Nodal Molecules Research Articles

Distinct PKA Signaling in Cytosolic and Mitochondrial Compartments in Electrically Paced Atrial Myocytes.

Protein kinase A (PKA) is a key nodal signaling molecule that regulates a wide range of cellular functions in the cytosol and mitochondria. The distribution of A-kinase anchoring proteins that tether PKA, the local interaction with degradation molecules, and regulation by Ca2+, may lead to distinct spatiotemporal cAMP/PKA signaling in these compartments. In this work, FRET-based sensors were used to investigate PKA signaling in the cytosol, outer mitochondrial membrane (OMM), and mitochondrial matrix (MM) and its crosstalk with Ca2+ in response to electrical stimulation of cultured rabbit atrial cells. A gradual decrease in PKA activity eliminating the ability of the atrial cells to respond to physiological electrical stimulation, was observed upon treatment of cells with H-89. Chelation of intracellular Ca2+ by BAPTA reduced PKA activity and diminished its response to forskolin, an AC stimulator. Under basal conditions, PKA activity in response to forskolin was lower in the OMM compared to the cytosol and MM. In response to electrical stimulation in the presence of ISO, distinct compartmentalization of PKA activity was observed, with higher activity in the cytosol and MM than in the OMM. Thus, distinct Ca2+-dependent spatiotemporal cAMP/PKA signaling exists in atrial cells, likely mediating its excitation and mitochondrial function.

In preprints: morphogens in motion.

In preprints: morphogens in motion.

Aberrant Expression of Nodal and Paranodal Molecules in Neuropathy Associated With IgM Monoclonal Gammopathy With Anti-Myelin-Associated Glycoprotein Antibodies.

To clarify the pathogenesis of anti-myelin-associated glycoprotein (MAG) antibody neuropathy associated with IgM monoclonal gammopathy (anti-MAG neuropathy), sural nerve biopsy specimens from 15 patients were investigated. Sodium channels, potassium channels, contactin-associated protein 1 (Caspr1), contactin 1, and neurofascin were evaluated by immunofluorescence in teased-fiber preparations. Immunoreactivity to the pan-sodium channel in both anti-MAG neuropathy patients and in normal controls was concentrated at the node of Ranvier unless there was demyelination, which was defined as the widening of the node of Ranvier. However, this immunoreactivity became weak or disappeared as demyelination progressed. In contrast, KCNQ2 immunostaining was nearly absent even in the absence of demyelination. The lengths of Caspr1, contactin 1, and pan-neurofascin immunostaining sites at the paranode were significantly increased compared with those of normal controls despite the absence of demyelination. The length of paranodal neurofascin staining correlated with the anti-MAG antibody titer, nerve conduction indices, the frequency of de/remyelination in teased-fiber preparations, and the frequency of widely spaced myelin (p < 0.05, p < 0.05, p < 0.01, and <0.05, respectively). These findings suggest that nodal and paranodal molecular alterations occur in early stages preceding the morphological changes associated with demyelination in anti-MAG neuropathy.

Accumulation of Neurofascin at Nodes of Ranvier Is Regulated by a Paranodal Switch.

