The Microbiome, Ocular Surface, and Corneal Disorders.

The ocular surface microbiome is an emerging field of study that seeks to understand how the community of microorganisms found on the ocular surface may help maintain homeostasis or can potentially lead to disease and dysbiosis. Initial questions include whether the organisms detected on the ocular surface inhabit that ecological niche and, if so, whether there exists a core microbiome found in most or all healthy eyes. Many questions have emerged around whether novel organisms and/or a redistribution of organisms play a role in disease pathogenesis, response to therapies, or convalescence. Although there is much enthusiasm about this topic, the ocular surface microbiome is a new field with many technical challenges. These challenges are discussed in this review as well as a need for standardization to adequately compare studies and advance the field. In addition, this review summarizes the current research on the microbiome of various ocular surface diseases and how these findings may impact treatments and clinical decision-making.

An Initial miRNA Profile of Persons With Persisting Neurobehavioral Impairments and States of Disordered Consciousness After Severe Traumatic Brain Injury.

OBJECTIVE: To examine the merits of using microRNAs (miRNAs) as biomarkers of disorders of consciousness (DoC) due to traumatic brain injury (TBI). SETTINGS: Acute and subacute beds. PARTICIPANTS: Patients remaining in vegetative and minimally conscious states (VS, MCS), an average of 1.5 years after TBI, and enrolled in a randomized clinical trial ( n = 6). Persons without a diagnosed central nervous system disorder, neurotypical controls ( n = 5). DESIGN: Comparison of whole blood miRNA profiles between patients and age/gender-matched controls. For patients, correlational analyses between miRNA profiles and measures of neurobehavioral function. MAIN MEASURES: Baseline measures of whole blood miRNAs isolated from the cellular and fluid components of blood and measured using miRNA-seq and real-time polymerase chain reaction (RT-PCR). Baseline neurobehavioral measures derived from 7 tests. RESULTS: For patients, relative to controls, 48 miRNA were significantly ( P < .05)/differentially expressed. Cluster analysis showed that neurotypical controls were most similar to each other and with 2 patients (VS: n = 1; and MCS: n = 1). Three patients, all in MCS, clustered separately. The only female in the sample, also in MCS, formed an independent group. For the 48 miRNAs, the enriched pathways identified are implicated in secondary brain damage and 26 miRNAs were significantly ( P < .05) correlated with measures of neurobehavioral function. CONCLUSIONS: Patients remaining in states of DoC an average of 1.5 years after TBI showed a different and reproducible pattern of miRNA expression relative to age/gender-matched neurotypical controls. The phenotypes, defined by miRNA profiles relative to persisting neurobehavioral impairments, provide the basis for future research to determine the miRNA profiles differentiating states of DoC and the basis for future research using miRNA to detect treatment effects, predict treatment responsiveness, and developing targeted interventions. If future research confirms and advances reported findings, then miRNA profiles will provide the foundation for patient-centric DoC neurorehabilitation.

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Focal Adhesion Kinase and ß-Catenin Cooperate to Induce Hepatocellular Carcinoma.

There is an urgent need to understand the molecular signaling pathways that drive or mediate the development of hepatocellular carcinoma (HCC). The focal adhesion kinase (FAK) gene protein tyrosine kinase 2 is amplified in 16.4% of The Cancer Genome Atlas HCC specimens, and its amplification leads to increased FAK mRNA expression. It is not known whether the overexpression of FAK alone is sufficient to induce HCC or whether it must cooperate in some ways with other oncogenes. In this study, we found that 34.8% of human HCC samples with FAK amplification also show ß-catenin mutations, suggesting a co-occurrence of FAK overexpression and ß-catenin mutations in HCC. We overexpressed FAK alone, constitutively active forms of ß-catenin (CAT) alone, or a combination of FAK and CAT in the livers of C57/BL6 mice. We found that overexpression of both FAK and CAT, but neither FAK nor CAT alone, in mouse livers was sufficient to lead to tumorigenesis. We further demonstrated that FAK's kinase activity is required for FAK/CAT-induced tumorigenesis. Furthermore, we performed RNA-sequencing analysis to identify the genes/signaling pathways regulated by FAK, CAT, or FAK/CAT. We found that FAK overexpression dramatically enhances binding of ß-catenin to the promoter of androgen receptor (AR), which leads to increased expression of AR in mouse livers. Moreover, ASC-J9, an AR degradation enhancer, suppressed FAK/CAT-induced HCC formation. Conclusion: FAK overexpression and ß-catenin mutations often co-occur in human HCC tissues. Co-overexpression of FAK and CAT leads to HCC formation in mice through increased expression of AR; this mouse model may be useful for further studies of the molecular mechanisms in the pathogenesis of HCC and could lead to the identification of therapeutic targets.

