Bile acids (BAs) have been established as ubiquitous regulatory molecules implicated in a large variety of healthy and pathological processes. However, the scope of BA heterogeneity is often underrepresented in current literature. This is due in part to inadequate detection methods, which fail to distinguish the individual constituents of the BA pool. Thus, the primary aim of this study was to develop a method that would allow the simultaneous analysis of specific C24 BA species, and to apply that method to biological systems of interest. Herein, we describe the generation and validation of an LC-MS/MS assay for quantification of numerous BAs in a variety of cell systems and relevant biofluids and tissue. These studies included the first baseline level assessment for planar BAs, including allocholic acid, in cell lines, biofluids, and tissue in a nonhuman primate (NHP) laboratory animal, Macaca mulatta, in healthy conditions. These results indicate that immortalized cell lines make poor models for the study of BA synthesis and metabolism, whereas human primary hepatocytes represent a promising alternative model system. We also characterized the BA pool of M. mulatta in detail. Our results support the use of NHP models for the study of BA metabolism and pathology in lieu of murine models. Moreover, the method developed here can be applied to the study of common and planar C24 BA species in other systems.

The analysis of T cell lipid raft proteome is challenging due to the highly dynamic nature of rafts and the hydrophobic character of raft-resident proteins. We explored an innovative strategy for bottom-up lipid raftomics based on suspension-trapping (S-Trap) sample preparation. Mouse T cells were prepared from splenocytes by negative immunoselection, and rafts were isolated by a detergent-free method and OptiPrep gradient ultracentrifugation. Microdomains enriched in flotillin-1, LAT, and cholesterol were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. Using this method, we identified 2,680 proteins in the raft-rich fraction and established a database of 894 T cell raft proteins. We then performed a differential analysis on the raft-rich fraction from nonstimulated versus anti-CD3/CD28 T cell receptor (TCR)-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other. For the first time, we performed a proteomic analysis on rafts from ex vivo T cells obtained from individual mice, before and after TCR activation. This work demonstrates that the proposed method utilizing an S-Trap-based approach for sample preparation increases the specificity and sensitivity of lipid raftomics.

Electrospray ionization is today the most widely used ionization technique in chemical and bio-chemical analysis. Interfaced with a mass spectrometer it allows to investigate the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized. There is no limitation in mass which enables even the investigation of large non-covalent protein complexes. Its high ionization efficiency profoundly changed bio-molecular sciences because proteins can be identified and quantified on trace amounts in a high throughput fashion. This review article focusses mainly on the exploration of the underlying ionization mechanism. Some ionization characteristics are discussed which are related to this mechanism. Typical spectra of peptides, proteins and non-covalent complexes are shown and the quantitative character of spectra is highlighted. Finally the possibilities and limitations in measuring the association constant of bivalent non-covalent complexes are described.

O-GlcNAcylation, the addition of a single N-acetylglucosamine residue to serine and threonine residues of cytoplasmic, nuclear, or mitochondrial proteins, is a widespread regulatory post-translational modification. It is involved in response to nutritional status and stress and its dysregulation is associated with diseases ranging from Alzheimer’s to diabetes.  While the modification was first detected over thirty-five years ago, research into the function of O-GlcNAcylation has accelerated dramatically in the last ten years due to the development of new enrichment and mass spectrometry techniques that facilitate its analysis.  This article summarizes methods for O-GlcNAc enrichment, key mass spectrometry instrumentation advancements, particularly those that allow modification site localization, and software tools that allow analysis of data from O-GlcNAc modified peptides.

OMer Richard Sharp teaches practical and relevant methods of sharing the Gospel with Portuguese young people at a youth camp.

Sequence variation observed in populations of pathogens can be used for important public health and evolutionary genomic analyses, especially outbreak analysis and transmission reconstruction. Identifying this variation is typically achieved by aligning sequence reads to a reference genome, but this approach is susceptible to reference biases and requires careful filtering of called genotypes. There is a need for tools that can process this growing volume of bacterial genome data, providing rapid results, but that remain simple so they can be used without highly trained bioinformaticians, expensive data analysis, and long-term storage and processing of large files. Here we describe split k-mer analysis (SKA2), a method that supports both reference-free and reference-based mapping to quickly and accurately genotype populations of bacteria using sequencing reads or genome assemblies. SKA2 is highly accurate for closely related samples, and in outbreak simulations, we show superior variant recall compared with reference-based methods, with no false positives. SKA2 can also accurately map variants to a reference and be used with recombination detection methods to rapidly reconstruct vertical evolutionary history. SKA2 is many times faster than comparable methods and can be used to add new genomes to an existing call set, allowing sequential use without the need to reanalyze entire collections. With an inherent absence of reference bias, high accuracy, and a robust implementation, SKA2 has the potential to become the tool of choice for genotyping bacteria. SKA2 is implemented in Rust and is freely available as open-source software.

