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.

On the expanding roles of tRNA fragments in modulating cell behavior.

The fragments that derive from transfer RNAs (tRNAs) are an emerging category of regulatory RNAs. Known as tRFs, these fragments were reported for the first time only a decade ago, making them a relatively recent addition to the ever-expanding pantheon of non-coding RNAs. tRFs are short, 16-35 nucleotides (nts) in length, and produced through cleavage of mature and precursor tRNAs at various positions. Both cleavage positions and relative tRF abundance depend strongly on context, including the tissue type, tissue state, and disease, as well as the sex, population of origin, and race/ethnicity of an individual. These dependencies increase the urgency to understand the regulatory roles of tRFs. Such efforts are gaining momentum, and comprise experimental and computational approaches. System-level studies across many tissues and thousands of samples have produced strong evidence that tRFs have important and multi-faceted roles. Here, we review the relevant literature on tRF biology in higher organisms, single cell eukaryotes, and prokaryotes.

Ribosomal RNA fragmentation into short RNAs (rRFs) is modulated in a sex- and population of origin-specific manner.

BACKGROUND: The advent of next generation sequencing (NGS) has allowed the discovery of short and long non-coding RNAs (ncRNAs) in an unbiased manner using reverse genetics approaches, enabling the discovery of multiple categories of ncRNAs and characterization of the way their expression is regulated. We previously showed that the identities and abundances of microRNA isoforms (isomiRs) and transfer RNA-derived fragments (tRFs) are tightly regulated, and that they depend on a person's sex and population origin, as well as on tissue type, tissue state, and disease type. Here, we characterize the regulation and distribution of fragments derived from ribosomal RNAs (rRNAs). rRNAs form a group that includes four (5S, 5.8S, 18S, 28S) rRNAs encoded by the human nuclear genome and two (12S, 16S) by the mitochondrial genome. rRNAs constitute the most abundant RNA type in eukaryotic cells. RESULTS: We analyzed rRNA-derived fragments (rRFs) across 434 transcriptomic datasets obtained from lymphoblastoid cell lines (LCLs) derived from healthy participants of the 1000 Genomes Project. The 434 datasets represent five human populations and both sexes. We examined each of the six rRNAs and their respective rRFs, and did so separately for each population and sex. Our analysis shows that all six rRNAs produce rRFs with unique identities, normalized abundances, and lengths. The rRFs arise from the 5'-end (5'-rRFs), the interior (i-rRFs), and the 3'-end (3'-rRFs) or straddle the 5' or 3' terminus of the parental rRNA (x-rRFs). Notably, a large number of rRFs are produced in a population-specific or sex-specific manner. Preliminary evidence suggests that rRF production is also tissue-dependent. Of note, we find that rRF production is not affected by the identity of the processing laboratory or the library preparation kit. CONCLUSIONS: Our findings suggest that rRFs are produced in a regimented manner by currently unknown processes that are influenced by both ubiquitous as well as population-specific and sex-specific factors. The properties of rRFs mirror the previously reported properties of isomiRs and tRFs and have implications for the study of homeostasis and disease.

MINTbase v2.0: a comprehensive database for tRNA-derived fragments that includes nuclear and mitochondrial fragments from all The Cancer Genome Atlas projects.

MINTbase is a repository that comprises nuclear and mitochondrial tRNA-derived fragments ('tRFs') found in multiple human tissues. The original version of MINTbase comprised tRFs obtained from 768 transcriptomic datasets. We used our deterministic and exhaustive tRF mining pipeline to process all of The Cancer Genome Atlas datasets (TCGA). We identified 23 413 tRFs with abundance of ≥ 1.0 reads-per-million (RPM). To facilitate further studies of tRFs by the community, we just released version 2.0 of MINTbase that contains information about 26 531 distinct human tRFs from 11 719 human datasets as of October 2017. Key new elements include: the ability to filter tRFs on-the-fly by minimum abundance thresholding; the ability to filter tRFs by tissue keywords; easy access to information about a tRF's maximum abundance and the datasets that contain it; the ability to generate relative abundance plots for tRFs across cancer types and convert them into embeddable figures; MODOMICS information about modifications of the parental tRNA, etc. Version 2.0 of MINTbase contains 15x more datasets and nearly 4x more distinct tRFs than the original version, yet continues to offer fast, interactive access to its contents. Version 2.0 is available freely at http://cm.jefferson.edu/MINTbase/.

