Single-Cell Transcriptomes Reveal a Complex Cellular Landscape in the Middle Ear and Differential Capacities for Acute Response to Infection.

Single-cell transcriptomics was used to profile cells of the normal murine middle ear. Clustering analysis of 6770 transcriptomes identified 17 cell clusters corresponding to distinct cell types: five epithelial, three stromal, three lymphocyte, two monocyte, two endothelial, one pericyte and one melanocyte cluster. Within some clusters, cell subtypes were identified. While many corresponded to those cell types known from prior studies, several novel types or subtypes were noted. The results indicate unexpected cellular diversity within the resting middle ear mucosa. The resolution of uncomplicated, acute, otitis media is too rapid for cognate immunity to play a major role. Thus innate immunity is likely responsible for normal recovery from middle ear infection. The need for rapid response to pathogens suggests that innate immune genes may be constitutively expressed by middle ear cells. We therefore assessed expression of innate immune genes across all cell types, to evaluate potential for rapid responses to middle ear infection. Resident monocytes/macrophages expressed the most such genes, including pathogen receptors, cytokines, chemokines and chemokine receptors. Other cell types displayed distinct innate immune gene profiles. Epithelial cells preferentially expressed pathogen receptors, bactericidal peptides and mucins. Stromal and endothelial cells expressed pathogen receptors. Pericytes expressed pro-inflammatory cytokines. Lymphocytes expressed chemokine receptors and antimicrobials. The results suggest that tissue monocytes, including macrophages, are the master regulators of the immediate middle ear response to infection, but that virtually all cell types act in concert to mount a defense against pathogens

FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy.

Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-β-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and β-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance

Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples.

Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens. IMPORTANCE: Malaria is a disease caused by Plasmodium parasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivax is the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodium parasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivax is challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivax genomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS

A new era for space life science: international standards for space omics processing

10 p.-2 fig.Space agencies have announced plans for human missions to the Moon to prepare for Mars. However, the space environment presents stressors that include radiation, microgravity, and isolation. Understanding how these factors affect biology is crucial for safe and effective crewed space exploration. There is a need to develop countermeasures, to adapt plants and microbes for nutrient sources and bioregenerative life support, and to limit pathogen infection. Scientists across the world are conducting space omics experiments on model organisms and, more recently, on humans. Optimal extraction of actionable scientific discoveries from these precious datasets will only occur at the collective level with improved standardization. To address this shortcoming, we established ISSOP (International Standards for Space Omics Processing), an international consortium of scientists who aim to enhance standard guidelines between space biologists at a global level. Here we introduce our consortium and share past lessons learned and future challenges related to spaceflight omics.European (D.B., H.C., N.J.S., R.H., and S. Giacomello) contribution is supported by ESA Topical Team “Space Omics: Towards an integrated ESA/NASA –omics database for spaceflight and ground facilities experiments” grant 4000131202/20/NL/PG/pt to R.H. S. Giacomello is supported by Formas grant 2017-01066_3. H.C. is supported by the Horizon Centre for Doctoral Training at the University of Nottingham (UKRI grant no. EP/S023305/1) and by the NASA GeneLab Animal Analysis Working Group. N.J.S. is supported by the National Aeronautics and Space Administration (NNX15AL16G). NASA GeneLab members (J.M.G., S.V.C., S.S.R., L.D., S. Gebre) are supported by the NASA Space Biology program within the NASA Science Mission Directorate's (SMD) Biological and Physical Sciences (BPS) Division. R.B. and S. Gilroy are supported by NASA (80NSSC19K0132). L.R. and M.M. represent the Omics Subgroup of Japan Society for the Promotion of Science (JSPS) KAKENHI funding group Living in Space and are supported by JP15K21745, JP15H05940, and JP20H03234. L.R. is supported by JSPS postdoctoral fellowship P20382. D.T. is supported by the Department of Biomedical and Health Informatics and The Children’s Hospital of Philadelphia Research Institute. K.F. is supported by the UC San Diego Department of Medicine and National Institutes of Health, grant UL1TR001442 of CTSA (Clinical and Translational Science Awards). C.E.M. is funded from the WorldQuant Foundation, The Pershing Square Sohn Cancer Research Alliance, and the National Institutes of Health (R01MH117406).Peer reviewe

Activation of NF-κB and p300/CBP potentiates cancer chemoimmunotherapy through induction of MHC-I antigen presentation

