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Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.

Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.

Small nucleotide variants in non-coding regions of the genome can alter transcriptional regulation, leading to changes in gene expression which can activate oncogenic gene regulatory networks. Melanoma is heavily burdened by non-coding variants, representing over 99% of total genetic variation, including the well-characterized TERT promoter mutation. However, the compendium of regulatory non-coding variants is likely still functionally under-characterized. We developed a pipeline to identify hotspots, i.e. recurrently mutated regions, in melanoma containing putatively functional non-coding somatic variants that are located within predicted melanoma-specific regulatory regions. We identified hundreds of statistically significant hotspots, including the hotspot containing the TERT promoter variants, and focused on a hotspot in the promoter of CDC20. We found that variants in the promoter of CDC20, which putatively disrupt an ETS motif, lead to lower transcriptional activity in reporter assays. Using CRISPR/Cas9, we generated an indel in the CDC20 promoter in human A375 melanoma cell lines and observed decreased expression of CDC20, changes in migration capabilities, increased growth of xenografts, and an altered transcriptional state previously associated with a more proliferative and less migratory state. Overall, our analysis prioritized several recurrent functional non-coding variants that, through downregulation of CDC20, led to perturbation of key melanoma phenotypes., (© 2023. The Author(s).)

Objective: To examine the effects of d-tagatose or stevia preloads on carbohydrate metabolism markers after an oral glucose load, as well as subjective and objective appetite in women with insulin resistance (IR)., Research Design and Methods: Randomized controlled crossover study. Women with IR without T2DM (n = 33; aged 23.4 ± 3.8; BMI 28.1 ± 3.4 kg × m -2 ) underwent three oral glucose loads (3 h each) on three different days. Ten min before oral glucose load, volunteers consumed a preload of 60 mL water (control), 60 mL water with stevia (15.3 mg), or d-tagatose (5000 mg). Serum glucose and C-peptide were evaluated at -10, 30-, 60-, 90-, 120-, and 180-min. Subjective appetite was determined with a visual analog scale. Food intake was measured at ad libitum buffet after 180 min., Results: C-peptide iAUC was significantly higher for stevia (median (IQR): 1033 (711-1293) ng × min × L -1 ) vs. d-tagatose (794 (366-1134) ng × min × L -1 ; P = 0.001) or control (730 (516-1078) ng × min × L -1 ; P = 0.012). At 30- and 60-min serum glucose was higher for stevia vs other conditions (P < 0.01). Volunteers reported greater satiety for stevia and d-tagatose vs. control at 60 min and greater desire to eat for stevia vs. control at 120- min (all P < 0.05). Objective appetite did not vary by condition (P = 0.06)., Conclusions: Our findings suggest that these NNS are not inert. Stevia intake produced an acute response on C-peptide release while increased serum glucose at earlier times. It is possible that NNS affects subjective but not objective appetite. This trial is registered at clinicaltrials.gov as NCT04327245., Clinical Trial Registry: NCT04327245., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2022 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.)

Transcriptional and epigenetic characterization of melanocytes and melanoma cells isolated from their in vivo context promises to unveil key differences between these developmentally related normal and cancer cell populations. We therefore engineered an enhanced Danio rerio (zebrafish) melanoma model with fluorescently labeled melanocytes to allow for isolation of normal (wild type) and premalignant (BRAFV600E-mutant) populations for comparison to fully transformed BRAFV600E-mutant, p53 loss-of-function melanoma cells. Using fluorescence-activated cell sorting to isolate these populations, we performed high-quality RNA- and ATAC-seq on sorted zebrafish melanocytes vs. melanoma cells, which we provide as a resource here. Melanocytes had consistent transcriptional and accessibility profiles, as did melanoma cells. Comparing melanocytes and melanoma, we note 4128 differentially expressed genes and 56,936 differentially accessible regions with overall gene expression profiles analogous to human melanocytes and the pigmentation melanoma subtype. Combining the RNA- and ATAC-seq data surprisingly revealed that increased chromatin accessibility did not always correspond with increased gene expression, suggesting that though there is widespread dysregulation in chromatin accessibility in melanoma, there is a potentially more refined gene expression program driving cancerous melanoma. These data serve as a resource to identify candidate regulators of the normal vs. diseased states in a genetically controlled in vivo context., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Millipedes are among the most diverse and abundant arthropods in terrestrial environments. However, little is known about their innate immune response against invading pathogenic microorganisms, which is very intriguing considering that the evolutionary success of millipedes is largely due to this complex and primitive defense system, since it allowed them to colonize a wide variety of microhabitats characterized by their high microbial proliferation. Accordingly, the aim of the present work was to determine the presence of antimicrobial peptides in the hemolymph of the millipede Rhinocricus sp. In total, four native peptides with potent antimicrobial activity against different microorganisms, lack of cytotoxicity against Vero cells and lack of hemolytic effects against human erythrocytes were isolated and named RP40-16, RP40-19, RP40-20/1 and RP40-20/2. The analysis with bioinformatics tools suggested that these peptides may be encrypted in large proteins present in the plasma: Hemocyanin and thioester-containing protein. Considering these results, it can be said that millipede hemolymph represents a promising source of molecules with potential for the development of non-conventional antibiotics. Therefore, in order to have a clearer notion of the biotechnological potential and the role of these peptides in the innate immune response of Rhinocricus sp., future studies should focus on elucidating their mechanisms of action, as well as additional biological properties., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The role of a neural crest developmental transcriptional program, which critically involves Sox10 upregulation, is a key conserved aspect of melanoma initiation in both humans and zebrafish, yet transcriptional regulation of sox10 expression is incompletely understood. Here we used ATAC-Seq analysis of multiple zebrafish melanoma tumors to identify recurrently open chromatin domains as putative melanoma-specific sox10 enhancers. Screening in vivo with EGFP reporter constructs revealed 9 of 11 putative sox10 enhancers with embryonic activity in zebrafish. Focusing on the most active enhancer region in melanoma, we identified a region 23 kilobases upstream of sox10, termed peak5, that drives EGFP reporter expression in a subset of neural crest cells, Kolmer-Agduhr neurons, and early melanoma patches and tumors with high specificity. A ~200 base pair region, conserved in Cyprinidae, within peak5 is required for transgenic reporter activity in neural crest and melanoma. This region contains dimeric SoxE/Sox10 dimeric binding sites essential for peak5 neural crest and melanoma activity. We show that deletion of the endogenous peak5 conserved genomic locus decreases embryonic sox10 expression and disrupts adult stripe patterning in our melanoma model background. Our work demonstrates the power of linking developmental and cancer models to better understand neural crest identity in melanoma.

