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Background: The vast majority of patients with ROS1 positive non-small cell lung cancer (NSCLC) derive clinical benefit from currently approved ROS1 therapies, including crizotinib and entrectinib. However, a small proportion of patients treated with ROS1 inhibitors fail to derive any clinical benefit and demonstrate rapid disease progression. The biological mechanisms underpinning intrinsic resistance remain poorly understood for oncogene-driven cancers. Methods: We generated a patient-derived cell line, CUTO33, from a ROS1 therapy naive patient with CD74-ROS1+ NSCLC, who ultimately did not respond to a ROS1 inhibitor. We evaluated a panel of ROS1+ patient-derived NSCLC cell lines and used cell-based assays to determine the mechanism of intrinsic resistance to ROS1 therapy. Results: The CUTO33 cell line expressed the CD74-ROS1 gene fusion at the RNA and protein level. The ROS1 fusion protein was phosphorylated at baseline consistent with the known intrinsic activity of this oncogene. ROS1 phosphorylation could be inhibited using a wide array of ROS1 inhibitors, however these inhibitors did not block cell proliferation, confirming intrinsic resistance in this model and consistent with the patient’s lack of response to a ROS1 inhibitor. CUTO33 expressed high levels of AXL, which has been associated with drug resistance. Combination of an AXL inhibitor or AXL knockdown with a ROS1 inhibitor partially reversed resistance. Conclusions: In summary, we demonstrate that AXL overexpression is a mechanism of intrinsic resistance to ROS1 inhibitors. [ABSTRACT FROM AUTHOR]

Cadherins (calcium-dependent adhesion proteins) are important in cellular adhesion and may play a role in the development and progression of renal cell carcinoma (RCC). This study investigated changes in cadherin 3 (CDH3; P-cadherin) mRNA expression, DNA methylation, and protein expression in RCC and compared the results with the histopathological and clinical characteristics of patients. The possible contribution of CDH3 to tumor cell invasiveness was tested in a functional assay using siRNA-based suppression of CDH3 expression and subsequent real-time impedance analysis using a Matrigel invasion model. Our analyses revealed a tumor-specific loss of CDH3 mRNA expression, CDH3 DNA hypermethylation, and loss of distal tubular and collecting duct CDH3 protein expression in RCC. A relatively higher methylation level in tumors was associated with a loss of cell differentiation and higher clinical stage. siRNA-induced suppression of CDH3 expression modulated the invasion characteristics of tumor cells in the impedance-based real-time cellular analysis. Our results indicate that loss of CDH3 expression is common in RCC and may contribute to the pathogenesis of a subset of RCC. Further studies to reveal the mechanisms of loss of expression and its effects on the invasive behavior of renal tumor cells are required. [ABSTRACT FROM AUTHOR]

Previous studies have conducted time course characterization of murine colitis models through transcriptional profiling of differential expression. We characterize the transcriptional landscape of acute and chronic models of dextran sodium sulfate (DSS) and adoptive transfer (AT) colitis to derive temporal gene expression and splicing signatures in blood and colonic tissue in order to capture dynamics of colitis remission and relapse. We identify sub networks of patient-derived causal networks that are enriched in these temporal signatures to distinguish acute and chronic disease components within the broader molecular landscape of IBD. The interaction between the DSS phenotype and chronological time-point naturally defines parsimonious temporal gene expression and splicing signatures associated with acute and chronic phases disease (as opposed to ordinary time-specific differential expression/splicing). We show these expression and splicing signatures are largely orthogonal, i.e. affect different genetic bodies, and that using machine learning, signatures are predictive of histopathological measures from both blood and intestinal data in murine colitis models as well as an independent cohort of IBD patients. Through access to longitudinal multi-scale profiling from disease tissue in IBD patient cohorts, we can apply this machine learning pipeline to generation of direct patient temporal multimodal regulatory signatures for prediction of histopathological outcomes. Longitudinal changes in gene expression, splicing patterns and adaptive immune repertoire in murine models of colitis are examined and associated with histologic inflammation in mice and in patients with inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

Septoria nodorum blotch (SNB) is a foliar disease of wheat caused by the necrotrophic fungal pathogen Parastagonospora nodorum. Research over the last two decades has shown that the wheat-P. nodorum pathosystem mostly follows an inverse gene-for-gene model. The fungus produces necrotrophic effectors (NEs) that interact with specific host gene products encoded by dominant sensitivity (S) genes. When a compatible interaction occurs, a 'defense response' in the host leads to programmed cell death thereby provided dead/dying cells from which the pathogen, being a necrotroph, can acquire nutrients allowing it to grow and sporulate. To date, nine S gene-NE interactions have been characterized in this pathosystem. Five NE-encoding genes, SnTox1, SnTox3, SnToxA, SnTox5, and SnTox267, have been cloned along with three host S genes, Tsn1, Snn1, and Snn3-D1. Studies have shown that P. nodorum hijacks multiple and diverse host targets to cause disease. SNB resistance is often quantitative in nature because multiple compatible interactions usually occur concomitantly. NE gene expression plays a key role in disease severity, and the effect of each compatible interaction can vary depending on the other existing compatible interactions. Numerous SNB-resistance QTL have been identified in addition to the known S genes, and more research is needed to understand the nature of these resistance loci. Marker-assisted elimination of S genes through conventional breeding practices and disruption of S genes using gene editing techniques are both effective strategies for the development of SNB-resistant wheat cultivars, which will become necessary as the global demand for sustenance grows. [ABSTRACT FROM AUTHOR]

Background: Cancer recurrence after tumor resection in early-stage non-small cell lung cancer (NSCLC) is common, yet difficult to predict. The lung microbiota and systemic immunity may be important modulators of risk for lung cancer recurrence, yet biomarkers from the lung microbiome and peripheral immune environment are understudied. Such markers may hold promise for prediction as well as improved etiologic understanding of lung cancer recurrence. Methods: In tumor and distant normal lung samples from 46 stage II NSCLC patients with curative resection (39 tumor samples, 41 normal lung samples), we conducted 16S rRNA gene sequencing. We also measured peripheral blood immune gene expression with nanoString®. We examined associations of lung microbiota and peripheral gene expression with recurrence-free survival (RFS) and disease-free survival (DFS) using 500 × 10-fold cross-validated elastic-net penalized Cox regression, and examined predictive accuracy using time-dependent receiver operating characteristic (ROC) curves. Results: Over a median of 4.8 years of follow-up (range 0.2–12.2 years), 43% of patients experienced a recurrence, and 50% died. In normal lung tissue, a higher abundance of classes Bacteroidia and Clostridia, and orders Bacteroidales and Clostridiales, were associated with worse RFS, while a higher abundance of classes Alphaproteobacteria and Betaproteobacteria, and orders Burkholderiales and Neisseriales, were associated with better RFS. In tumor tissue, a higher abundance of orders Actinomycetales and Pseudomonadales were associated with worse DFS. Among these taxa, normal lung Clostridiales and Bacteroidales were also related to worse survival in a previous small pilot study and an additional independent validation cohort. In peripheral blood, higher expression of genes TAP1, TAPBP, CSF2RB, and IFITM2 were associated with better DFS. Analysis of ROC curves revealed that lung microbiome and peripheral gene expression biomarkers provided significant additional recurrence risk discrimination over standard demographic and clinical covariates, with microbiome biomarkers contributing more to short-term (1-year) prediction and gene biomarkers contributing to longer-term (2–5-year) prediction. Conclusions: We identified compelling biomarkers in under-explored data types, the lung microbiome, and peripheral blood gene expression, which may improve risk prediction of recurrence in early-stage NSCLC patients. These findings will require validation in a larger cohort. [ABSTRACT FROM AUTHOR]