Many cancers evade immune rejection by suppressing major histocompatibility class I (MHC-I) antigen processing and presentation (AgPP). Such cancers do not respond to immune checkpoint inhibitor therapies (ICIT) such as PD-1/PD-L1 [PD-(L)1] blockade. Certain chemotherapeutic drugs augment tumor control by PD-(L)1 inhibitors through potentiation of T-cell priming but whether and how chemotherapy enhances MHC-I-dependent cancer cell recognition by cytotoxic T cells (CTLs) is not entirely clear. We now show that the lysine acetyl transferases p300/CREB binding protein (CBP) control MHC-I AgPPM expression and neoantigen amounts in human cancers. Moreover, we found that two distinct DNA damaging drugs, the platinoid oxaliplatin and the topoisomerase inhibitor mitoxantrone, strongly up-regulate MHC-I AgPP in a manner dependent on activation of nuclear factor kappa B (NF-κB), p300/CBP, and other transcription factors, but independently of autocrine IFNγ signaling. Accordingly, NF-κB and p300 ablations prevent chemotherapy-induced MHC-I AgPP and abrogate rejection of low MHC-I-expressing tumors by reinvigorated CD8+ CTLs. Drugs like oxaliplatin and mitoxantrone may be used to overcome resistance to PD-(L)1 inhibitors in tumors that had "epigenetically down-regulated," but had not permanently lost MHC-I AgPP activity

Novel Small-Molecule Inhibitors of Hepatitis C Virus Entry Block Viral Spread and Promote Viral Clearance in Cell Culture

Combinations of direct-acting anti-virals offer the potential to improve the efficacy, tolerability and duration of the current treatment regimen for hepatitis C virus (HCV) infection. Viral entry represents a distinct therapeutic target that has been validated clinically for a number of pathogenic viruses. To discover novel inhibitors of HCV entry, we conducted a high throughput screen of a proprietary small-molecule compound library using HCV pseudoviral particle (HCVpp) technology. We independently discovered and optimized a series of 1,3,5-triazine compounds that are potent, selective and non-cytotoxic inhibitors of HCV entry. Representative compounds fully suppress both cell-free virus and cell-to-cell spread of HCV in vitro. We demonstrate, for the first time, that long term treatment of an HCV cell culture with a potent entry inhibitor promotes sustained viral clearance in vitro. We have confirmed that a single amino acid variant, V719G, in the transmembrane domain of E2 is sufficient to confer resistance to multiple compounds from the triazine series. Resistance studies were extended by evaluating both the fusogenic properties and growth kinetics of drug-induced and natural amino acid variants in the HCVpp and HCV cell culture assays. Our results indicate that amino acid variations at position 719 incur a significant fitness penalty. Introduction of I719 into a genotype 1b envelope sequence did not affect HCV entry; however, the overall level of HCV replication was reduced compared to the parental genotype 1b/2a HCV strain. Consistent with these findings, I719 represents a significant fraction of the naturally occurring genotype 1b sequences. Importantly, I719, the most relevant natural polymorphism, did not significantly alter the susceptibility of HCV to the triazine compounds. The preclinical properties of these triazine compounds support further investigation of entry inhibitors as a potential novel therapy for HCV infection

Focal adhesion is associated with lithium response in bipolar disorder: evidence from a network-based multi-omics analysis

Lithium (Li) is one of the most effective drugs for treating bipolar disorder (BD), however, there is presently no way to predict response to guide treatment. The aim of this study is to identify functional genes and pathways that distinguish BD Li responders (LR) from BD Li non-responders (NR). An initial Pharmacogenomics of Bipolar Disorder study (PGBD) GWAS of lithium response did not provide any significant results. As a result, we then employed network-based integrative analysis of transcriptomic and genomic data. In transcriptomic study of iPSC-derived neurons, 41 significantly differentially expressed (DE) genes were identified in LR vs NR regardless of lithium exposure. In the PGBD, post-GWAS gene prioritization using the GWA-boosting (GWAB) approach identified 1119 candidate genes. Following DE-derived network propagation, there was a highly significant overlap of genes between the top 500- and top 2000-proximal gene networks and the GWAB gene list (Phypergeometric = 1.28E–09 and 4.10E–18, respectively). Functional enrichment analyses of the top 500 proximal network genes identified focal adhesion and the extracellular matrix (ECM) as the most significant functions. Our findings suggest that the difference between LR and NR was a much greater effect than that of lithium. The direct impact of dysregulation of focal adhesion on axon guidance and neuronal circuits could underpin mechanisms of response to lithium, as well as underlying BD. It also highlights the power of integrative multi-omics analysis of transcriptomic and genomic profiling to gain molecular insights into lithium response in BD.publishedVersio

Genetic Variation in the Platelet Endothelial Aggregation Receptor 1 Gene Results in Endothelial Dysfunction