The paranodal junctions flank mature nodes of Ranvier and provide a barrier between ion channels at the nodes and juxtaparanodes. These junctions also promote node assembly and maintenance by mechanisms that are poorly understood. Here, we examine their role in the accumulation of NF186, a key adhesion molecule of PNS and CNS nodes. We previously showed that NF186 is initially targeted/accumulates via its ectodomain to forming PNS (hemi)nodes by diffusion trapping, whereas it is later targeted to mature nodes by a transport-dependent mechanism mediated by its cytoplasmic segment. To address the role of the paranodes in this switch, we compared accumulation of NF186 ectodomain and cytoplasmic domain constructs in WT versus paranode defective (i.e., Caspr-null) mice. Both pathways are affected in the paranodal mutants. In the PNS of Caspr-null mice, diffusion trapping mediated by the NF186 ectodomain aberrantly persists into adulthood, whereas the cytoplasmic domain/transport-dependent targeting is impaired. In contrast, accumulation of NF186 at CNS nodes does not undergo a switch; it is predominantly targeted to both forming and mature CNS nodes via its cytoplasmic domain and requires intact paranodes. Fluorescence recovery after photobleaching analysis indicates that the paranodes provide a membrane diffusion barrier that normally precludes diffusion of NF186 to nodes. Linkage of paranodal proteins to the underlying cytoskeleton likely contributes to this diffusion barrier based on 4.1B and βII spectrin expression in Caspr-null mice. Together, these results implicate the paranodes as membrane diffusion barriers that regulate targeting to nodes and highlight differences in the assembly of PNS and CNS nodes.SIGNIFICANCE STATEMENT Nodes of Ranvier are essential for effective saltatory conduction along myelinated axons. A major question is how the various axonal proteins that comprise the multimeric nodal complex accumulate at this site. Here we examine how targeting of NF186, a key nodal adhesion molecule, is regulated by the flanking paranodal junctions. We show that the transition from diffusion-trapping to transport-dependent accumulation of NF186 requires the paranodal junctions. We also demonstrate that these junctions are a barrier to diffusion of axonal proteins into the node and highlight differences in PNS and CNS node assembly. These results provide new insights into the mechanism of node assembly and the pathophysiology of neurologic disorders in which impaired paranodal function contributes to clinical disability.

Physiological Disturbance in Fatty Liver Energy Metabolism Converges on IGFBP2 Abundance and Regulation in Mice and Men.

Fatty liver occurs from simple steatosis with accumulated hepatic lipids and hepatic insulin resistance to severe steatohepatitis, with aggravated lipid accumulation and systemic insulin resistance, but this progression is still poorly understood. Analyses of hepatic gene expression patterns from alb-SREBP-1c mice with moderate, or aP2-SREBP-1c mice with aggravated, hepatic lipid accumulation revealed IGFBP2 as key nodal molecule differing between moderate and aggravated fatty liver. Reduced IGFBP2 expression in aggravated fatty liver was paralleled with promoter hypermethylation, reduced hepatic IGFBP2 secretion and IGFBP2 circulating in plasma. Physiologically, the decrease of IGFBP2 was accompanied with reduced fatty acid oxidation and increased de novo lipogenesis potentially mediated by IGF1 in primary hepatocytes. Furthermore, methyltransferase and sirtuin activities were enhanced. In humans, IGFBP2 serum concentration was lower in obese men with non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) compared to non-obese controls, and liver fat reduction by weight-loss intervention correlated with an increase of IGFBP2 serum levels. In conclusion, hepatic IGFBP2 abundance correlates to its circulating level and is related to hepatic energy metabolism and de novo lipogenesis. This designates IGFBP2 as non-invasive biomarker for fatty liver disease progression and might further provide an additional variable for risk prediction for pathogenesis of fatty liver in diabetes subtype clusters.

The Role of FAT10 in Alcoholic Hepatitis Pathogenesis

BackgroundMorphologic features of Alcoholic Hepatitis (AH) balloon cells forming Mallory‐Denk bodies and bile duct metaplasia are two morphologic features consistently being found in AH. Increased FAT10 expression may induce increasing mitotic non‐disjunction and chromosome instability leading to tumorigenesis. FAT10 expression is highly up‐regulated by pro‐inflammatory cytokines IFNγ and TNFα in all cell types and tissues.ObjectiveTo clarify the role of FAT10 in alcoholic hepatitis pathogenesis, we studied FAT10 expression in liver biopsy samples from patients with AH.MethodsWe use gene and protein array to detect the potential changes during alcoholic hepatitis pathogenesis. The potential target, FAT10, were tested at protein expression level by using western blot, immunofluorescent (IF) staining and quantitation. And FAT10 was also studied by using knockout animal models.ResultsThe results show that FAT10 is essential to maintain the function of liver cell protein quality control and Mallory‐Denk body (MDB) formation. This is consistent with the finding with fat10−/− mice which fail to form MDBs. FAT10 also signals a switch in the 26s proteasome’s proteases to cause the proteins accumulation, overexpression, and then forming MDBs. FAT10 overexpression in AH causes an extensive change in protein content in the liver leading to balloon degeneration and MDB aggregation formation, all of which is prevented in fat10−/− mice. The pathway that increases FAT10 expression includes TNFα, IFNγ, followed by NFκB and STAT3, which were all up‐regulated in AH. Using proteins expression quantitation by measuring the intensity of immunofluorescent (IF) staining, it showed the FAT10 protein expression was 4.5 fold increased in the AH livers but not in the NASH or control livers.DiscussionMilk thistle derivatives were used to treat FAT10 overexpression experimentally. Flavanone derivatives of milk thistle inhibit IFNγ/TNFα, NFκB and STAT3 then inhibits the expression of FAT10. NFκB is the key nodal hub of the IFNα/TNFα‐response genes. Silibinin derived from milk thistle is reported to be a powerful antioxidant and has anti‐carcinoma effects including against TNFα‐induced tumor growth. Studies on Silibinin or other milk thistle derivatives to treat AH confirms that overexpressed FAT10 with NFκB and TNFα are the major key nodal molecules in these networks.ConclusionOverexpressed FAT10 with over‐activated NFκB and TNFα are the major key nodal molecules in alcoholic hepatitis pathogenesis. These molecules might be potential therapeutic targets.

