Explore the vast range of titles available.

Diabetes is associated with a higher incidence of myocardial infarction (MI) and increased risk for adverse vascular and fibrogenic events post-MI. Bone marrow-derived progenitor cell (BMPC) therapy has been shown to promote neovascularization, decrease infarct area and attenuate left ventricular (LV) dysfunction after MI. Unlike vascular effects, the anti-fibrosis mechanisms of BMPC, specifically under diabetic conditions, are poorly understood. We demonstrated that intramyocardial delivery of BMPCs in infarcted diabetic db/db mice significantly down-regulates profibrotic miRNA-155 in the myocardium and improves LV remodeling and function. Furthermore, inhibition of paracrine factor hepatocyte growth factor (HGF) signaling in vivo suppressed the BMPC-mediated inhibition of miR-155 expression and the associated protective effect on cardiac fibrosis and function. In vitro studies confirmed that the conditioned media of BMPC inhibited miR-155 expression and profibrotic signaling in mouse cardiac fibroblasts under diabetic conditions. However, neutralizing antibodies directed against HGF blocked these effects. Furthermore, miR-155 over-expression in mouse cardiac fibroblasts inhibited antifibrotic Sloan-Kettering Institute proto-oncogene (Ski) and Ski-related novel gene, non-Alu-containing (SnoN) signaling and abrogated antifibrogenic response of HGF. Together, our data demonstrates that paracrine regulation of cardiac miRNAs by transplanted BMPCs contributes to the antifibrotic effects of BMPC therapy. BMPCs release HGF, which inhibits miR-155-mediated profibrosis signaling, thereby preventing cardiac fibrosis. These data suggest that targeting miR-155 might serve as a potential therapy against cardiac fibrosis in the diabetic heart.

Mature neocortical pyramidal cells functionally express two sodium channel (Na V ) isoforms: Na V 1.2 and Na V 1.6. These isoforms are differentially localized to pyramidal cell compartments, and as such are thought to contribute to different aspects of neuronal excitability. But determining their precise roles in pyramidal cell excitability has been hampered by a lack of tools that allow for selective, acute block of each isoform individually. Here, we leveraged aryl sulfonamide-based molecule (ASC) inhibitors of Na V channels that exhibit state-dependent block of both Na V 1.2 and Na V 1.6, along with knock-in mice with changes in Na V 1.2 or Na V 1.6 structure that prevents ASC binding. This allowed for acute, potent, and reversible block of individual isoforms that permitted dissection of the unique contributions of Na V 1.2 and Na V 1.6 in pyramidal cell excitability. Remarkably, block of each isoform had contrasting—and in some situations, opposing—effects on neuronal action potential output, with Na V 1.6 block decreasing and Na V 1.2 block increasing output. Thus, Na V isoforms have unique roles in regulating different aspects of pyramidal cell excitability, and our work may help guide the development of therapeutics designed to temper hyperexcitability through selective Na V isoform blockade.

Precise dosing of warfarin is important to achieve therapeutic benefit without adverse effects. Pharmacogenomics explains some interindividual variability in warfarin response, but less attention has been paid to drug‐drug interactions in the context of genetic factors. We investigated retrospectively the combined effects of cytochrome P450 (CYP)2C9 and vitamin K epoxide reductase complex (VKORC)1 genotypes and concurrent exposure to CYP2C9‐interacting drugs on long‐term measures of warfarin anticoagulation. Study participants predicted to be sensitive responders to warfarin based on CYP2C9 and VKORC1 genotypes, had significantly greater international normalized ratio (INR) variability over time. Participants who were concurrently taking CYP2C9‐interacting drugs were found to have greater INR variability and lesser time in therapeutic range. The associations of INR variability with genotype were driven by the subgroup not exposed to interacting drugs, whereas the effect of interacting drug exposure was driven by the subgroup categorized as normal responders. Our findings emphasize the importance of considering drug interactions in pharmacogenomic studies.

