Marine sediments are the largest carbon sink on earth. Nearly half of dark carbon fixation in the oceans occurs in coastal sediments, but the microorganisms responsible are largely unknown. By ...integrating the 16S rRNA approach, single-cell genomics, metagenomics and transcriptomics with (14)C-carbon assimilation experiments, we show that uncultured Gammaproteobacteria account for 70-86% of dark carbon fixation in coastal sediments. First, we surveyed the bacterial 16S rRNA gene diversity of 13 tidal and sublittoral sediments across Europe and Australia to identify ubiquitous core groups of Gammaproteobacteria mainly affiliating with sulfur-oxidizing bacteria. These also accounted for a substantial fraction of the microbial community in anoxic, 490-cm-deep subsurface sediments. We then quantified dark carbon fixation by scintillography of specific microbial populations extracted and flow-sorted from sediments that were short-term incubated with (14)C-bicarbonate. We identified three distinct gammaproteobacterial clades covering diversity ranges on family to order level (the Acidiferrobacter, JTB255 and SSr clades) that made up >50% of dark carbon fixation in a tidal sediment. Consistent with these activity measurements, environmental transcripts of sulfur oxidation and carbon fixation genes mainly affiliated with those of sulfur-oxidizing Gammaproteobacteria. The co-localization of key genes of sulfur and hydrogen oxidation pathways and their expression in genomes of uncultured Gammaproteobacteria illustrates an unknown metabolic plasticity for sulfur oxidizers in marine sediments. Given their global distribution and high abundance, we propose that a stable assemblage of metabolically flexible Gammaproteobacteria drives important parts of marine carbon and sulfur cycles.
Summary
Members of the Bacteroidetes, formerly known as the Cytophaga‐Flavobacteria‐Bacteroides (CFB) phylum, are among the major taxa of marine heterotrophic bacterioplankton frequently found on ...macroscopic organic matter particles (marine snow). In addition, they have been shown to also represent a significant part of free‐living microbial assemblages in nutrient‐rich microenvironments. Their abundance and distribution pattern in combination with enzymatic activity studies has led to the notion that organisms of this group are specialists for degradation of high molecular weight compounds in both the dissolved and particulate fraction of the marine organic matter pool, implying a major role of Bacteroidetes in the marine carbon cycle. Despite their ecological importance, comprehensive molecular data on organisms of this group have been scarce so far. Here we report on the first whole genome analysis of a marine Bacteroidetes representative, ‘Gramella forsetii’ KT0803. Functional analysis of the predicted proteome disclosed several traits which in joint consideration suggest a clear adaptation of this marine Bacteroidetes representative to the degradation of high molecular weight organic matter, such as a substantial suite of genes encoding hydrolytic enzymes, a predicted preference for polymeric carbon sources and a distinct capability for surface adhesion.
Members of the Bacteroidetes, formerly known as the Cytophaga-Flavobacteria-Bacteroides (CFB) phylum, are among the major taxa of marine heterotrophic bacterioplankton frequently found on macroscopic ...organic matter particles (marine snow). In addition, they have been shown to also represent a significant part of free-living microbial assemblages in nutrient-rich microenvironments. Their abundance and distribution pattern in combination with enzymatic activity studies has led to the notion that organisms of this group are specialists for degradation of high molecular weight compounds in both the dissolved and particulate fraction of the marine organic matter pool, implying a major role of Bacteroidetes in the marine carbon cycle. Despite their ecological importance, comprehensive molecular data on organisms of this group have been scarce so far. Here we report on the first whole genome analysis of a marine Bacteroidetes representative, 'Gramella forsetii' KT0803. Functional analysis of the predicted proteome disclosed several traits which in joint consideration suggest a clear adaptation of this marine Bacteroidetes representative to the degradation of high molecular weight organic matter, such as a substantial suite of genes encoding hydrolytic enzymes, a predicted preference for polymeric carbon sources and a distinct capability for surface adhesion.
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•26E01 is a novel inhibitor of TRPV3 channels.•TRPV3 is functionally expressed in the DLD-1 colon carcinoma cell line.•26E01 can inhibit TRPV3 in primary colonic epithelial cells.