Exosomal miR-21a-5p mediates cardioprotection by mesenchymal stem cells.

Though experimental, stem cell transplantation has the potential to improve the condition of the heart after myocardial infarction. It does so by reducing infarct size and inducing repair of heart muscle and its blood supply. Mesenchymal stem cells (MSC) have been found to be effective in pre-clinical animal models and clinical trials, but the mechanisms by which they induce cardioprotection and repair are still not fully understood. Small extracellular vesicles known as exosomes are now recognized to be key mediators of beneficial MSC paracrine effects, and the concept that they transfer miRNA to change gene expression in recipient cells is of current therapeutic interest. We present complete deep miRNA sequencing of MSC exosome cargo, and found that of several cardioprotective miRNAs, miR-21a-5p was the most abundant. Because miR-21a-5p is a well-known cardioprotective miRNA, we investigated the hypothesis that MSC exosomes can cardioprotect the heart by increasing the level of miR-21a-5p in recipient cardiac cells, thereby downregulating expression of the pro-apoptotic gene products PDCD4, PTEN, Peli1 and FasL in the myocardium. Using miR-21 mimic transfection and treatment with wild type and miR-21a knockout MSC exosomes, we confirmed that exosomal miR-21a-5p is transferred into myocardium and is a major cardioprotective paracrine factor produced by MSCs acting via synergistic activity on multiple pathways. The data supports that residual cardioprotective effect may be due to other ncRNA or protein cargo. In silico analyses support that MSC exosomes may also contribute to angiogenesis, cell proliferation and other aspects of cardiac repair.

Cardiac inflammation in genetic dilated cardiomyopathy caused by MYBPC3 mutation.

Cardiomyopathies are a leading cause of heart failure and are often caused by mutations in sarcomeric genes, resulting in contractile dysfunction and cellular damage. This may stimulate the production of a robust proinflammatory response. To determine whether myocardial inflammation is associated with cardiac dysfunction in dilated cardiomyopathy (DCM) caused by MYBPC3 mutation, we used the well-characterized cMyBP-C(t/t) mouse model of DCM at 3months of age. Compared to wild type (WT) mice, DCM mice exhibited significantly decreased fractional shortening (36.4±2% vs. 15.5±1.0%, p<0.0001) and significantly increased spleen weight (5.3±0.3 vs. 7.2±0.4mg/mm, p=0.002). Intriguingly, flow cytometry analysis revealed a significant increase in total (CD45+CD11b+Ly6C-MHCII+F480+) macrophages (6.5±1.4% vs. 14.8±1.4%, p=0.002) and classically activated (CD45+CD11b+Ly6C-MHCII+F480+CD206-) proinflammatory (M1) macrophages (3.4±0.8% vs. 10.3±1.2%, p=0.0009) in DCM hearts as compared with WT hearts. These results were further confirmed by immunofluorescence analysis of heart tissue sections. Splenic red pulp (CD11b+Ly6C+MHCIIlowF480hi) macrophages were significantly elevated (1.3±0.1% vs. 2.4±0.1%, p=0.0001) in DCM compared to WT animals. Serum cytokine analysis in DCM animals exhibited a significant increase (0.65±0.2 vs. 2.175±0.5pg/mL, p=0.02) in interleukin (IL)-6 compared to WT animals. Furthermore, RNA-seq analysis revealed the upregulation of inflammatory pathways in the DCM hearts. Together, these data indicate a robust proinflammatory response in DCM hearts, likely in response to cellular damage triggered by MYBPC3 mutation and resultant contractile dysfunction.

