atmosphere depends strongly on product type and use (table S4).

      Emissions from volatile chemical products was determined by looking at the composition of chemicals in a particular product and how readily those chemicals evaporate into the air. The authors compiled information from various other researchers to perform these calculations.

    6. In 2012, the amount of oil and natural gas used as fuel in the United States was ~15 times the amount used as chemical feedstocks (Fig. 1A).

      United States government data was compiled to look at the amount of oil and natural gas supplied and what these sources are used for. See Table S1 of the supporting information to see the breakdown.

      Table S1 can be found on page 22 of the Supplementary Materials document

    7. Although U.S. sales of VCPs are substantially smaller than for gasoline and diesel fuel, VOC emissions from VCPs (7.6 ± 1.5 Tg) are twice as large as from mobile sources (3.5 ± 1.1 Tg) (Fig. 1E, light green, dark green, and blue bars) because of differences in emission factors.

      The authors use different calculations to determine VOC emissions from mobile sources and VCPs. This is due to the different end uses. Oil and natural gas used for mobile sources undergo combustion that causes much of the carbon to be emitted as carbon dioxide and not VOC. VCPs are primarily used directly and remain as intact organic molecules.

    1. formed on the PET film upon culturing

      After collecting the samples the authors provided the samples with water, an appropriate temperature, and a food source of PET film. They later used microscopy to observe what samples were able to use the PET film as an energy source.

    1. Fig. 2. Changing bumble bee community composition, bumble bee tongue length distributions, and tube depth distributions of visited flowers over time. (A and B) Bumble bee community composition. (C and D) Bumble bee tongue length. (E and F) Flower tube depth distribution. Bombus species abundance in alpine communities is indicated by the proportion of total foragers (15). Species are ordered by increasing tongue length [in (A), species’ names follow (18)]. Bimodality of the density functions (15) indicates that bumble bee communities contain two predominant phenotypes, short-tongued and long-tongued [(C) and (D)]. (E) and (F) show the tube depth density functions for flowers visited by, respectively, B. balteatus and B. sylvicola in the Front Range [Mount Evans and Niwot Ridge (15)]. For tongue length [(C) and (D)] and tube depth [(E) and (F)], representative density functions for simulated communities (15) are shown.

      These results suggest that bumble bee tongue lengths are decreasing and corolla tube lengths are also decreasing in response to climate change.

    2. On Pennsylvania Mountain, alpine bumble bees forage over hundreds of meters to provision their nests (28). To ask how warming has affected floral resources at this scale, we measured PFD of six bumble bee host plants from 1977–1980 and 2012–2014 in five habitats along a 400-m altitudinal span (table S5). Land surface area decreases with altitude above tree line in the Rocky Mountains (29), declining by more than an order of magnitude on Pennsylvania Mountain, where 58% of habitable terrain is found below 3800 m and only 4% above 3938 m on the summit (Fig. 3Aand table S5). Because bumble bees forage across the 400-m altitudinal range (28), we evaluated the temporal change in flower production at this landscape scale. For each habitat, we multiplied PFD (flowers per square meter) within sampling plots by surface area (square meters) to estimate of total flower production (15)

      From 2012-2014, researchers analyzed the change of flower production of six bumble bee host plants over time at a altitude of 400m. This is where the bumble bees most commonly forage. The flower abundance was measured as flowers per square meter across five different habitats. It was then compared to data from 1977-1984 to determine change over time.

    3. Climate records from Niwot Ridge show warming summer minimum temperatures over the past 56 years (27).We see similar changes on Mount Evans (R2 = 0.383, t1,52 = 5.68, P < 0.0001) and Pennsylvania Mountain (R2 = 0.341, t1,52 = 5.20, P < 0.0001) (fig. S3, A and B), where summer minimums have increased ~2°C since 1960. We used a nonlinear model to characterize the relationship between peak flower density (PFD; flowers per square meter) and summer minimum temperature.

      Over the past 56 years, climate change has caused summer temperatures at Niwot Ridge, Mount Evans, and Pennsylvania Mountain to rise. To understand the relationship between summer temperatures and flower density, four bumble bee host species were analyzed for change in average flower density between 1977 and 2014.