Since cost-effective osteoanabolic treatment options remain to be established, it is relevant to identify specific molecules physiologically regulating osteoblast differentiation and/or activity that are principally accessible as drug targets. Specific or predominant gene expression in a given cell type often predicts a relevant function in the respective tissue. Thus, we aimed to identify genes encoding membrane-associated proteins with selective expression in differentiated osteoblasts. We therefore applied an unbiased approach, i.e. Affymetrix Gene Chip hybridization, to compare global gene expression in primary murine osteoblasts at two stages of differentiation. For the most strongly induced genes we analyzed their expression pattern in different tissues, which led us to identify known and unknown osteoblast differentiation markers with predominant expression in bone. One of these genes was Panx3 , encoding a transmembrane hemichannel with ill-defined function in skeletal remodeling. To decipher the role of Panx3 in osteoblasts we first generated Panx3-fl/fl mice carrying a Runx2-Cre transgene. Using undecalcified histology followed by bone-specific histomorphometry we did not observe any significant difference between 24 weeks old Cre-negative and Cre-positive littermates. We additionally generated and analyzed mice with ubiquitous Panx3 deletion, where a delay of endochondral ossification did not translate into a detectable skeletal phenotype after weaning, possibly explained by compensatory induction of Panx1. Of note, newborn Panx3 -deficient mice displayed significantly reduced serum glucose levels, which was not the case in older animals. Our findings demonstrate that Panx3 expression in osteoblasts is not required for postnatal bone remodeling, which essentially rules out its suitability as a target protein for osteoanabolic medication. • Panx3 was identified as a gene with predominant expression in bone using an unbiased screening approach. • Among the known Panx genes, only Panx3 displays selective expression in mature osteoblasts. • Mice with an osteoblast-specific inactivation of Panx3 do not display impaired bone remodeling. • Panx3-deficient mice display delayed endochondral ossification, increased postnatal mortality, but no skeletal phenotype after weaning. [ABSTRACT FROM AUTHOR]

Might the 2018 birth of two designer babies in China write the opening paragraph for the next chapter in the history of eugenics? The worldwide scientific community has tacitly put a moratorium on human clinical application of CRISPR gene editing, waiting until unknown risks can become known. But this ethical agreement has been breached, and calls are now being heard for more rigorous regulations. Perhaps religious and spiritual leaders can join the bioethical chant: the yellow light of caution is flashing. [ABSTRACT FROM AUTHOR]

Chronic inflammation in inflammatory bowel disease (IBD) results from a breakdown of intestinal immune homeostasis and compromise of the intestinal barrier. Genome-wide association studies have identified over 200 genetic loci associated with risk for IBD, but the functional mechanisms of most of these genetic variants remain unknown. Polymorphisms at the TNFSF15 locus, which encodes the TNF superfamily cytokine commonly known as TL1A, are associated with susceptibility to IBD in multiple ethnic groups. In a wide variety of murine models of inflammation including models of IBD, TNFSF15 promotes immunopathology by signaling through its receptor DR3. Such evidence has led to the hypothesis that expression of this lymphocyte costimulatory cytokine increases risk for IBD. In contrast, here we show that the IBD-risk haplotype at TNFSF15 is associated with decreased expression of the gene by peripheral blood monocytes in both healthy volunteers and IBD patients. This association persists under various stimulation conditions at both the RNA and protein levels and is maintained after macrophage differentiation. Utilizing a “recall-by-genotype” bioresource for allele-specific expression measurements in a functional fine-mapping assay, we localize the polymorphism controlling TNFSF15 expression to the regulatory region upstream of the gene. Through a T cell costimulation assay, we demonstrate that genetically regulated TNFSF15 has functional relevance. These findings indicate that genetically enhanced expression of TNFSF15 in specific cell types may confer protection against the development of IBD. [ABSTRACT FROM AUTHOR]

Metabolic engineering increasingly depends upon RNA technology to customly rewire the metabolism to maximize production. To this end, pure riboregulators allow dynamic gene repression without the need of a potentially burdensome coexpressed protein like typical Hfq binding small RNAs and clustered regularly interspaced short palindromic repeats technology. Despite this clear advantage, no clear general design principles are available to de novo develop repressing riboregulators, limiting the availability and the reliable development of these type of riboregulators. Here, to overcome this lack of knowledge on the functionality of repressing riboregulators, translation inhibiting RNAs are developed from scratch. These de novo developed riboregulators explore features related to thermodynamical and structural factors previously attributed to translation initiation modulation. In total, 12 structural and thermodynamic features were defined of which six features were retained after removing correlations from an in silico generated riboregulator library. From this translation inhibiting RNA library, 18 riboregulators were selected using a experimental design and subsequently constructed and co-expressed with two target untranslated regions to link the translation inhibiting RNA features to functionality. The pure riboregulators in the design of experiments showed repression down to 6% of the original protein expression levels, which could only be partially explained by a ordinary least squares regression model. To allow reliable forward engineering, a partial least squares regression model was constructed and validated to link the properties of translation inhibiting RNA riboregulators to gene repression. In this model both structural and thermodynamic features were important for efficient gene repression by pure riboregulators. This approach enables a more reliable de novo forward engineering of effective pure riboregulators, which further expands the RNA toolbox for gene expression modulation. [ABSTRACT FROM AUTHOR]

The main mechanisms of pathogenesis of clear cell renal cell carcinoma (CCRCC) are realized through the PI3K-AKT-mTOR and Ras-RAF-ERK signaling pathways. Targeted therapy is directed primarily at the genes and their encoded products that are components of these pathways. The levels of expression and coexpression of target genes were determined, and the difference in the functioning of the genes of one of the two major signaling pathways in tumors of CCRCC patients with different life duration (more and less than 3.5 years) and the relationship of the VEGFA gene expression level with the life duration was revealed. [ABSTRACT FROM AUTHOR]

The cyclin-dependent kinase inhibitor p21 is an important player in stress pathways exhibiting both tumor-suppressive and oncogenic functions. Thus, expression of p21 has to be tightly controlled, which is achieved by numerous mechanisms at the transcriptional, translational, and posttranslational level. Performing immunoprecipitation of bromouridine-labeled p21 mRNAs that had been incubated before with cytoplasmic extracts of untreated HCT116 colon carcinoma cells, we identified the DEAD-box RNA helicase DDX41 as a novel regulator of p21 expression. DDX41 specifically precipitates with the 3'UTR, but not with the 5'UTR, of p21 mRNA. Knockdown of DDX41 increases basal and γ irradiation-induced p21 protein levels without affecting p21 mRNA expression. Conversely, overexpression of DDX41 strongly inhibits expression of a FLAG-p21 and a luciferase construct, but only in the presence of the p21 3'UTR. Together, these data suggest that this helicase regulates p21 expression at the translational level independent of the transcriptional activity of p53. However, knockdown of DDX41 completely fails to increase p21 protein levels in p53-deficient HCT116 cells. Moreover, posttranslational up-regulation of p21 achieved in both p53+/+ and p53-/- HCT116 cells in response to pharmaceutical inhibition of the proteasome (by MG-132) or p90 ribosomal S6 kinases (by BI-D1870) is further increased by knockdown of DDX41 only in p53-proficient but not in p53-deficient cells. Although our data demonstrate that DDX41 suppresses p21 translation without disturbing the function of p53 to directly induce p21 mRNA expression, this process indirectly requires p53, perhaps in the form of another p53 target gene or as a still undefined posttranscriptional function of p53. [ABSTRACT FROM AUTHOR]