Incontinence medication response relates to the female urinary microbiota.

INTRODUCTION AND HYPOTHESIS: Many adult women have resident urinary bacteria (urinary microbiome/microbiota). In adult women affected by urinary urgency incontinence (UUI), the etiologic and/or therapeutic role of the urinary microbiome/microbiota remains unknown. We hypothesized that microbiome/microbiota characteristics would relate to clinically relevant treatment response to UUI medication per os. METHODS: Adult women initiating medication treatment orally for UUI and a comparator group of unaffected women were recruited in a tertiary care health-care system. All participants provided baseline clinical data and urine samples. Women with UUI were given 5 mg solifenacin, with potential dose escalation to 10 mg for inadequate UUI symptom control at 4 weeks. Additional data and urine samples were collected from women with UUI at 4 and 12 weeks. The samples were assessed using 16S ribosomal RNA (rRNA) gene sequencing and enhanced quantitative urine culturing. The primary outcome was treatment response as measured by the validated Patient Global Symptom Control (PGSC) questionnaire. Clinically relevant UUI symptom control was defined as a 4 or 5 score on the PGSC. RESULTS: Diversity and composition of the urinary microbiome/microbiota of women with and without UUI differed at baseline. Women with UUI had more bacteria and a more diverse microbiome/microbiota. The clinical response to solifenacin in UUI participants was related to baseline microbiome/microbiota, with responders more likely to have fewer bacteria and a less diverse community at baseline. Nonresponders had a more diverse community that often included bacteria not typically found in responders. CONCLUSIONS: Knowledge of an individual's urinary microbiome/microbiota may help refine UUI treatment. Complementary tools, DNA sequencing, and expanded urine culture provide information about bacteria that appear to be related to UUI incontinence status and treatment response in this population of adult women.

The Gene Expression Barcode 3.0: improved data processing and mining tools.

The Gene Expression Barcode project, http://barcode.luhs.org, seeks to determine the genes expressed for every tissue and cell type in humans and mice. Understanding the absolute expression of genes across tissues and cell types has applications in basic cell biology, hypothesis generation for gene function and clinical predictions using gene expression signatures. In its current version, this project uses the abundant publicly available microarray data sets combined with a suite of single-array preprocessing, quality control and analysis methods. In this article, we present the improvements that have been made since the previous version of the Gene Expression Barcode in 2011. These include a variety of new data mining tools and summaries, estimated transcriptomes and curated annotations.

The female urinary microbiome in urgency urinary incontinence.

OBJECTIVE: The purpose of this study was to characterize the urinary microbiota in women who are planning treatment for urgency urinary incontinence and to describe clinical associations with urinary symptoms, urinary tract infection, and treatment outcomes. STUDY DESIGN: Catheterized urine samples were collected from multisite randomized trial participants who had no clinical evidence of urinary tract infection; 16S ribosomal RNA gene sequencing was used to dichotomize participants as either DNA sequence-positive or sequence-negative. Associations with demographics, urinary symptoms, urinary tract infection risk, and treatment outcomes were determined. In sequence-positive samples, microbiotas were characterized on the basis of their dominant microorganisms. RESULTS: More than one-half (51.1%; 93/182) of the participants' urine samples were sequence-positive. Sequence-positive participants were younger (55.8 vs 61.3 years old; P = .0007), had a higher body mass index (33.7 vs 30.1 kg/m(2); P = .0009), had a higher mean baseline daily urgency urinary incontinence episodes (5.7 vs 4.2 episodes; P < .0001), responded better to treatment (decrease in urgency urinary incontinence episodes, -4.4 vs -3.3; P = .0013), and were less likely to experience urinary tract infection (9% vs 27%; P = .0011). In sequence-positive samples, 8 major bacterial clusters were identified; 7 clusters were dominated not only by a single genus, most commonly Lactobacillus (45%) or Gardnerella (17%), but also by other taxa (25%). The remaining cluster had no dominant genus (13%). CONCLUSION: DNA sequencing confirmed urinary bacterial DNA in many women with urgency urinary incontinence who had no signs of infection. Sequence status was associated with baseline urgency urinary incontinence episodes, treatment response, and posttreatment urinary tract infection risk.