Proteomic and transcriptomic profiling of Pten gene-knockout mouse model of prostate cancer.

BackgroundThe prostate‐specific phosphatase and tensin homolog deleted on chromosome 10 (Pten) gene‐conditional knockout (KO) mouse carcinogenesis model is highly desirable for studies of prostate cancer biology and chemoprevention due to its close resemblance of primary molecular defect and many histopathological features of human prostate cancer including androgen response and disease progression from prostatic intraepithelial neoplasia to invasive adenocarcinoma. Here, we profiled the proteome and transcriptome of the Pten‐KO mouse prostate tumors for global macromolecular expression alterations for signaling changes and biomarker signatures.MethodsFor proteomics, four pairs of whole prostates from tissue‐specific conditional knockout Pten‐KO mice (12‐15 weeks of age) and their respective wild‐type littermates housed in the same cages were analyzed by 8‐plex isobaric tags for relative and absolute quantitation iTRAQ. For microarray transcriptomic analysis, three additional matched pairs of prostate/tumor specimens from respective mice at 20 to 22 weeks of age were used. Real‐time quantitative reverse transcription‐polymerase chain reaction was used to verify the trends of protein and RNA expression changes. Gene Set Enrichment Analysis and Ingenuity Pathway Analysis were carried out for bioinformatic characterizations of pathways and networks.ResultsAt the macromolecular level, proteomic and transcriptomic analyses complement and cross‐validate to reveal overexpression signatures including inflammation and immune alterations, in particular, neutrophil/myeloid lineage suppressor cell features, chromatin/histones, ion and nutrient transporters, and select glutathione peroxidases and transferases in Pten‐KO prostate tumors. Suppressed expression patterns in the Pten‐KO prostate tumors included glandular differentiation such as secretory proteins and androgen receptor targets, smooth muscle features, and endoplasmic reticulum stress proteins. Bioinformatic analyses identified immune and inflammation responses as the most profound macromolecular landscape changes, and the predicted key nodal activities through Akt, nuclear factor‐kappaB, and P53 in the Pten‐KO prostate tumor. Comparison with other genetically modified mouse prostate carcinogenesis models revealed notable molecular distinctions, especially the dominance of immune and inflammation features in the Pten‐KO prostate tumors.ConclusionsOur work identified prominent macromolecular signatures and key nodal molecules that help to illuminate the patho‐ and immunobiology of Pten‐loss driven prostate cancer and can facilitate the choice of biomarkers for chemoprevention and interception studies in this clinically relevant mouse prostate cancer model.

