The Lineage-Defining Transcription Factors SOX2 and NKX2-1 Determine Lung Cancer Cell Fate and Shape the Tumor Immune Microenvironment.

The major types of non-small-cell lung cancer (NSCLC)-squamous cell carcinoma and adenocarcinoma-have distinct immune microenvironments. We developed a genetic model of squamous NSCLC on the basis of overexpression of the transcription factor Sox2, which specifies lung basal cell fate, and loss of the tumor suppressor Lkb1 (SL mice). SL tumors recapitulated gene-expression and immune-infiltrate features of human squamous NSCLC; such features included enrichment of tumor-associated neutrophils (TANs) and decreased expression of NKX2-1, a transcriptional regulator that specifies alveolar cell fate. In Kras-driven adenocarcinomas, mis-expression of Sox2 or loss of Nkx2-1 led to TAN recruitment. TAN recruitment involved SOX2-mediated production of the chemokine CXCL5. Deletion of Nkx2-1 in SL mice (SNL) revealed that NKX2-1 suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Depletion of TANs in SNL mice reduced squamous tumors, suggesting that TANs foster squamous cell fate. Thus, lineage-defining transcription factors determine the tumor immune microenvironment, which in turn might impact the nature of the tumor.

Effect of benralizumab on histopathology and inflammatory signatures in a clinical cohort of eosinophilic esophagitis.

A preliminary report from the recent phase 3 trial of benralizumab, a monoclonal antibody that binds to interleukin-5 receptor alpha (IL5Rα), in patients with EoE revealed that medication use led to tissue eosinophil eradication but did not meet the clinical endpoint of symptom resolution. Here, we characterized the clinical, endoscopic, histologic, and transcriptional changes in patients with active EoE following benralizumab treatment. We retrospectively examined patients with EoE treated with benralizumab at the University of Utah (n = 11) and reviewed reported clinical symptoms, circulating and tissue eosinophilia, and endoscopic and histologic scores. Gene expression profiles from available esophageal tissue from benralizumab-treated patients were compared to those from patients with remission EoE (n = 5), active EoE (n = 10), and controls (n = 22). Benralizumab treatment resulted in partial symptom improvement and significant reduction in tissue eosinophilia, and endoscopic and histologic disease scoring (P < 0.01). Histologic score reductions were driven by eosinophil feature scores, while scores for epithelial features (basal cell hyperplasia and dilated intercellular spaces) were similar to those in active EoE. The gene signatures in benralizumab-treated patients mimicked those of active EoE (e.g. upregulation of POSTN, CDH26, CCL26, and downregulation of DSG1). RNA profiles and pathways support histologic findings of impaired epithelial function that persists despite benralizumab treatment. In conclusion, despite eosinophil eradication, patients treated with benralizumab had persistent epithelial injury at the histologic and transcriptional level. In this cohort, benralizumab therapy failed to eradicate inflammation and epithelial dysfunction showing that interleukin-5 receptor alpha blockade monotherapy is insufficient to control EoE.

Platelet gene expression and function in patients with COVID-19.