Knowledge about the presence or absence of miRNA isoforms (isomiRs) can successfully discriminate amongst 32 TCGA cancer types.

Isoforms of human miRNAs (isomiRs) are constitutively expressed with tissue- and disease-subtype-dependencies. We studied 10 271 tumor datasets from The Cancer Genome Atlas (TCGA) to evaluate whether isomiRs can distinguish amongst 32 TCGA cancers. Unlike previous approaches, we built a classifier that relied solely on 'binarized' isomiR profiles: each isomiR is simply labeled as 'present' or 'absent'. The resulting classifier successfully labeled tumor datasets with an average sensitivity of 90% and a false discovery rate (FDR) of 3%, surpassing the performance of expression-based classification. The classifier maintained its power even after a 15× reduction in the number of isomiRs that were used for training. Notably, the classifier could correctly predict the cancer type in non-TCGA datasets from diverse platforms. Our analysis revealed that the most discriminatory isomiRs happen to also be differentially expressed between normal tissue and cancer. Even so, we find that these highly discriminating isomiRs have not been attracting the most research attention in the literature. Given their ability to successfully classify datasets from 32 cancers, isomiRs and our resulting 'Pan-cancer Atlas' of isomiR expression could serve as a suitable framework to explore novel cancer biomarkers.

Threshold-seq: a tool for determining the threshold in short RNA-seq datasets.

SUMMARY: We present 'Threshold-seq,' a new approach for determining thresholds in deep-sequencing datasets of short RNA transcripts. Threshold-seq addresses the critical question of how many reads need to support a short RNA molecule in a given dataset before it can be considered different from 'background.' The proposed scheme is easy to implement and incorporate into existing pipelines. AVAILABILITY AND IMPLEMENTATION: Source code of Threshold-seq is freely available as an R package at: http://cm.jefferson.edu/threshold-seq/. CONTACT: isidore.rigoutsos@jefferson.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Scaling and Root Planning in a Patient Taking Chronic Corticosteroid Therapy for Lupus Erythematosus.

The dental provider should be aware of the oral manifestations of systemic lupus erythematosus (SLE). Patients with SLE may be on chronic oral corticosteroids, which can increase the risk for periodontitis and opportunistic oral infections in addition to inducing multiple systemic adverse effects. Disease complications such as lupus nephritis and comorbid antiphospholipid antibody syndrome can further impact dental decision-making including around medications to prescribe or hemostatic measures to employ during treatment. Patients with SLE on systemic corticosteroid therapy usually do not require steroid supplementation before or after non-surgical or surgical dental treatment.

Trsper: a web-based application for Archimedes spiral analysis.

BACKGROUND: We built a web-based application of the Archimedes spiral exam that implements clinically validated spiral metrics and tested drawing instructions to define a clinical workflow. METHODS: We designed an HTML5 and Javascript implementation of the spiral exam to run on mobile touchscreen devices. We then recruited 10 volunteers each for 2 experiments designed to validate the programmed spiral metrics and assess how instructions or drawing implement affect the results. In task one, volunteers drew 5 spirals each while following 6 different instruction sets (n=30 spirals each, n=300 spirals total) that varied by support of the drawing hand and tracing condition (either tracing a spiral template, drawing in-between it, or freehand). In task two, volunteers drew 5 spirals each while following 2 instruction sets and drawing using a stylus or their dominant index finger (n=20 spirals each, n=200 spirals total). RESULTS: Principal components analysis of calculated metrics revealed that the experiments grouped by instruction set and by subject. Mean Euclidean distance between experiments represented as 11-dimensional vectors revealed that consistency varied among instruction tasks and that drawing with a stylus produced more consistent results than did using the dominant index finger. Using experimental data and simulated abnormal spirals, we designed a decision support system that accurately identifies potentially abnormal spirals. CONCLUSIONS: We built and validated a robust digital implementation of the Archimedes spiral exam and recommend a sensitive and specific workflow on the basis of data gathered from healthy volunteers.

A retrospective, single-center cohort study on complications after dental extractions in patients taking biologic agents.

