Demonstration of Membrane Transport of Histidine using Goat Intestinal Inverted Sacs: An Experiential Pedagogical Tool for Undergraduates.

Histidine is an essential amino acid that is also a precursor for metabolites implicated in the immune system, pulmonary ventilation, and vascular circulation. Absorption of dietary histidine relies largely on the sodium-coupled neutral amino acid transport by the Broad neutral amino acid transporter (B0AT) present on the apical membrane of the enterocyte. Here, we demonstrate the absorption of histidine by the intestinal villus enterocytes from the lumen using goat jejunal inverted sacs. The jejunal sacs exposed to varying concentrations of sodium and histidine were assayed to determine the concentration of histidine inside the sacs as a function of time. The results show active histidine absorption. Increasing the concentration of salt resulted in higher absorption of histidine, suggesting a symport of sodium and histidine absorption in goat intestinal inverted sacs. This protocol may be applied to visualize the intestinal mobility of amino acids or other metabolites with appropriate modifications. We propose this experiment as an experiential pedagogical tool that can help undergraduate students comprehend the concept of membrane transport.

Single-Cell Histone Modification Profiling with Cell Enrichment Using sortChIC.

Histone post-translational modifications (PTMs) influence the overall structure of the chromatin and gene expression. Over the course of cell differentiation, the distribution of histone modifications is remodeled, resulting in cell type-specific patterns. In the past, their study was limited to abundant cell types that could be purified in necessary numbers. However, studying these cell type-specific dynamic changes in heterogeneous in vivo settings requires sensitive single-cell methods. Current advances in single-cell sequencing methods remove these limitations, allowing the study of nonpurifiable cell types. One complicating factor is that some of the most biologically interesting cell types, including stem and progenitor cells that undergo differentiation, only make up a small fraction of cells in a tissue. This makes whole-tissue analysis rather inefficient. In this chapter, we present a sort-assisted single-cell Chromatin ImmunoCleavage sequencing technique (sortChIC) to map histone PTMs in single cells. This technique combines the mapping of histone PTM location in combination with surface staining-based enrichment, to allow the integration of established strategies for rare cell type enrichment. In general terms, this will enable researchers to quantify local and global chromatin changes in dynamic complex biological systems and can provide additional information on their contribution to lineage and cell-type specification in physiological conditions and disease.

Acute multi-level response to defective de novo chromatin assembly in S-phase.

Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these defects is unknown. Here, we combine acute degradation of Chromatin Assembly Factor 1 (CAF-1), a key player in de novo chromatin assembly, with single-cell genomics, quantitative proteomics, and live-microscopy to uncover these initiating mechanisms in human cells. CAF-1 loss immediately slows down DNA replication speed and renders nascent DNA hyperaccessible. A rapid cellular response, distinct from canonical DNA damage signaling, is triggered and lowers histone mRNAs. As a result, histone variants usage and their modifications are altered, limiting transcriptional fidelity and delaying chromatin maturation within a single S-phase. This multi-level response induces a cell-cycle arrest after mitosis. Our work reveals the immediate consequences of defective de novo chromatin assembly during DNA replication, explaining how at later times the epigenome and cell fate can be altered.

An enhanced diabetes prediction amidst COVID-19 using ensemble models.

In the contemporary landscape of healthcare, the early and accurate prediction of diabetes has garnered paramount importance, especially in the wake of the COVID-19 pandemic where individuals with diabetes exhibit increased vulnerability. This research embarked on a mission to enhance diabetes prediction by employing state-of-the-art machine learning techniques. Initial evaluations highlighted the Support Vector Machines (SVM) classifier as a promising candidate with an accuracy of 76.62%. To further optimize predictions, the study delved into advanced feature engineering techniques, generating interaction and polynomial features that unearthed hidden patterns in the data. Subsequent correlation analyses, visualized through heatmaps, revealed significant correlations, especially with attributes like Glucose. By integrating the strengths of Decision Trees, Gradient Boosting, and SVM in an ensemble model, we achieved an accuracy of 93.2%, showcasing the potential of harmonizing diverse algorithms. This research offers a robust blueprint for diabetes prediction, holding profound implications for early diagnosis, personalized treatments, and preventive care in the context of global health challenges and with the goal of increasing life expectancy.