There is an urgent need to understand the pathogenesis of coronavirus disease 2019 (COVID-19). In particular, thrombotic complications in patients with COVID-19 are common and contribute to organ failure and mortality. Patients with severe COVID-19 present with hemostatic abnormalities that mimic disseminated intravascular coagulopathy associated with sepsis, with the major difference being increased risk of thrombosis rather than bleeding. However, whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters platelet function to contribute to the pathophysiology of COVID-19 remains unknown. In this study, we report altered platelet gene expression and functional responses in patients infected with SARS-CoV-2. RNA sequencing demonstrated distinct changes in the gene-expression profile of circulating platelets of COVID-19 patients. Pathway analysis revealed differential gene-expression changes in pathways associated with protein ubiquitination, antigen presentation, and mitochondrial dysfunction. The receptor for SARS-CoV-2 binding, angiotensin-converting enzyme 2 (ACE2), was not detected by messenger RNA (mRNA) or protein in platelets. Surprisingly, mRNA from the SARS-CoV-2 N1 gene was detected in platelets from 2 of 25 COVID-19 patients, suggesting that platelets may take-up SARS-COV-2 mRNA independent of ACE2. Resting platelets from COVID-19 patients had increased P-selectin expression basally and upon activation. Circulating platelet-neutrophil, -monocyte, and -T-cell aggregates were all significantly elevated in COVID-19 patients compared with healthy donors. Furthermore, platelets from COVID-19 patients aggregated faster and showed increased spreading on both fibrinogen and collagen. The increase in platelet activation and aggregation could partially be attributed to increased MAPK pathway activation and thromboxane generation. These findings demonstrate that SARS-CoV-2 infection is associated with platelet hyperreactivity, which may contribute to COVID-19 pathophysiology.

Reciprocal regulation of pancreatic ductal adenocarcinoma growth and molecular subtype by HNF4α and SIX1/4.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a 5-year survival of less than 5%. Transcriptomic analysis has identified two clinically relevant molecular subtypes of PDAC: classical and basal-like. The classical subtype is characterised by a more favourable prognosis and better response to chemotherapy than the basal-like subtype. The classical subtype also expresses higher levels of lineage specifiers that regulate endodermal differentiation, including the nuclear receptor hepatocyte nuclear factor 4 α (HNF4α). The objective of this study is to evaluate the role of HNF4α, SIX4 and SIX1 in regulating the growth and molecular subtype of PDAC. DESIGN: We manipulate the expression of HNF4α, SIX4 and SIX1 in multiple in vitro and in vivo PDAC models. We determine the consequences of manipulating these genes on PDAC growth, differentiation and molecular subtype using functional assays, gene expression analysis and cross-species comparisons with human datasets. RESULTS: We show that HNF4α restrains tumour growth and drives tumour cells toward an epithelial identity. Gene expression analysis of murine models and human tumours shows that HNF4α activates expression of genes associated with the classical subtype. HNF4α also directly represses SIX4 and SIX1, two mesodermal/neuronal lineage specifiers expressed in the basal-like subtype. Finally, SIX4 and SIX1 drive proliferation and regulate differentiation in HNF4α-negative PDAC. CONCLUSION: Our data show that HNF4α regulates the growth and molecular subtype of PDAC by multiple mechanisms, including activation of the classical gene expression programme and repression of SIX4 and SIX1, which may represent novel dependencies of the basal-like subtype.

Postseptic Cognitive Impairment and Expression of APOE in Peripheral Blood: The Cognition After SepsiS (CASS) Observational Pilot Study.

BACKGROUND: Cognitive impairment after sepsis is an important clinical problem. Determinants of postseptic cognitive impairment are not well understood. We thus undertook a systems biology approach to exploring a possible role for apolipoprotein E (APOE) in postseptic cognitive impairment. DESIGN: Prospective, observational cohort. SETTING: Intermountain Medical Center, a tertiary referral center in Utah. PATIENTS/PARTICIPANTS: Patients with sepsis admitted to study intensive care units. INTERVENTIONS: None. METHODS: We obtained peripheral blood for deep sequencing of RNA and followed up survivors at 6 months with a battery of cognitive instruments. We defined cognitive impairment based on the 6-month Hayling test of executive function. In our primary analysis, we employed weighted network analysis. Secondarily, we compared variation in gene expression between patients with normal versus impaired cognition. MEASUREMENTS AND MAIN RESULTS: We enrolled 40 patients, of whom 34 were follow-up eligible and 31 (91%) completed follow-up; 1 patient's RNA sample was degraded-the final analytic cohort was 30 patients. Mean Hayling test score was 5.8 (standard deviation 1.1), which represented 20% with impaired executive function. The network module containing APOE was dominated by low-expression genes, with no association on primary analysis (P = .8). Secondary analyses suggested several potential lines of future investigation, including oxidative stress. CONCLUSIONS: In this prospective pilot cohort, executive dysfunction affected 1 in 5 survivors of sepsis. The APOE gene was sparsely transcribed in peripheral leukocytes and not associated with cognitive impairment. Future lines of research are suggested.

