Comparative transcriptomics of multidrug-resistant Acinetobacter baumannii in response to antibiotic treatments

Abstract Multidrug-resistant Acinetobacter baumannii, a major hospital-acquired pathogen, is a serious health threat and poses a great challenge to healthcare providers. Although there have been many genomic studies on the evolution and antibiotic resistance of this species, there have been very limited transcriptome studies on its responses to antibiotics. We conducted a comparative transcriptomic study on 12 strains with different growth rates and antibiotic resistance profiles, including 3 fast-growing pan-drug-resistant strains, under separate treatment with 3 antibiotics, namely amikacin, imipenem, and meropenem. We performed deep sequencing using a strand-specific RNA-sequencing protocol, and used de novo transcriptome assembly to analyze gene expression in the form of polycistronic transcripts. Our results indicated that genes associated with transposable elements generally showed higher levels of expression under antibiotic-treated conditions, and many of these transposon-associated genes have previously been linked to drug resistance. Using co-expressed transposon genes as markers, we further identified and experimentally validated two novel genes of which overexpression conferred significant increases in amikacin resistance. To the best of our knowledge, this study represents the first comparative transcriptomic analysis of multidrug-resistant A. baumannii under different antibiotic treatments, and revealed a new relationship between transposons and antibiotic resistance

A targeted gene panel that covers coding, non-coding and short tandem repeat regions improves the diagnosis of patients with neurodegenerative diseases

Genetic testing for neurodegenerative diseases (NDs) is highly challenging because of genetic heterogeneity and overlapping manifestations. Targeted-gene panels (TGPs), coupled with next-generation sequencing (NGS), can facilitate the profiling of a large repertoire of ND-related genes. Due to the technical limitations inherent in NGS and TGPs, short tandem repeat (STR) variations are often ignored. However, STR expansions are known to cause such NDs as Huntington\u27s disease and spinocerebellar ataxias type 3 (SCA3). Here, we studied the clinical utility of a custom-made TGP that targets 199 NDs and 311 ND-associated genes on 118 undiagnosed patients. At least one known or likely pathogenic variation was found in 54 patients; 27 patients demonstrated clinical profiles that matched the variants; and 16 patients whose original diagnosis were refined. A high concordance of variant calling were observed when comparing the results from TGP and whole-exome sequencing of four patients. Our in-house STR detection algorithm has reached a specificity of 0.88 and a sensitivity of 0.82 in our SCA3 cohort. This study also uncovered a trove of novel and recurrent variants that may enrich the repertoire of ND-related genetic markers. We propose that a combined comprehensive TGPs-bioinformatics pipeline can improve the clinical diagnosis of NDs

Macrophage depletion blocks congenital SARM1-dependent neuropathy

Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified 2 rare NMNAT2 missense variants in 2 brothers afflicted with a progressive neuropathy syndrome. The polymorphisms resulted in amino acid substitutions V98M and R232Q, which reduced NMNAT2 NAD+-synthetase activity. We generated a mouse model to mirror the human syndrome and found that Nmnat2V98M/R232Q compound-heterozygous CRISPR mice survived to adulthood but developed progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes were all SARM1-dependent. Remarkably, macrophage depletion therapy blocked and reversed neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induced inflammatory neuropathy and highlight the potential of immune therapy as a treatment for this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation

The draft genome, transcriptome, and microbiome of Dermatophagoides farinae reveal a broad spectrum of dust mite allergens

© 2014 The Authors. Published by Elsevier Inc. Background A sequenced house dust mite (HDM) genome would advance our understanding of HDM allergens, a common cause of human allergies. Objective We sought to produce an annotated Dermatophagoides farinae draft genome and develop a combined genomic-transcriptomic-proteomic approach for elucidation of HDM allergens. Methods A D farinae draft genome and transcriptome were assembled with high-throughput sequencing, accommodating microbiome sequences. The allergen gene structures were validated by means of Sanger sequencing. The mite's microbiome composition was determined, and the predominant genus was validated immunohistochemically. The allergenicity of a ubiquinol-cytochrome c reductase binding protein homologue was evaluated with immunoblotting, immunosorbent assays, and skin prick tests. Results The full gene structures of 20 canonical allergens and 7 noncanonical allergen homologues were produced. A novel major allergen, ubiquinol-cytochrome c reductase binding protein-like protein, was found and designated Der f 24. All 40 sera samples from patients with mite allergy had IgE antibodies against rDer f 24. Of 10 patients tested, 5 had positive skin reactions. The predominant bacterial genus among 100 identified species was Enterobacter (63.4%). An intron was found in the 13.8-kDa D farinae bacteriolytic enzyme gene, indicating that it is of HDM origin. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed a phototransduction pathway in D farinae, as well as thiamine and amino acid synthesis pathways, which is suggestive of an endosymbiotic relationship between D farinae and its microbiome. Conclusion An HDM genome draft produced from genomic, transcriptomic, and proteomic experiments revealed allergen genes and a diverse endosymbiotic microbiome, providing a tool for further identification and characterization of HDM allergens and development of diagnostics and immunotherapeutic vaccines.Link_to_subscribed_fulltex

