Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation.

Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.

exRNA Atlas Analysis Reveals Distinct Extracellular RNA Cargo Types and Their Carriers Present across Human Biofluids.

To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study by deconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of the exRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.

Comparative analysis of the transcriptome across distant species.

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.

Architecture of the human regulatory network derived from ENCODE data.

Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.

Annotating non-coding regions of the genome.

Most of the human genome consists of non-protein-coding DNA. Recently, progress has been made in annotating these non-coding regions through the interpretation of functional genomics experiments and comparative sequence analysis. One can conceptualize functional genomics analysis as involving a sequence of steps: turning the output of an experiment into a 'signal' at each base pair of the genome; smoothing this signal and segmenting it into small blocks of initial annotation; and then clustering these small blocks into larger derived annotations and networks. Finally, one can relate functional genomics annotations to conserved units and measures of conservation derived from comparative sequence analysis.

Understanding transcriptional regulation by integrative analysis of transcription factor binding data.

Statistical models have been used to quantify the relationship between gene expression and transcription factor (TF) binding signals. Here we apply the models to the large-scale data generated by the ENCODE project to study transcriptional regulation by TFs. Our results reveal a notable difference in the prediction accuracy of expression levels of transcription start sites (TSSs) captured by different technologies and RNA extraction protocols. In general, the expression levels of TSSs with high CpG content are more predictable than those with low CpG content. For genes with alternative TSSs, the expression levels of downstream TSSs are more predictable than those of the upstream ones. Different TF categories and specific TFs vary substantially in their contributions to predicting expression. Between two cell lines, the differential expression of TSS can be precisely reflected by the difference of TF-binding signals in a quantitative manner, arguing against the conventional on-and-off model of TF binding. Finally, we explore the relationships between TF-binding signals and other chromatin features such as histone modifications and DNase hypersensitivity for determining expression. The models imply that these features regulate transcription in a highly coordinated manner.

The EV antibody database: An interactive database of curated antibodies for extracellular vesicle and nanoparticle research.

Antibodies are critical tools for research into extracellular vesicles (EVs) and other extracellular nanoparticles (ENPs), where they can be used for their identification, characterization, and isolation. However, the lack of a centralized antibody platform where researchers can share validation results thus minimizing wasted personnel time and reagents, has been a significant obstacle. Moreover, because the performance of antibodies varies among assay types and conditions, detailed information on assay variables and protocols is also of value. To facilitate sharing of results on antibodies that are relevant to EV/ENP research, the EV Antibody Database has been developed by the investigators of the Extracellular RNA Communication Consortium (ERCC). Hosted by the ExRNA Portal (https://exrna.org/resources/evabdb/), this interactive database aggregates and shares results from antibodies that have been tested by research groups in the EV/ENP field. Currently, the EV Antibody Database includes modules for antibodies tested for western Blot, EV Flow Cytometry, and EV Sandwich Assays, and holds 110 records contributed by 6 laboratories from the ERCC. Detailed information on antibody sources, assay conditions, and results is provided, including negative results. We encourage ongoing expert input and community feedback to enhance the database's utility, making it a valuable resource for comprehensive validation data on antibodies and protocols in EV biology.

Comparing genomes to computer operating systems in terms of the topology and evolution of their regulatory control networks.

The genome has often been called the operating system (OS) for a living organism. A computer OS is described by a regulatory control network termed the call graph, which is analogous to the transcriptional regulatory network in a cell. To apply our firsthand knowledge of the architecture of software systems to understand cellular design principles, we present a comparison between the transcriptional regulatory network of a well-studied bacterium (Escherichia coli) and the call graph of a canonical OS (Linux) in terms of topology and evolution. We show that both networks have a fundamentally hierarchical layout, but there is a key difference: The transcriptional regulatory network possesses a few global regulators at the top and many targets at the bottom; conversely, the call graph has many regulators controlling a small set of generic functions. This top-heavy organization leads to highly overlapping functional modules in the call graph, in contrast to the relatively independent modules in the regulatory network. We further develop a way to measure evolutionary rates comparably between the two networks and explain this difference in terms of network evolution. The process of biological evolution via random mutation and subsequent selection tightly constrains the evolution of regulatory network hubs. The call graph, however, exhibits rapid evolution of its highly connected generic components, made possible by designers' continual fine-tuning. These findings stem from the design principles of the two systems: robustness for biological systems and cost effectiveness (reuse) for software systems.

exRNA-eCLIP intersection analysis reveals a map of extracellular RNA binding proteins and associated RNAs across major human biofluids and carriers.