Identification of novel markers for mouse CD4(+) T follicular helper cells.

CD4(+) T follicular helper (TFH) cells are central for generation of long-term B-cell immunity. A defining phenotypic attribute of TFH cells is the expression of the chemokine R CXCR5, and TFH cells are typically identified by co-expression of CXCR5 together with other markers such as PD-1, ICOS, and Bcl-6. Herein, we report high-level expression of the nutrient transporter folate R 4 (FR4) on TFH cells in acute viral infection. Distinct from the expression profile of conventional TFH markers, FR4 was highly expressed by naive CD4(+) T cells, was downregulated after activation and subsequently re-expressed on TFH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory TFH cells. Comparative gene expression profiling of FR4(hi) versus FR4(lo) Ag-specific CD4(+) effector T cells revealed a molecular signature consistent with TFH and TH1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto-enzyme CD73, were enriched in TFH cells compared with TH1 cells, and phenotypic analysis confirmed expression of CD73 on TFH cells. As there is now considerable interest in developing vaccines that would induce optimal TFH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of TFH cells following vaccination and infection.

Urine is not sterile: use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder.

Our previous study showed that bacterial genomes can be identified using 16S rRNA sequencing in urine specimens of both symptomatic and asymptomatic patients who are culture negative according to standard urine culture protocols. In the present study, we used a modified culture protocol that included plating larger volumes of urine, incubation under varied atmospheric conditions, and prolonged incubation times to demonstrate that many of the organisms identified in urine by 16S rRNA gene sequencing are, in fact, cultivable using an expanded quantitative urine culture (EQUC) protocol. Sixty-five urine specimens (from 41 patients with overactive bladder and 24 controls) were examined using both the standard and EQUC culture techniques. Fifty-two of the 65 urine samples (80%) grew bacterial species using EQUC, while the majority of these (48/52 [92%]) were reported as no growth at 10(3) CFU/ml by the clinical microbiology laboratory using the standard urine culture protocol. Thirty-five different genera and 85 different species were identified by EQUC. The most prevalent genera isolated were Lactobacillus (15%), followed by Corynebacterium (14.2%), Streptococcus (11.9%), Actinomyces (6.9%), and Staphylococcus (6.9%). Other genera commonly isolated include Aerococcus, Gardnerella, Bifidobacterium, and Actinobaculum. Our current study demonstrates that urine contains communities of living bacteria that comprise a resident female urine microbiota.

Comparative analysis of simian immunodeficiency virus gag-specific effector and memory CD8+ T cells induced by different adenovirus vectors.

