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1. Small Particle Fluorescence and Light Scatter Calibration Using FCM PASS Software

   https://doi.org/10.1002/cpcy.79  

   Welsh, Joshua A. ; Jones, Jennifer C. ( September 2020 , Current Protocols in Cytometry)

   Use of flow cytometry to analyze small particles has been implemented for several decades. More recently, small particle analysis has become increasingly utilized owing to the increased sensitivity of conventional and commercially available flow cytometers along with growing interest in small particles such as extracellular vesicles (EVs). Despite an increase in small particle flow cytometry utilization, a lack of standardization in data reporting has resulted in a growing body of literature regarding EVs that cannot be easily interpreted, validated, or reproduced. Methods for fluorescence and light scatter standardization are well established, and the reagents to perform these analyses are commercially available. Here, we describe FCM_PASS, a software package for performing fluorescence and light scatter calibration of small particles while generating standard reports conforming to the MIFlowCyt‐EV standard reporting framework. This article covers the workflow of implementing calibration using FCM_PASSas follows: acquisition of fluorescence and light scatter calibration materials, cataloguing the reference materials for use in the software, creating cytometer databases and datasets to associate calibration data and fcs files, importing fcs files for calibration, inputting fluorescence calibration parameters, inputting light scatter calibration parameters, and applying the calibration to fcs files. Published 2020. U.S. Government.

   Basic Protocol 1: Acquisition and gating of light scatter calibration materials

   Basic Protocol 2: Acquisition and gating of fluorescence calibration materials

   Alternate Protocol: Cross‐calibration of fluorescence reference materials

   Basic Protocol 3: Cataloguing light scatter calibration materials

   Basic Protocol 4: Cataloguing fluorescence calibration materials

   Basic Protocol 5: Creating cytometer databases and datasets

   Basic Protocol 6: Importing fcs files

   Basic Protocol 7: Fluorescence calibration

   Basic Protocol 8: Light scatter calibration

   Basic Protocol 9: Performing and reporting fcs file calibration

    

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2. Re‐Assessing the Need for Apatite‐ and Dolomite‐Specific Calibrations of the Carbonate Clumped Isotope Thermometer

   https://doi.org/10.1029/2023GC011049  

   Anderson, N. T. ; Bonifacie, M. ; Jost, A. B. ; Siebert, J. ; Bontognali, T. ; Horita, J. ; Müller, I. A. ; Bernasconi, S. M. ; Bergmann, K. D. ( January 2024 , Geochemistry, Geophysics, Geosystems)

   A few key methodological uncertainties remain for the carbonate clumped isotope community. One is how to compare data among published data sets that are not anchored to the InterCarb Carbon Dioxide Equilibrium Scale (I‐CDES). A second is how temperature calibrations of calcite compare to those of other carbonate minerals in the I‐CDES—particularly dolomite and apatite—which can elucidate several Earth system dynamics. Previous calibrations of the clumped isotope thermometer for dolomite are discrepant from one another and variably (dis)agree with calibrations developed for calcite; apatite calibrations have not yet been compared between laboratories using carbonate‐based standardization. Here we report I‐CDES standardized values for a suite of 11 carbonates that are commonly measured by the clumped isotope community to aid future comparisons of non‐I‐CDES data sets. In addition, 17 dolomite samples (25–1,200°C) and five apatite samples (1–38°C) of known precipitation temperature were measured using carbonate‐based standardization. Excellent agreement between calcites and dolomites heated to similar temperatures (1,100–1,200°C) suggests no mineral‐specific differences in absolute acid fractionation factor. We show that calcite and dolomite regressions largely agree but are sensitive to sample characteristics, regression method, and how equations are statistically compared. We suggest that there is no need for a dolomite‐specific clumped isotope calibration, although our results suggest that further work is necessary to determine the influence of sample characteristics on this relationship. The apatite calibration equation defined in this study is statistically indistinguishable from calcite‐based calibrations; we corroborate previous findings that an apatite‐specific calibration is unnecessary.

    

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3. Minority language maintenance and the production‐prescription interface: Number agreement in New York Yiddish

   https://doi.org/10.1111/josl.12539  

   Bleaman, Isaac L. ( December 2021 , Journal of Sociolinguistics)

   Standardization is a focus of language maintenance efforts in many, but not all, minority language communities. What is the impact of this choice on interspeaker variation in maintained languages? This study investigates variable number agreement in Yiddish, a minority language spoken by two distinct communities in the New York area: (1) Hasidic Jews, who maintain the language without standardization, and (2) Yiddishists, who are overtly committed to maintaining a “correct” Yiddish. An analysis of data from 40 sociolinguistic interviews shows that Yiddishists have significantly higher rates of normative agreement than Hasidim do. The Yiddishists’ standard language ideology has also contributed to a leveling of the differences across grammatical constructions, a predictor that is more robust among the Hasidic speakers. These community‐based differences in speech reflect differences in speakers’ prescriptive judgments, which were elicited through a novel postinterview text editing task.