Comprehensive analysis of transcriptome profiles in hepatocellular carcinoma

BackgroundHepatocellular carcinoma is the second most deadly cancer with late presentation and limited treatment options, highlighting an urgent need to better understand HCC to facilitate the identification of early-stage biomarkers and uncover therapeutic targets for the development of novel therapies for HCC.MethodsDeep transcriptome sequencing of tumor and paired non-tumor liver tissues was performed to comprehensively evaluate the profiles of both the host and HBV transcripts in HCC patients. Differential gene expression patterns and the dys-regulated genes associated with clinical outcomes were analyzed. Somatic mutations were identified from the sequencing data and the deleterious mutations were predicted. Lastly, human-HBV chimeric transcripts were identified, and their distribution, potential function and expression association were analyzed.ResultsExpression profiling identified the significantly upregulated TP73 as a nodal molecule modulating expression of apoptotic genes. Approximately 2.5% of dysregulated genes significantly correlated with HCC clinical characteristics. Of the 110 identified genes, those involved in post-translational modification, cell division and/or transcriptional regulation were upregulated, while those involved in redox reactions were downregulated in tumors of patients with poor prognosis. Mutation signature analysis identified that somatic mutations in HCC tumors were mainly non-synonymous, frequently affecting genes in the micro-environment and cancer pathways. Recurrent mutations occur mainly in ribosomal genes. The most frequently mutated genes were generally associated with a poorer clinical prognosis. Lastly, transcriptome sequencing suggest that HBV replication in the tumors of HCC patients is rare. HBV-human fusion transcripts are a common observation, with favored HBV and host insertion sites being the HBx C-terminus and gene introns (in tumors) and introns/intergenic-regions (in non-tumors), respectively. HBV-fused genes in tumors were mainly involved in RNA binding while those in non-tumors tissues varied widely. These observations suggest that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts may occur during tumorigenesis.ConclusionsTranscriptome sequencing of HCC patients reveals key cancer molecules and clinically relevant pathways deregulated/mutated in HCC patients and suggests that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts likely occur during the process of tumorigenesis.

Clinical and diagnostic characteristics of peripheral vasculopathy biomarkers in systemic scleroderma

Vascular lesion is a hallmark of systemic scleroderma (SS). Vasculopathy occurs in every patient with SS. It can be the earliest clinical manifestation or cause the main life‑threatening complications of the disease, and thus determine morbidity and mortality. In SS, progressive vascular damage is characterized by constant activation/damage and apoptosis of endothelial cells, intimal thickening and vasoconstriction, leading to the lumen obliteration. Vascular remodeling leads to a violation of their tone and a decrease in capillary blood flow, followed by tissue ischemia and chronic hypoxia. These phenomena are often accompanied by altered levels of vascular factors. Among the most significant biomarkers of vasculopathy in SS with the greatest evidence base we define anti‑AT1R and anti‑ETAR, soluble intercellular adhesion molecules (sICAM‑1), soluble molecules of adhesion of thrombocyte endothelial cells, placental growth factor (PlGF), pro‑angiogenic angiopoietin‑like protein‑3 (ANGPTL‑3), apelin, chemokines (CXCL5), galectin‑3, E‑selectin, tissue kallikrein, interleukins (IL)‑13 and ‑33, vascular endothelial growth factor (VEGF165b), matrix metalloproteinases‑12, endothelin‑1, nodal adhesion molecules which are associated with the development of either digital ulcers or changes on videocapillaroscopy typical for SS. This shows their active role in the pathogenesis of SS, especially during the disease manifestation. Data is presented on the most significant vascular biomarkers and the main connections between vascular biomarkers and capillaroscopic parameters and/or the presence of digital ulcers in SS. Vascular biomarkers can become prognostic factors of vascular damage at SS, which allows early treatment of vascular complications.

Dual Targeting of PI3Kdelta and BRD4 Sensitizes Acute Lymphoblastic Leukemia to Chemotherapy

