Simultaneous detection of pathogens and antimicrobial resistance genes with the open source, cloud-based, CZ ID pipeline.

Antimicrobial resistant (AMR) pathogens represent urgent threats to human health, and their surveillance is of paramount importance. Metagenomic next generation sequencing (mNGS) has revolutionized such efforts, but remains challenging due to the lack of open-access bioinformatics tools capable of simultaneously analyzing both microbial and AMR gene sequences. To address this need, we developed the CZ ID AMR module, an open-access, cloud-based workflow designed to integrate detection of both microbes and AMR genes in mNGS and whole-genome sequencing (WGS) data. It leverages the Comprehensive Antibiotic Resistance Database and associated Resistance Gene Identifier software, and works synergistically with the CZ ID short-read mNGS module to enable broad detection of both microbes and AMR genes. We highlight diverse applications of the AMR module through analysis of both publicly available and newly generated mNGS and WGS data from four clinical cohort studies and an environmental surveillance project. Through genomic investigations of bacterial sepsis and pneumonia cases, hospital outbreaks, and wastewater surveillance data, we gain a deeper understanding of infectious agents and their resistomes, highlighting the value of integrating microbial identification and AMR profiling for both research and public health. We leverage additional functionalities of the CZ ID mNGS platform to couple resistome profiling with the assessment of phylogenetic relationships between nosocomial pathogens, and further demonstrate the potential to capture the longitudinal dynamics of pathogen and AMR genes in hospital acquired bacterial infections. In sum, the new AMR module advances the capabilities of the open-access CZ ID microbial bioinformatics platform by integrating pathogen detection and AMR profiling from mNGS and WGS data. Its development represents a critical step toward democratizing pathogen genomic analysis and supporting collaborative efforts to combat the growing threat of AMR.

Simultaneous detection of pathogens and antimicrobial resistance genes with the open source, cloud-based, CZ ID pipeline.

Antimicrobial resistant (AMR) pathogens represent urgent threats to human health, and their surveillance is of paramount importance. Metagenomic next generation sequencing (mNGS) has revolutionized such efforts, but remains challenging due to the lack of open-access bioinformatics tools capable of simultaneously analyzing both microbial and AMR gene sequences. To address this need, we developed the Chan Zuckerberg ID (CZ ID) AMR module, an open-access, cloud-based workflow designed to integrate detection of both microbes and AMR genes in mNGS and whole-genome sequencing (WGS) data. It leverages the Comprehensive Antibiotic Resistance Database and associated Resistance Gene Identifier software, and works synergistically with the CZ ID short-read mNGS module to enable broad detection of both microbes and AMR genes. We highlight diverse applications of the AMR module through analysis of both publicly available and newly generated mNGS and WGS data from four clinical cohort studies and an environmental surveillance project. Through genomic investigations of bacterial sepsis and pneumonia cases, hospital outbreaks, and wastewater surveillance data, we gain a deeper understanding of infectious agents and their resistomes, highlighting the value of integrating microbial identification and AMR profiling for both research and public health. We leverage additional functionalities of the CZ ID mNGS platform to couple resistome profiling with the assessment of phylogenetic relationships between nosocomial pathogens, and further demonstrate the potential to capture the longitudinal dynamics of pathogen and AMR genes in hospital acquired bacterial infections. In sum, the new AMR module advances the capabilities of the open-access CZ ID microbial bioinformatics platform by integrating pathogen detection and AMR profiling from mNGS and WGS data. Its development represents a critical step toward democratizing pathogen genomic analysis and supporting collaborative efforts to combat the growing threat of AMR.

Recent Research Trends in Neuroinflammatory and Neurodegenerative Disorders.

Neuroinflammatory and neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), traumatic brain injury (TBI) and Amyotrophic lateral sclerosis (ALS) are chronic major health disorders. The exact mechanism of the neuroimmune dysfunctions of these disease pathogeneses is currently not clearly understood. These disorders show dysregulated neuroimmune and inflammatory responses, including activation of neurons, glial cells, and neurovascular unit damage associated with excessive release of proinflammatory cytokines, chemokines, neurotoxic mediators, and infiltration of peripheral immune cells into the brain, as well as entry of inflammatory mediators through damaged neurovascular endothelial cells, blood-brain barrier and tight junction proteins. Activation of glial cells and immune cells leads to the release of many inflammatory and neurotoxic molecules that cause neuroinflammation and neurodegeneration. Gulf War Illness (GWI) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) are chronic disorders that are also associated with neuroimmune dysfunctions. Currently, there are no effective disease-modifying therapeutic options available for these diseases. Human induced pluripotent stem cell (iPSC)-derived neurons, astrocytes, microglia, endothelial cells and pericytes are currently used for many disease models for drug discovery. This review highlights certain recent trends in neuroinflammatory responses and iPSC-derived brain cell applications in neuroinflammatory disorders.