...TRPV3 is a Ca2+-permeable cation channel, prominently expressed by keratinocytes where it contributes to maintaining the skin barrier, skin regeneration, and keratinocyte differentiation. However, much less is known about its physiological function in other tissues and there is still a need for identifying novel and efficient TRPV3 channel blockers. By screening a compound library, we identified 26E01 as a novel TRPV3 blocker. 26E01 blocks heterologously expressed TRPV3 channels overexpressed in HEK293 cells as assessed by fluorometric intracellular free Ca2+ assays (IC50 = 8.6 μM) but does not affect TRPV1, TRPV2 or TRPV4 channels. Electrophysiological whole-cell recordings confirmed the reversible block of TRPV3 currents by 26E01, which was also effective in excised inside-out patches, hinting to a rather direct mode of action. 26E01 suppresses endogenous TRPV3 currents in the mouse 308 keratinocyte cell line and in the human DLD-1 colon carcinoma cell line (IC50 = 12 μM). In sections of the gastrointestinal epithelium of mice, the expression of TRPV3 mRNA follows a gradient along the gastrointestinal tract, with the highest expression in the distal colon. 26E01 efficiently attenuates 2-aminoethoxydiphenyl borate-induced calcium influx in primary colonic epithelial cells isolated from the distal colon. As 26E01 neither shows toxic effects on DLD-1 cells at concentrations of up to 100 μM in MTT assays nor on mouse primary colonic crypts as assessed by calcein-AM/propidium iodide co-staining, it may serve as a useful tool to further study the physiological function of TRPV3 in various tissues.
BackgroundSkin cancers are known for their strong immunogenicity, which may contribute to a high treatment efficacy of immune checkpoint inhibition (ICI). However, a considerable proportion of ...patients with skin cancer is immuno-compromised by concomitant diseases. Due to their previous exclusion from clinical trials, the ICI treatment efficacy is poorly investigated in these patients. The present study analyzed the ICI treatment outcome in advanced patients with skin cancer with a concomitant hematological malignancy.MethodsThis retrospective multicenter study included patients who were treated with ICI for locally advanced or metastatic melanoma (MM), cutaneous squamous cell carcinoma (cSCC), or Merkel cell carcinoma (MCC), and had a previous diagnosis of a hematological malignancy irrespective of disease activity or need of therapy at ICI treatment start. Comparator patient cohorts without concomitant hematological malignancy were extracted from the prospective multicenter skin cancer registry ADOREG. Treatment outcome was measured as best overall response, progression-free (PFS), and overall survival (OS).Results84 patients (MM, n=52; cSCC, n=15; MCC, n=17) with concomitant hematological malignancy were identified at 20 skin cancer centers. The most frequent concomitant hematological malignancies were non-Hodgkin’s lymphoma (n=70), with chronic lymphocytic leukemia (n=32) being the largest entity. While 9 patients received ICI in an adjuvant setting, 75 patients were treated for advanced non-resectable disease (55 anti-PD-1; 8 anti-PD-L1; 5 anti-CTLA-4; 7 combinations). In the latter 75 patients, best objective response (complete response+partial response) was 28.0%, disease stabilization was 25.3%, and 38.6% showed progressive disease (PD). Subdivided by skin cancer entity, best objective response was 31.1% (MM), 26.7% (cSCC), and 18.8% (MCC). Median PFS was 8.4 months (MM), 4.0 months (cSCC), and 5.7 months (MCC). 1-year OS rates were 78.4% (MM), 65.8% (cSCC), and 47.4% (MCC). Comparison with respective ADOREG patient cohorts without hematological malignancy (n=392) revealed no relevant differences in ICI therapy outcome for MM and MCC, but a significantly reduced PFS for cSCC (p=0.002).ConclusionsICI therapy showed efficacy in advanced patients with skin cancer with a concomitant hematological malignancy. Compared with patients without hematological malignancy, the observed ICI therapy outcome was impaired in cSCC, but not in MM or MCC patients.
Nuclear orphan receptor NR4A1 exerts an essential tumor suppressor function in aggressive lymphomas. In this study, we investigated the hypothesized contribution of the related NR4A family member ...NR4A3 to lymphomagenesis. In aggressive lymphoma patients, low expression of NR4A3 was associated with poor survival. Ectopic expression or pharmacological activation of NR4A3 in lymphoma cell lines led to a significantly higher proportion of apoptotic cells. In a mouse NSG xenograft model of lymphoma (stably transduced SuDHL4 cells), NR4A3 expression abrogated tumor growth, compared with vector control and uninduced cells that formed massive tumors. Transcript analysis of four different aggressive lymphoma cell lines overexpressing either NR4A3 or NR4A1 revealed that apoptosis was driven similarly by induction of BAK, Puma, BIK, BIM, BID, and Trail. Overall, our results showed that NR4A3 possesses robust tumor suppressor functions of similar impact to NR4A1 in aggressive lymphomas.