Environmental enrichment (EE) is widely used in the life sciences to study effects of environment on the brain. In pigs, despite lack of EE being a key welfare issue there is little understanding of brain effects of EE in pigs. This project aimed to study the effects of exposure to an EE arena on piglet behaviours and on brain gene expression levels with a focus on IGF-1 and related genes. Eight litters of large white × landrace × Hampshire piglets were farrowed and raised in a free farrowing system (PigSAFE). At 42 days of age, 6 piglets per litter were given access to an enriched arena with plentiful peat, straw and space, (in groups of 4 made up of stable pairs) for 15 min per day on 5 consecutive days to allow them to habituate to the apparatus. Piglet behaviours were recorded in the arena for 15 min periods on 3 consecutive days. On the final day only one pair of test piglets per litter was given access to the arena. Brain tissue was collected within 45 min of the test from piglets exposed to the arena on the day and their non-exposed littermate controls. RNA was extracted from the frontal cortex and QRT-PCR for selected genes run on a Stratgene MX3005P. In both the home pen and the EE arena litters spent the largest proportion of time engaging in foraging behaviour which was significantly increased in the enriched arena ( t 7 = 5.35, df = 6, p = 0.001). There were decreases in non-running play ( t 7 = 4.82, p = 0.002) and inactivity ( t 7 = 4.6, p = 0.002) in the arena. A significant fold change increase (FC = 1.07, t = 4.42, p = 0.002) was observed in IGF-1 gene expression in the frontal cortex of piglets exposed to the enriched arena compared to those not exposed on the day of culling. No change in expression was observed in CSF1 , the IGF-1 receptor gene nor in any of the binding proteins tested ( IGFBP1-6 ). There was a weak tendency for increased expression of the neurotrophic factor BDNF1 (fold change: 1.03; t 7 = 1.54, p = 0.1). We believe this work is the first to explore effects of EE on pig brain physiology and development, and also points to a potential role for IGF-1 in brain effects of EE. [ABSTRACT FROM AUTHOR]

Aim To evaluate the effects of two types of calcium silicate cements on viability, angiogenic growth factor release, and angiogenic and inflammation-related gene expression in human stem cells from the apical papilla ( SCAP). Methodology SCAPs were grown for 7 days with either ProRoot mineral trioxide aggregate ( MTA) or Biodentine ( BD). Cell viability and media concentrations of vascular endothelial growth factor ( VEGF/ VEGFA) and angiopoietin 1 ( ANGPT1) were measured. The expression of genes related to angiogenic potential and inflammatory response was measured by quantitative reverse transcription-polymerase chain reaction ( qRT- PCR). One-way and two-way analyses of variance with multiple comparisons Tukey's test were performed ( P < 0.05). Results Cells in contact with either cement were associated with increased cell viability compared with the no-treatment group at day 1 but there were no differences amongst groups at days 3 and 7. Exposure to either cement significantly increased VEGF concentrations at day 3; however, ANGPT-1 levels decreased significantly compared with the no-treatment group at day 3. Exposure to MTA and BD stimulated expression of VEGFA and FIGF/ VEGFD. Furthermore, exposure to both cements significantly decreased the mRNA levels of ANGPT1 and FGF2 relative to the no-treatment group. Conclusions Both MTA and BD stimulated the expression of angiogenic genes and release of VEGF, inducing similar expression patterns; however, they appeared to inhibit the expression of specific genes, including ANGPT1 and FGF2. [ABSTRACT FROM AUTHOR]

Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1 -deleted individuals carrying 3–4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression—a situation resembling the human condition in asymptomatic SMN1 -deleted individuals—rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA. [ABSTRACT FROM AUTHOR]

We analyzed association of the levels of VEGFA, RAF1, and mTOR gene expression in the tissue of clear-cell renal cell carcinoma (ccRCC) with tumor metastasizing. Significant association with metastases was found only for VEGFA gene: OR=6.641, 95%CI=2.111-20.696. The risk of metastasis associated with reduced expression of VEGFA gene — 2.467, 95%CI=1.238-4.915. An association of VEGFA gene expression with the time to the metastasis appearance was revealed (p=0.0005). Reduced expression of the VEGFA gene is associated with reduction of the time to metastasis appearance; the median of this time is shifted from 46 to 2 months. Analysis of tumor samples with reduced expression of the VEGFA gene revealed association of increased expression of RAF1 (p=0.003) and mTOR genes (p=0.038) with metastasis. [ABSTRACT FROM AUTHOR]

Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. [ABSTRACT FROM AUTHOR]

Genome-wide association studies (GWAS) have transformed our understanding of the genetics of complex traits such as autoimmune diseases, but how risk variants contribute to pathogenesis remains largely unknown. Identifying genetic variants that affect gene expression (expression quantitative trait loci, or eQTLs) is crucial to addressing this. eQTLs vary between tissues and following in vitro cellular activation, but have not been examined in the context of human inflammatory diseases. We performed eQTL mapping in five primary immune cell types from patients with active inflammatory bowel disease (n = 91), anti-neutrophil cytoplasmic antibody-associated vasculitis (n = 46) and healthy controls (n = 43), revealing eQTLs present only in the context of active inflammatory disease. Moreover, we show that following treatment a proportion of these eQTLs disappear. Through joint analysis of expression data from multiple cell types, we reveal that previous estimates of eQTL immune cell-type specificity are likely to have been exaggerated. Finally, by analysing gene expression data from multiple cell types, we find eQTLs not previously identified by database mining at 34 inflammatory bowel disease-associated loci. In summary, this parallel eQTL analysis in multiple leucocyte subsets from patients with active disease provides new insights into the genetic basis of immune-mediated diseases. [ABSTRACT FROM AUTHOR]