Mining locus tags in PubMed Central to improve microbial gene annotation.

BACKGROUND: The scientific literature contains millions of microbial gene identifiers within the full text and tables, but these annotations rarely get incorporated into public sequence databases. We propose to utilize the Open Access (OA) subset of PubMed Central (PMC) as a gene annotation database and have developed an R package called pmcXML to automatically mine and extract locus tags from full text, tables and supplements. RESULTS: We mined locus tags from 1835 OA publications in ten microbial genomes and extracted tags mentioned in 30,891 sentences in main text and 20,489 rows in tables. We identified locus tag pairs marking the start and end of a region such as an operon or genomic island and expanded these ranges to add another 13,043 tags. We also searched for locus tags in supplementary tables and publications outside the OA subset in Burkholderia pseudomallei K96243 for comparison. There were 168 publications containing 48,470 locus tags and 83% of mentions were from supplementary materials and 9% from publications outside the OA subset. CONCLUSIONS: B. pseudomallei locus tags within the full text and tables of OA publications represent only a small fraction of the total mentions in the literature. For microbial genomes with very few functionally characterized proteins, the locus tags mentioned in supplementary tables and within ranges like genomic islands contain the majority of locus tags. Significantly, the functions in the R package provide access to additional resources in the OA subset that are not currently indexed or returned by searching PMC.

Differential expression of small RNAs from Burkholderia thailandensis in response to varying environmental and stress conditions.

BACKGROUND: Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with downstream target mRNAs to attenuate translation of mRNA into protein at the post-transcriptional level. In response to specific environmental changes, sRNAs can modulate the expression levels of target genes, thus enabling adaptation of cellular physiology. RESULTS: We profiled sRNA expression in the Gram-negative bacteria Burkholderia thailandensis cultured under 54 distinct growth conditions using a Burkholderia-specific microarray that contains probe sets to all intergenic regions greater than 90 bases. We identified 38 novel sRNAs and performed experimental validation on five sRNAs that play a role in adaptation of Burkholderia to cell stressors. In particular, the trans-encoded BTH_s1 and s39 exhibited differential expression profiles dependent on growth phase and cell stimuli, such as antibiotics and serum. Furthermore, knockdown of the highly-expressed BTH_s39 by antisense transcripts reduced B. thailandensis cell growth and attenuated host immune response upon infection, indicating that BTH_s39 functions in bacterial metabolism and adaptation to the host. In addition, expression of cis-encoded BTH_s13 and s19 found in the 5' untranslated regions of their cognate genes correlated with tight regulation of gene transcript levels. This sRNA-mediated downregulation of gene expression may be a conserved mechanism of post-transcriptional gene dosage control. CONCLUSIONS: These studies provide a broad analysis of differential Burkholderia sRNA expression profiles and illustrate the complexity of bacterial gene regulation in response to different environmental stress conditions.

MicroRNA Signatures Associated with Basal Cell Carcinoma Subtypes.