Although the role of RNA binding proteins (RBPs) in extracellular RNA (exRNA) biology is well established, their exRNA cargo and distribution across biofluids are largely unknown. To address this gap, we extend the exRNA Atlas resource by mapping exRNAs carried by extracellular RBPs (exRBPs). This map was developed through an integrative analysis of ENCODE enhanced crosslinking and immunoprecipitation (eCLIP) data (150 RBPs) and human exRNA profiles (6,930 samples). Computational analysis and experimental validation identified exRBPs in plasma, serum, saliva, urine, cerebrospinal fluid, and cell-culture-conditioned medium. exRBPs carry exRNA transcripts from small non-coding RNA biotypes, including microRNA (miRNA), piRNA, tRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), Y RNA, and lncRNA, as well as protein-coding mRNA fragments. Computational deconvolution of exRBP RNA cargo reveals associations of exRBPs with extracellular vesicles, lipoproteins, and ribonucleoproteins across human biofluids. Overall, we mapped the distribution of exRBPs across human biofluids, presenting a resource for the community.

RodZ, a component of the bacterial core morphogenic apparatus.

The molecular basis of bacterial cell morphogenesis remains largely an open question. Here we discover a morphogenic protein, RodZ, which is widely conserved across the bacterial kingdom. In Caulobacter crescentus, RodZ is essential for viability and is involved in all aspects of this organism's complex morphology. Depletion or over-production of RodZ results in grossly misshapen cells with stalk defects. RodZ exhibits a localization pattern during the cell cycle corresponding to sites of active peptidoglycan synthesis. The temporal transition of RodZ between patchy/helical and mid-cell localization mimics and depends on the actin-like MreB cytoskeleton. In Escherichia coli, an organism with a distinct mode of growth and MreB localization dynamics, RodZ follows MreB and retains its crucial role in cell morphogenesis, demonstrating conservation of function. Genomic analysis shows that RodZ represents an ancient function unique to bacteria. Multiple sequence alignment of 143 RodZ sequences from species across bacterial phyla identifies an N-terminal cytoplasmic domain with a helix-turn-helix motif, a transmembrane sequence, and a previously unidentified, conserved periplasmic or extracellular C-terminal domain. Both the N- and C-terminal domains are important for function, with the N-terminal domain containing localization determinants. This study uncovers a key missing player in the cytoskeleton-based growth machinery enabling heritable and defined cellular forms in bacteria.

Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research.

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.

Summary of the new 2010 American Heart Association Guidelines for Basic Life Support (CPR).

Approximately every 5 years, American Heart Association (AHA) experts review emerging scientific evidence and recent clinical experiences and update the AHA guidelines for basic and advanced life support procedures for in-hospital and out-of-hospital victims of life-threatening cardiac events. This article summarizes many of the 2010 changes in those guidelines as they apply to dental healthcare providers (HCP). More detailed information will be available in the near future as these guidelines are fully implemented and instructional materials are released by the AHA. Until they are trained in future AHA or American Red Cross (ARC) basic or advanced cardiac life support (BLS, ACLS) courses in 2011, dentists, dental assistants, dental hygienists, and office staff should continue to rely on the training and information they received in their most recent basic (and/or advanced cardiac) life support training course.

Open Problems in Extracellular RNA Data Analysis: Insights From an ERCC Online Workshop.

We now know RNA can survive the harsh environment of biofluids when encapsulated in vesicles or by associating with lipoproteins or RNA binding proteins. These extracellular RNA (exRNA) play a role in intercellular signaling, serve as biomarkers of disease, and form the basis of new strategies for disease treatment. The Extracellular RNA Communication Consortium (ERCC) hosted a two-day online workshop (April 19-20, 2021) on the unique challenges of exRNA data analysis. The goal was to foster an open dialog about best practices and discuss open problems in the field, focusing initially on small exRNA sequencing data. Video recordings of workshop presentations and discussions are available (https://exRNA.org/exRNAdata2021-videos/). There were three target audiences: experimentalists who generate exRNA sequencing data, computational and data scientists who work with those groups to analyze their data, and experimental and data scientists new to the field. Here we summarize issues explored during the workshop, including progress on an effort to develop an exRNA data analysis challenge to engage the community in solving some of these open problems.

Is your office prepared for an accidental needlestick or other unexpected exposure incident?

Recommendations and mandatory guidelines for preventing and managing needlestick incidents and other accidental exposures to bloodborne pathogens in healthcare facilities have been published by the Occupational Safety and Health Administration (OSHA) and the Centers for Disease Control and Prevention (CDC) for more than 2 decades. Over the years, the incidence of official enforcement actions has declined and a complacency about the standards may have evolved in some dental offices. Some practitioners may not have written an occupational exposure incident protocol or made appropriate arrangements for medical laboratory testing and postexposure medical evaluation following an unexpected needlestick or other exposure incident in the office. When an unexpected event occurs, practitioners may become confused regarding the steps to be taken, and may turn to their local dental society or fellow practitioners for guidance. The provided information may or may not be complete, accurate and/or current. Implementation of periodic personnel training to prevent exposure incidents is extremely important and could ultimately save a dental practice thousands of dollars in expenses related to the occurrence of even one exposure incident, as well as save the life and/or career of a dental healthcare provider. This article does not comprehensively detail all infection control and bloodborne pathogen transmission prevention requirements for dental offices. Rather, the article provides suggestions for dental practitioners regarding the step by step management of exposure incidents, and provides resource information for additional steps that can be taken towards prevention, improved office compliance, and improved litigation protection.