Background: Long non-coding RNAs (lncRNAs) are a class of RNA molecules with diverse regulatory functions during embryonic development, normal life, and disease in higher organisms. However, research on the role of lncRNAs in cardiovascular diseases and in particular heart failure is still in its infancy. The exceptionally well conserved nuclear lncRNA Metastasis associated in lung adenocarcinoma transcript 1 (Malat-1) is a regulator of mRNA splicing and highly expressed in the heart. Malat-1 modulates hypoxia-induced vessel growth, activates ERK/MAPK signaling, and scavenges the anti-hypertrophic microRNA-133. We therefore hypothesized that Malat-1 may act as regulator of cardiac hypertrophy and failure during cardiac pressure overload induced by thoracic aortic constriction (TAC) in mice. Results: Absence of Malat-1 did not affect cardiac hypertrophy upon pressure overload: Heart weight to tibia length ratio significantly increased in WT mice (sham: 5.78±0.55, TAC 9.79±1.82 g/mm; p<0.001) but to a similar extend also in Malat-1 knockout (KO) mice (sham: 6.21±1.12, TAC 8.91±1.74 g/mm; p<0.01) with no significant difference between genotypes. As expected, TAC significantly reduced left ventricular fractional shortening in WT (sham: 38.81±6.53%, TAC: 23.14±11.99%; p<0.01) but to a comparable degree also in KO mice (sham: 37.01±4.19%, TAC: 25.98±9.75%; p<0.05). Histological hallmarks of myocardial remodeling, such as cardiomyocyte hypertrophy, increased interstitial fibrosis, reduced capillary density, and immune cell infiltration, did not differ significantly between WT and KO mice after TAC. In line, the absence of Malat-1 did not significantly affect angiotensin II-induced cardiac hypertrophy, dysfunction, and overall remodeling. Above that, pressure overload by TAC significantly induced mRNA levels of the hypertrophy marker genes Nppa, Nppb and Acta1, to a similar extend in both genotypes. Alternative splicing of Ndrg2 after TAC was apparent in WT (isoform ratio; sham: 2.97±0.26, TAC 1.57±0.40; p<0.0001) and KO mice (sham: 3.64±0.37; TAC: 2.24±0.76; p<0.0001) and interestingly differed between genotypes both at baseline and after pressure overload (p<0.05 each). Conclusion: These findings confirm a role for the lncRNA Malat-1 in mRNA splicing. However, no critical role for Malat-1 was found in pressure overload-induced heart failure in mice, despite its reported role in vascularization, ERK/MAPK signaling, and regulation of miR-133. [ABSTRACT FROM AUTHOR]

Altitude exposure and exercise elicit oxidative stress in blood; however, exercise recovery at 5000 m attenuates oxidative stress. The purpose was to determine the altitude threshold at which blood oxidative stress is blunted during exercise recovery. Twelve males 18–28 years performed four-cycle ergometry bouts (60 min, 70% VO2max, at 975 m). In a randomised counterbalanced crossover design, participants recovered 6 h at 0, 1667, 3333 and 5000 m in a normobaric hypoxia chamber (recovery altitudes were simulated by using a computerised system in an environmental chamber by lowering the partial pressure of oxygen to match that of the respective altitude). Oxygen saturation was monitored throughout exercise recovery. Blood samples obtained pre-, post-, 1 h post- and 5 h post-exercise were assayed for ferric-reducing antioxidant plasma, Trolox equivalent antioxidant capacity, uric acid, lipid hydroperoxides and protein carbonyls. Muscle biopsies obtained pre and 6 h were analysed by real-time polymerase chain reaction to quantify expression of hemeoxgenase 1, superoxide dismutase 2 and nuclear factor (euthyroid-derived 2)-like factor. Pulse oximetry data were similar during exercise, but decreased for the three highest recovery elevations (0 m = 0%, 1667 m = −3%; 3333 m = −7%; 5000 m = −17%). A time-dependent oxidative stress occurred following exercise for all variables, but the two highest recovery altitudes partially attenuated the lipid hydroperoxide response (0 m = +135%, 1667 m = +251%, 3333 m = +99%; 5000 m = +108%). Data may indicate an altitude threshold between 1667 and 3333 m, above which the oxidative stress response is blunted during exercise recovery. [ABSTRACT FROM AUTHOR]

Synthetic biology, in close concert with systems biology, is revolutionizing the field of metabolic engineering by providing novel tools and technologies to rationally, in a standardized way, reroute metabolism with a view to optimally converting renewable resources into a broad range of bio-products, bio-materials and bio-energy. Increasingly, these novel synthetic biology tools are exploiting the extensive programmable nature of RNA, vis-à-vis DNA- and protein-based devices, to rationally design standardized, composable, and orthogonal parts, which can be scaled and tuned promptly and at will. This review gives an extensive overview of the recently developed parts and tools for i) modulating gene expression ii) building genetic circuits iii) detecting molecules, iv) reporting cellular processes and v) building RNA nanostructures. These parts and tools are becoming necessary armamentarium for contemporary metabolic engineering. Furthermore, the design criteria, technological challenges, and recent metabolic engineering success stories of the use of RNA devices are highlighted. Finally, the future trends in transforming metabolism through RNA engineering are critically evaluated and summarized. [ABSTRACT FROM AUTHOR]

Histidine-containing dipeptides like carnosine and anserine have protective functions in both health and disease. Animal studies suggest that carnosine can be metabolized within the kidney. The goal of this study was to obtain evidence of carnosine metabolism in the human kidney and to provide insight with regards to diabetic nephropathy. Expression, distribution, and localization of carnosinase-1 (CNDP1), carnosine synthase (CARNS), and taurine transporters (TauT) were measured in human kidneys. CNDP1 and CARNS activities were measured in vitro. CNDP1 and CARNS were located primarily in distal and proximal tubules, respectively. Specifically, CNDP1 levels were high in tubular cells and podocytes (20.3 ± 3.4 and 15 ± 3.2 ng/mg, respectively) and considerably lower in endothelial cells (0.5 ± 0.1 ng/mg). CNDP1 expression was correlated with the degradation of carnosine and anserine ( r = 0.88 and 0.81, respectively). Anserine and carnosine were also detectable by HPLC in the renal cortex. Finally, TauT mRNA and protein were found in all renal epithelial cells. In diabetic patients, CNDP1 seemed to be reallocated to proximal tubules. We report compelling evidence that the kidney has an intrinsic capacity to metabolize carnosine. Both CNDP1 and CARNS are expressed in glomeruli and tubular cells. Carnosine-synthesizing and carnosine-hydrolyzing enzymes are localized in distinct compartments in the nephron and increased CNDP1 levels suggest a higher CNDP1 activity in diabetic kidneys. [ABSTRACT FROM AUTHOR]

Modest protocols of repetitive acute intermittent hypoxia (rAIH) enhance motor function in patients with chronic incomplete spinal injury. Since chronic intermittent hypoxia (CIH) elicits neuroinflammation, there is potential for rAIH to have similar effects. Thus, we tested the hypothesis that rAIH has minimal impact on microglial inflammatory gene expression, but up-regulates key neurotrophic factor expression in a CNS region-specific manner. Using real time PCR, we evaluated mRNA levels of inflammatory and neurotrophic factors in immunomagnetically-isolated microglia from rat frontal cortex, brainstem and upper and lower cervical spinal cord following rAIH (ten, 5-min episodes, thrice weekly, 4 weeks). In agreement with our hypothesis, rAIH had no significant impact on microglial inflammatory gene expression in any region studied. On the other hand, neurotrophic factor expression was altered in a gene- and region-specific pattern. These results have important implications for the safety of rAIH as a potential therapy to enhance neuroplasticity and motor function in patients with spinal injury or other neurologic disorders. [ABSTRACT FROM AUTHOR]

The role of the nuclear hormone receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) in carcinogenesis is controversial because conflicting studies indicate that it both inhibits and promotes tumorigenesis. In this review, we focus on recent studies on PPARβ/δ including the significance of increased or decreased PPARβ/δ expression in cancers; a range of opposing mechanisms describing how PPARβ/δ agonists, antagonists, and inverse agonists regulate tumorigenesis and/or whether there may be cell context-specific mechanisms; and whether activating or inhibiting PPARβ/δ is feasible for cancer chemoprevention and/or therapy. Research questions that need to be addressed are highlighted to establish whether PPARβ/δ can be effectively targeted for cancer chemoprevention. [ABSTRACT FROM AUTHOR]