The COVID-19 pandemic has highlighted the critical role of genomic surveillance for guiding policy and control. Timeliness is key, but sequence alignment and phylogeny slow most surveillance techniques. Millions of SARS-CoV-2 genomes have been assembled. Phylogenetic methods are ill equipped to handle this sheer scale. We introduce a pangenomic measure that examines the information diversity of a k-mer library drawn from a country's complete set of clinical, pooled, or wastewater sequence. Quantifying diversity is central to ecology. Hill numbers, or the effective number of species in a sample, provide a simple metric for comparing species diversity across environments. The more diverse the sample, the higher the Hill number. We adopt this ecological approach and consider each k-mer an individual and each genome a transect in the pangenome of the species. Structured in this way, Hill numbers summarize the temporal trajectory of pandemic variants, collapsing each day's assemblies into genome equivalents. For pooled or wastewater sequence, we instead compare days using survey sequence divorced from individual infections. Across data from the UK, USA, and South Africa, we trace the ascendance of new variants of concern as they emerge in local populations well before these variants are named and added to phylogenetic databases. Using data from San Diego wastewater, we monitor these same population changes from raw, unassembled sequence. This history of emerging variants senses all available data as it is sequenced, intimating variant sweeps to dominance or declines to extinction at the leading edge of the COVID-19 pandemic.

High-throughput sequencing (HTS) technologies have been instrumental in investigating biological questions at the bulk and single-cell levels. Comparative analysis of two HTS data sets often relies on testing the statistical significance for the difference of two negative binomial distributions (DOTNB). Although negative binomial distributions are well studied, the theoretical results for DOTNB remain largely unexplored. Here, we derive basic analytical results for DOTNB and examine its asymptotic properties. As a state-of-the-art application of DOTNB, we introduce DEGage, a computational method for detecting differentially expressed genes (DEGs) in scRNA-seq data. DEGage calculates the mean of the sample-wise differences of gene expression levels as the test statistic and determines significant differential expression by computing the P-value with DOTNB. Extensive validation using simulated and real scRNA-seq data sets demonstrates that DEGage outperforms five popular DEG analysis tools: DEGseq2, DEsingle, edgeR, Monocle3, and scDD. DEGage is robust against high dropout levels and exhibits superior sensitivity when applied to balanced and imbalanced data sets, even with small sample sizes. We utilize DEGage to analyze prostate cancer scRNA-seq data sets and identify marker genes for 17 cell types. Furthermore, we apply DEGage to scRNA-seq data sets of mouse neurons with and without fear memory and reveal eight potential memory-related genes overlooked in previous analyses. The theoretical results and supporting software for DOTNB can be widely applied to comparative analyses of dispersed count data in HTS and broad research questions.

Mapping transcriptomic variations using either short- or long-read RNA sequencing is a staple of genomic research. Long reads are able to capture entire isoforms and overcome repetitive regions, whereas short reads still provide improved coverage and error rates. Yet, open questions remain, such as how to quantitatively compare the technologies, can we combine them, and what is the benefit of such a combined view? We tackle these questions by first creating a pipeline to assess matched long- and short-read data using a variety of transcriptome statistics. We find that across data sets, algorithms, and technologies, matched short-read data detects ~30% more splice junctions, such that ~10%–30% of the splice junctions included at ≥20% by short reads are missed by long reads. In contrast, long reads detect many more intron-retention events and can detect full isoforms, pointing to the benefit of combining the technologies. We introduce MAJIQ-L, an extension of the MAJIQ software, to enable a unified view of transcriptome variations from both technologies and demonstrate its benefits. Our software can be used to assess any future long-read technology or algorithm and can be combined with short-read data for improved transcriptome analysis.

Most studies of genome organization have focused on intrachromosomal (cis) contacts because they harbor key features such as DNA loops and topologically associating domains. Interchromosomal (trans) contacts have received much less attention, and tools for interrogating potential biologically relevant trans structures are lacking. Here, we develop a computational framework that uses Hi-C data to identify sets of loci that jointly interact in trans. This method, trans-C, initiates probabilistic random walks with restarts from a set of seed loci to traverse an input Hi-C contact network, thereby identifying sets of trans-contacting loci. We validate trans-C in three increasingly complex models of established trans contacts: the Plasmodium falciparum var genes, the mouse olfactory receptor "Greek islands," and the human RBM20 cardiac splicing factory. We then apply trans-C to systematically test the hypothesis that genes coregulated by the same trans-acting element (i.e., a transcription or splicing factor) colocalize in three dimensions to form "RNA factories" that maximize the efficiency and accuracy of RNA biogenesis. We find that many loci with multiple binding sites of the same DNA-binding proteins interact with one another in trans, especially those bound by factors with intrinsically disordered domains. Similarly, clustered binding of a subset of RNA-binding proteins correlates with trans interaction of the encoding loci. We observe that these trans-interacting loci are close to nuclear speckles. These findings support the existence of trans-interacting chromatin domains (TIDs) driven by RNA biogenesis. Trans-C provides an efficient computational framework for studying these and other types of trans interactions, empowering studies of a poorly understood aspect of genome architecture.