Introduction: Acute lymphoblastic leukemia is the most prevalent form of cancer affecting children with 2,500-3,500 new cases per year. More effective targeted therapies have yielded a current five-year survival rate of at least 85% for children; however relapsed disease, as well as harsh side effects of treatment, remain prevalent hurdles for many patients. Cells harbored by the bone marrow comprise minimal residual disease that may contribute to later re-expansion of the tumor population following treatment, also known as cell adhesion mediated drug resistance (CAM-DR).Bone marrow stromal cell contact has been shown to upregulate phosphorylated AKT, promoting survival of ALL cells. We investigate a new dual targeted therapy, Morpholinothienopyrane (SF2535), which inhibits both PI3Kdelta and BRD4, each key molecules in signal transduction pathways between microenvironment and leukemia cells. PI3Kdelta is a central nodal molecule in outside-in pathways including cell-cell interactions mediated by integrins. BRD4, a member of the bromodomain and extraterminal domain (BET) family of proteins which bind acetylated lysines at promoter and enhancer regions, is another key player involved in regulation of transcription of myc family transcription factors. In addition to transcriptional regulation by BRD4, myc is also regulated by PI3kdelta which inhibits GSK3beta-mediated degradation of myc. We hypothesize that this dual inhibitor, SF2535, will disrupt signaling between leukemia cells and the bone marrow microenvironment, thus addressing CAM-DR.Methods/Results:In vitro and in vivo BRD4 and PI3Kdelta target downregulation was measured by western blot including assessment of myc, AKT, and phosphorylated AKT. Cells were starved in vitro in serum-free conditions for 18 hours followed by treatment with SF2535 and stimulation with serum. Levels of phosphorylated AKT were noticeably decreased in SF2535 treated cells compared with DMSO control or JQ1 (BET inhibitor) treatment alone.Truncation of microenvironmental outside-in signaling via inhibition of PI3Kdelta and BRD4 was measured via transwell migration assay. Primary ALL cells were cultured on top of a porous membrane with SDF-1alpha in the chamber below. Significantly less primary ALL cells migrated toward SDF-1alpha when treated with SF2535 compared to DMSO, CAL101 (PI3Kdelta inhibitor), or JQ1 (BET inhibitor).The combination of SF2535 with vincristine, dexamethasone, and L-asparaginase (VDL), the backbone of induction therapy for many ALL patients, was evaluated in vitro. Primary tumor cells were co-cultured on irradiated OP-9 murine stromal cells and treated with SF2535, VDL, or SF2535 and VDL combined. Apoptosis in response to treatment was measured via AnnexinV and 7-AAD flow cytometry. The combination of SF2535 and VDL showed significantly more cell death compared to either SF2535 or VDL alone at three concentrations of each treatment. Additionally, CalcuSyn software was used to assess putative additive versus synergistic effects of this combination. CalcuSyn analysis of VDL and SF2535 combination compared to either SF2535 or VDL treatment alone shows synergy (defined as a combination index of 0.3-0.7) between these two therapies used on sample LAX7R after 1 day of treatment.Discussion: Thus far we have shown that this novel inhibitor decreases expression of downstream targets of PI3Kdelta (phosphorylated AKT) and BRD4 (C-myc), inhibits migration of ALL toward bone marrow stromal factors, and increases apoptosis of primary patient samples when combined with VDL compared to VDL alone. Most notably, we have determined a synergistic relationship between SF2535 and VDL in vitro, suggesting a role for this novel therapy in sensitizing cells to chemotherapy. The use of a single agent against both PI3Kdelta and BRD4 ultimately aimed at downregulation of myc levels provides the possibility of more effective, less toxic therapeutic option that addresses CAM-DR faced by many patients. While ongoing experiments in vitro and in vivo will provide a more robust picture of the efficacy of this drug, our preliminary data suggest promise for this novel application of BRD4 and PI3Kdelta inhibition in the context of acute lymphoblastic leukemia. DisclosuresDurden:SignalRx Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Study of the Treatment Effects of Compound Tufuling Granules in Hyperuricemic Rats Using Serum Metabolomics.

