Project description:Cognitive dysfunction (CD) in heart failure (HF) adversely affects treatment compliance and quality of life. Although, ryanodine receptor type 2 (RyR2) has been linked to cardiac muscle dysfunction, its role in CD remains unclear. Here, we show in hippocampal neurons from patients and mice with HF that the ryanodine receptor type 2 (RyR2)/intracellular Ca 2+ release channels were post-translationally modified (PTM) and leaky. RyR2 PTM was caused by hyper-adrenergic stress and activation of the transforming growth factor (TGF-β) pathway. HF mice treated with either a RyR2 stabilizer drug (S107), beta-blocker (propranolol) or TGF-β inhibitor (SD-208), or mice insensitive RyR2 Ca 2+ leak (RyR2-S2808A), were protected against HF-induced CD. Taken together, we propose that HF is a systemic illness that include cardiogenic dementia, and intracellular Ca 2+ leak is a common effector in multiple components of HF.
Project description:Posttraumatic stress disorder (PTSD) is a prevalent psychiatric disorder. Several studies have attempted to characterize molecular alterations associated with PTSD, but most findings were limited to the investigation of specific cellular markers in the periphery or defined brain regions. In the current study, we aimed to unravel affected molecular pathways/mechanisms in the fear circuitry associated with PTSD. We interrogated a foot shock induced PTSD mouse model by integrating proteomics and metabolomics profiling data. Alterations at the proteome level were analyzed using in vivo 15N metabolic labeling combined with mass spectrometry in prelimbic cortex (PrL), anterior cingulate cortex (ACC), basolateral amygdala (BLA), central nucleus of amygdala (CeA) and CA1 of hippocampus between shocked and non-shocked (control) mice, with and without fluoxetine treatment.
Project description:MEIS2 has an important role in development and organogenesis, and is implicated in the pathogenesis of human cancer. The molecular basis of MEIS2 action in tumorigenesis is not clear. Here, we show that MEIS2 is highly expressed in human neuroblastoma cell lines and is required for neuroblastoma cell survival and proliferation. Depletion of MEIS2 in neuroblastoma cells leads to M phase arrest and mitotic catastrophe, whereas ectopic expression of MEIS2 markedly enhances neuroblastoma cell proliferation, anchorage-independent growth, and tumorigenicity. Gene expression profiling reveals an essential role of MEIS2 in maintaining the expression of a large number of late cell cycle genes, including those required for DNA replication, G2-M checkpoint control and M phase progression. Importantly, we identify MEIS2 as a transcription activator of the MuvB-BMYB-FOXM1 complex that functions as a master regulator of mitotic gene expression. Further, we show that FOXM1 is a direct target gene of MEIS2 and is required for MEIS2 to upregulate mitotic genes. These findings link a development gene to the control of cell cycle progression and suggest that high MEIS2 expression is a molecular mechanism for high expression of mitotic genes that is commonly observed in cancers of poor prognosis. Affymetrix microarray assays were performed according to the manufacturer's directions on total RNA isolated from three independent samples of BE(2)-C cells infected with lentiviruses expressing either shGFP or shMEIS2-43 for 48 hours.
Project description:The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide health emergency. Patients infected with SARS-CoV-2 present with diverse symptoms relating to the severity of the disease. Determining the proteomic changes associated with these diverse symptoms and in different stages of infection is beneficial for clinical diagnosis and management. Here, we performed a Tandem Mass Tag (TMT)-labeling proteomic study on the plasma of healthy controls and COVID-19 patients, including those with asymptomatic infection (NS), mild syndrome (MS), and severe syndrome in the early phase (SSEP) and the later phase (SSLP). While the number of patients included in each group is low, our comparative proteomic analysis revealed that complement and coagulation cascades, cholesterol metabolism and glycolysis-related proteins were affected after infection with SARS-CoV-2. Compared to healthy controls, ELISA analysis confirmed that SOD1, PRDX2 and LDHA levels were increased in the patients with severe symptoms. Both gene set enrichment analysis and receiver operator characteristic analysis indicated that SOD1 could be a pivotal indicator for the severity of COVID-19. Our results indicated that plasma proteome changes differed based on symptoms and disease stages and SOD1 could be a predictor protein for indicating COVID-19 progression. These results may also provide new understanding for COVID-19 diagnosis and treatment.