Given the increasing interest in their use as disease biomarkers, the establishment of reproducible, accurate, sensitive, and specific platforms for microRNA (miRNA) quantification in biofluids is of high priority. We compare four platforms for these characteristics: small RNA sequencing (RNA-seq), FirePlex, EdgeSeq, and nCounter. For a pool of synthetic miRNAs, coefficients of variation for technical replicates are lower for EdgeSeq (6.9%) and RNA-seq (8.2%) than for FirePlex (22.4%); nCounter replicates are not performed. Receiver operating characteristic analysis for distinguishing present versus absent miRNAs shows small RNA-seq (area under curve 0.99) is superior to EdgeSeq (0.97), nCounter (0.94), and FirePlex (0.81). Expected differences in expression of placenta-associated miRNAs in plasma from pregnant and non-pregnant women are observed with RNA-seq and EdgeSeq, but not FirePlex or nCounter. These results indicate that differences in performance among miRNA profiling platforms impact ability to detect biological differences among samples and thus their relative utility for research and clinical use., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the version of this article initially published, grant PF-17-201-01-TBG from the American Cancer Society to author Erica C. Pehrsson was not included in the Acknowledgements. The error has been corrected in the HTML and PDF versions of the article.

Transposable elements (TEs) are an abundant and rich genetic resource of regulatory sequences 1-3 . Cryptic regulatory elements within TEs can be epigenetically reactivated in cancer to influence oncogenesis in a process termed onco-exaptation 4 . However, the prevalence and impact of TE onco-exaptation events across cancer types are poorly characterized. Here, we analyzed 7,769 tumors and 625 normal datasets from 15 cancer types, identifying 129 TE cryptic promoter-activation events involving 106 oncogenes across 3,864 tumors. Furthermore, we interrogated the AluJb-LIN28B candidate: the genetic deletion of the TE eliminated oncogene expression, while dynamic DNA methylation modulated promoter activity, illustrating the necessity and sufficiency of a TE for oncogene activation. Collectively, our results characterize the global profile of TE onco-exaptation and highlight this prevalent phenomenon as an important mechanism for promiscuous oncogene activation and ultimately tumorigenesis.

Extracellular microRNAs (miRNAs) and other small RNAs are implicated in cellular communication and may be useful as disease biomarkers. We systematically compared small RNAs in 12 human biofluid types using RNA sequencing (RNA-seq). miRNAs and tRNA-derived RNAs (tDRs) accounted for the majority of mapped reads in all biofluids, but the ratio of miRNA to tDR reads varied from 72 in plasma to 0.004 in bile. miRNA levels were highly correlated across all biofluids, but levels of some miRNAs differed markedly between biofluids. tDR populations differed extensively between biofluids. Y RNA fragments were seen in all biofluids and accounted for >10% of reads in blood plasma, serum, and cerebrospinal fluid (CSF). Reads mapping exclusively to Piwi-interacting RNAs (piRNAs) were very rare, except in seminal plasma. These results demonstrate extensive differences in small RNAs between human biofluids and provide a useful resource for investigating extracellular RNA biology and developing biomarkers., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