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Dilated cardiomyopathy (DCM) is characterized by ventricular chamber enlargement and impaired myocardial function. Endomyocardial biopsies (EMB) enable immunohistochemical and molecular ...characterization of this disease. However, knowledge about specific molecular patterns and their relation to cardiac function in both ventricles is rare. Therefore, we performed a mass spectrometric analysis of 28 paired EMBs of left (LV) and right ventricles (RV) of patients with DCM or suspected myocarditis allowing quantitative profiling of 743 proteins. We analysed associations between protein abundance of LV and RV as well as the echocardiographic parameters LVEF, TAPSE, LVEDDI, and RVEDDI by linear regression models.
Overall, more LV than RV proteins were associated with LV parameters or with RVEDDI. Most LV and RV proteins increasing in level with impairing of LVEF were annotated to structural components of cardiac tissue. Additionally, a high proportion of LV proteins with metabolic functions decreased in level with decreasing LVEF. Results were validated with LV heart sections of a genetic murine heart failure model.
The study shows, that remodelling and systolic dysfunction in DCM is mirrored by distinct alterations in protein composition of both ventricles. Loss of LV systolic function is reflected predominantly by alterations in proteins assigned to metabolic functions in the LV whereas structural remodelling was more obvious in the RV. Alterations related to intermediate filaments were seen in both ventricles and highlight such proteins as early indicators of LV loss of function.
The present study report protein sets in the RV and the LV being associated with ventricular function and remodelling in DCM. Protein abundances in the LV and the RV emphasize and expand current knowledge on pathophysiological changes in heart failure and DCM. While RV and LV EMBs do not differ concerning diagnostic assessment of inflammatory status and virus persistence, additional information reflecting disease severity associated protein alterations can be gained by EMB protein profiling. RV and LV protein data provided complementary information. The protein pattern of the LV reflects metabolic changes and an impaired energy production, which is associated with the degree of LV systolic dysfunction and remodelling and may yield important information about the disease status in DCM. On the other hand, at this disease stage of DCM with still preserved RV function, RV alterations in structural proteins may reflect myocardial compensatory protective mechanisms for maintenance of structure and cellular function.
The study highlight particular proteins being of interest as heart failure biomarkers in both ventricles which seem to reflect the severity of the disease. Further comparative studies between different HF aetiologies have to evaluate those proteins as markers specific for DCM.
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•Protein profiling of LV and RV EMBs allows molecular insights in the DCM heart.•Linear regression analyses highlight associations between proteins and LVEF or LVEDDI.•DCM is characterized by adaptations of structural proteins in both ventricles.•Impaired LVEF associates with loss of energy producing enzymes in LV only.•Data are a potential starting point for the selection of new therapeutic targets.
Mucous membrane pemphigoid (MMP) is a rare, chronic and often aggressive subepidermal autoimmune blistering disease potentially affecting several mucous membranes with blisters and secondary erosions ...and scars. The pathogenesis of MMP is poorly understood, and the contribution of genetic predispositions, other than HLA class II allele variants to MMP, is unknown. The objective of this study is to identify susceptibility genes for MMP in a British cohort of MMP patients. A GWAS was conducted in a British cohort of 106 MMP patients. Publicly available genotypes of 2900 blood donors of the UK Blood Service and of 6740 individuals of the 1958 British Birth Cohort served as control. Subsequently, putative susceptibility genes were independently replicated in a German cohort of 42 MMP patients. The GWAS found 38 SNPs in 28 haploblocks with an odds ratio >2 reaching genomewide significance (P < 5.7 × 10−7). Replication confirmed an association of MMP with SNPs in rs17203398 (OR: 3.9), located intronically in the β‐galactocerebrosidase gene (GALC) on chromosome 14 and with recessive polymorphisms in rs9936045 (OR: 3.1) in the intergenic region between CASC16 and CHD9 on chromosome 16. The risk of developing MMP is partially genetically determined. SNPs in GALC enhance the risk for MMP, indicating that β‐galactocerebrosidase may be involved in the pathogenesis of MMP. Likewise, impacts of polymorphisms in the intergenic region between CASC16 and CHD9 on the activity of neighbouring genes may facilitate the emergence of MMP. The putative role of both polymorphisms requires functional studies in the future.
Introduction and Aim:
We recently described a reduced NR4A1 and NR4A3 expression chronic lymphocytic leukemia, follicular lymphoma, and diffuse large B-cell lymphoma compared to normal controls. Our ...survival analysis of aggressive lymphomas revealed that low NR4A1 expression was associated with poor cancer specific survival. Over-expression of NR4A1 in lymphoma cell lines led to a significantly higher proportion of lymphoma cells undergoing apoptosis and abrogated tumor growth in xenografts1. The aim of this study is to define the role of NR4A1 as a tumor suppressor in the development of lymphoid malignancies in vivo.