Age is a major risk factor for neurodegenerative diseases. Shortening of leucocyte telomeres with advancing age, arguably a measure of "biological" age, is a known phenomenon and epidemiologically correlated with age‐related disease. The main mechanism of telomere shortening is cell division, rendering telomere length in post‐mitotic cells presumably stable. Longitudinal measurement of human brain telomere length is not feasible, and cross‐sectional cortical brain samples so far indicated no attrition with age. Hence, age‐related changes in telomere length in the brain and the association between telomere length and neurodegenerative diseases remain unknown. Here, we demonstrate that mean telomere length in the putamen, a part of the basal ganglia, physiologically shortens with age, like leukocyte telomeres. This was achieved by using matched brain and leukocyte‐rich spleen samples from 98 post‐mortem healthy human donors. Using spleen telomeres as a reference, we further found that mean telomere length was brain region‐specific, as telomeres in the putamen were significantly shorter than in the cerebellum. Expression analyses of genes involved in telomere length regulation and oxidative phosphorylation revealed that both region‐ and age‐dependent expression pattern corresponded with region‐dependent telomere length dynamics. Collectively, our results indicate that mean telomere length in the human putamen physiologically shortens with advancing age and that both local and temporal gene expression dynamics correlate with this, pointing at a potential mechanism for the selective, age‐related vulnerability of the nigro‐striatal network. [ABSTRACT FROM AUTHOR]

The mammalian and avian auditory brainstem likely arose by independent evolution. To compare the underlying molecular mechanisms, we focused on Atoh7, as its expression pattern in the mammalian hindbrain is restricted to bushy cells in the ventral cochlear nucleus. We thereby took advantage of an Atoh7 centered gene regulatory network (GRN) in the retina including upstream regulators, Hes1 and Pax6, and downstream targets, Ebf3 and Eya2. In situ hybridization demonstrated for the latter four genes broad expression in all three murine cochlear nuclei at postnatal days (P) 4 and P30, contrasting the restricted expression of Atoh7. In chicken, all five transcription factors were expressed in all auditory hindbrain nuclei at embryonic day (E) 13 and P14. Notably, all five genes showed graded expression in the embryonic nucleus magnocellularis (NM). Atoh7 was highly expressed in caudally located neurons, whereas the other four transcription factors were highly expressed in rostrally located neurons. Thus, Atoh7 shows a strikingly different expression between the mammalian and avian auditory hindbrain. This together with the consistent absence of graded expression of GRN components in developing mammalian nuclei provide the first molecular support to the current view of convergent evolution as a major mechanism in the amniote auditory hindbrain. The graded expression of five transcription factors specifically in the developing NM confirms this nucleus as a central organizer of tonotopic features in birds. Finally, the expression of all five retinal GRN components in the auditory system suggests co-options of genes for development of sensory systems of distinct modalities. [ABSTRACT FROM AUTHOR]

Normal levels of the methyl CpG–binding protein 2 (MeCP2) are critical to neurologic functioning, and slight alterations result in intellectual disability and autistic features. It was hypothesized that children with MECP2 duplication (overexpression of MeCP2) and Rett syndrome (underexpression of MeCP2) would exhibit distinct electroencephalographic (EEG) indices of auditory stimulus discrimination. In this study, gamma-band oscillatory responses to familiar and novel voices were examined and related to social functioning in 17 children (3-11 years old) with MECP2 duplication (n = 12) and Rett syndrome (n = 5). Relative to the stranger’s voice, gamma activity in response to the mother’s voice was increased in MECP2 duplication but decreased in Rett syndrome. In MECP2 duplication, greater mother versus stranger differences in gamma activity were associated with higher social functioning. For the first time, brain responses in a passive voice discrimination paradigm show that overexpression and underexpression of MeCP2 have differential effects on cortical information processing. [ABSTRACT FROM PUBLISHER]

Aging is associated with declines in memory and cognitive function. Here, we evaluate the effects of HT-0712 on memory formation and on cAMP response element-binding protein (CREB)-regulated genes in aged mice. HT-0712 enhanced long-term memory formation in normal young mice at brain concentrations similar to those found to increase CRE-mediated gene expression in hippocampal neurons. Aged mice showed significantly poorer contextual and trace conditioning compared with young-adult mice. In aged mice, a single injection of HT-0712 significantly boosted contextual and trace long-term memory. Additional effects of HT-0712 were seen in a spatial memory task. Our parallel biochemical experiments revealed that inductions of the CREB-regulated genes, cFos, Zif268, and Bdnf, after fear conditioning were diminished in aged mice. HT-0712 facilitated expression of these CREB-regulated genes in aged hippocampus, indicating that the drug engages a CREB-regulated mechanism in vivo. These findings corroborate and extend our previous results on the mechanism of action of HT-0712 and its efficacy to enhance memory formation. Our data also indicate that HT-0712 may be effective to treat age-associated memory impairment in humans. [ABSTRACT FROM AUTHOR]

Thanks to the discovery of novel technologies and sophisticated analysis tools we can now 'see' molecules, genes and even patterns of gene expression, which have resulted in major advances in many areas of biology. Recently, similar technologies have been developed for behavioral studies. However, the wide implementation of such technological progress in behavioral research remains behind, as if there are inhibiting factors for accepting and adopting available innovations. The methods of the majority of studies measuring and interpreting behavior of laboratory animals seem to have frozen in time somewhere in the last century. As an example of the so-called classical tests, we will present the history and shortcomings of one of the most frequently used tests, the open field. Similar objections and critical remarks, however, can be made with regard to the elevated plus maze, light-dark box, various other mazes, object recognition tests, etc. Possible solutions and recommendations on how progress in behavioral neuroscience can be achieved and accelerated will be discussed in the second part of this review. [ABSTRACT FROM AUTHOR]

Genomic imprinting is an epigenetic phenomenon that results in monoallelic gene expression according to parental origin. It has long been established that imprinted genes have major effects on development and placental biology before birth. More recently, it has become evident that imprinted genes also have important roles after birth. In this Review, I bring together studies of the effects of imprinted genes from the prenatal period onwards. Recent work on postnatal stages shows that imprinted genes influence an extraordinarily wide-ranging array of biological processes, the effects of which extend into adulthood, and play important parts in common diseases that range from obesity to psychiatric disorders. [ABSTRACT FROM AUTHOR]

Heart failure as a consequence of ischemic, hypertensive, infectious, or hereditary heart disease is a major challenge in cardiology and topic of intense research. Recently, new players appeared in this field and promise deeper insights into cardiac development, function, and disease. Long non-coding RNAs are a novel class of transcripts that can regulate gene expression and may have many more functions inside the cell. Here, we present examples on long non-coding RNA (lncRNA) function in cardiac development and give suggestions on how lncRNAs may be involved in cardiomyocyte dysfunction, myocardial fibrosis, and inflammation, three hallmarks of the failing heart. Above that, we point out opportunities as well as challenges that should be considered in the endeavor to investigate cardiac lncRNAs. [ABSTRACT FROM AUTHOR]