Basal cell carcinoma (BCC) is classified histologically into subtypes that determine treatment decisions. MicroRNAs (miRs) are short noncoding RNAs that may serve as diagnostic biomarkers. We investigated if particular miRs could distinguish BCC subtypes. We sequenced miRs from 55 archival BCC and 9 control skin specimens and then validated these miRs by qRT-PCR assay on a second BCC cohort (18 superficial, 16 nodular, 15 infiltrative) and control skin (n = 12). Expression values for individual miRs were normalized to miR-16-5p, which was the least variant among the control skin and BCC samples. We found that (i) miR-383-5p and miR-145-5p are downregulated in all BCC subtypes compared with control skin, (ii) miR-181c-5p is downregulated in superficial compared with invasive (nodular/infiltrative) BCC, and (iii) miR-22-5p and miR-708-5p are upregulated in infiltrative compared with superficial/nodular BCC and miR-30c-5p is downregulated in infiltrative compared with nodular BCC. Receiver operating characteristic analysis demonstrated excellent capacity of these miRs to discriminate between BCC and control skin (area under the curve, 0.94-0.98), whereas the capacity to discriminate between superficial and invasive subtypes was less robust (area under the curve, 0.7-0.8). Future prospective studies may determine the utility of these miRs as diagnostic biomarkers to guide biopsy and treatment of BCC.

Prospective, international, multisite comparison of platelet isolation techniques for genome-wide transcriptomics: communication from the SSC of the ISTH.

Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committees, we assessed how 3 of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALL filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. Moreover, all 3 isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbß3 activation is reduced by anti-CD45 bead isolation compared with washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.

A zebrafish model of combined saposin deficiency identifies acid sphingomyelinase as a potential therapeutic target.

Sphingolipidoses are a subcategory of lysosomal storage diseases (LSDs) caused by mutations in enzymes of the sphingolipid catabolic pathway. Like many LSDs, neurological involvement in sphingolipidoses leads to early mortality with limited treatment options. Given the role of myelin loss as a major contributor toward LSD-associated neurodegeneration, we investigated the pathways contributing to demyelination in a CRISPR-Cas9-generated zebrafish model of combined saposin (psap) deficiency. psap knockout (KO) zebrafish recapitulated major LSD pathologies, including reduced lifespan, reduced lipid storage, impaired locomotion and severe myelin loss; loss of myelin basic protein a (mbpa) mRNA was progressive, with no changes in additional markers of oligodendrocyte differentiation. Brain transcriptomics revealed dysregulated mTORC1 signaling and elevated neuroinflammation, where increased proinflammatory cytokine expression preceded and mTORC1 signaling changes followed mbpa loss. We examined pharmacological and genetic rescue strategies via water tank administration of the multiple sclerosis drug monomethylfumarate (MMF), and crossing the psap KO line into an acid sphingomyelinase (smpd1) deficiency model. smpd1 mutagenesis, but not MMF treatment, prolonged lifespan in psap KO zebrafish, highlighting the modulation of acid sphingomyelinase activity as a potential path toward sphingolipidosis treatment.

miRNA-1 promotes acute myeloid leukemia cell pathogenesis through metabolic regulation.

Acute myeloid leukemia (AML) is a heterogeneous and deadly disease characterized by uncontrolled expansion of malignant blasts. Altered metabolism and dysregulated microRNA (miRNA) expression profiles are both characteristic of AML. However, there is a paucity of studies exploring how changes in the metabolic state of the leukemic cells regulate miRNA expression leading to altered cellular behavior. Here, we blocked pyruvate entry into mitochondria by deleting the Mitochondria Pyruvate Carrier (MPC1) gene in human AML cell lines, which decreased Oxidative Phosphorylation (OXPHOS). This metabolic shift also led to increased expression of miR-1 in the human AML cell lines tested. AML patient sample datasets showed that higher miR-1 expression correlates with reduced survival. Transcriptional and metabolic profiling of miR-1 overexpressing AML cells revealed that miR-1 increased OXPHOS, along with key metabolites that fuel the TCA cycle such as glutamine and fumaric acid. Inhibition of glutaminolysis decreased OXPHOS in miR-1 overexpressing MV4-11 cells, highlighting that miR-1 promotes OXPHOS through glutaminolysis. Finally, overexpression of miR-1 in AML cells exacerbated disease in a mouse xenograft model. Together, our work expands current knowledge within the field by uncovering novel connections between AML cell metabolism and miRNA expression that facilitates disease progression. Further, our work points to miR-1 as a potential new therapeutic target that may be used to disrupt AML cell metabolism and thus pathogenesis in the clinic.