Adenovirus (Ad) vectors are widely used as experimental vaccines against several infectious diseases, but the magnitude, phenotype, and functionality of CD8(+) T cell responses induced by different adenovirus serotypes have not been compared. To address this question, we have analyzed simian immunodeficiency virus Gag-specific CD8(+) T cell responses in mice following vaccination with Ad5, Ad26, and Ad35. Our results show that although Ad5 is more immunogenic than Ad26 and Ad35, the phenotype, function, and recall potential of memory CD8(+) T cells elicited by these vectors are substantially different. Ad26 and Ad35 vectors generated CD8(+) T cells that display the phenotype and function of long-lived memory T cells, whereas Ad5 vector-elicited CD8(+) T cells are of a more terminally differentiated phenotype. In addition, hepatic memory CD8(+) T cells elicited by Ad26 and Ad35 mounted more robust recall proliferation following secondary challenge than those induced by Ad5. Furthermore, the boosting potential was higher following priming with alternative-serotype Ad vectors than with Ad5 vectors in heterologous prime-boost regimens. Anamnestic CD8(+) T cell responses were further enhanced when the duration between priming and boosting was extended from 30 to 60 days. Our results demonstrate that heterologous prime-boost vaccine regimens with alternative-serotype Ad vectors elicited more functional memory CD8(+) T cells than any of the regimens containing Ad5. In summary, these results suggest that alternative-serotype Ad vectors will prove useful as candidates for vaccine development against human immunodeficiency virus type 1 and other pathogens and also emphasize the importance of a longer rest period between prime and boost for generating optimal CD8(+) T cell immunity.

A Multi-Omics Approach to Defining Target Organ Injury in Youth with Primary Hypertension.

BACKGROUND: Primary hypertension in childhood tracks into adulthood and may be associated with increased cardiovascular risk. Studies conducted in children and adolescents provide an opportunity to explore the early cardiovascular target organ injury (CV-TOI) in a population free from many of the co-morbid cardiovascular disease risk factors that confound studies in adults. METHODS: Youths (n=132, mean age 15.8 years) were stratified by blood pressure (BP) as low, elevated, and high-BP and by left ventricular mass index (LVMI) as low- and high-LVMI. Systemic circulating RNA, miRNA, and methylation profiles in peripheral blood mononuclear cells and deep proteome profiles in serum were determined using high-throughput sequencing techniques. RESULTS: VASH1 gene expression was elevated in youths with high-BP with and without high-LVMI. VASH1 expression levels positively correlated with systolic BP (r=0.3143, p=0.0034). The expression of hsa-miR-335-5p, one of the VASH1- predicted miRNAs, was downregulated in high-BP with high-LVMI youths and was inversely correlated with systolic BP (r=-0.1891, p=0.0489). GSE1 hypermethylation, circulating PROZ upregulation (log 2 FC=0.61, p=0.0049 and log 2 FC=0.62, p=0.0064), and SOD3 downregulation (log 2 FC=-0.70, p=0.0042 and log 2 FC=-0.64, p=0.010) were observed in youths with elevated BP and high-BP with high-LVMI. Comparing the transcriptomic and proteomic profiles revealed elevated HYAL1 levels in youths displaying high-BP and high-LVMI. CONCLUSIONS: The findings are compatible with a novel blood pressure-associated mechanism that may occur through impaired angiogenesis and extracellular matrix degradation through dysregulation of Vasohibin-1 and Hyaluronidase1 was identified as a possible mediator of CV-TOI in youth with high-BP and suggests strategies for ameliorating TOI in adult-onset primary hypertension.

Phenotype, function, and gene expression profiles of programmed death-1(hi) CD8 T cells in healthy human adults.

T cell dysfunction is an important feature of many chronic viral infections. In particular, it was shown that programmed death-1 (PD-1) regulates T cell dysfunction during chronic lymphocytic choriomeningitis virus infection in mice, and PD-1(hi) cells exhibit an intense exhausted gene signature. These findings were extended to human chronic infections such as HIV, hepatitis C virus, and hepatitis B virus. However, it is not known if PD-1(hi) cells of healthy humans have the traits of exhausted cells. In this study, we provide a comprehensive description of phenotype, function, and gene expression profiles of PD-1(hi) versus PD-1(lo) CD8 T cells in the peripheral blood of healthy human adults as follows: 1) the percentage of naive and memory CD8 T cells varied widely in the peripheral blood cells of healthy humans, and PD-1 was expressed by the memory CD8 T cells; 2) PD-1(hi) CD8 T cells in healthy humans did not significantly correlate with the PD-1(hi) exhausted gene signature of HIV-specific human CD8 T cells or chronic lymphocytic choriomeningitis virus-specific CD8 T cells from mice; 3) PD-1 expression did not directly affect the ability of CD8 T cells to secrete cytokines in healthy adults; 4) PD-1 was expressed by the effector memory compared with terminally differentiated effector CD8 T cells; and 5) finally, an interesting inverse relationship between CD45RA and PD-1 expression was observed. In conclusion, our study shows that most PD-1(hi) CD8 T cells in healthy adult humans are effector memory cells rather than exhausted cells.