This article presents an overview of intramuscular gluteal augmentation involving primary and secondary augmentation with implants. Although gluteal augmentation is a well-studied procedure, few reports have described the intramuscular technique or this technique in association with lipofilling. This article presents experience with a combined technique; this recent innovation combines placement of an anatomic silicone gel implant underneath the gluteus maximus and immediate fat grafting. Primary and secondary gluteal augmentations using implants resulted in satisfactory outcomes. A majority of complications were minor, predictable, and did not affect aesthetic outcome or normal postoperative recovery. Success depends on patient selection and careful intraoperative and postoperative management., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The profiles of proteins present in the exoantigens of Brazilian Sporothrix schenckii isolates were studied and compared by electrophoresis (SDS-PAGE). Thirteen isolates from five different regions of Brazil (1,000 to 2,000 km apart) and ten from a more limited region (200 to 400 km apart within the state of São Paulo) were cultured in Sabouraud, M199 and minimum (MM) media. Qualitative and quantitative differences in the expression of proteins, which varied according to the medium and the isolate, were observed. Fractions with the same MW but varying in intensity were detected, as well as fractions present in 1 isolate but absent in others. Dendrograms were constructed and isolates grouped based on the fractions obtained, irrespective of the intensity. The results showed that Brazilian S. schenckii isolates express different protein profiles, a feature also present in isolates from a more restricted region. The exoantigens were found to have a maximum of 15 protein fractions, ranging in MW from 19-220 KDaltons depending on the medium used for the cultures. These data show the great heterogeneity of Brazilian S. schenckii protein expression.

Background: Numerous techniques have been described for the treatment of breast hypertrophy and ptosis. Unfortunately, recurrent ptosis after mammaplasty can occur regardless of the technique used. To avoid this problem, different kinds of supporting devices have been described with variable rates of success. However, the true implications of incorporating prosthetic materials into breast surgery have never been clarified. Therefore, surgeons have traditionally been reluctant to apply any kind of prosthetic material to the breast, fearing inflammation, an unfavorable aesthetic outcome, palpable or visible deformities, and interference with the mammographic evaluation of breast cancer. This study analyzed the aesthetic, clinical, and mammographic implications of using mesh as a supportive device in periareolar breast surgery., Methods: For this study, 18 patients (mean age, 42 years) with breast hypertrophy, ptosis, or both were managed with the double-skin periareolar mammaplasty technique, with placement of mixed (60% Polyglactine and 40% polyester) mesh. Clinical assessment was performed by three breast surgeons actively working on cancer surveillance who knew that the patients had experienced mesh application. After a mean follow-up period of 30 months, a standard mammogram was performed for each patient and analyzed by both the surgeons and an expert radiologist. The evaluated factors were hyperemia, calcifications, contour irregularities, capsular contraction, thickening or widening of the scar with extrusion of the mesh, and any palpable or hardened areas., Results: According to the authors' clinical observations, there were no mesh-related abnormalities in the breast; the mesh was not palpable after the operation; and there was no recurrent ptosis. In terms of mammographic imaging, the mesh was visible as a very fine line in the periphery of the breast's parenchyma (measuring 0.2 mm on the lateral views) in three patients (17%). The mesh did not interfere with the visualization and analysis of the breast's parenchyma. In seven patients (39%), benign localized microcalcifications were detected in the breast and no further investigation was performed. In two patients (11%), grouped calcifications were detected and biopsied, with histopathologic analysis demonstrating epithelial hyperplasia with atypia. In two patients (11%), nodules smaller than 1 cm were detected and biopsied, with histopathologic analysis demonstrating a fibroadenoma in one patient and an invasive ductal carcinoma in the other., Conclusions: The use of mesh support in breast surgery can enhance the aesthetic results without inducing visible or palpable deformities or mammographic abnormalities. In terms of surveillance mammograms, the presence of the mesh did not interfere with the diagnosis and treatment of minute lesions such as calcifications and small nodules.

An epidemiologic and seroprevalence survey was conducted (n=830) to assess the proportion of persons exposed to hantavirus in IX Region Chile, which accounts for 25% of reported cases of hantavirus cardiopulmonary syndrome. This region has three geographic areas with different disease incidences and a high proportion of aboriginals. Serum samples were tested for immunoglobulin (Ig) G antibodies by enzyme-linked immunosorbent assay against Sin Nombre virus N antigen by strip immunoblot assay against Sin Nombre, Puumala, Río Mamoré, and Seoul N antigens. Samples from six patients were positive for IgG antibodies reactive with Andes virus; all patients lived in the Andes Mountains. Foresting was also associated with seropositivity; but not sex, age, race, rodent exposure, or farming activities. Exposure to hantavirus varies in different communities of IX Region. Absence of history of pneumonia or hospital admission in persons with specific IgG antibodies suggests that infection is clinically inapparent.