BMPER is a marker of adipose progenitors and adipocytes and a positive modulator of adipogenesis.

Autocrine and paracrine signaling regulating adipogenesis in white adipose tissue remains largely unclear. Here we used single-cell RNA-sequencing (RNA-seq) and single nuclei RNA-sequencing (snRNA-seq) to identify markers of adipose progenitor cells (APCs) and adipogenic modulators in visceral adipose tissue (VAT) of humans and mice. Our study confirmed the presence of major cellular clusters in humans and mice and established important sex and diet-specific dissimilarities in cell proportions. Here we show that bone morphogenetic protein (BMP)-binding endothelial regulator (BMPER) is a conserved marker for APCs and adipocytes in VAT in humans and mice. Further, BMPER is highly enriched in lineage negative stromal vascular cells and its expression is significantly higher in visceral compared to subcutaneous APCs in mice. BMPER expression and release peaked by day four post-differentiation in 3T3-L1 preadipocytes. We reveal that BMPER is required for adipogenesis both in 3T3-L1 preadipocytes and in mouse APCs. Together, this study identified BMPER as a positive modulator of adipogenesis.

Senescence-induced immune remodeling facilitates metastatic adrenal cancer in a sex-dimorphic manner.

Aging markedly increases cancer risk, yet our mechanistic understanding of how aging influences cancer initiation is limited. Here we demonstrate that the loss of ZNRF3, an inhibitor of Wnt signaling that is frequently mutated in adrenocortical carcinoma, leads to the induction of cellular senescence that remodels the tissue microenvironment and ultimately permits metastatic adrenal cancer in old animals. The effects are sexually dimorphic, with males exhibiting earlier senescence activation and a greater innate immune response, driven in part by androgens, resulting in high myeloid cell accumulation and lower incidence of malignancy. Conversely, females present a dampened immune response and increased susceptibility to metastatic cancer. Senescence-recruited myeloid cells become depleted as tumors progress, which is recapitulated in patients in whom a low myeloid signature is associated with worse outcomes. Our study uncovers a role for myeloid cells in restraining adrenal cancer with substantial prognostic value and provides a model for interrogating pleiotropic effects of cellular senescence in cancer.

ARF6-dependent endocytic trafficking of the Interferon-γ receptor drives adaptive immune resistance in cancer.

Adaptive immune resistance (AIR) is a protective process used by cancer to escape elimination by CD8+ T cells. Inhibition of immune checkpoints PD-1 and CTLA-4 specifically target Interferon-gamma (IFNγ)-driven AIR. AIR begins at the plasma membrane where tumor cell-intrinsic cytokine signaling is initiated. Thus, plasma membrane remodeling by endomembrane trafficking could regulate AIR. Herein we report that the trafficking protein ADP-Ribosylation Factor 6 (ARF6) is critical for IFNγ-driven AIR. ARF6 prevents transport of the receptor to the lysosome, augmenting IFNγR expression, tumor intrinsic IFNγ signaling and downstream expression of immunosuppressive genes. In murine melanoma, loss of ARF6 causes resistance to immune checkpoint blockade (ICB). Likewise, low expression of ARF6 in patient tumors correlates with inferior outcomes with ICB. Our data provide new mechanistic insights into tumor immune escape, defined by ARF6-dependent AIR, and support that ARF6-dependent endomembrane trafficking of the IFNγ receptor influences outcomes of ICB.

FoxA1 and FoxA2 control growth and cellular identity in NKX2-1-positive lung adenocarcinoma.