The Gene Expression Barcode: leveraging public data repositories to begin cataloging the human and murine transcriptomes.

Various databases have harnessed the wealth of publicly available microarray data to address biological questions ranging from across-tissue differential expression to homologous gene expression. Despite their practical value, these databases rely on relative measures of expression and are unable to address the most fundamental question--which genes are expressed in a given cell type. The Gene Expression Barcode is the first database to provide reliable absolute measures of expression for most annotated genes for 131 human and 89 mouse tissue types, including diseased tissue. This is made possible by a novel algorithm that leverages information from the GEO and ArrayExpress public repositories to build statistical models that permit converting data from a single microarray into expressed/unexpressed calls for each gene. For selected platforms, users may upload data and obtain results in a matter of seconds. The raw data, curated annotation, and code used to create our resource are also available at http://rafalab.jhsph.edu/barcode.

Assessing the ocular surface microbiome in severe ocular surface diseases.

PURPOSE: There is growing evidence for a critical role of the microbiome in ocular health and disease. We performed a prospective, observational study to characterize the ocular surface microbiome (OSM) in four chronic ocular surface diseases (OSDs) and healthy controls. METHODS: Sterile swabs were used to collect samples from each eye of 39 patients (78 eyes). Sterile technique and multiple controls were used to assess contamination during DNA extraction, amplification and sequencing. Concurrent use of topical antibiotics, steroids, and bandage contact lenses (BCLs) was documented. RESULTS: Despite the low biomass of the ocular surface, 47/78 (60%) eyes sampled had positive sequencing reads. We observed that half of patients (8/17, 47%) had distinct microbiomes in each eye. Healthy controls had a Lactobacillus/Streptococcus mixture or significant Corynebacterium. Staphylococcus predominated in 4/7 (57%) patients with Stevens-Johnson Syndrome (SJS) in at least one eye, compared to 0/10 healthy controls. Interestingly, 8/11 (73%) eyes with SJS were using BCLs, including 4/5 (80%) eyes dominated by Staphylococcus. Lax eyelid syndrome (LES) and Dry Eye Disease (DED) patients had similar OSMs, with Corynebacterium being the most prevalent bacteria. Alpha diversity was higher in controls and ocular graft-vs-host (oGVHD) patients compared to the other OSDs. CONCLUSIONS: Only 50% of the 39 patients had similar microbiomes in each eye. A majority of healthy eyes had a Lactobacillus/Streptococcus mix or Corynebacterium microbiome. Staphylococcus predominated in SJS, Lactobacillus in oGVHD, and Corynebacterium in DED and LES. There may be an association between different OSDs and the microbiome.

Identification of human CD4+ T cell populations with distinct antitumor activity.

How naturally arising human CD4+ T helper subsets affect cancer immunotherapy is unclear. We reported that human CD4+CD26high T cells elicit potent immunity against solid tumors. As CD26high T cells are often categorized as TH17 cells for their IL-17 production and high CD26 expression, we posited these populations would have similar molecular properties. Here, we reveal that CD26high T cells are epigenetically and transcriptionally distinct from TH17 cells. Of clinical importance, CD26high and TH17 cells engineered with a chimeric antigen receptor (CAR) regressed large human tumors to a greater extent than enriched TH1 or TH2 cells. Only human CD26high T cells mediated curative responses, even when redirected with a suboptimal CAR and without aid by CD8+ CAR T cells. CD26high T cells cosecreted effector cytokines, produced cytotoxic molecules, and persisted long term. Collectively, our work underscores the promise of CD4+ T cell populations to improve durability of solid tumor therapies.