    4. (i) decreasing body size, (ii) coevolution with floral traits, (iii) competition from subalpine invaders, and (iv) diminishing floral resources.

      The author listed four possible processes responsible for the tongue length changes in the bees studied:

      1) Decreased body size over time has correlated to a shorter tongue - The authors compared the body size measurements with the tongue length measurements over time.

      2) Coevolution between the flowers and bees - The tube depth of the flowers were measured and compared to the tongue length of the bees over time.

      3) Competition from other bees in the same region affected tongue length - The authors compared other bee species to the bees studied and determine which species had the advantage and if these advantaged affected the other species.

      4) Diminishing floral resources - The authors analyzed the effects of lower amounts of flowers due to increased temperatures on the foraging habits of bees. They then concluded if this could have impacted tongue length.

    1. JH synthesis was analyzed

      Scientists started using fluorescent tags as a natural way to accurately detect low concentration of metabolites in insects. This method proved to be sensitive and effective and most importantly stable. Being stable was an important factor in this method because if it was not stable it would start to degrade after about 15 minutes. The new method has lasted over an hour, which is in benefit of being detectable by HPLC-FD (High-Performance Light Chromatography with Fluorescent Detection).

    2. JH and insulin regulate reproductive output in mosquitoes; both hormones are involved in a complex regulatory network, in which they influence each other and in which the mosquito's nutritional status is a crucial determinant of the network's output.

      The hypothesis is saying the mosquitoes' nutrition has an effect on "insulin sensitivity" and "juvenile hormone synthesis." Insulin is a hormone that works with the amount of glucose (sugar) in blood. A juvenile hormone is a hormone in insects that work with maturation and reproduction. So the researchers are saying that the amount of nutrition (food) the mosquitoes eat will determine the amount of these two hormones, insulin and juvenile, are produced.

      Since the researchers hypothesize that how much the mosquitoes eat determines how much insulin and juvenile hormones work, this means how much insects reproduce is also affected. So the researchers are saying if the insects eat enough, they will reproduce with better conditions than if they were not eating enough. This is because the hormones that control reproduction are controlled by the insects' nutrition.

    1. we measured the position and bearing of the fish swimming at a constant velocity with no acceleration; the position and bearing at time zero was then converted into Cartesian coordinates using the range of the center acoustic beam as a reference distance.

      Certain variables in the frame of these barracuda, as they were being detected, was used through complicated physics and mathematics to calculate their stride lengths.

    2. ensuring stimulation of the white muscle tissue

      Ensuring the white muscle tissue were stimulated by the electric pulse to contract, tighten.

    3. time from initial lure strike to landing was minimized and never exceeded 15 min.

      The time it took to hook a fish, reel it in, and brought on board was never more than 15 minutes.

    4. x-axis is percentage of total length

      The x-axis shows where along the length of each fish the contraction times were collected from, starting from the head (0%) to the tail (100%).

    5. P<0.001

      In statistics, 'P' is the p-value. The p-value is the probability that the null hypothesis is true, and if it's below .05 that usually means the null hypothesis (there is no correlation between the two variables being observed) is rejected as not true.

      In the case of this section of the paper, a statistical test was run to see if the mean temperatures of the fishes (taken from the muscles being observed) were significantly different from each other. It was found that the probability of this was so low (below .05), they were not significantly different.

      This is important because it implies that all the muscles of all the fish had close enough temperatures to each other that the researchers wouldn't have to worry about slightly different muscle temperatures making the contraction times collected in each fish faster or slower in relation to each other.

      Basically, they checked that the muscle temperatures in each fish were close enough to each other that the variable temperature would be (close enough) to being a "constant" in all the tested subjects in the experiment.

    6. forces needed to reach a certain swimming speed

      This refers primarily to effects of strong and weak water currents, as discussed in the last few annotations.

    7. Here we investigated maximum speed of sailfish, and three other large marine

      Sailfish, barracuda, little tunny, and dorado are four marine predatory fish species known for their extremely high swimming speeds.

      The researchers decided to reconsider how accurate the previously predicted estimates of each of those species' maximum swimming speeds truly is, hypothesizing that they may be over-estimations.

      They did this by using a different method than that used to make the estimates they're investigating. Their method was to measure the time it takes for every contraction of swimming muscle (that doesn't use oxygen) when it twitches.