The study aimed to investigate the mechanism of the effect of Compound Tufuling Granules (CTG) to lower the serum uric acid level in a rat model of hyperuricemia. The rat model was established by administering hypoxanthine through oral gavage and potassium oxonate through intraperitoneal injection. Rats were divided into the normal group, model group, CTG group, and allopurinol group. Serum uric acid, creatinine, urea nitrogen, and inflammatory cytokine levels were determined in each group. In the model group, ultrahigh performance liquid chromatography-mass spectrometry was used to analyze the metabolic profiles and delineate the action mechanism of CTG; in addition, the orthogonal projection method was used to perform latent structure-discrimination analysis to screen the related metabolites. The results indicated significant differences in the metabolic profiles between the model and normal groups. A total of seven related metabolites were identified through screening in the model group, mainly related to the pathways of bile secretion, pyrimidine, purine, and phenylalanine metabolism, pantothenate and CoA biosynthesis, and pentose and glucuronate interconversions; these related pathways were reversed in the CTG group. In the metabolic networks, uracil and acetyl-coenzyme A were the nodal molecules. In addition, the test results of the evaluation of serum biochemical and inflammatory factors confirmed that CTG had significant effect in reducing the levels of serum uric acid and protecting renal function. These results confirmed that CTG primarily regulated the recruitment of nodal molecules to achieve anti-inflammatory effects, reduced uric acid level, and renal protection.

Genome-wide miRNA response to anacardic acid in breast cancer cells.

MicroRNAs are biomarkers and potential therapeutic targets for breast cancer. Anacardic acid (AnAc) is a dietary phenolic lipid that inhibits both MCF-7 estrogen receptor α (ERα) positive and MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation with IC50s of 13.5 and 35 μM, respectively. To identify potential mediators of AnAc action in breast cancer, we profiled the genome-wide microRNA transcriptome (microRNAome) in these two cell lines altered by the AnAc 24:1n5 congener. Whole genome expression profiling (RNA-seq) and subsequent network analysis in MetaCore Gene Ontology (GO) algorithm was used to characterize the biological pathways altered by AnAc. In MCF-7 cells, 69 AnAc-responsive miRNAs were identified, e.g., increased let-7a and reduced miR-584. Fewer, i.e., 37 AnAc-responsive miRNAs were identified in MDA-MB-231 cells, e.g., decreased miR-23b and increased miR-1257. Only two miRNAs were increased by AnAc in both cell lines: miR-612 and miR-20b; however, opposite miRNA arm preference was noted: miR-20b-3p and miR-20b-5p were upregulated in MCF-7 and MDA-MB-231, respectively. miR-20b-5p target EFNB2 transcript levels were reduced by AnAc in MDA-MB-231 cells. AnAc reduced miR-378g that targets VIM (vimentin) and VIM mRNA transcript expression was increased in AnAc-treated MCF-7 cells, suggesting a reciprocal relationship. The top three enriched GO terms for AnAc-treated MCF-7 cells were B cell receptor signaling pathway and ribosomal large subunit biogenesis and S-adenosylmethionine metabolic process for AnAc-treated MDA-MB-231 cells. The pathways modulated by these AnAc-regulated miRNAs suggest that key nodal molecules, e.g., Cyclin D1, MYC, c-FOS, PPARγ, and SIN3, are targets of AnAc activity.

Transcriptomic dissection reveals wide spread differential expression in chickpea during early time points of Fusarium oxysporum f. sp. ciceri Race 1 attack.

Plants’ reaction to underground microorganisms is complex as sessile nature of plants compels them to prioritize their responses to diverse microorganisms both pathogenic and symbiotic. Roots of important crops are directly exposed to diverse microorganisms, but investigations involving root pathogens are significantly less. Thus, more studies involving root pathogens and their target crops are necessitated to enrich the understanding of underground interactions. Present study reported the molecular complexities in chickpea during Fusarium oxysporum f. sp. ciceri Race 1 (Foc1) infection. Transcriptomic dissections using RNA-seq showed significantly differential expression of molecular transcripts between infected and control plants of both susceptible and resistant genotypes. Radar plot analyses showed maximum expressional undulations after infection in both susceptible and resistant plants. Gene ontology and functional clustering showed large number of transcripts controlling basic metabolism of plants. Network analyses demonstrated defense components like peptidyl cis/trans isomerase, MAP kinase, beta 1,3 glucanase, serine threonine kinase, patatin like protein, lactolylglutathione lyase, coproporphyrinogen III oxidase, sulfotransferases; reactive oxygen species regulating components like respiratory burst oxidase, superoxide dismutases, cytochrome b5 reductase, glutathione reductase, thioredoxin reductase, ATPase; metabolism regulating components, myo inositol phosphate, carboxylate synthase; transport related gamma tonoplast intrinsic protein, and structural component, ubiquitins to serve as important nodals of defense signaling network. These nodal molecules probably served as hub controllers of defense signaling. Functional characterization of these hub molecules would not only help in developing better understanding of chickpea-Foc1 interaction but also place them as promising candidates for resistance management programs against vascular wilt of legumes.