Molecular Phenotypes of Acute Respiratory Distress Syndrome in the ROSE Trial Have Differential Outcomes and Gene Expression Patterns That Differ at Baseline and Longitudinally over Time.

Rationale: Two molecular phenotypes have been identified in acute respiratory distress syndrome (ARDS). In the ROSE (Reevaluation of Systemic Early Neuromuscular Blockade) trial of cisatracurium in moderate to severe ARDS, we addressed three unanswered questions: 1) Do the same phenotypes emerge in a more severe ARDS cohort with earlier recruitment; 2) Do phenotypes respond differently to neuromuscular blockade? and 3) What biological pathways most differentiate inflammatory phenotypes?Methods: We performed latent class analysis in ROSE using preenrollment clinical and protein biomarkers. In a subset of patients (n = 134), we sequenced whole-blood RNA using enrollment and Day 2 samples and performed differential gene expression and pathway analyses. Informed by the differential gene expression analysis, we measured additional plasma proteins and evaluated their abundance relative to gene expression amounts.Measurements and Main Results: In ROSE, we identified the hypoinflammatory (60.4%) and hyperinflammatory (39.6%) phenotypes with similar biological and clinical characteristics as prior studies, including higher mortality at Day 90 for the hyperinflammatory phenotype (30.3% vs. 61.6%; P < 0.0001). We observed no treatment interaction between the phenotypes and randomized groups for mortality. The hyperinflammatory phenotype was enriched for genes associated with innate immune response, tissue remodeling, and zinc metabolism at Day 0 and collagen synthesis and neutrophil degranulation at Day 2. Longitudinal changes in gene expression patterns differed dependent on survivorship. For most highly expressed genes, we observed correlations with their corresponding plasma proteins' abundance. However, for the class-defining plasma proteins in the latent class analysis, no correlation was observed with their corresponding genes' expression.Conclusions: The hyperinflammatory and hypoinflammatory phenotypes have different clinical, protein, and dynamic transcriptional characteristics. These findings support the clinical and biological potential of molecular phenotypes to advance precision care in ARDS.

In host evolution of beta lactam resistance during active treatment for Pseudomonas aeruginosa bacteremia.

Multidrug-resistant (MDR) Pseudomonas aeruginosa has been declared a serious threat by the United States Centers for Disease Control and Prevention. Here, we used whole genome sequencing (WGS) to investigate recurrent P. aeruginosa bloodstream infections in a severely immunocompromised patient. The infections demonstrated unusual, progressive increases in resistance to beta lactam antibiotics in the setting of active treatment with appropriate, guideline-directed agents. WGS followed by comparative genomic analysis of isolates collected over 44 days demonstrated in host evolution of a single P. aeruginosa isolate characterized by stepwise acquisition of two de-novo genetic resistance mechanisms over the course of treatment. We found a novel deletion affecting the ampC repressor ampD and neighboring gene ampE, which associated with initial cefepime treatment failure. This was followed by acquisition of a porin nonsense mutation, OprD, associated with resistance to carbapenems. This study highlights the potential for in-host evolution of P. aeruginosa during bloodstream infections in severely immunocompromised patients despite appropriate antimicrobial therapy. In addition, it demonstrates the utility of WGS for understanding unusual resistance patterns in the clinical context.

Whole-genome sequencing rule-out of suspected hospital-onset Rhizopus outbreaks.

Two independent temporal-spatial clusters of hospital-onset Rhizopus infections were evaluated using whole-genome sequencing (WGS). Phylogenetic analysis confirmed that isolates within each cluster were unrelated despite epidemiological suspicion of outbreaks. The ITS1 region alone was insufficient for accurate analysis. WGS has utility for rapid rule-out of suspected nosocomial Rhizopus outbreaks.

Prolonged silent carriage, genomic virulence potential and transmission between staff and patients characterize a neonatal intensive care unit (NICU) outbreak of methicillin-resistant Staphylococcus aureus (MRSA).

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in neonatal intensive care units (NICU) that confers significant morbidity and mortality. OBJECTIVE: Improving our understanding of MRSA transmission dynamics, especially among high-risk patients, is an infection prevention priority. METHODS: We investigated a cluster of clinical MRSA cases in the NICU using a combination of epidemiologic review and whole-genome sequencing (WGS) of isolates from clinical and surveillance cultures obtained from patients and healthcare personnel (HCP). RESULTS: Phylogenetic analysis identified 2 genetically distinct phylogenetic clades and revealed multiple silent-transmission events between HCP and infants. The predominant outbreak strain harbored multiple virulence factors. Epidemiologic investigation and genomic analysis identified a HCP colonized with the dominant MRSA outbreak strain who cared for most NICU patients who were infected or colonized with the same strain, including 1 NICU patient with severe infection 7 months before the described outbreak. These results guided implementation of infection prevention interventions that prevented further transmission events. CONCLUSIONS: Silent transmission of MRSA between HCP and NICU patients likely contributed to a NICU outbreak involving a virulent MRSA strain. WGS enabled data-driven decision making to inform implementation of infection control policies that mitigated the outbreak. Prospective WGS coupled with epidemiologic analysis can be used to detect transmission events and prompt early implementation of control strategies.

