Epilipidomics platform for holistic profiling of oxidized complex lipids in blood plasma of obese individuals

Lipids are a structurally diverse class of biomolecules which can undergo a variety of chemical modifications. Among them, lipid (per)oxidation attracts most of the attention due to its significance in regulation of inflammation, cell proliferation and death programs. Despite their apparent regulatory significance, the molecular repertoire of oxidized lipids remains largely elusive as accurate annotation of lipid modifications is challenged by their low abundance and largely unknown, biological context-dependent structural diversity. Here we provide a holistic workflow based on the combination of bioinformatics and LC-MS/MS technologies to support identification and relative quantification of oxidized complex lipids in a modification type- and position-specific manner. The developed methodology was used to identify epilipidomics signatures of lean and obese individuals with and without type II diabetes. Characteristic signature of lipid modifications in lean individuals, dominated by the presence of modified octadecanoid acyl chains in phospho- and neutral lipids, was drastically shifted towards lipid peroxidation-driven accumulation of oxidized eicosanoids, suggesting significant alteration of endocrine signalling by oxidized lipids in metabolic disorders.

Advances in the Relationship Between Pyroptosis and Diabetic Neuropathy

Pyroptosis is a novel programmed cell death process that promotes the release of interleukin-1β (IL-1β) and interleukin-18 (IL-18) by activating inflammasomes and gasdermin D (GSDMD), leading to cell swelling and rupture. Pyroptosis is involved in the regulation of the occurrence and development of cardiovascular and cerebrovascular diseases, tumors, and nerve injury. Diabetes is a metabolic disorder characterized by long-term hyperglycemia, insulin resistance, and chronic inflammation. The people have paid more and more attention to the relationship between pyroptosis, diabetes, and its complications, especially its important regulatory significance in diabetic neurological diseases, such as diabetic encephalopathy (DE) and diabetic peripheral neuropathy (DPN). This article will give an in-depth overview of the relationship between pyroptosis, diabetes, and its related neuropathy, and discuss the regulatory pathway and significance of pyroptosis in diabetes-associated neuropathy.

At the Intersection of Major and Minor Spliceosomes: Crosstalk Mechanisms and Their Impact on Gene Expression

Many eukaryotic species contain two separate molecular machineries for removing non-coding intron sequences from pre-mRNA molecules. The majority of introns (more than 99.5% in humans) are recognized and excised by the major spliceosome, which utilizes relatively poorly conserved sequence elements at the 5′ and 3′ ends of the intron that are used for intron recognition and in subsequent catalysis. In contrast, the minor spliceosome targets a rare group of introns (approximately 0.5% in humans) with highly conserved sequences at the 5′ and 3′ ends of the intron. Minor introns coexist in the same genes with major introns and while the two intron types are spliced by separate spliceosomes, the two splicing machineries can interact with one another to shape mRNA processing events in genes containing minor introns. Here, we review known cooperative and competitive interactions between the two spliceosomes and discuss the mechanistic basis of the spliceosome crosstalk, its regulatory significance, and impact on spliceosome diseases.

Polymorphic locus rs1061624 of the ТNFR2 gene is associated with the development of arterial hypertension in males

Aim To study the involvement of cytokine polymorphous loci in development of arterial hypertension (AH) in men from the Central Black Earth region of Russia.Materials and methods 821 men were evaluated, including 564 patients with AH and 257 individuals of the control group. Analysis of 8 cytokine mononucleotide polymorphisms (MNP) was performed using the real-time polymerase chain reaction with TagMan probes. Statistical analysis was performed with the STATISTICA (v.10.0) and PLINK (v.1.06) software. The regulatory potential of MNP was analyzed with the HaploReg (v.4.1) service (http://archive.broadinstitute.org).Results The rs1061624 ТNFR2 polymorphous locus was associated with development of AH in men in recessive (odd ratio (OR), 0.33; 95 % confidence interval (CI): 0.18–0.61, рperm=0.0004) and additive (OR, 0.50, 95 % CI: 0.34–0.74, рperm=0.0006) genetic models and exerted a protective effect in development of AH. The rs1061624 MNP of the ТNFR2 gene has a regulatory significance; it is located in the DNA sites hypersensitive to the action of DNAase 1 and in binding sites for transcriptional factors and histones that mark enhancers and promoters in different organs and tissues.Conclusion The rs1061624 ТNFR2 gene polymorphism is involved in the development of AH in men of the Central Black Earth region of Russia.

H2S and reactive sulfur signaling at the host-bacterial pathogen interface

Bacterial pathogens that cause invasive disease in the vertebrate host must adapt to host efforts to cripple their viability. Major host insults are reactive oxygen and reactive nitrogen species as well as cellular stress induced by antibiotics. Hydrogen sulfide (H2S) is emerging as an important player in cytoprotection against these stressors, which may well be attributed to downstream more oxidized sulfur species termed reactive sulfur species (RSS). In this review, we summarize recent work that suggests that H2S/RSS impacts bacterial survival in infected cells and animals. We discuss the mechanisms of biogenesis and clearance of RSS in the context of a bacterial H2S/RSS homeostasis model and the bacterial transcriptional regulatory proteins that act as “sensors” of cellular RSS that maintain H2S/RSS homeostasis. In addition, we cover fluorescence imaging– and MS–based approaches used to detect and quantify RSS in bacterial cells. Last, we discuss proteome persulfidation (S-sulfuration) as a potential mediator of H2S/RSS signaling in bacteria in the context of the writer-reader-eraser paradigm, and progress toward ascribing regulatory significance to this widespread post-translational modification.