Draft Genome Sequence of Clinical Isolate Alcaligenaceae sp. Strain 429.

Here, we present the 3.53-Mb genome for Alcaligenaceae sp. strain 429, isolated from a patient with unknown etiology. While the 16S rRNA gene most closely resembles Paenalcaligenes species, average amino acid identity (AAI) analysis did not meet the threshold to classify our strain as a species of this family.

Altered C10 domain in cardiac myosin binding protein-C results in hypertrophic cardiomyopathy.

AIMS: A 25-base pair deletion in the cardiac myosin binding protein-C (cMyBP-C) gene (MYBPC3), proposed to skip exon 33, modifies the C10 domain (cMyBP-CΔC10mut) and is associated with hypertrophic cardiomyopathy (HCM) and heart failure, affecting approximately 100 million South Asians. However, the molecular mechanisms underlying the pathogenicity of cMyBP-CΔC10mutin vivo are unknown. We hypothesized that expression of cMyBP-CΔC10mut exerts a poison polypeptide effect leading to improper assembly of cardiac sarcomeres and the development of HCM. METHODS AND RESULTS: To determine whether expression of cMyBP-CΔC10mut is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-CΔC10mut at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-CΔC10mut (heart weight/body weight ratio: 4.43 ± 0.11 mg/g non-transgenic (NTG) vs. 5.34 ± 0.25 mg/g cMyBP-CΔC10mut, P < 0.05). Furthermore, haematoxylin and eosin, Masson's trichrome staining, as well as second-harmonic generation imaging revealed the presence of significant fibrosis and a greater relative nuclear area in cMyBP-CΔC10mut hearts compared with NTG controls. M-mode echocardiography analysis revealed hypercontractile hearts (EF: 53.4%±2.9% NTG vs. 66.4% ± 4.7% cMyBP-CΔC10mut; P < 0.05) and early diastolic dysfunction (E/E': 28.7 ± 3.7 NTG vs. 46.3 ± 8.4 cMyBP-CΔC10mut; P < 0.05), indicating the presence of an HCM phenotype. To assess whether these changes manifested at the myofilament level, contractile function of single skinned cardiomyocytes was measured. Preserved maximum force generation and increased Ca2+-sensitivity of force generation were observed in cardiomyocytes from cMyBP-CΔC10mut mice compared with NTG controls (EC50: 3.6 ± 0.02 µM NTG vs. 2.90 ± 0.01 µM cMyBP-CΔC10mut; P < 0.0001). CONCLUSION: Expression of cMyBP-C protein with a modified C10 domain is sufficient to cause contractile dysfunction and HCM in vivo.

Pro-Survival Lipid Sphingosine-1-Phosphate Metabolically Programs T Cells to Limit Anti-tumor Activity.

Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid generated by sphingosine kinase 1 (SphK1), regulates lymphocyte egress into circulation via S1P receptor 1 (S1PR1) signaling, and it controls the differentiation of regulatory T cells (Tregs) and T helper-17 cells. However, the mechanisms by which receptor-independent SphK1-mediated intracellular S1P levels modulate T cell functionality remains unknown. We show here that SphK1-deficient T cells maintain central memory phenotype and exhibit higher mitochondrial respiration and reduced differentiation to Tregs. Mechanistically, we discovered a direct correlation between SphK1-generated S1P and lipid transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) activity, which in turn regulates lipolysis in T cells. Genetic and pharmacologic inhibition of SphK1 improved metabolic fitness and anti-tumor activity of T cells against murine melanoma. Further, inhibition of SphK1 and PD1 together led to improved control of melanoma. Overall, these data highlight the clinical potential of limiting SphK1/S1P signaling for enhancing anti-tumor-adoptive T cell therapy.