A 2-Gene Host Signature for Improved Accuracy of COVID-19 Diagnosis Agnostic to Viral Variants.

The continued emergence of SARS-CoV-2 variants is one of several factors that may cause false-negative viral PCR test results. Such tests are also susceptible to false-positive results due to trace contamination from high viral titer samples. Host immune response markers provide an orthogonal indication of infection that can mitigate these concerns when combined with direct viral detection. Here, we leverage nasopharyngeal swab RNA-seq data from patients with COVID-19, other viral acute respiratory illnesses, and nonviral conditions (n = 318) to develop support vector machine classifiers that rely on a parsimonious 2-gene host signature to diagnose COVID-19. We find that optimal classifiers include an interferon-stimulated gene that is strongly induced in COVID-19 compared with nonviral conditions, such as IFI6, and a second immune-response gene that is more strongly induced in other viral infections, such as GBP5. The IFI6+GBP5 classifier achieves an area under the receiver operating characteristic curve (AUC) greater than 0.9 when evaluated on an independent RNA-seq cohort (n = 553). We further provide proof-of-concept demonstration that the classifier can be implemented in a clinically relevant RT-qPCR assay. Finally, we show that its performance is robust across common SARS-CoV-2 variants and is unaffected by cross-contamination, demonstrating its utility for improved accuracy of COVID-19 diagnostics. IMPORTANCE In this work, we study upper respiratory tract gene expression to develop and validate a 2-gene host-based COVID-19 diagnostic classifier and then demonstrate its implementation in a clinically practical qPCR assay. We find that the host classifier has utility for mitigating false-negative results, for example due to SARS-CoV-2 variants harboring mutations at primer target sites, and for mitigating false-positive viral PCR results due to laboratory cross-contamination. Both types of error carry serious consequences of either unrecognized viral transmission or unnecessary isolation and contact tracing. This work is directly relevant to the ongoing COVID-19 pandemic given the continued emergence of viral variants and the continued challenges of false-positive PCR assays. It also suggests the feasibility of pan-respiratory virus host-based diagnostics that would have value in congregate settings, such as hospitals and nursing homes, where unrecognized respiratory viral transmission is of particular concern.

Extracellular signal-regulated kinase-1 phosphorylates early growth response-1 at serine 26.

Egr-1, an immediate-early gene product and master regulator was originally described as a phosphoprotein following its discovery in the 1980s. However specific residue(s) phosphorylated in Egr-1 remain elusive. Here we phosphorylated recombinant Egr-1 in vitro with ERK1 prior to mass spectrometry, which identified phosphorylation of Ser12 and Ser26 with the latter ∼12 times more abundant than Ser12. Phosphorylation of wild-type recombinant Egr-1 (as compared with Ser26>Ala26 mutant Egr-1) revealed that Ser26 accounts for the majority of phosphorylation of Egr-1 by ERK1. N-FGSFPH(pS)PTMDNYC-C was used as an antigen to generate mouse monoclonal antibodies (pS26 MAb). pS26 MAb recognised ERK1-phosphorylated Egr-1 but not Egr-1 bearing a point mutation at Ser26. pS26 MAb recognised inducible ∼75 kDa and 100 kDa species in nuclear extracts of cells exposed to FGF-2. Peptide blocking revealed both inducible species were phosphosite-specific. Immunoprecipitation of nuclear extracts of cells exposed to FGF-2 with pS26 MAb followed by SDS-PAGE and mass spectrometry identified Egr-1 sequences corresponding to the ∼75 kDa species but not ∼100 kDa species. This study identifies a specific amino acid phosphorylated in endogenous Egr-1.

IL-1beta signals through the EGF receptor and activates Egr-1 through MMP-ADAM.

The immediate-early gene Egr-1 controls the inducible expression of many genes implicated in the pathogenesis of a range of vascular disorders, yet our understanding of the mechanisms controlling the rapid expression of this prototypic zinc finger transcription factor is poor. Here we show that Egr-1 expression induced by IL-1beta is dependent on metalloproteinases (MMP) and a disintegrin and a metalloproteinase (ADAM). Pharmacologic MMP/ADAM inhibitors and siRNA knockdown prevent IL-1beta induction of Egr-1. Further, IL-1beta activates Egr-1 via the epidermal growth factor receptor (EGFR). This is blocked by EGFR tyrosine kinase inhibition and EGFR knockdown. IL-1beta induction of Egr-1 expression is reduced in murine embryonic fibroblasts (mEFs) deficient in ADAM17 despite unbiased expression of EGFR and IL-1RI in ADAM17-deficient and wild-type mEFs. Finally, we show that IL-1beta-inducible wound repair after mechanical injury requires both EGFR and MMP/ADAM. This study reports for the first time that Egr-1 induction by IL-1beta involves EGFR and MMP/ADAM-dependent EGFR phosphorylation.