Long-acting erythromycins: assessing their role in treating outpatient odontogenic infections.

Erythromycins have been part of our armamentarium against selected bacterial infections since they were discovered in 1952 and approved by the Food and Drug Administration (FDA) in 1964. In 1991, two newer, long-acting erythromycin analogues, azythromycin (brand name: Zithromax) and clarithromycin (brand name: Biaxin) were approved by the FDA. They were joined a few years later by a third long-acting form, dirithromycin (brand name: Dynabac).

Prevalencia de conductos en C de segundos molares mandibulares evaluados en tomografía de haz cónico / Prevalence of C-ducts of Mandibular Second Molars Evaluated on Cone Beam Tomography

Introducción: El conocimiento adecuado de la configuración de conductos radiculares es fundamental en endodoncia; la evaluación tomográfica permite una correcta evaluación de su disposición radicular. Objetivo: Determinar la prevalencia de conductos en C de segundos molares mandibulares, evaluados en tomografía de haz cónico. Métodos: Se realizó un estudio descriptivo y de corte transversal; la muestra estuvo conformada por 200 segundos molares mandibulares permanentes de una población peruana, observadas en tomografías cone beam, donde se registró la presencia del conducto en C, su configuración según la clasificación de Fan y el sexo del paciente. Resultados: La prevalencia de la configuración radicular en forma de C en segundos molares inferiores fue del 65,5 por ciento; según la Clasificación de Fan se observó mayor prevalencia en el tercio cervical del conducto radicular el tipo C1 con 85,7 por ciento; en el tercio medio el tipo C2 con 42,9 por ciento; a nivel apical fue el tipo C3C con 72,1 por ciento; según el sexo, el 65,2 por ciento de los conductos en C correspondió al femenino. Conclusión: La prevalencia de los conductos en C de los segundos molares mandibulares evaluados en tomografías de haz cónico fue de 65,5 por ciento con mayor predominio en el sexo femenino. La evaluación tomográfica permite una mejor identificación y configuración interna de los conductos radiculares(AU)

Introduction: Adequate knowledge of the configuration of root canals is fundamental in endodontics; tomographic evaluation allows a correct assessment of their radicular arrangement. Objective: To determine the prevalence of C-shaped canals in mandibular second molars, evaluated by cone beam tomography. Methods: A descriptive and cross-sectional study was carried out; the sample consisted of 200 permanent mandibular second molars from a Peruvian population, observed in cone beam tomography, where the presence of the C-shaped canal, its configuration according to Fan's classification and the patient's gender were recorded. Results: The prevalence of the C-shaped root canal configuration in lower second molars was 65.5 percent; according to the Fan classification, the highest prevalence was observed in the cervical third of the root canal, type C1 with 85.7 percent; in the middle third, type C2 with 42.9 percent; at the apical level it was type C3C with 72.1 percent; according to gender, 65.2 percent of the C-shaped canals corresponded to females. Conclusion: The prevalence of C-shaped canals in mandibular second molars evaluated in cone beam tomography was 65.5% with a higher predominance in the female gender. The tomographic evaluation allows a better identification and internal configuration of the root canals(AU)

Overview of Protocols for Studying Extracellular RNA and Extracellular Vesicles.

Understanding the role of extracellular RNA (exRNA) has emerged as an exciting avenue for biomarker, therapeutic, as well as basic cell-cell communication applications and discoveries. Multiple protocols, kits, and procedures have been developed in the last decade to allow fractionation as well as isolation of subpopulations of macromolecules of interest found in biofluids. Here, we introduce the protocols decision tree developed by the Extracellular RNA Communication Consortium and available on their website (exRNA portal), and compare all methods currently available to the exRNA field and report pros and cons for each platform.

Comparative genomic and protein sequence analyses of a complex system controlling bacterial chemotaxis.

Molecular machinery governing bacterial chemotaxis consists of the CheA-CheY two-component system, an array of specialized chemoreceptors, and several auxiliary proteins. It has been studied extensively in Escherichia coli and, to a significantly lesser extent, in several other microbial species. Emerging evidence suggests that homologous signal transduction pathways regulate not only chemotaxis, but several other cellular functions in various bacterial species. The availability of genome sequence data for hundreds of organisms enables productive study of this system using comparative genomics and protein sequence analysis. This chapter describes advances in genomics of the chemotaxis signal transduction system, provides information on relevant bioinformatics tools and resources, and outlines approaches toward developing a computational framework for predicting important biological functions from raw genomic data based on available experimental evidence.