Impact of Boron Carbide Particles and Weight Percentage on the Mechanical and Wear Characterization of Al2011 Alloy Metal Composites.

Micron-sized B4C addition to the Al2011 alloy was investigated for its impact on mechanical and wear performance. The stir-casting method was used to manufacture the Al2011 alloy metal matrix composites reinforced with varying percentages of B4C particulates (2, 4, and 6). The microstructural, mechanical, and wear properties of the synthesized composites were tested. scanning electronic microscope (SEM) microscopy and XRD patterns were used to characterize the microstructure of the samples that were obtained. The XRD patterns confirmed the presence of B4C particles. The addition of B4C reinforcement increased the metal composite's hardness, tensile strength, and compressive strength. Incorporating the reinforcement resulted in a decrease in elongation for the Al2011 alloy composite. The wear behavior of the prepared samples was examined under various load and speed conditions. In terms of wear resistance, the microcomposites were far superior. SEM observations of the Al2011-B4C composites revealed numerous fracture and wear mechanisms.

Slow Heat-Based Hybrid Simulated Annealing Algorithm in Vehicular Ad Hoc Network.

Vehicular ad hoc networks (VANETs) using reliable protocols of routing have become crucial in identifying the changes to topology on a continuous basis for a large collection of vehicles. For this purpose, it becomes important to identify an optimal configuration of these protocols. There are several possible configurations that have been preventing the configuration of efficient protocols that do not make use of automatic and intelligent design tools. It can further motivate using the techniques of metaheuristics like the tools, which are well-suited to be able to solve these problems. The glowworm swarm optimization (GSO), simulated annealing (SA), and slow heat-based SA-GSO algorithms have been proposed in this work. The SA is a method of optimization, which imitates the manner in which the thermal system has been frozen down to its lowest state of energy. In the GSO, there is guidance to the rules of feasibility, where the swarm converges to its feasible regions very fast. Additionally, for overcoming any premature convergence, there is a local search strategy that is based on the SA and is used for making a search that is near to its true optimum solutions. Finally, this sluggish temperature-based SA-GSO algorithm will be employed to solve routing problems and problems of heat transfer. There is a hybrid slow heat SA-GSO algorithm with a faster speed of convergence and higher precision of computation that is more effective in solving problems of constrained engineering.

Single-cell sortChIC identifies hierarchical chromatin dynamics during hematopoiesis.

Post-translational histone modifications modulate chromatin activity to affect gene expression. How chromatin states underlie lineage choice in single cells is relatively unexplored. We develop sort-assisted single-cell chromatin immunocleavage (sortChIC) and map active (H3K4me1 and H3K4me3) and repressive (H3K27me3 and H3K9me3) histone modifications in the mouse bone marrow. During differentiation, hematopoietic stem and progenitor cells (HSPCs) acquire active chromatin states mediated by cell-type-specifying transcription factors, which are unique for each lineage. By contrast, most alterations in repressive marks during differentiation occur independent of the final cell type. Chromatin trajectory analysis shows that lineage choice at the chromatin level occurs at the progenitor stage. Joint profiling of H3K4me1 and H3K9me3 demonstrates that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin states. This implies a hierarchical regulation of chromatin during hematopoiesis: heterochromatin dynamics distinguish differentiation trajectories and lineages, while euchromatin dynamics reflect cell types within lineages.

Creating clear and informative image-based figures for scientific publications.