Objective Interpretation of clinical genetic testing, which identifies a potential genetic etiology in 25% of children with epilepsy, is limited by variants of uncertain significance. Understanding functional consequences of variants can help distinguish pathogenic from benign alleles. We combined automated patch clamp recording with neurophysiological simulations to discern genotype‐function‐phenotype correlations in a real‐world cohort of children with SCN1A‐associated epilepsy. Methods Clinical data were extracted for children with SCN1A variants identified by clinical genetic testing. Functional properties of non‐truncating NaV1.1 variant channels were determined using automated patch clamp recording. Functional data were incorporated into a parvalbumin‐positive (PV+) interneuron computer model to predict variant effects on neuron firing and were compared with longitudinal clinical data describing epilepsy types, neurocognitive outcomes, and medication response. Results Twelve SCN1A variants were identified (nine non‐truncating). Six non‐truncating variants exhibited no measurable sodium current in heterologous cells consistent with complete loss of function (LoF). Two variants caused either partial LoF (L479P) or a mixture of gain and loss of function (I1356M). The remaining non‐truncating variant (T1250M) exhibited normal function. Functional data changed classification of pathogenicity for six variants. Complete LoF variants were universally associated with seizure onset before one year of age and febrile seizures, and were often associated with drug resistant epilepsy and below average cognitive outcomes. Simulations demonstrated abnormal firing in heterozygous model neurons containing dysfunctional variants. Interpretation In SCN1A‐associated epilepsy, functional analysis and neuron simulation studies resolved variants of uncertain significance and correlated with aspects of phenotype and medication response.

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

Objective We identified a novel de novo SCN2A variant (M1879T) associated with infantile‐onset epilepsy that responded dramatically to sodium channel blocker antiepileptic drugs. We analyzed the functional and pharmacological consequences of this variant to establish pathogenicity, and to correlate genotype with phenotype and clinical drug response. Methods The clinical and genetic features of an infant boy with epilepsy are presented. We investigated the effect of the variant using heterologously expressed recombinant human NaV1.2 channels. We performed whole‐cell patch clamp recording to determine the functional consequences and response to carbamazepine. Results The M1879T variant caused disturbances in channel inactivation including substantially depolarized voltage dependence of inactivation, slower time course of inactivation, and enhanced resurgent current that collectively represent a gain‐of‐function. Carbamazepine partially normalized the voltage dependence of inactivation and produced use‐dependent block of the variant channel at high pulsing frequencies. Carbamazepine also suppresses resurgent current conducted by M1879T channels, but this effect was explained primarily by reducing the peak transient current. Molecular modeling suggests that the M1879T variant disrupts contacts with nearby residues in the C‐terminal domain of the channel. Interpretation Our study demonstrates the value of conducting functional analyses of SCN2A variants of unknown significance to establish pathogenicity and genotype–phenotype correlations. We also show concordance of in vitro pharmacology using heterologous cells with the drug response observed clinically in a case of SCN2A‐associated epilepsy.

BackgroundBacteria play a role in inflammatory bowel disease and other forms of intestinal inflammation. Although much attention has focused on the search for a pathogen or inciting inflammatory bacteria, another possibility is a lack of beneficial bacteria that normally confer anti-inflammatory properties in the gut. The purpose of this study was to determine whether normal commensal bacteria could inhibit inflammatory pathways important in intestinal inflammation.MethodsConditioned media from Lactobacillus plantarum (Lp-CM) and other gut bacteria was used to treat intestinal epithelial cell (YAMC) and macrophage (RAW 264.7) or primary culture murine dendritic cells. NF-κB was activated through TNF-Receptor, MyD88-dependent and -independent pathways and effects of Lp-CM on the NF-κB pathway were assessed. NF-κB binding activity was measured using ELISA and EMSA. 1κB expression was assessed by Western blot analysis, and proteasome activity determined using fluorescence-based proteasome assays. MCP-1 release was determined by ELISA.ResultsLp-CM inhibited NF-κB binding activity, degradation of IκBα and the chymotrypsin-like activity of the proteasome. Moreover, Lp-CM directly inhibited the activity of purified mouse proteasomes. This effect was specific, since conditioned media from other bacteria had no inhibitory effect. Unlike other proteasome inhibitors, Lp-CM was not toxic in cell death assays. Lp-CM inhibited MCP-1 release in all cell types tested.ConclusionsThese studies confirm, and provide a mechanism for, the anti-inflammatory effects of the probiotic and commensal bacterium Lactobacillus plantarum. The use of bacteria-free Lp-CM provides a novel strategy for treatment of intestinal inflammation which would eliminate the risk of bacteremia reported with conventional probiotics.