Changes in cellular identity (also known as histologic transformation or lineage plasticity) can drive malignant progression and resistance to therapy in many cancers, including lung adenocarcinoma (LUAD). The lineage-specifying transcription factors FoxA1 and FoxA2 (FoxA1/2) control identity in NKX2-1/TTF1-negative LUAD. However, their role in NKX2-1-positive LUAD has not been systematically investigated. We find that Foxa1/2 knockout severely impairs tumorigenesis in KRAS-driven genetically engineered mouse models and human cell lines. Loss of FoxA1/2 leads to the collapse of a dual-identity state, marked by co-expression of pulmonary and gastrointestinal transcriptional programs, which has been implicated in LUAD progression. Mechanistically, FoxA1/2 loss leads to aberrant NKX2-1 activity and genomic localization, which in turn actively inhibits tumorigenesis and drives alternative cellular identity programs that are associated with non-proliferative states. This work demonstrates that FoxA1/2 expression is a lineage-specific vulnerability in NKX2-1-positive LUAD and identifies mechanisms of response and resistance to targeting FoxA1/2 in this disease.

IFITM3 regulates fibrinogen endocytosis and platelet reactivity in nonviral sepsis.

Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We determined whether nonviral sepsis induces differential platelet gene expression and reactivity. Nonviral sepsis upregulated IFN-induced transmembrane protein 3 (IFITM3), an IFN-responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we determined whether IFITM3 promoted endocytosis of α-granule proteins. IFN stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacted with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo IFN administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from IFN-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on IFN-α and IFITMs. Platelets from patients with nonviral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.

Nitric oxide releasing nanomatrix gel treatment inhibits venous intimal hyperplasia and improves vascular remodeling in a rodent arteriovenous fistula.

Vascular access is the lifeline for hemodialysis patients and the single most important component of the hemodialysis procedure. Arteriovenous fistula (AVF) is the preferred vascular access for hemodialysis patients, but nearly 60% of AVFs created fail to successfully mature due to early intimal hyperplasia development and poor outward remodeling. There are currently no therapies available to prevent AVF maturation failure. First, we showed the important regulatory role of nitric oxide (NO) on AVF development by demonstrating that intimal hyperplasia development was reduced in an overexpressed endothelial nitric oxide synthase (NOS3) mouse AVF model. This supported the rationale for the potential application of NO to the AVF. Thus, we developed a self-assembled NO releasing nanomatrix gel and applied it perivascularly at the arteriovenous anastomosis immediately following rat AVF creation to investigate its therapeutic effect on AVF development. We demonstrated that the NO releasing nanomatrix gel inhibited intimal hyperplasia formation (more than 70% reduction), as well as improved vascular outward remodeling (increased vein diameter) and hemodynamic adaptation (lower wall shear stress approaching the preoperative level and less vorticity). Therefore, direct application of the NO releasing nanomatrix gel to the AVF anastomosis immediately following AVF creation may enhance AVF development, thereby providing long-term and durable vascular access for hemodialysis.

FGF21 (Fibroblast Growth Factor 21) Defines a Potential Cardiohepatic Signaling Circuit in End-Stage Heart Failure.

BACKGROUND: Extrinsic control of cardiomyocyte metabolism is poorly understood in heart failure (HF). FGF21 (Fibroblast growth factor 21), a hormonal regulator of metabolism produced mainly in the liver and adipose tissue, is a prime candidate for such signaling. METHODS: To investigate this further, we examined blood and tissue obtained from human subjects with end-stage HF with reduced ejection fraction at the time of left ventricular assist device implantation and correlated serum FGF21 levels with cardiac gene expression, immunohistochemistry, and clinical parameters. RESULTS: Circulating FGF21 levels were substantially elevated in HF with reduced ejection fraction, compared with healthy subjects (HF with reduced ejection fraction: 834.4 [95% CI, 628.4-1040.3] pg/mL, n=40; controls: 146.0 [86.3-205.7] pg/mL, n=20, P=1.9×10-5). There was clear FGF21 staining in diseased cardiomyocytes, and circulating FGF21 levels negatively correlated with the expression of cardiac genes involved in ketone metabolism, consistent with cardiac FGF21 signaling. FGF21 gene expression was very low in failing and nonfailing hearts, suggesting extracardiac production of the circulating hormone. Circulating FGF21 levels were correlated with BNP (B-type natriuretic peptide) and total bilirubin, markers of chronic cardiac and hepatic congestion. CONCLUSIONS: Circulating FGF21 levels are elevated in HF with reduced ejection fraction and appear to bind to the heart. The liver is likely the main extracardiac source. This supports a model of hepatic FGF21 communication to diseased cardiomyocytes, defining a potential cardiohepatic signaling circuit in human HF.