Scientists routinely use images to display data. Readers often examine figures first; therefore, it is important that figures are accessible to a broad audience. Many resources discuss fraudulent image manipulation and technical specifications for image acquisition; however, data on the legibility and interpretability of images are scarce. We systematically examined these factors in non-blot images published in the top 15 journals in 3 fields; plant sciences, cell biology, and physiology (n = 580 papers). Common problems included missing scale bars, misplaced or poorly marked insets, images or labels that were not accessible to colorblind readers, and insufficient explanations of colors, labels, annotations, or the species and tissue or object depicted in the image. Papers that met all good practice criteria examined for all image-based figures were uncommon (physiology 16%, cell biology 12%, plant sciences 2%). We present detailed descriptions and visual examples to help scientists avoid common pitfalls when publishing images. Our recommendations address image magnification, scale information, insets, annotation, and color and may encourage discussion about quality standards for bioimage publishing.

pyGenomeTracks: reproducible plots for multivariate genomic datasets.

MOTIVATION: Generating publication ready plots to display multiple genomic tracks can pose a serious challenge. Making desirable and accurate figures requires considerable effort. This is usually done by hand or using a vector graphic software. RESULTS: pyGenomeTracks (PGT) is a modular plotting tool that easily combines multiple tracks. It enables a reproducible and standardized generation of highly customizable and publication ready images. AVAILABILITY AND IMPLEMENTATION: PGT is available through a graphical interface on https://usegalaxy.eu and through the command line. It is provided on conda via the bioconda channel, on pip and it is openly developed on github: https://github.com/deeptools/pyGenomeTracks. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Neural metabolic imbalance induced by MOF dysfunction triggers pericyte activation and breakdown of vasculature.

Mutations in chromatin-modifying complexes and metabolic enzymes commonly underlie complex human developmental syndromes affecting multiple organs. A major challenge is to determine how disease-causing genetic lesions cause deregulation of homeostasis in unique cell types. Here we show that neural-specific depletion of three members of the non-specific lethal (NSL) chromatin complex-Mof, Kansl2 or Kansl3-unexpectedly leads to severe vascular defects and brain haemorrhaging. Deregulation of the epigenetic landscape induced by the loss of the NSL complex in neural cells causes widespread metabolic defects, including an accumulation of free long-chain fatty acids (LCFAs). Free LCFAs induce a Toll-like receptor 4 (TLR4)-NFκB-dependent pro-inflammatory signalling cascade in neighbouring vascular pericytes that is rescued by TLR4 inhibition. Pericytes display functional changes in response to LCFA-induced activation that result in vascular breakdown. Our work establishes that neurovascular function is determined by the neural metabolic environment.

MAPCap allows high-resolution detection and differential expression analysis of transcription start sites.

The position, shape and number of transcription start sites (TSS) are critical determinants of gene regulation. Most methods developed to detect TSSs and study promoter usage are, however, of limited use in studies that demand quantification of expression changes between two or more groups. In this study, we combine high-resolution detection of transcription start sites and differential expression analysis using a simplified TSS quantification protocol, MAPCap (Multiplexed Affinity Purification of Capped RNA) along with the software icetea . Applying MAPCap on developing Drosophila melanogaster embryos and larvae, we detected stage and sex-specific promoter and enhancer activity and quantify the effect of mutants of maleless (MLE) helicase at X-chromosomal promoters. We observe that MLE mutation leads to a median 1.9 fold drop in expression of X-chromosome promoters and affects the expression of several TSSs with a sexually dimorphic expression on autosomes. Our results provide quantitative insights into promoter activity during dosage compensation.

snakePipes: facilitating flexible, scalable and integrative epigenomic analysis.