    

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4. Unification of miRNA and isomiR research: the mirGFF3 format and the mirtop API

   https://doi.org/10.1093/bioinformatics/btz675  

   Desvignes, Thomas ; Loher, Phillipe ; Eilbeck, Karen ; Ma, Jeffery ; Urgese, Gianvito ; Fromm, Bastian ; Sydes, Jason ; Aparicio-Puerta, Ernesto ; Barrera, Victor ; Espín, Roderic ; et al ( August 2019 , Bioinformatics)

   MicroRNAs (miRNAs) are small RNA molecules (∼22 nucleotide long) involved in post-transcriptional gene regulation. Advances in high-throughput sequencing technologies led to the discovery of isomiRs, which are miRNA sequence variants. While many miRNA-seq analysis tools exist, the diversity of output formats hinders accurate comparisons between tools and precludes data sharing and the development of common downstream analysis methods.

   To overcome this situation, we present here a community-based project, miRNA Transcriptomic Open Project (miRTOP) working towards the optimization of miRNA analyses. The aim of miRTOP is to promote the development of downstream isomiR analysis tools that are compatible with existing detection and quantification tools. Based on the existing GFF3 format, we first created a new standard format, mirGFF3, for the output of miRNA/isomiR detection and quantification results from small RNA-seq data. Additionally, we developed a command line Python tool, mirtop, to create and manage the mirGFF3 format. Currently, mirtop can convert into mirGFF3 the outputs of commonly used pipelines, such as seqbuster, isomiR-SEA, sRNAbench, Prost! as well as BAM files. Some tools have also incorporated the mirGFF3 format directly into their code, such as, miRge2.0, IsoMIRmap and OptimiR. Its open architecture enables any tool or pipeline to output or convert results into mirGFF3. Collectively, this isomiR categorization system, along with the accompanying mirGFF3 and mirtop API, provide a comprehensive solution for the standardization of miRNA and isomiR annotation, enabling data sharing, reporting, comparative analyses and benchmarking, while promoting the development of common miRNA methods focusing on downstream steps of miRNA detection, annotation and quantification.

   https://github.com/miRTop/mirGFF3/ and https://github.com/miRTop/mirtop.

   desvignes@uoneuro.uoregon.edu or lpantano@iscb.org

   Supplementary data are available at Bioinformatics online.

    

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5. specificity: an R package for analysis of feature specificity to environmental and higher dimensional variables, applied to microbiome species data

   https://doi.org/10.1186/s40793-022-00426-0  

   Darcy, John L. ; Amend, Anthony S. ; Swift, Sean O. I. ; Sommers, Pacifica S. ; Lozupone, Catherine A. ( June 2022 , Environmental Microbiome)

   Understanding the factors that influence microbes’ environmental distributions is important for determining drivers of microbial community composition. These include environmental variables like temperature and pH, and higher-dimensional variables like geographic distance and host species phylogeny. In microbial ecology, “specificity” is often described in the context of symbiotic or host parasitic interactions, but specificity can be more broadly used to describe the extent to which a species occupies a narrower range of an environmental variable than expected by chance. Using a standardization we describe here, Rao’s (Theor Popul Biol, 1982. https://doi.org/10.1016/0040-5809(82)90004-1, Sankhya A, 2010. https://doi.org/10.1007/s13171-010-0016-3 ) Quadratic Entropy can be conveniently applied to calculate specificity of a feature, such as a species, to many different environmental variables.

   We present our R packagespecificityfor performing the above analyses, and apply it to four real-life microbial data sets to demonstrate its application. We found that many fungi within the leaves of native Hawaiian plants had strong specificity to rainfall and elevation, even though these variables showed minimal importance in a previous analysis of fungal beta-diversity. In Antarctic cryoconite holes, our tool revealed that many bacteria have specificity to co-occurring algal community composition. Similarly, in the human gut microbiome, many bacteria showed specificity to the composition of bile acids. Finally, our analysis of the Earth Microbiome Project data set showed that most bacteria show strong ontological specificity to sample type. Our software performed as expected on synthetic data as well.

   specificityis well-suited to analysis of microbiome data, both in synthetic test cases, and across multiple environment types and experimental designs. The analysis and software we present here can reveal patterns in microbial taxa that may not be evident from a community-level perspective. These insights can also be visualized and interactively shared among researchers usingspecificity’s companion package,specificity.shiny.

    

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