Embryonic Stem Cells not only hold a lot of potential for use in regenerative medicine, but also provide an elegant and efficient way to study specific developmental processes and pathways in mammals when whole animal gene knock out experiments fail. We have investigated a pathway through which HDAC1 affects cardiovascular and more specifically cardiomyocyte differentiation in ES cells by controlling expression of SOX17 and BMP2 during early differentiation. This data explains current discrepancies in the role of HDAC1 in cardiovascular differentiation and sheds light into a new pathway through which ES cells determine cardiovascular cell fate.

Background Radiation therapy (RT) results in pain relief for about 6 of 10 patients with cancer induced bone pain (CIBP) caused by bone metastases. The high number of non-responders, the long median time from RT to pain response and the risk of adverse effects, makes it important to determine predictors of treatment response. Clinical features such as cancer type, performance status and pain intensity, and biomarkers for osteoclast activity are proposed as predictors of response to RT. However, results are inconsistent and there is a need for better predictors of RT response. A similar argument can be stated for the development of cachexia; there are currently no predictors that can identify patients who will develop cachexia later in the cancer disease trajectory. Experimental and preclinical studies show that pain, depression and cachexia are related to inflammation. However, it is not known if inflammatory biomarkers can predict CIBP, depression or development of cachexia. Methods This multicenter, multinational longitudinal observational study will include 600 adult patients receiving RT for CIBP. Demographic data, clinical variables, osteoclast and inflammatory biomarkers will be assessed before start of RT, and 3, 8, 16, 24 and 52 weeks after last course of RT. The primary aim of the study is to identify potential predictors for pain relief from RT. Secondary aims are to explore potential predictors for development of cachexia, the longitudinal relationship between pain intensity and depression, and if inflammatory biomarkers are associated with changes in pain intensity, cachexia and depression during one-year follow up. Discussion The immediate clinical implication of the PRAIS study is to identify potential predictive factors for a RT response on CIBP, and thereby reduce non-efficacious RT. Patient benefits are fewer hospital visits, reduced risk of adverse effects and more individualized pain treatment. The long-term clinical implication of the PRAIS study is to improve the knowledge about inflammation in relation to CIBP, cachexia and depression and potentially identify associations and mechanisms that can be targeted for treatment. Trial registration ClinicalTrials.gov NCT02107664 , date of registration April 8, 2014 (retrospectively registered). Trial sponsor The European Palliative Care Research Centre (PRC), Department of Clinical and Molecular Medicine, NTNU, Faculty of medicine and Health Sciences, Trondheim, N-7491, Norway.

Krüppel-like factor 1(KLF1) is a hematopoietic-specific zinc finger transcription factor essential for erythroid gene expression. In concert with the transacting factor GATA1, KLF1 modulates the coordinate expression of the genes encoding the multi-enzyme heme biosynthetic pathway during erythroid differentiation. To explore the mechanisms underpinning KLF1 action at the gene loci regulating the first 3 steps in this process, we have exploited the K1-ERp erythroid cell line, in which KLF1 translocates rapidly to the nucleus in response to treatment with 4-OH-Tamoxifen (4-OHT). KLF1 acts as a differentiation-independent transcriptional co-regulator of delta-aminolevulinic acid dehydratase (Alad), but not 5-aminolevulinate synthase gene (Alas2) or porphobilinogen deaminase (Pbgd). Similar to its role at the β-globin promoter, KLF1 induces factor recruitment and chromatin changes at the Alad1b promoter in a temporally-specific manner. In contrast to these changes, we observed a distinct mechanism of histone eviction at the Alad1b promoter. Furthermore, KLF1-dependent events were not modulated by GATA1 factor promoter co-occupancy alone. These results not only enhance our understanding of erythroid-specific modulation of heme biosynthetic regulation by KLF1, but provide a model that will facilitate the elucidation of novel KLF1-dependent events at erythroid gene loci that are independent of GATA1 activity.