A unique microRNA profile in end-stage heart failure indicates alterations in specific cardiovascular signaling networks.

It is well established that the gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, however, less is known about the reasons behind such global differences. MicroRNAs (miRNAs) are short non-coding RNAs that can target multiple molecules to regulate wide array of proteins in diverse pathways. The goal of the study was to profile alterations in miRNA expression using end-stage human heart failure samples with an aim to build signaling network pathways using predicted targets for the altered miRNA and to determine nodal molecules regulating individual networks. Profiling of miRNAs using custom designed microarray and validation with an independent set of samples identified eight miRNAs that are altered in human heart failure including one novel miRNA yet to be implicated in cardiac pathology. To gain an unbiased perspective on global regulation by top eight altered miRNAs, functional relationship of predicted targets for these eight miRNAs were examined by network analysis. Ingenuity Pathways Analysis network algorithm was used to build global signaling networks based on the targets of altered miRNAs which allowed us to identify participating networks and nodal molecules that could contribute to cardiac pathophysiology. Majority of the nodal molecules identified in our analysis are targets of altered miRNAs and known regulators of cardiovascular signaling. Cardio-genomics heart failure gene expression public data base was used to analyze trends in expression pattern for target nodal molecules and indeed changes in expression of nodal molecules inversely correlated to miRNA alterations. We have used NF kappa B network as an example to show that targeting other molecules in the network could alter the nodal NF kappa B despite not being a miRNA target suggesting an integrated network response. Thus, using network analysis we show that altering key functional target proteins may regulate expression of the myriad signaling pathways underlying the cardiac pathology.

Ultrastructural anatomy of nodes of Ranvier in the peripheral nervous system as revealed by STED microscopy

We used stimulated emission depletion (STED) superresolution microscopy to analyze the nanoscale organization of 12 glial and axonal proteins at the nodes of Ranvier of teased sciatic nerve fibers. Cytoskeletal proteins of the axon (betaIV spectrin, ankyrin G) exhibit a high degree of one-dimensional longitudinal order at nodal gaps. In contrast, axonal and glial nodal adhesion molecules [neurofascin-186, neuron glial-related cell adhesion molecule (NrCAM)] can arrange in a more complex, 2D hexagonal-like lattice but still feature a ∼190-nm periodicity. Such a lattice-like organization is also found for glial actin. Sodium and potassium channels exhibit a one-dimensional periodicity, with the Nav channels appearing to have a lower degree of organization. At paranodes, both axonal proteins (betaII spectrin, Caspr) and glial proteins (neurofascin-155, ankyrin B) form periodic quasi-one-dimensional arrangements, with a high degree of interdependence between the position of the axonal and the glial proteins. The results indicate the presence of mechanisms that finely align the cytoskeleton of the axon with the one of the Schwann cells, both at paranodal junctions (with myelin loops) and at nodal gaps (with microvilli). Taken together, our observations reveal the importance of the lateral organization of proteins at the nodes of Ranvier and pave the way for deeper investigations of the molecular ultrastructural mechanisms involved in action potential propagation, the formation of the nodes, axon-glia interactions, and demyelination diseases.

Effects of a novel Nodal-targeting monoclonal antibody in melanoma.

Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.