Nascent transcript folding plays a major role in determining RNA polymerase elongation rates

Transcription elongation rates are important for RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S.cerevisiae. Analysis of Miller chromatin spreads and mapping RNAPI using UV crosslinking, revealed a marked 5' bias and strikingly uneven local polymerase occupancy, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution; folding energy and G+C content. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high G+C within the transcription bubble slows elongation. We developed a mathematical model for RNAPI elongation, which confirmed the importance of nascent RNA folding in transcription. RNAPI from S.pombe was similarly sensitive to transcript folding, as were S.cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage indicating regulatory significance for transcript folding.

Withdrawal Symptoms From E-Cigarette Abstinence Among Adult Never-Smokers: A Pilot Experimental Study

Introduction Use of e-cigarettes among never-smokers has substantially increased; yet there are few descriptions of the consequences of such use. We assessed whether adult never-smokers can have withdrawal from cessation of e-cigarettes. Methods In an un-blinded pre-post clinical trial, 30 never-smoker daily e-cigarette users used their own nicotine-containing e-cigarette for 7 days followed by 6 days of biologically confirmed abstinence. Participants monitored symptoms of nicotine withdrawal nightly via an Interactive Voice Response system. They attended three lab visits/week to provide expired carbon monoxide and urine samples to determine compliance. Findings Abstinence increased all the DSM5 symptoms of tobacco withdrawal and this occurred in the majority of participants. The increase in severity of withdrawal was small and rarely impaired functioning. Conclusions Our finding suggests that withdrawal symptoms can occur in never-smokers who stop e-cigarettes abruptly. However, the severity of withdrawal appears to be small and may not be of clinical or regulatory significance. Although our sample size was small and thus replication tests of our results are indicated, it may be prudent to warn never-smokers that withdrawal symptoms may occur. Implications This study indicates that withdrawal symptoms can occur in never-smokers who are daily e-cigarette users. However, the severity of withdrawal from e-cigarette abstinence in never-smokers appears to be small and may not be of clinical or regulatory significance. Given our small sample size, replication of our results is warranted. Nevertheless, it might be prudent to warn never-smokers of addiction to e-cigarettes. Clinical Trial Registration = NCT02825459

Generics and Biosimilars

The generic pharmaceutical industry is arguably the most important player in the quest for affordable healthcare, especially in the United States. Since the inception of the industry following the enactment of the Drug Price Competition and Patent Term Restoration Act in 1984, generics have grown by leaps and bounds, and by 2015 they accounted for almost 90 percent of prescriptions filled in the United States. This chapter describes the history, industrial organization, regulatory approval process, price competition, and strategic behavior in this vitally important sector. It also provides an in-depth look at the issue of drug shortages, which affect older, generic products the most. The last part of the chapter examines the rising financial and regulatory significance of biosimilar drugs, which are analogous to generics for biological drugs, in the United States and Europe.

Long-read sequencing of nascent RNA reveals coupling among RNA processing events

Pre-mRNA splicing is accomplished by the spliceosome, a megadalton complex that assembles de novo on each intron. Because spliceosome assembly and catalysis occur co-transcriptionally, we hypothesized that introns are removed in the order of their transcription in genomes dominated by constitutive splicing. Remarkably little is known about splicing order and the regulatory potential of nascent transcript remodeling by splicing, due to the limitations of existing methods that focus on analysis of mature splicing products (mRNAs) rather than substrates and intermediates. Here, we overcome this obstacle through long-read RNA sequencing of nascent, multi-intron transcripts in the fission yeast Schizosaccharomyces pombe. Most multi-intron transcripts were fully spliced, consistent with rapid co-transcriptional splicing. However, an unexpectedly high proportion of transcripts were either fully spliced or fully unspliced, suggesting that splicing of any given intron is dependent on the splicing status of other introns in the transcript. Supporting this, mild inhibition of splicing by a temperature-sensitive mutation in Prp2, the homolog of vertebrate U2AF65, increased the frequency of fully unspliced transcripts. Importantly, fully unspliced transcripts displayed transcriptional read-through at the polyA site and were degraded co-transcriptionally by the nuclear exosome. Finally, we show that cellular mRNA levels were reduced in genes with a high number of unspliced nascent transcripts during caffeine treatment, showing regulatory significance of co-transcriptional splicing. Therefore, overall splicing of individual nascent transcripts, 3’ end formation, and mRNA half-life depend on the splicing status of neighboring introns, suggesting crosstalk among spliceosomes and the polyA cleavage machinery during transcription elongation.