BACKGROUND: Although biologic agents represent a growing class of therapeutics, little is known about how these agents affect the provision of dental treatment. METHODS: This retrospective case-control study analyzed patients undergoing dental extraction treated with biologic agents from 2017 through 2020. Complications within 30 days postextraction were recorded. RESULTS: One-hundred twenty-one patients were treated during 147 encounters. Fifteen patients experienced complications during 16 encounters. Notable or excessive pain was most common (14/16; 88%). Patients who experienced complications were treated with 7 biologic agents: dulaglutide, belimumab, adalimumab, aflibercept, tofacitinib, ranibizumab, and ixekizumab. Complication after extraction-specifically, pain-was elevated for patients receiving aflibercept and ranibizumab. When grouped by class, complications were more common with vascular endothelial growth factor antagonism. CONCLUSIONS: The impact of biologics on the provision of and recovery after dental treatment remains unknown. Pain was most commonly reported. Patients treated with vascular endothelial growth factor antagonists experienced an elevated rate of complications. PRACTICAL IMPLICATIONS: This study provides preliminary data on how patients taking biologic agents heal after dental extraction. It is limited by small sample sizes. Further work will build on this data to determine appropriate management of patients taking biologics in the dental setting.

Gender Disparity in Evaluation of Internal Medicine Clerkship Performance.

Importance: Women studying medicine currently equal men in number, but evidence suggests that men and women might not be evaluated equally throughout their education. Objective: To examine whether there are differences associated with gender in either objective or subjective evaluations of medical students in an internal medicine clerkship. Design, Setting, and Participants: This single-center retrospective cohort study evaluated data from 277 third-year medical students completing internal medicine clerkships in the 2017 to 2018 academic year at an academic hospital and its affiliates in Pennsylvania. Data were analyzed from September to November 2020. Exposure: Gender, presumed based on pronouns used in evaluations. Main Outcomes and Measures: Likert scale evaluations of clinical skills, standardized examination scores, and written evaluations were analyzed. Univariate and multivariate linear regression were used to observe trends in measures. Word embeddings were analyzed for narrative evaluations. Results: Analyses of 277 third-year medical students completing an internal medicine clerkship (140 women [51%] with a mean [SD] age of 25.5 [2.3] years and 137 [49%] presumed men with a mean [SD] age of 25.9 [2.7] years) detected no difference in final grade distribution. However, women outperformed men in 5 of 8 domains of clinical performance, including patient interaction (difference, 0.07 [95% CI, 0.04-0.13]), growth mindset (difference, 0.08 [95% CI, 0.01-0.11]), communication (difference, 0.05 [95% CI, 0-0.12]), compassion (difference, 0.125 [95% CI, 0.03-0.11]), and professionalism (difference, 0.07 [95% CI, 0-0.11]). With no difference in examination scores or subjective knowledge evaluation, there was a positive correlation between these variables for both genders (women: r = 0.35; men: r = 0.26) but different elevations for the line of best fit (P < .001). Multivariate regression analyses revealed associations between final grade and patient interaction (women: coefficient, 6.64 [95% CI, 2.16-11.12]; P = .004; men: coefficient, 7.11 [95% CI, 2.94-11.28]; P < .001), subjective knowledge evaluation (women: coefficient, 6.66 [95% CI, 3.87-9.45]; P < .001; men: coefficient, 5.45 [95% CI, 2.43-8.43]; P < .001), reported time spent with the student (women: coefficient, 5.35 [95% CI, 2.62-8.08]; P < .001; men: coefficient, 3.65 [95% CI, 0.83-6.47]; P = .01), and communication (women: coefficient, 6.32 [95% CI, 3.12-9.51]; P < .001; men: coefficient, 4.21 [95% CI, 0.92-7.49]; P = .01). The model based on the men's data also included growth mindset as a significant variable (coefficient, 4.09 [95% CI, 0.67-7.50]; P = .02). For narrative evaluations, words in context with "he or him" and "she or her" differed, with agentic terms used in descriptions of men and personality descriptors used more often for women. Conclusions and Relevance: Despite no difference in final grade, women scored higher than men on various domains of clinical performance, and performance in these domains was associated with evaluators' suggested final grade. The content of narrative evaluations significantly differed by student gender. This work supports the hypothesis that how students are evaluated in clinical clerkships is associated with gender.