Expression of Nodal on Bronchial Epithelial Cells Influenced by Lung Microbes Through DNA Methylation Modulates the Differentiation of T-Helper Cells

Background/Aims: The previous study in our lab showed that Nodal molecule on bronchial epithelial cells (BECs) was modulated by all kinds of lung microbes. The present study was designed to determine the effects of Nodal on proliferation of BECs and BECs-induced differentiation of T-helper (Th) cells. The epigenetic mechanisms of Nodal expression following treatments of different lung microbes were also identified. Methods: Real-time polymerization chain reaction (PCR) and western blot were used to determine the expression of Nodal. Flow cytometry was used to observe the effects of proliferation of BECs and subsequent BECs-induced differentiation of Th cells. Methylation levels of CpG islands in Nodal promoters were also analyzed by time of flight mass spectrometry. Results: The results showed that Nodal promoted proliferation of BECs and BECs-induced differentiation of Th cell from Th1 to Th2 and Th17. Nodal promoter showed a hyper-methylation in normal BECs. Through methylation modification in the promoter, P. aeruginosa or A.baumanni inhibited the expression of Nodal while RSV promoted the expression of Nodal. Conclusions: Our data showed that Nodal promoted Th2 and Th17 differentiation and inhibited Th1 differentiation which may cause imbalance of airway microenvironment. P. aeruginosa or A.baumanni may be hopeful for the treatment of airway hyperresponsveness by inhibition Nodal expression through DNA methylation modification in the promoter.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

MicroRNAs miR-1, miR-133a, miR-133b, miR-208a and miR-208b are dysregulated in Chronic Chagas disease Cardiomyopathy

Background/methodsChagas disease is caused by an intracellular parasite, Trypanosoma cruzi, and it is a leading cause of heart failure in Latin America. The main clinical consequence of the infection is the development of a Chronic Chagas disease Cardiomyopathy (CCC), which is characterized by myocarditis, hypertrophy and fibrosis and affects about 30% of infected patients. CCC has a worse prognosis than other cardiomyopathies, like idiopathic dilated cardiomyopathy (DCM). It is well established that myocardial gene expression patterns are altered in CCC, but the molecular mechanisms underlying these differences are not clear. MicroRNAs are recently discovered regulators of gene expression, and are recognized as important factors in heart development and cardiovascular disorders (CD). We analyzed the expression of nine different miRNAs in myocardial tissue samples of CCC patients in comparison to DCM patients and samples from heart transplant donors. Using the results of a cDNA microarray database on CCC and DCM myocardium, signaling networks were built and nodal molecules were identified. ResultsWe observed that five miRNAs were significantly altered in CCC and three in DCM; importantly, three miRNAs were significantly reduced in CCC as compared to DCM. We observed that multiple gene targets of the differentially expressed miRNAs showed a concordant inverse expression in CCC. Significantly, most gene targets and involved networks belong to crucial disease-related signaling pathways. ConclusionThese results suggest that miRNAs may play a major role in the regulation of gene expression in CCC pathogenesis, with potential implication as diagnostic and prognostic tools.

Diabetic Hyperglycemia Acutely Affects Action Potentials and Ionic Currents through CaMKII Activation on Rat Ventricular Myocytes

Diabetes mellitus is a complex disease that involves cardiomyopathy and neuropathy. Ca2+-Calmodulin dependent protein kinase II (CaMKII) is a nodal molecule that participates in many physiological and pathological processes in the heart. Diabetic hyperglycemia has been shown to activate CaMKII, through a novel modification of O-linked N-acetylglucosamine (O-GlcNAc) at S279, leading to cardiac arrhythmias at the whole heart/animal level. However, acute hyperglycemia affects specific ion channels and thus action potentials (APs) through O-GlcNAc activated CaMKII are still unclear. To investigate this question, we measured APs and ionic currents on freshly isolated rat ventricular myocytes under acute diabetic hyperglycemia challenge. Glucose (30mM) perfusion significantly reduced the AP amplitude (82.7±5.7% of control, n=8, p=0.011), depolarized the resting membrane potential (−79.8±0.8 mV for control vs. −73.4±1.8 mV for glucose, n=8, p=0.002), and prolonged the AP duration (117.3±7.1% of control, n=8, p=0.001) on rat myocytes whereas osmolality matched mannitol application did not change any AP parameters. KN-93 (10uM) pre-incubation abolished the glucose effects, indicating that acute glucose application changes cellular electrical activities via a CaMKII-dependent manner. Together, these data provide evidence for the arrhythmogenesis of acute hyperglycemia at the cellular level and suggest that CaMKII-modulated ionic currents are responsible for the hyperglycemic effects on APs.