The proliferation and interleukin-2 production of CD4CD25 αβ T cells were inhibited in a cell-contact manner by Vδ2 γδ T cells. The transcription factor Helios was constitutively expressed in about one-third of circulating γδ T cells and was upregulated by CD28-signaling. Our data suggest that Helios could serve as a marker for differential activation status rather than for regulatory T cells (Treg). Our findings also indicate that the interaction of CD86 on activated Vδ2 T cells and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) on activated αβ T cells mediated the suppression because the suppressive effect was abolished by blocking the CD86:CTLA-4 interaction. Pre-treatment of Vδ2 T cells with Toll-like receptor 2 ligands enhanced phosphorylation of MAPKs, Akt, and NF-κB and partially abrogated the suppressive capacity, whereas on co-cultured responder T cells inhibitory molecules were downregulated and Akt and NF-κB phosphorylation was restored. Our results suggest that the regulation of αβ T cell proliferation by activated Vδ2 T cells might contribute to fine-tuning of αβ T cell responses. [ABSTRACT FROM AUTHOR]

Specific product formation rates and cellular growth rates are important maximization targets in biotechnology and microbial evolution. Maximization of a specific rate (i.e. a rate expressed per unit biomass amount) requires the expression of particular metabolic pathways at optimal enzyme concentrations. In contrast to the prediction of maximal product yields, any prediction of optimal specific rates at the genome scale is currently computationally intractable, even if the kinetic properties of all enzymes are available. In the present study, we characterize maximal-specific-rate states of metabolic networks of arbitrary size and complexity, including genome-scale kinetic models. We report that optimal states are elementary flux modes, which are minimal metabolic networks operating at a thermodynamically-feasible steady state with one independent flux. Remarkably, elementary flux modes rely only on reaction stoichiometry, yet they function as the optimal states of mathematical models incorporating enzyme kinetics. Our results pave the way for the optimization of genome-scale kinetic models because they offer huge simplifications to overcome the concomitant computational problems. [ABSTRACT FROM AUTHOR]

The yellow-pigmented soil bacterium Corynebacterium glutamicum ATCC13032 is accumulating the cyclic C carotenoid decaprenoxanthin and its glucosides. Carotenoid pathway engineering was previously shown to allow for efficient lycopene production. Here, engineering of C. glutamicum for production of endogenous decaprenoxanthin as well as of the heterologous C carotenoids C.p.450 and sarcinaxanthin is described. Plasmid-borne overexpression of genes for lycopene cyclization and hydroxylation from C. glutamicum, Dietzia sp., and Micrococcus luteus, in a lycopene-producing platform strain constructed here, resulted in accumulation of these three C carotenoids to concentrations of about 3-4 mg/g CDW. Chromosomal deletion of a putative carotenoid glycosyltransferase gene cg0730/ crtX in these strains entailed production of non-glucosylated derivatives of decaprenoxanthin, C.p.450, and sarcinaxanthin, respectively. Upon introduction of glucosyltransferase genes from M. luteus, C. glutamicum, and Pantoea ananatis, these hydroxylated C carotenoids were glucosylated. We here also demonstrate production of the C carotenoids β-carotene and zeaxanthin in recombinant C. glutamicum strains and co-expression of the P. ananatis crtX gene was used to obtain glucosylated zeaxanthin. Together, our results show that C. glutamicum is a potentially valuable host for production of a wide range of glucosylated C and C carotenoids. [ABSTRACT FROM AUTHOR]

AIRmax (maximal inflammation) and AIRmin (minimal inflammation) mice show distinct susceptibilities to pristane-induced arthritis (PIA). The Slc11a1 gene, which regulates macrophage and neutrophil activity, is involved in this infirmity. AIRmaxSS mice homozygous for the non-functional Slc11a1 S (gly169asp) allele obtained by genotype-assisted crosses from AIRmax and AIRmin mice are more susceptible than mice homozygous for the Slc11a1 resistant (R) allele. The present work sought to identify the quantitative trait loci (QTL) regulating PIA and to examine the interactions of these QTL with Slc11a1 alleles in modulating PIA. Mice were given two ip injections of 0.5 mL pristane at 60 day intervals, and the incidence and severity of PIA was scored up to 160 days. Genome-wide linkage studies were performed to search for arthritis QTL in an F2 (AIRmax × AIRmin, n = 290) population. Significant arthritis QTL (LODscore>4) were detected on chromosomes 5 and 8, and suggestive QTL on chromosomes 7, 17 and 19. Global gene expression analyses performed on Affymetrix mouse 1.0 ST bioarrays (27k genes) using RNA from arthritic or control mice paws showed 419 differentially expressed genes between AIRmax and AIRmin mice and demonstrated significantly (P<0.001) over-represented genes related to inflammatory responses and chemotaxis. Up-regulation of the chemokine genes Cxcl1, Cxcl9, Cxcl5, Cxcl13 on chromosome 5 was higher in AIRmaxSS than in the other lines. Macrophage scavenger receptor 1 and hemeoxigenase (decycling) 1 genes on chromosome 8 were also expressed at higher levels in AIRmaxSS mice. Our results show that the gene expression profiles of the two arthritis QTL (on chromosomes 5 and 8) correlate with Slc11a1 alleles, resulting in enhanced AIRmaxSS mice susceptibility to PIA. [ABSTRACT FROM AUTHOR]

Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the bestcharacterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells. [ABSTRACT FROM AUTHOR]

Myoblast differentiation is mediated by a cascade of changes in gene expression including transcription factors such as myogenin. Subsequent to myoblast differentiation, there is an increase in expression of the transmembrane protein NADPH oxidase (Nox). Nox is one of the primary factors for the generation of reactive oxygen species (ROS) in myogenic (C2C12) cells. Recently, ROS have been shown to be important regulators of several intracellular signaling pathways, and the full extent of their regulatory roles is yet to be discovered. In the present study, qRT PCR analysis demonstrated that Nox4 isoform is primarily expressed in differentiating C2C12 cells and contributes to the generation of ROS in C2C12 myoblast during differentiation. Over-expression and silencing of Nox4 expression during myoblast differentiation was accompanied by a reduction in intracellular ROS concentrations and an alteration in the expression patterns of Myf5, Pax7, MyoD1, and myogenin. This modulation was found to be associated with ERK1/2 phosphorylation. In both over-expression and reduced expression of Nox4, we found significant reductions in ERK1/2 phosphorylation. This indicates that cellular differentiation may be affected by Nox4-mediated endogenous ROS generation. These data suggest a new opportunity to study the temporal expression of Nox4 in the generation of ROS accompanying changes in myogenic differentiation. [ABSTRACT FROM AUTHOR]

In addition to their role in absorption and secretion, epithelial cells play an important role in the protection of the colon mucosa from the resident microbiota and are important for the maintenance of homeostasis. Microarray analysis of intact colon samples is widely used to gain an overview of the cellular pathways and processes that are active in the colon during inflammation. Laser microdissection of colon epithelial cells allows a more targeted analysis of molecular pathways in the mucosa, preceding and during inflammation, with potentially increased sensitivity to changes in specific cell populations. The aim of this study was to investigate the molecular changes that occur in early and late inflammation stages in colon epithelium of a mouse model of inflammatory bowel diseases. Microarray analysis of intact colon samples and microdissected colon epithelial cell samples from interleukin-10 gene deficient and control mice at 6 and 12 weeks of age was undertaken. Results of gene set enrichment analysis showed that more immune-related pathways were identified between interleukin-10 gene deficient and control mice at 6 weeks of age in epithelial cells than intact colon. This suggests that targeting epithelial cells could increase sensitivity for detecting immune changes that occur early in the inflammatory process. However, in the later stages of inflammation, microarray analyses of intact colon and epithelium both provide a similar overview of gene expression changes in the colon mucosa at the pathway level. [ABSTRACT FROM AUTHOR]