We gratefully acknowledge our Amish liaisons and field workers and the extraordinary cooperation and support of the Amish community, without which these studies would not have been possible. We also acknowledge Dr. Alan Shuldiner for his impactful insights and guidance.Platelet Endothelial Aggregation Receptor 1 (PEAR1) is a newly identified membrane protein reported to be involved in multiple vascular and thrombotic processes. While most studies to date have focused on the effects of this receptor in platelets, PEAR1 is located in multiple tissues including the endothelium, where it is most highly expressed. Our first objective was to evaluate the role of PEAR1 in endothelial function by examining flow-mediated dilation of the brachial artery in 641 participants from the Heredity and Phenotype Intervention Heart Study. Our second objective was to further define the impact of PEAR1 on cardiovascular disease computationally through meta-analysis of 75,000 microarrays, yielding insights regarding PEAR1 function, and predictions of phenotypes and diseases affected by PEAR1 dysregulation. Based on the results of this meta-analysis we examined whether genetic variation in PEAR1 influences endothelial function using an ex vivo assay of endothelial cell migration. We observed a significant association between rs12041331 and flow-mediated dilation in participants of the Heredity and Phenotype Intervention Heart Study (P = 0.02). Meta-analysis results revealed that PEAR1 expression is highly correlated with several genes (e.g. ANG2, ACVRL1, ENG) and phenotypes (e.g. endothelial cell migration, angiogenesis) that are integral to endothelial function. Functional validation of these results revealed that PEAR1 rs12041331 is significantly associated with endothelial migration (P = 0.04). Our results suggest for the first time that genetic variation of PEAR1 is a significant determinant of endothelial function through pathways implicated in cardiovascular disease.Yeshttp://www.plosone.org/static/editorial#pee

Characterization of 24 Microsatellite Loci in Delta Smelt, \u3ci\u3eHypomesus transpacificus\u3c/i\u3e, and Their Cross-Species Amplification in Two Other Smelt Species of the Osmeridae Family

We characterized 24 polymorphic tetranucleotide microsatellite loci for delta smelt (Hypomesus transpacificus) endemic to the San Francisco Bay Estuary, California, USA. Screening of samples (n = 30) yielded two to 26 alleles per locus with observed levels of heterozygosity ranging from 0.17 to 1.0. Only one locus deviated from Hardy–Weinberg equilibrium, suggesting these individuals originate from a single panmictic population. Linkage disequilibrium was found in two pairs of loci after excluding the locus out of Hardy–Weinberg equilibrium. Twenty-two primer pairs cross-amplified in wakasagi smelt (Hypomesus nipponensis), and 15 primer pairs cross-amplified in longfin smelt (Spirinchus thaleichthys)

Increased Fibrogenic Gene Expression in Multifidus Muscles of Patients With Chronic Versus Acute Lumbar Spine Pathology.

Study designProspective observational study-basic science (Level 1).ObjectiveThe aim of this study was to compare expression of functional groups of genes within the atrophic, myogenic, fibrogenic, adipogenic, and inflammatory pathways between paraspinal muscle biopsies from individuals with acute and chronic lumbar spine pathology.Summary of background dataLow back pain is a complex and multifactorial condition that affects a majority of the general population annually. Changes in muscle tissue composition (i.e., fatty and fibrotic infiltration) are a common feature in individuals with lumbar spine pathology associated with low back pain, which often results in functional loss. Understanding the molecular underpinnings of these degenerative changes in different phases of disease progression may improve disease prevention and treatment specificity.MethodsIntraoperative biopsies of the multifidus muscle were obtained from individuals undergoing surgery for acute (<6-month duration) or chronic (>6-month duration) lumbar spine pathology. Expression of 42 genes related to myogenesis, atrophy, adipogenesis, metabolism, inflammation, and fibrosis were measured in 33 samples (eight acute, 25 chronic) using qPCR, and tissue composition of fat, muscle, and fibrosis was quantified using histology.ResultsWe found that tissue composition of the biopsies was heterogeneous, resulting in a trend toward lower RNA yields in biopsies with higher proportions of fat (r <-0.39, P < 0.1). There were no significant differences in gene expression patterns for atrophy (P > 0.635), adipogenesis (P > 0.317), myogenesis (P > 0.320), or inflammatory (P > 0.413) genes after adjusting for the proportion of muscle, fat, and connective tissue. However, in the fibrogenesis pathway, we found significant upregulation of CTGF (P = 0.046), and trends for upregulation of COL1A1 (P = 0.061), and downregulation of MMP1 and MMP9 (P = 0.061) in the chronic group.ConclusionThere is increased fibrogenic gene expression in individuals with chronic disease when compared to acute disease, without significant differences in atrophic, myogenic, adipogenic, or inflammatory pathways, suggesting increased efforts should be made to prevent or reverse fibrogenesis to improve patient function in this population.Level of evidenceN/A