Nivel de satisfacción y factores sociodemográficos de usuarios atendidos en los servicios forenses de la Unidad Médico Legal II Amazonas / Level of satisfaction and sociodemographic factors of users attended in the forensic services of the Medical Legal Unit II Amazonas

Resumen Introducción: La insatisfacción en la atención de los servicios forenses públicos, representa una preocupación de las entidades estatales hacia la atención brindada a los usuarios, siendo necesario su identificación. La investigación determino la relación entre el nivel de satisfacción y factores sociodemográficos de usuarios atendidos en la Unidad Médico Legal II Amazonas. Materiales y métodos: Estudio descriptivo-correlacional, de enfoque cuantitativo y diseño no experimental, en una muestra de 126 usuarios, seleccionados por muestreo probabilístico aleatorio estratificado, se aplicó un cuestionario mediante encuesta, validado por juicio de expertos (V de Alkin, 94.38) y confiabilidad (alfa de Cronbach, 0.824). Resultados: Mediante prueba Chi-cuadrado de Pearson, no se encontró relación entre la variable nivel de satisfacción y características sociodemográficas (p>0.05); al evaluarse por servicios, se encontró en mesa de partes satisfacción indiferente (44.4%) e insatisfacción (27%), en Medicina legal, satisfacción (35%) e indiferente (55%), en Odontología forense, tuvo satisfacción (84.1%) y el servicio de Psicología forense satisfacción (78.6%). Conclusiones: El nivel de satisfacción y los factores sociodemográficos no se relacionan, es decir las variables se disocian; en general los usuarios atendidos mostraron satisfacción (56.6%), insatisfacción (11%) y satisfacción indiferente (32.6%), por ello se requieren de planes de mejora para la atención al usuario.

Abstract Introduction: The dissatisfaction of the attention of the public forensic services, represent a concern of the state entities towards the attention of the users, being necessary their identification. The research determined the relationship between the level of satisfaction and sociodemographic factors of users attended in the Medical Legal Unit II Amazonas. Materials and methods: Descriptive-correlational study, with a quantitative approach and non-experimental design, in a sample of 126 users, selected by stratified random probability sampling, a questionnaire was applied by means of a survey, validated by expert judgment (V de Alkin, 94.38) and reliability (Cronbach's alpha, 0.824). Results: Using Pearson's Chi-square test, no relationship was found between the level of satisfaction variable and sociodemographic characteristics (p> 0.05); When evaluated by services, indifferent satisfaction (44.4%) and dissatisfaction (27%) were found at the party table, in Legal Medicine, satisfaction (35%) and indifferent (55%), in Forensic Dentistry, there was satisfaction (84.1%) and the forensic psychology service satisfaction (78.6%). Conclusions: The level of satisfaction and the sociodemographic factors are not related, that is, the variables are dissociated; In general, the users attended showed satisfaction (56.6%), dissatisfaction (11%) and indifferent satisfaction (32.6%), for this reason improvement plans are required for user service.

Extending gene ontology in the context of extracellular RNA and vesicle communication.

BACKGROUND: To address the lack of standard terminology to describe extracellular RNA (exRNA) data/metadata, we have launched an inter-community effort to extend the Gene Ontology (GO) with subcellular structure concepts relevant to the exRNA domain. By extending GO in this manner, the exRNA data/metadata will be more easily annotated and queried because it will be based on a shared set of terms and relationships relevant to extracellular research. METHODS: By following a consensus-building process, we have worked with several academic societies/consortia, including ERCC, ISEV, and ASEMV, to identify and approve a set of exRNA and extracellular vesicle-related terms and relationships that have been incorporated into GO. In addition, we have initiated an ongoing process of extractions of gene product annotations associated with these terms from Vesiclepedia and ExoCarta, conversion of the extracted annotations to Gene Association File (GAF) format for batch submission to GO, and curation of the submitted annotations by the GO Consortium. As a use case, we have incorporated some of the GO terms into annotations of samples from the exRNA Atlas and implemented a faceted search interface based on such annotations. RESULTS: We have added 7 new terms and modified 9 existing terms (along with their synonyms and relationships) to GO. Additionally, 18,695 unique coding gene products (mRNAs and proteins) and 963 unique non-coding gene products (ncRNAs) which are associated with the terms: "extracellular vesicle", "extracellular exosome", "apoptotic body", and "microvesicle" were extracted from ExoCarta and Vesiclepedia. These annotations are currently being processed for submission to GO. CONCLUSIONS: As an inter-community effort, we have made a substantial update to GO in the exRNA context. We have also demonstrated the utility of some of the new GO terms for sample annotation and metadata search.