IsomiRs and tRNA-derived fragments are associated with metastasis and patient survival in uveal melanoma.

Uveal melanoma (UVM) is the most common primary intraocular malignancy in adults. With over 50% of patients developing metastatic disease, there is an unmet need for improved diagnostic and therapeutic options. Efforts to understand the molecular biology of the disease have revealed several markers that correlate with patient prognosis, including the copy number of chromosome 3, genetic alterations in the BAP1, EIF1AX and SF3B1 genes, and other transcriptional features. Here, we expand upon previous reports by comprehensively characterizing the short RNA-ome in 80 primary UVM tumor samples. In particular, we describe a previously unseen complex network involving numerous regulatory molecules that comprise microRNA (miRNAs), novel UVM-specific miRNA loci, miRNA isoforms (isomiRs), and tRNA-derived fragments (tRFs). Importantly, we show that the abundance profiles of isomiRs and tRFs associate with various molecular phenotypes, metastatic disease, and patient survival. Our findings suggest deep involvement of isomiRs and tRFs in the disease etiology of UVM. We posit that further study and characterization of these novel molecules will improve understanding of the mechanisms underlying UVM, and lead to the development of new diagnostic and therapeutic approaches.

Energy recycling by co-combustion of coal and recovered paint solids from automobile paint operations.

During the past decade, there has been substantial interest in recovering energy from many unwanted byproducts from industries and municipalities. Co-combustion of these products with coal seems to be the most cost-effective approach. The combustion process typically results in emissions of pollutants, especially fine particles and trace elements. This paper presents the results of an experimental study of particulate emission and the fate of 13 trace elements (arsenic [As], barium [Ba], cadmium [Cd], chromium [Cr], copper [Cu], cobalt [Co], manganese [Mn], molybdenum [Mo], nickel [Ni], lead [Pb], mercury [Hg], vanadium [V], and zinc [Zn]) during combustion tests of recovered paint solids (RPS) and coal. The emissions from combustions of coal or RPS alone were compared with those of co-combustion of RPS with subbituminous coal. The distribution/partitioning of these toxic elements between a coarse-mode ash (particle diameter [dp] > 0.5 microm), a submicrometer-mode ash (dp < 0.5 microm), and flue gases was also evaluated. Submicrometer particles generated by combustion of RPS alone were lower in concentration and smaller in size than that from combustion of coal. However, co-combustion of RPS and coal increased the formation of submicrometer-sized particles because of the higher reducing environment in the vicinity of burning particles and the higher volatile chlorine species. Hg was completely volatilized in all cases; however, the fraction in the oxidized state increased with co-combustion. Most trace elements, except Zn, were retained in ash during combustion of RPS alone. Mo was mostly retained in all samples. The behavior of elements, except Mn and Mo, varied depending on the fuel samples. As, Ba, Cr, Co, Cu, and Pb were vaporized to a greater extent from cocombustion of RPS and coal than from combustion of either fuel. Evidence of the enrichment of certain toxic elements in submicrometer particles has also been observed for As, Cd, Cr, Cu, and Ni during co-combustion.

TRNA-derived fragments as sex-dependent circulating candidate biomarkers for Parkinson's disease.

INTRODUCTION: Parkinson's Disease (PD) is diagnosed clinically. Reliable non-invasive PD biomarkers are actively sought. Transfer RNAs produce short non-coding RNAs, the tRNA-derived fragments (tRF). tRF have been shown to play diverse roles, including in amyotrophic lateral sclerosis, and the response to ischemic stroke. Rich tRF populations are being reported in biofluids. We explored the possibility that tRF can serve as non-invasive biomarkers for PD. METHODS: We collected existing RNA-seq samples and re-analyzed a total of 254 legacy datasets from 3 previous studies, from male and female PD patients and controls that belong to three categories: prefrontal cortex samples from 29 patients and 33 controls; cerebrospinal fluid (CSF) samples from 63 patients and 64 controls; and, serum samples from 34 patients and 31 controls. First, we identified tRF exhaustively and deterministically in every dataset. Second, we determined tRF that are differentially abundant (DA) between PD and control samples, using uncorrected t-tests. Lastly, we assessed all the DA tRF from the previous step with Partial Least Squares - Discriminant Analysis (PLS-DA) to stringently sub-select tRF that can distinguish PD patients from controls. RESULTS: We show that PLS-DA identified tRF from prefrontal cortex, CSF, and serum that can distinguish PD patients from controls. A handful of identified tRF were previously investigated in neurological contexts. Signatures built from relatively few tRF suffice to distinguish PD from control in each category of samples with high sensitivity (89-100%) and specificity (79-98%). CONCLUSION: tRF-based signatures are promising candidates that warrant further evaluation as non-invasive PD biomarkers.