Determining hitherto uninvestigated and safe targets to halt the aging process is important in our aging society. Graying is a hallmark of the aging process and may be used to identify aging tissue for comparative analysis. Here we analyzed differential gene expressions between pigmented, gray, and white human scalp skin hair follicles (HFs) from identical donors. Forming intersections between five donors identified 194/192 downregulated and 186/177 upregulated genes in gray/white HFs. These included melanogenesis (tyrosinase; tyrosinase-related protein 1)- and melanosome structure (Melan-A; Pmel17)-associated genes and regulation of melanocyte relevant tyrosine kinases. Alongside these expected changes, regulated genes included nonmelanocyte-related genes associated with aging as well as nonaging-related genes associated with melanocytes. Intriguingly, among them, genes associated with energy metabolism (i.e., glutaminase) and axon guidance (plexin C1) were altered. These results were reflected by pathway analysis and exemplarily confirmed by PCR and immunohistochemical studies. Supplementing cultured HFs with glutamine or plexin C1 revealed biological relevance and pharmacointerventional potential of these microarray results in altering the HF aging process. Together, we present intriguing data obtained from intra-individual sample comparison that suggest the graying HF to be a valid aging model and a promising target for testing therapeutic interventions. [ABSTRACT FROM AUTHOR]

Innate lymphoid cells (ILCs) are effectors of innate immunity and regulators of tissue modeling. Recently identified ILC populations have a cytokine expression pattern that resembles that of the helper T cell subsets TH2, TH17 and TH22. Here we describe a distinct ILC subset similar to TH1 cells, which we call 'ILC1'. ILC1 cells expressed the transcription factor T-bet and responded to interleukin 12 (IL-12) by producing interferon-γ (IFN-γ). ILC1 cells were distinct from natural killer (NK) cells as they lacked perforin, granzyme B and the NK cell markers CD56, CD16 and CD94, and could develop from RORγt+ ILC3 under the influence of IL-12. The frequency of the ILC1 subset was much higher in inflamed intestine of people with Crohn's disease, which indicated a role for these IFN-γ-producing ILC1 cells in the pathogenesis of gut mucosal inflammation. [ABSTRACT FROM AUTHOR]

Purpose: The (pro)renin receptor ((P)RR) is receptor that has been shown to be involved in developmental processes. Adult neurogenesis shares many similarities with fetal and embryonic neuronal development, but is restricted to some brain areas, including the hippocampus. We therefore investigated the expression of the (P)RR within the adult hippocampal formation and investigated whether (P)PR is expressed by adult and newly generated neurons in the dentate gyrus. Methods: (P)PR protein expressing cells in the hippocampus were analyzed using immunohistochemistry. Double-labeling with markers for adult neurogenesis was used to investigate whether newly formed cells also express (P)PR. Results: (P)PR is expressed by neuronal cells with the hippocampus. (P)RR protein is expressed during different stages of adult neurogenesis within the dentate gyrus (DG). (P)RR is not expressed by Sox2 positive neuronal stem cells, but by doublecortin positive cells located both in the subgranular zone and the granular layer of the DG. Conclusions: The results indicate that (P)PR is mainly expressed by adult neurons in the hippocampus as well as in late stages of adult neurogenesis within the hippocampus. However, to clarify the involvement of this receptor in adult hippocampal neurogenesis and neuronal cell differentiation in detail, functional analyses needed to be performed. [ABSTRACT FROM AUTHOR]

The aim of this experiment was to demonstrate the ability of feeding serotonin (5-HT; 5-hydroxytryptamine) precursors to increase 5-HT production during the transition from pregnancy to lactation and the effects this has on maternal energy metabolism in the liver and mammary gland. Pregnant rats (n = 45) were fed one of three diets: I) control (CON), II) CON supplemented with 0.2% 5-hydroxytryptophan (5-HTP) or III) CON supplemented with 1.35% L-tryptophan (L-TRP), beginning on d13 of pregnancy through d9 of lactation (d9). Serum (pre and post-partum), milk (daily), liver and mammary gland tissue (d9) were collected. Serum 5-HT was increased in the 5-HTP fed dams beginning on d20 of gestation and remained elevated through d9, while it was only increased on d9 in the L-TRP fed dams. 5-HT levels were increased in mammary gland and liver of both groups. Additionally, 5-HTP fed dams had serum and milk glucose levels similar to the CON, while L-TRP had decreased serum (d9) and milk glucose (all dates evaluated). Feeding 5-HTP resulted in increased mRNA expression of key gluconeogenic and glycolytic enzymes in liver and glucose transporters 1 and 8 (GLUT-1, -8) in the mammary gland. We demonstrated the location of GLUT-8 in the mammary gland both in the epithelial and vascular endothelial cells. Finally, phosphorylated 5′ AMP-activated protein kinase (pAMPK), a known regulator of intracellular energy status, was elevated in mammary glands of 5-HTP fed dams. Our results suggest that increasing 5-HT production during the transition from pregnancy to lactation increases mRNA expression of enzymes involved in energy metabolism in the liver, and mRNA abundance and distribution of glucose transporters within the mammary gland. This suggests the possibility that 5-HT may be involved in regulating energy metabolism during the transition from pregnancy to lactation. [ABSTRACT FROM AUTHOR]

Abstract: Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug–drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. Vmax for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and Km was 3.41 and 2.66μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC50 of 5.63 and 6.26μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses. [Copyright &y& Elsevier]

Background: Over the past years, dust of green coffee beans has become known to be a relevant cause for occupational type I allergies. Up to now, allergy diagnostics is based on native green coffee bean extract which exhibits insufficient specificity due to interfering substances as well as batch-to-batch variations. No coffee allergen has been described on the molecular level so far. The aim of this study was to identify the first allergen of green coffee. Methods: The allergenicity of native green coffee bean extracts was analyzed by means of ImmunoCAP in sera of 17 symptomatic coffee workers. A Coffea arabica pJuFo cDNA phage display library was constructed and screened for IgE binding to coffee proteins with 2 sera from allergic coffee workers. By sequence analysis, a new coffee allergen (Cof a 1) was identified, expressed in Escherichia coli, and evaluated by Western blots. The frequency of sensitization was investigated by ELISA screening. Results: The Cof a 1 cDNA encoded a 32-kDa C. arabica class III chitinase. Serum IgE antibodies to the recombinant allergen were found in 3 out of 17 symptomatic coffee workers (18%), whereas only 2 of them reacted to the commercial allergy test. Conclusions: A class III chitinase of C. arabica was identified to be the first known coffee allergen Cof a 1. It may have a relevant potential for the specific diagnosis of coffee sensitization. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small non-coding RNAs of ∼20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located ∼2 kb upstream of the 5′ stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers. [ABSTRACT FROM AUTHOR]