Steps toward broad-spectrum therapeutics: discovering virulence-associated genes present in diverse human pathogens.

BACKGROUND: New and improved antimicrobial countermeasures are urgently needed to counteract increased resistance to existing antimicrobial treatments and to combat currently untreatable or new emerging infectious diseases. We demonstrate that computational comparative genomics, together with experimental screening, can identify potential generic (i.e., conserved across multiple pathogen species) and novel virulence-associated genes that may serve as targets for broad-spectrum countermeasures. RESULTS: Using phylogenetic profiles of protein clusters from completed microbial genome sequences, we identified seventeen protein candidates that are common to diverse human pathogens and absent or uncommon in non-pathogens. Mutants of 13 of these candidates were successfully generated in Yersinia pseudotuberculosis and the potential role of the proteins in virulence was assayed in an animal model. Six candidate proteins are suggested to be involved in the virulence of Y. pseudotuberculosis, none of which have previously been implicated in the virulence of Y. pseudotuberculosis and three have no record of involvement in the virulence of any bacteria. CONCLUSION: This work demonstrates a strategy for the identification of potential virulence factors that are conserved across a number of human pathogenic bacterial species, confirming the usefulness of this tool.

Disuse-induced insulin resistance susceptibility coincides with a dysregulated skeletal muscle metabolic transcriptome.

Short-term muscle disuse is characterized by skeletal muscle insulin resistance, although this response is divergent across subjects. The mechanisms regulating inactivity-induced insulin resistance between populations that are more or less susceptible to disuse-induced insulin resistance are not known. RNA sequencing was conducted on vastus lateralis muscle biopsies from subjects before and after bed rest (n = 26) to describe the transcriptome of inactivity-induced insulin resistance. Subjects were separated into Low (n = 14) or High (n = 12) Susceptibility Groups based on the magnitude of change in insulin sensitivity after 5 days of bed rest. Both groups became insulin-resistant after bed rest, and there were no differences between groups in nonmetabolic characteristics (body mass, body mass index, fat mass, and lean mass). The High Susceptibility Group had more genes altered >1.5-fold (426 high versus 391 low) and more than twofold (73 high versus 55 low). Twenty-four genes were altered more than twofold in the High Susceptibility Group that did not change in the Low Susceptibility Group. 95 gene changes correlated with the changes in insulin sensitivity; 6 of these genes changed more than twofold in the High Susceptibility Group. Participants in the High Susceptibility Group were uniquely characterized with muscle gene responses described by a decrease in pathways responsible for lipid uptake and oxidation, decreased capacity for triglyceride export (APOB), increased lipogenesis (i.e., PFKFB3, FASN), and increased amino acid export (SLC43A1). These transcriptomic data provide a comprehensive examination of pathways and genes that may be useful biomarkers, or novel targets to offset muscle disuse-induced insulin resistance. NEW & NOTEWORTHY Short-term muscle disuse results in skeletal muscle insulin resistance through mechanisms that are not fully understood. Following a 5-day bed rest intervention, subjects were divided into High and Low Susceptibility Groups to inactivity-induced insulin resistance. This was followed by a genome-wide transcriptional analysis on muscle biopsy samples to gain insight on divergent insulin sensitivity responses. Our primary finding was that the skeletal muscle of subjects who experienced the most inactivity-induced insulin resistance (high susceptibility) was characterized by a decreased preference for lipid oxidation, increased lipogenesis, and increased amino acid export.