Disentangling Glial Diversity in Peripheral Nerves at Single Nuclei Resolution

The ability to discern gene expression at single cell level is revolutionizing our understanding of both basic biology and human health. Peripheral nerves are essential communicators between the outside world and the CNS, as evidenced by the devastating effects of diseases that disrupt them, such as ALS, Charcot-Marie-Tooth Syndrome and diabetic neuropathy. Understanding peripheral nerve dysfunction at a mechanistic level is of considerable interest due to the increasing prevalence and associated patient care costs of these disorders. Although most research of the peripheral nerve has focused on glial-axonal interactions, the important contributions of other cell types besides Schwann cells, such as fibroblasts and immune cells, are increasingly appreciated. This more comprehensive focus on the cellular components of peripheral nerve stimulated a number of studies using single cell sequencing approaches for cell characterization. While interesting information has been gleaned from these efforts, they all largely failed to identify myelinating Schwann cells in their samples due to extreme capture bias secondary to their complex morphology and the close apposition of these glial cells to the axon. For my dissertation, I set out to generate a comprehensive single nuclei atlas of peripheral nerves with the hope to unbiasedly characterize the glial population and to understand how glia contributes to the homeostasis of peripheral nerves. My work began with the development of a fluorescent-activated cell sorting (FACS)-based nuclei isolation approach to avoid issues with capture bias, and to allow an examination of the potential diversity of peripheral glia and other cell types across multiple types of peripheral nerves. Our atlases highlight over 20 cell types in mouse sciatic, peroneal, sural and vagus nerves and show that Schwann cell, as the principal glia, are consistently the predominant cell type across all nerves. In-depth analysis of the immune populations at single cell level has revealed two PNS macrophage populations with unique microglia signatures. I next performed extensive transcriptional characterization of PNS macrophages and showed that, through comparative analytics with CNS diseased microglia data, PNS macrophages constitutively expressed gene previously identified to be upregulated by activated microglia during aging, neurodegeneration, or loss of Sall1. Interestingly, myelinating Schwann cell is the source of IL-34 which is required for the maintenance of PNS macrophages in peripheral nerves. Finally, through the peripheral nerve atlases, I identified multiple Schwann cell sub-populations, including two non-myelinating and four myelinating subtypes. Specifically, a distinct myelinating Schwann cell subtype that expresses Cldn14, Adamtsl1 and Pmp2 exhibits unique transcriptional signature with high NAD and pyruvate metabolisms, and preferentially ensheath large motor axons that innervate fast twitch muscle fiber. The number of these motor-associated, Pmp2+ SCs is significantly reduced in motor neuropathy models and human ALS nerve samples. Collectively, these findings reveal the diversity of SCs and other cell types in peripheral nerve and serve as a reference for future studies of nerve biology and disease

Single-Cell RNA-Seq Uncovers a Robust Transcriptional Response to Morphine by Glia

Summary: Molecular and behavioral responses to opioids are thought to be primarily mediated by neurons, although there is accumulating evidence that other cell types play a prominent role in drug addiction. To investigate cell-type-specific opioid responses, we performed single-cell RNA sequencing (scRNA-seq) of the nucleus accumbens of mice following acute morphine treatment. Differential expression analysis uncovered unique morphine-dependent transcriptional responses by oligodendrocytes and astrocytes. We examined the expression of selected genes, including Cdkn1a and Sgk1, by FISH, confirming their induction by morphine in oligodendrocytes. Further analysis using RNA-seq of FACS-purified oligodendrocytes revealed a large cohort of morphine-regulated genes. The affected genes are enriched for roles in cellular pathways intimately linked to oligodendrocyte maturation and myelination, including the unfolded protein response. Altogether, our data illuminate the morphine-dependent transcriptional response by oligodendrocytes and offer mechanistic insights into myelination defects associated with opioid abuse. : Avey et al. use single-cell RNA-seq to uncover cell-type-specific responses to morphine in the brains of mice, revealing a unique and robust response by oligodendrocytes, which they further validate by multiple approaches. These analyses offer insights into the molecular mechanisms underlying oligodendrocyte dysfunction in the context of opioid abuse. Keywords: opioid, morphine, addiction, single-cell, RNA-seq, oligodendrocyte, UPR, glucocorticoid, nucleus accumbens, myeli