tRNA Fragments Show Intertwining with mRNAs of Specific Repeat Content and Have Links to Disparities.

tRNA-derived fragments (tRF) are a class of potent regulatory RNAs. We mined the datasets from The Cancer Genome Atlas (TCGA) representing 32 cancer types with a deterministic and exhaustive pipeline for tRNA fragments. We found that mitochondrial tRNAs contribute disproportionally more tRFs than nuclear tRNAs. Through integrative analyses, we uncovered a multitude of statistically significant and context-dependent associations between the identified tRFs and mRNAs. In many of the 32 cancer types, these associations involve mRNAs from developmental processes, receptor tyrosine kinase signaling, the proteasome, and metabolic pathways that include glycolysis, oxidative phosphorylation, and ATP synthesis. Even though the pathways are common to multiple cancers, the association of specific mRNAs with tRFs depends on and differs from cancer to cancer. The associations between tRFs and mRNAs extend to genomic properties as well; specifically, tRFs are positively correlated with shorter genes that have a higher density in repeats, such as ALUs, MIRs, and ERVLs. Conversely, tRFs are negatively correlated with longer genes that have a lower repeat density, suggesting a possible dichotomy between cell proliferation and differentiation. Analyses of bladder, lung, and kidney cancer data indicate that the tRF-mRNA wiring can also depend on a patient's sex. Sex-dependent associations involve cyclin-dependent kinases in bladder cancer, the MAPK signaling pathway in lung cancer, and purine metabolism in kidney cancer. Taken together, these findings suggest diverse and wide-ranging roles for tRFs and highlight the extensive interconnections of tRFs with key cellular processes and human genomic architecture. SIGNIFICANCE: Across 32 TCGA cancer contexts, nuclear and mitochondrial tRNA fragments exhibit associations with mRNAs that belong to concrete pathways, encode proteins with particular destinations, have a biased repeat content, and are sex dependent.

Profiles of miRNA Isoforms and tRNA Fragments in Prostate Cancer.

MicroRNA (miRNA) isoforms ("isomiRs") and tRNA-derived fragments ("tRFs") are powerful regulatory non-coding RNAs (ncRNAs). In human tissues, both types of molecules are abundant, with expression patterns that depend on a person's race, sex and population origin. Here, we present our analyses of the Prostate Cancer (PRAD) datasets of The Cancer Genome Atlas (TCGA) from the standpoint of isomiRs and tRFs. This study represents the first simultaneous examination of isomiRs and tRFs in a large cohort of PRAD patients. We find that isomiRs and tRFs have extensive correlations with messenger RNAs (mRNAs). These correlations are disrupted in PRAD, which suggests disruptions of the regulatory network in the disease state. Notably, we find that the profiles of isomiRs and tRFs differ in patients belonging to different races. We hope that the presented findings can lay the groundwork for future research efforts aimed at elucidating the functional roles of the numerous and distinct members of these two categories of ncRNAs that are present in PRAD.

Assessment of isomiR Discrimination Using Commercial qPCR Methods.

We sought to determine whether commercial quantitative polymerase chain reaction (qPCR) methods are capable of distinguishing isomiRs: variants of mature microRNAs (miRNAs) with sequence endpoint differences. We used two commercially available miRNA qPCR methods to quantify miR-21-5p in both synthetic and real cell contexts. We find that although these miRNA qPCR methods possess high sensitivity for specific sequences, they also pick up background signals from closely related isomiRs, which influences the reliable quantification of individual isomiRs. We conclude that these methods do not possess the requisite specificity for reliable isomiR quantification.