Methods:
To identify, whether the loss of NR4A1 has an impact in Myc driven lymphomagenesis we crossed EµMyc mice with NR4A1-/- mice and performed phenotypical analysis including measurement of tumor development, survival and immunophenotypic determination of the newly developed lymphomas by FACS analysis. To further investigate the impact of NR4A1 loss on the oncogenic potential of EµMyc lymphoma cells we isolated viable tumor cells (B220+ and 7AAD-) and cultured them for 72h with or without lipopolysaccharide (LPS) and determined the number of viable cells and their viability (B220 and 7AAD-staining by flow cytometry analysis) after 24h, 48h and 72h. Finally, expression levels of NR4A1, NR4A3 and Myc with or without NR4A1 loss were evaluated by using RT-qPCR.
Results:
EµMyc mice with NR4A1 loss (EµMyc NR4A1-/-, n=46) developed visible tumors significantly faster compared to EµMyc mice with NR4A1 (EµMyc NR4A1+/+, n=75) (median = 44 days for EµMyc NR4A1 -/- vs. 107 days for EµMyc NR4A1+/+; p<0.001). Additionally, EµMyc NR4A1-/- mice showed a significantly shorter life span (median survival = 77 days) compared to EµMyc NR4A1 +/+ mice (median survival = 156 days; p<0.001). By comparing the immunophenotype of the newly developed lymphoma between the two groups (EµMyc NR4A1+/+, n=17 and EµMyc NR4A1 /- , n=19), no significant difference was observed. Interestingly, EµMyc NR4A1-/- mice showed an increased frequency of strong CD93 expression (10 of 18, respectively, vs. 2 of 17 EµMyc NR4A1 +/+ mice, p=0.004). Since most of the EµMyc NR4A1-/- lymphoma were IgM negative (7 of 10) it might be speculated that NR4A1 loss leads to a more immature phenotype of the lymphoma. The number of viable B220+ lymphoma cells isolated from EµMyc NR4A1-/- mice was higher compared to B220+ lymphoma cells isolated from EµMyc NR4A1+/+ mice after 72h in culture with or without LPS (p=0.056; p=0,052). This was accompagnied by a higher in vitro proliferation rate as demonstrated by a higher percentage of BrdU positive cells of the EµMyc NR4A1-/- mice compared to B220+ EµMyc NR4A1+/+ cells with and without LPS stimulation (p= 0,064, p=0,038). Interestingly, we detected a 12 fold higher NR4A3 mRNA expression (p=0,038) in EµMyc NR4A1-/- tumors compared to EµMyc NR4A1+/+.
Conclusion: Our data demonstrate that NR4A1 possesses tumor suppressive properties and that loss of NR4A1 accelerates Myc driven lymphomagenesis. Furthermore, this study indicates that deletion of NR4A1 confers a more aggressive behavior and increases the oncogenic potential of EµMyc driven lymphoma cells.
1. Deutsch AJ, Rinner B, Wenzl K, et al. NR4A1-mediated apoptosis suppresses lymphomagenesis and is associated with a favorable cancer specific survival in patients with aggressive B-cell lymphomas. Blood. 2014.
No relevant conflicts of interest to declare.
Pharmaceutical compounds enter the body via several major natural gateways; i.e. the lung, the skin and the gastrointestinal tract. Drug application during surgical operations can lead to severe ...impairment of gastrointestinal motility, which can contribute to a paralytic ileus. Here we investigated an ex vivo perfused small intestine model that allows us to ascertain the influence of pharmaceuticals upon the gut.
Corresponding segments from the proximal jejunum of adult rats were used. Their mesenteric arteries and veins were cannulated and the jejunal segment excised. The individual segments were placed in a custom designed perfusion chamber and perfusion performed through the intestinal lumen as well as the mesenteric superior artery. Three test drugs, which are commonly used in anesthesiology; i.e. pentobarbital, propofol and ketamine were administered via the blood vessels. Their effects upon gastrointestinal motility patterns were evaluated by optical measurements. Longitudinal and pendular movements were distinguishable and separately analyzed.
Pharmacological effects of the individual substances could be investigated. Propofol (50–200μg/ml) was found to decrease intestinal motility, especially longitudinal movements in a dose dependent manner. Pentobarbital decreased intestinal motility only at high concentrations, above 2.5mg/ml. A dose of 2.5mg/ml lead to an increase in longitudinal- and pendular movements in comparison to control, while ketamine (2.5–10mg/ml) did not alter intestinal motility at all. Histological examination of the perfused segments revealed only minor changes in tissue morphology after perfusion.
The perfusion approach shown here allows for the identification of compounds which interfere with gut motility in a highly sophisticated way. It is suitable for characterization of drug and dose specific changes in motility patterns and can be used in drug development and preclinical studies.
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