Genistein inhibits PDGF-stimulated proteoglycan synthesis in vascular smooth muscle without blocking PDGFß receptor phosphorylation.

The signaling pathways that regulate the synthesis and structure of proteoglycans secreted by vascular smooth muscle cells are potential therapeutic targets for preventing lipid deposition in the early stage of atherosclerosis. PDGF stimulates both core protein expression and elongation of glycosaminoglycan (GAG) chains on proteoglycans. In this study we investigated the effects of the tyrosine kinase inhibitor genistein on PDGF mediated receptor phosphorylation and proteoglycan synthesis in human vascular smooth muscle cells. We demonstrate that genistein does not block phosphorylation of the activation site of the PDGF receptor at Tyr(857) and two other downstream sites Tyr(751) and Tyr(1021). Genistein blocked PDGF-mediated proteoglycan core protein synthesis however it had no effect on GAG chain elongation. These results differ markedly to two other tyrosine kinase inhibitors, imatinib and Ki11502, that block PDGF receptor phosphorylation and PDGF mediated GAG elongation. We conclude that the action of genistein on core protein synthesis does not involve the PDGF receptor and that PDGF mediates GAG elongation via the PDGF receptor.

Repression of PDGF-R-α after cellular injury involves TNF-α, formation of a c-Fos-YY1 complex, and negative regulation by HDAC.

Wound healing is a complex dynamic process involving a variety of cell types, including fibroblasts that express and respond to cytokines and growth factors in the local microenvironment. The mechanisms controlling gene expression after injury at a transcriptional level are poorly understood. Here we show that decreased expression of a key receptor, PDGF-receptor (R)-α, after fibroblast injury is due to the release and paracrine activity of TNF-α. TNF-α inhibits PDGF-R-α expression and this involves formation of a c-Fos-Yin Yang 1 (YY1) complex and histone deacetylase (HDAC) activity. c-Fos, induced by TNF-α, negatively regulates PDGF-R-α transcription. Small interfering RNA (siRNA) targeting c-Fos or the zinc finger transcription factor YY1 inhibits TNF-α suppression of PDGF-R-α expression. Coimmunoprecipitation studies show that TNF-α stimulates the formation of a complex between c-Fos with YY1. Furthermore, chromatin immunoprecipitation (ChIP) analysis reveals the enrichment of c-Fos, YY1, and HDAC-1 at the PDGF-R-α promoter in cells exposed to TNF-α. With suberoylanilide hydroxamic acid (SAHA) and HDAC-1 siRNA, we demonstrate that HDAC mediates TNF-α repression of PDGF-R-α. These findings demonstrate that transcriptional repression of PDGF-R-α after fibroblast injury involves paracrine activity of endogenous TNF-α, the formation of a c-Fos-YY1 complex, and negative regulatory activity by HDAC.

Angiotensin II induction of PDGF-C expression is mediated by AT1 receptor-dependent Egr-1 transactivation.

Platelet-derived growth factors are a family of mitogens and chemoattractants comprising of four ligand genes (A-, B-, C-, D-chains) implicated in many physiologic and pathophysiologic processes, including atherosclerosis, fibrosis and tumorigenesis. Our understanding of the molecular mechanisms, which regulate PDGF-C transcription remains incomplete. Transient transfection analysis, conventional and quantitative real-time PCR revealed the induction of PDGF-C transcription and mRNA expression in smooth muscle cells (SMCs) exposed to the peptide hormone angiotensin (ATII), which induces Egr-1. Occupancy of a G + C-rich element in the proximal region of the PDGF-C promoter was unaffected by ATII. Instead we discovered, using both nuclear extracts and recombinant proteins with EMSA and ChIP analyses, the existence of a second Egr-1-binding element located 500 bp upstream. ATII induction of PDGF-C transcription is mediated by the angiotensin type 1 receptor (AT1R) and Egr-1 activation through this upstream element. DNAzyme ED5 targeting Egr-1 blocked ATII-inducible PDGF-C expression. Moreover, increased PDGF-C expression after exposure to ATII depends upon the differentiation state of the SMCs. This study demonstrates the existence of this novel ATII-AT1R-Egr-1-PDGF-C axis in SMCs of neonatal origin, but not in adult SMCs, where ATII induces Egr-1 but not PDGF-C.