The rising obesity epidemic is a phenomenon that has gained increasing attention from health providers and health policy makers. This led to recognition of nonalcoholic fatty liver disease (MASLD). The standard for its assessment has been histologic, which is neither practical nor acceptable by patients. Subsequently, a number of noninvasive assessment methods have been developed. However, despite ease of implementation, their confounding variables do hinder their accuracy. Nonetheless, the development of the liver stiffness measurement (LSM) and incorporation of other biological parameters has minimized but not eliminated the need for liver biopsy. Imaging methods are useful in evaluation, estimation, and following the progression of steatosis and fibrosis with particular attention to controlled attenuation parameter (CAP) and MRI–Proton Density Fat Fraction (MRI-PDFF). The choices for the family physician are broad and rely on tests’ availability, cost, and patient acceptance. Great efforts have been undertaken to produce more robust and novel noninvasive markers that indicate fibrinogenesis directly in an implementable and cost-effective way.

ABSTRACTIntroduction:Emergency contraceptive pills (ECPs) are effective and can be used safely at any age repeatedly within the same cycle. They are often favored by youth yet are underutilized. Private facilities can increase ECP access but present barriers including cost. Identifying effective public-sector ECP distribution models can help ensure equitable access. The Malawi Ministry of Health developed a strategy to improve ECP access in 2020. We documented ECP provision through select public, youth-serving channels recommended by the strategy: general and youth-specific outreach, paid and unpaid community health workers (CHWs), and youth clubs.Methods:We conducted this mixed methods study from November 2022–March 2023 in 2 rural districts (Mchinji and Phalombe) implementing the strategy. We conducted qualitative interviews with 10 national stakeholders, 46 providers, and 24 clients aged 15–24 years about ECP service delivery. Additionally, 25 providers collected quantitative tally data about clients seeking ECPs. We analyzed qualitative data using grounded theory and quantitative data descriptively.Results:Stakeholders and providers reported ECP uptake increased in geographies where the strategy was implemented, especially among youth. Providers documented 3,988 client visits for ECPs over 3 months. Of these visits, 26% were from male clients, 36% were from clients aged younger than 20 years, and 64% received ECPs for the first time. Across channels, youth club leaders and unpaid CHWs reported the most client visits per provider and served the youngest clients. However, no ECPs were dispensed during 29% of visits due to stock-outs. While many providers were supportive of youth accessing ECPs, most held unfavorable attitudes toward repeat use.Conclusion:ECP access should be expanded through provision in the studied channels, especially youth clubs and CHWs. However, to meet demand, the supply chain must be strengthened. We recommend addressing providers’ attitudes about repeat use to ensure informed method choice.

Affibody molecules are small (6-kDa) affinity proteins folded in a three-helical bundle and generated by directed evolution for specific binding to various target molecules. The most advanced affibody molecules are currently tested in the clinic, and data from more than 300 subjects show excellent activity and safety profiles. The generation of affibody molecules against a particular target starts with the generation of an affibody library, which can then be used for panning using multiple methods and selection systems. This protocol describes the molecular cloning of DNA-encoded affibody libraries to a display vector of choice, for either phage, Escherichia coli, or Staphylococcus carnosus display. The DNA library can come from different sources, such as error-prone polymerase chain reaction (PCR), molecular shuffling of mutations from previous selections, or, more commonly, from DNA synthesis using various methods. Restriction enzyme-based subcloning is the most common strategy for affibody libraries of higher diversity (e.g., >10⁷ variants) and is described here.

Imaging methods are fundamental tools to detect and diagnose lung diseases, monitor their treatment and detect possible complications. Each modality, starting from classical chest radiographs and computed tomography, as well as the ever more popular and easily available thoracic ultrasound, magnetic resonance imaging and nuclear medicine methods, and new techniques such as photon counting computed tomography, radiomics and application of artificial intelligence, has its strong and weak points, which we should be familiar with to properly choose between the methods and interpret their results. In this review, we present the indications, strengths and main limitations of methods for chest imaging.

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Announcement Number: DOE-20-EIA-ES-00885
Closing Date: 23 March 2020

In the fast-paced world of social media, boosting engagement is crucial for people aiming to make an impact. With platforms constantly evolving, understanding advanced strategies can set your posts apart. This article explores effective methods to enhance interaction on your social

Cham, Switzerland : Springer, [2016]

A marriage between a couple of different backrounds can be a beautiful and rewarding encounter, however it is not really without its obstacles. It is important just for couples to keep yourself informed of these possible risks and take the necessary precautions in order to avoid them. A new study from University of Washington found […]

A Data Space is a protect, centralized site for posting information and documents with multiple stakeholders related to a small business transaction. These types of transactions can easily involve M&A or be for every other business activity that will need the transfer of very sensitive data to parties beyond the organization. Handling these trades can […]

The decline in the groundwater level is a major issue for agriculturists in all four districts of Shekhawati — Sikar, Jhunjhunu, Churu, and Neem Ka Thana

Boston : Birkhäuser, [2005]

New York, NY : Routledge , 2024.

Cheltenham, UK ; Northampton, MA, USA : Edward Elgar Publishing, [2023]