SUMMARY: Due to the rapidly increasing scale and diversity of epigenomic data, modular and scalable analysis workflows are of wide interest. Here we present snakePipes, a workflow package for processing and downstream analysis of data from common epigenomic assays: ChIP-seq, RNA-seq, Bisulfite-seq, ATAC-seq, Hi-C and single-cell RNA-seq. snakePipes enables users to assemble variants of each workflow and to easily install and upgrade the underlying tools, via its simple command-line wrappers and yaml files. AVAILABILITY AND IMPLEMENTATION: snakePipes can be installed via conda: `conda install -c mpi-ie -c bioconda -c conda-forge snakePipes'. Source code (https://github.com/maxplanck-ie/snakepipes) and documentation (https://snakepipes.readthedocs.io/en/latest/) are available online. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The NSL complex-mediated nucleosome landscape is required to maintain transcription fidelity and suppression of transcription noise.

Nucleosomal organization at gene promoters is critical for transcription, with a nucleosome-depleted region (NDR) at transcription start sites (TSSs) being required for transcription initiation. How NDRs and the precise positioning of the +1 nucleosomes are maintained on active genes remains unclear. Here, we report that the Drosophila nonspecific lethal (NSL) complex is necessary to maintain this stereotypical nucleosomal organization at promoters. Upon NSL1 depletion, nucleosomes invade the NDRs at TSSs of NSL-bound genes. NSL complex member NSL3 binds to TATA-less promoters in a sequence-dependent manner. The NSL complex interacts with the NURF chromatin remodeling complex and is necessary and sufficient to recruit NURF to target promoters. Not only is the NSL complex essential for transcription, but it is required for accurate TSS selection for genes with multiple TSSs. Furthermore, loss of the NSL complex leads to an increase in transcriptional noise. Thus, the NSL complex establishes a canonical nucleosomal organization that enables transcription and determines TSS fidelity.

Galaxy HiCExplorer: a web server for reproducible Hi-C data analysis, quality control and visualization.

Galaxy HiCExplorer is a web server that facilitates the study of the 3D conformation of chromatin by allowing Hi-C data processing, analysis and visualization. With the Galaxy HiCExplorer web server, users with little bioinformatic background can perform every step of the analysis in one workflow: mapping of the raw sequence data, creation of Hi-C contact matrices, quality assessment, correction of contact matrices and identification of topological associated domains (TADs) and A/B compartments. Users can create publication ready plots of the contact matrix, A/B compartments, and TADs on a selected genomic locus, along with additional information like gene tracks or ChIP-seq signals. Galaxy HiCExplorer is freely usable at: https://hicexplorer.usegalaxy.eu and is available as a Docker container: https://github.com/deeptools/docker-galaxy-hicexplorer.

Community-Driven Data Analysis Training for Biology.

The primary problem with the explosion of biomedical datasets is not the data, not computational resources, and not the required storage space, but the general lack of trained and skilled researchers to manipulate and analyze these data. Eliminating this problem requires development of comprehensive educational resources. Here we present a community-driven framework that enables modern, interactive teaching of data analytics in life sciences and facilitates the development of training materials. The key feature of our system is that it is not a static but a continuously improved collection of tutorials. By coupling tutorials with a web-based analysis framework, biomedical researchers can learn by performing computation themselves through a web browser without the need to install software or search for example datasets. Our ultimate goal is to expand the breadth of training materials to include fundamental statistical and data science topics and to precipitate a complete re-engineering of undergraduate and graduate curricula in life sciences. This project is accessible at https://training.galaxyproject.org.

High-resolution TADs reveal DNA sequences underlying genome organization in flies.

Despite an abundance of new studies about topologically associating domains (TADs), the role of genetic information in TAD formation is still not fully understood. Here we use our software, HiCExplorer (hicexplorer.readthedocs.io) to annotate >2800 high-resolution (570 bp) TAD boundaries in Drosophila melanogaster. We identify eight DNA motifs enriched at boundaries, including a motif bound by the M1BP protein, and two new boundary motifs. In contrast to mammals, the CTCF motif is only enriched on a small fraction of boundaries flanking inactive chromatin while most active boundaries contain the motifs bound by the M1BP or Beaf-32 proteins. We demonstrate that boundaries can be accurately predicted using only the motif sequences at open chromatin sites. We propose that DNA sequence guides the genome architecture by allocation of boundary proteins in the genome. Finally, we present an interactive online database to access and explore the spatial organization of fly, mouse and human genomes, available at http://chorogenome.ie-freiburg.mpg.de .

DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome.

Transposable elements are viewed as 'selfish genetic elements', yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human genome consists of transposable elements. Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability. Alu elements are the main targets of the RNA-editing enzyme ADAR and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant nuclear RNA helicase, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.

Alu-miRNA interactions modulate transcript isoform diversity in stress response and reveal signatures of positive selection.

Primate-specific Alus harbor different regulatory features, including miRNA targets. In this study, we provide evidence for miRNA-mediated modulation of transcript isoform levels during heat-shock response through exaptation of Alu-miRNA sites in mature mRNA. We performed genome-wide expression profiling coupled with functional validation of miRNA target sites within exonized Alus, and analyzed conservation of these targets across primates. We observed that two miRNAs (miR-15a-3p and miR-302d-3p) elevated in stress response, target RAD1, GTSE1, NR2C1, FKBP9 and UBE2I exclusively within Alu. These genes map onto the p53 regulatory network. Ectopic overexpression of miR-15a-3p downregulates GTSE1 and RAD1 at the protein level and enhances cell survival. This Alu-mediated fine-tuning seems to be unique to humans as evident from the absence of orthologous sites in other primate lineages. We further analyzed signatures of selection on Alu-miRNA targets in the genome, using 1000 Genomes Phase-I data. We found that 198 out of 3177 Alu-exonized genes exhibit signatures of selection within Alu-miRNA sites, with 60 of them containing SNPs supported by multiple evidences (global-FST > 0.3, pair-wise-FST > 0.5, Fay-Wu's H < -20, iHS > 2.0, high ΔDAF) and implicated in p53 network. We propose that by affecting multiple genes, Alu-miRNA interactions have the potential to facilitate population-level adaptations in response to environmental challenges.

deepTools2: a next generation web server for deep-sequencing data analysis.

We present an update to our Galaxy-based web server for processing and visualizing deeply sequenced data. Its core tool set, deepTools, allows users to perform complete bioinformatic workflows ranging from quality controls and normalizations of aligned reads to integrative analyses, including clustering and visualization approaches. Since we first described our deepTools Galaxy server in 2014, we have implemented new solutions for many requests from the community and our users. Here, we introduce significant enhancements and new tools to further improve data visualization and interpretation. deepTools continue to be open to all users and freely available as a web service at deeptools.ie-freiburg.mpg.de The new deepTools2 suite can be easily deployed within any Galaxy framework via the toolshed repository, and we also provide source code for command line usage under Linux and Mac OS X. A public and documented API for access to deepTools functionality is also available.

The Zebrafish GenomeWiki: a crowdsourcing approach to connect the long tail for zebrafish gene annotation.

A large repertoire of gene-centric data has been generated in the field of zebrafish biology. Although the bulk of these data are available in the public domain, most of them are not readily accessible or available in nonstandard formats. One major challenge is to unify and integrate these widely scattered data sources. We tested the hypothesis that active community participation could be a viable option to address this challenge. We present here our approach to create standards for assimilation and sharing of information and a system of open standards for database intercommunication. We have attempted to address this challenge by creating a community-centric solution for zebrafish gene annotation. The Zebrafish GenomeWiki is a 'wiki'-based resource, which aims to provide an altruistic shared environment for collective annotation of the zebrafish genes. The Zebrafish GenomeWiki has features that enable users to comment, annotate, edit and rate this gene-centric information. The credits for contributions can be tracked through a transparent microattribution system. In contrast to other wikis, the Zebrafish GenomeWiki is a 'structured wiki' or rather a 'semantic wiki'. The Zebrafish GenomeWiki implements a semantically linked data structure, which in the future would be amenable to semantic search. Database URL: http://genome.igib.res.in/twiki.