Proteins that coat the lipid droplets (also known as PAT proteins or perilipin (PLIN) family proteins) have diverse functions that are not well elucidated in many tissues. In skeletal muscle, there is even less known about the functions or characteristics of these proteins or how they might change in response to perturbations that alter both intramyocellular lipid (IMCL) content and fat utilization and oxidation. Therefore, the purpose of this study was to examine the human muscle content and gene expression of the four skeletal muscle PLIN proteins in both lean and obese men and women and how this was changed following a 12-week endurance training protocol. PLIN2-PLIN5 proteins were all more abundant in women than in men ( p = 0.037 and p < 0.0001, respectively), consistent with higher IMCL content observed in female skeletal muscle. PLIN5 (previously known as OXPAT) is of particular interest because it has previously been associated primarily with oxidative tissues that rely heavily on fat oxidation for energy production. Although PLIN5 was not different between lean and obese subjects, it was the only PLIN protein to increase in response to endurance training in both sexes. PLIN5 correlated with IMCL volume ( p < 0.0001), but in general, the other PLIN proteins did not correlate well with IMCL volume, suggesting that the relationship between lipid accumulation and PLIN family protein content is not a simple one. Although more work is necessary, it is clear that PLIN5 likely plays an important role in IMCL accumulation and oxidation, both of which increase with endurance training in human skeletal muscle. [ABSTRACT FROM AUTHOR]

Virtually all diabetes‐related foot ulcers (DRFUs) will become colonized by microorganisms that may increase the risk of developing an infection. The reasons why some ulcerations develop acute clinical infections (AI‐DRFUs) whilst others develop chronic infection (CI‐DRFUs) and the preceding host‐microbe interactions in vivo remain largely unknown. Establishing that acute and chronic infections are distinct processes requires demonstrating that these are two different strategies employed by microbes when interacting with a host. In this study, dual‐RNA seq was employed to differentiate the host‐microbe metatranscriptome between DRFUs that had localized chronic infection or acute clinical infection. Comparison of the host metatranscriptome in AI‐DRFUs relative to CI‐DRFUs identified upregulated differentially expressed genes (DEGs) that functioned as regulators of vascular lymphatic inflammatory responses, T‐cell signalling and olfactory receptors. Conversely, CI‐DRFUs upregulated DEGs responsible for cellular homeostasis. Gene set enrichment analysis using Hallmark annotations revealed enrichment of immune and inflammatory profiles in CI‐DRFUs relative to AI‐DRFUs. Analysis of the microbial metatranscriptome identified the DEGs being enriched within AI‐DRFUs relative to CI‐DRFUs included several toxins, two‐component systems, bacterial motility, secretion systems and genes encoding for energy metabolism. Functions relevant to DRFU pathology were further explored, including biofilm and bacterial pathogenesis. This identified that the expression of biofilm‐associated genes was higher within CI‐DRFUs compared to that of AI‐DRFUs, with mucR being the most highly expressed gene. Collectively, these data provide insights into the host‐microbe function in two clinically‐distinct infective phenotypes that affect DRFUs. The data reveal that bacteria in acutely infected DRFUs prioritize motility over biofilm and demonstrate greater pathogenicity and mechanisms, which likely subvert host cellular and immune pathways to establish infection. Upregulation of genes for key vascular inflammatory mediators in acutely infected ulcers may contribute, in part, to the clinical picture of a red, hot, swollen foot, which differentiates an acutely infected ulcer from that of a chronic infection. [ABSTRACT FROM AUTHOR]

Abstract: In the adrenal gland all components of the renin–angiotensin system (RAS) are expressed in both the adrenal cortex and the adrenal medulla. In this review evidence shall be presented that a local secretory RAS exists in the adrenal cortex that stimulates aldosterone production and serves as an amplification system for circulating angiotensin (ANG) II. The regulation of the secretory adrenal RAS clearly differs from the regulation of the circulatory RAS in terms of renin expression as well as of renin secretion. For example under potassium load the activity of the renal and circulatory RAS is suppressed whereas the activity of the adrenal RAS is stimulated. Thus the activity of the adrenal RAS but not of the circulating RAS correlates well with the regulation of aldosterone production by potassium. The present review also summarizes the knowledge about the expression and functions of an additional renin transcript that has recently been discovered. This transcript encodes for a non-secretory cytosolic renin isoform. The cytosolic renin may be a basis for the existence of an intracellular renin system in the adrenal gland that has long been proposed. The present state of knowledge shall be discussed indicating that such an intracellular system modulates cell survival and cell death such as apoptosis and necrosis or cell functions such as aldosterone production. [Copyright &y& Elsevier]

Abstract: The pathogenesis of canine sino-nasal aspergillosis (SNA) and lymphoplasmacytic rhinitis (LPR) remains poorly understood. The innate immune system is implicated in the etiology of human chronic rhinosinusitis. Therefore, we hypothesized that dysfunction in innate immunity could be implicated in the pathogenesis of SNA and LPR. Expression of messenger RNA (mRNA) encoding Toll-like receptors (TLRs) 1–10 and NOD-like receptors (NODs) 1 and 2 in nasal mucosal biopsies from SNA or LPR dogs was compared with mucosa from healthy controls. Gene expression was quantified using quantitative real-time reverse transcriptase polymerase chain reaction normalized against multiple housekeeper genes. All TLR and NOD genes were quantified in all samples. SNA was associated with significantly increased expression of TLRs 1–4, 6–10; and NOD2, relative to controls. LPR was associated with significantly increased expression of TLRs 1, 2, 6–8, relative to controls. There was significantly more expression of TLRs 1, 4, 6–10 and NOD2 in SNA dogs than in LPR dogs. The significance of these differences in the pathogenesis of these diseases is yet to be determined. [Copyright &y& Elsevier]

Background: Gene duplication is a major force that contributes to the evolution of new metabolic functions in all organisms. Rhodobacter sphaeroides 2.4.1 is a bacterium that displays a wide degree of metabolic versatility and genome complexity and therefore is a fitting model for the study of gene duplications in bacteria. A comprehensive analysis of 234 duplicate gene-pairs in R. sphaeroides was performed using structural constraint and expression analysis. Results: The results revealed that most gene-pairs in in-paralogs are maintained under negative selection (ω≤0.3), but the strength of selection differed among in-paralog gene-pairs. Although in-paralogs located on different replicons are maintained under purifying selection, the duplicated genes distributed between the primary chromosome (CI) and the second chromosome (CII) are relatively less selectively constrained than the gene-pairs located within each chromosome. The mRNA expression patterns of duplicate gene-pairs were examined through microarray analysis of this organism grown under seven different growth conditions. Results revealed that ~62% of paralogs have similar expression patterns (cosine ≥ 0.90) over all of these growth conditions, while only ~7% of paralogs are very different in their expression patterns (cosine<0.50). Conclusions: The overall findings of the study suggest that only a small proportion of paralogs contribute to the metabolic diversity and the evolution of novel metabolic functions in R. sphaeroides. In addition, the lack of relationships between structural constraints and gene-pair expression suggests that patterns of gene-pair expression are likely associated with conservation or divergence of gene-pair promoter regions and other coregulation mechanisms. [ABSTRACT FROM AUTHOR]