Project description:Chronic alcohol consumption can lead to alchohol-related brain damage (ARBD). Despite the well known acute effects of alcohol the mechanism responsible for chronic brain damage is largely unknown. Pathologically the major change is the loss of white matter while neuronal loss is mild and restricted to a few areas such as the prefrontal cortex. In order to improve our understanding of ARBD pathogenesis we used microarrays to explore the white matter transcriptome of alcoholics and controls. Our results suggest that hepatic encephalopathy, along with two confounders, gray matter contamination and low RNA quality, are major drivers of gene expression in ARBD. All three exceeded the effects of alcohol itself. In particular, low quality RNA samples were characterized by an upregulation of protein translation machinery while hepatic encephalopathy was associated with a downregulation of mitochondrial energy metabolism pathways. The findings in HE alcoholics are consistent with the metabolic acidosis seen in this condition. In contrast non-HE alcoholics had widespread but only subtle changes in gene expression in their white matter. The initial cohort was compromised of four alcoholics without hepatic encephalopathy (non-HE alcoholics), three alcoholics with HE (HE alcoholics) and three neurologically normal controls. For each indvidual frozen white matter was sampled in the superior frontal gyrus (prefrontal cortex) and the precentral gyrus (motor cortex). These two cortices experience either moderate (prefrontal cortex) or no neuronal loss (motor cortex) with alcohol-related brain damage. Each white matter sample was divided in two before RNA was extracted to give two 'biological' repeats and a total of 40 samples. Subsequently eight duplicates were removed due to their gray matter contamination or low RNA quality to leave a 32-sample cohort (23 alcoholic (including eight with HE ) and nine control samples.
Project description:Transgenic KrasG12D mice can recapitulate pancreas intra-epithelial neoplasia (PanIN). Caerulein is a cholecystokinin analogue and induces acute pancreatitis when injected intra-abdominally. Caerulein-induced acute pancreatitis will accelerate PanIN progression in KrasG12D mice. We compared mRNA profile changes between KrasG12D mice with acute caerulein-induced pancreatitis and wild-type mice without acute pancreatitis. The experiment had two groups. Experiment group: KrasG12D mice with acute caerulein-induced pancreatitis (N=6). Three mice in experiment group received 1-week caerulein injection, and the other three mice received 2-week caerulein injection. All experiment group mice started to receive caerulein injection at 1-month of age, and were sacrificed at the last day of caerulein injection. Control group: wild-type mice without acute pancreatitis (N=6). The mice were sacrificed at 1.5-month of age. Whole pancreas tissue lysate samples were subjected to mRNA array assay.
Project description:A major problem in cancer research is the lack of a tractable model for delayed metastasis. Herein we show that cancer cells suppressed by SISgel, a gel-forming normal ECM material derived from Small Intestine Submucosa, in flank xenografts show properties of suppression and re-activation that are very similar to normal delayed metastasis and suggest they can serve as a novel model for developing therapeutics to target micrometastases or suppressed cancer cells. Co-injection with SISgel suppressed the malignant phenotype of highly invasive J82 and T24 bladder cancer cells and highly metastatic JB-V cells in flank xenografts. Cells could remain viable up to 120 days without forming tumors and appeared much more highly differentiated and less atypical than tumors from cells co-injected with Matrigel. In 40% of SISgel xenografts, growth resumed in the malignant phenotype after a period of suppression or dormancy for at least 30 days and was more likely with implantation of 3 million cells or more cells. Ordinary Type I collagen did not suppress malignant growth, and tumors developed about as well with collagen as with Matrigel. A clear signal in gene expression over different cell lines was not seen, but in contrast, Reverse Phase Protein Analysis of 250 proteins across 4 cell lines identified a clear signal at the protein level involving Integrin Linked Kinase (ILK) signaling that was confirmed by an ILK inhibitor. We suggest that cancer cells suppressed on SISgel could serve as a model for dormancy and re-awakening to develop therapeutic targets for micrometastases. Earlier we demonstrated that the phenotype of bladder cancer cells was radically different in 3-dimensional organotypic culture when grown on a normal extracellular matrix preparation (SISgel) as compared to that observed on a cancer-modulated permissive extracellular matrix preparation (Matrigel). SISgel is a gel-forming material derived from acellular small intestine submucosa, whereas Matrigel is a basement membrane preparation obtained from a mouse sarcoma. On Matrigel the bladder cancer cells recapitulated the phenotype reported for the original tumor; in sharp contrast, most of the malignant properties were lost when the cells were grown on SISgel. Cell lines derived from papillomas formed a layered structure reminiscent of normal urothelium, whereas cell lines derived from higher grade tumors formed a noninvasive layer of cells. These findings suggested that growth of cancer cells on normal ECM could provide a model to investigate the phenomenon of suppression of malignancy by normal ECM in metastasis and recurrence. In this study we explored whether the phenotypic suppression seen in organotypic culture of bladder cancer cells on SISgel also is observed in vivo. Positive findings support the use of SISgel as a model for investigations of the dormant or suppressed tumor cell phenotype and of mechanisms by which the normal ECM exerts an inhibitory influence on tumorigenesis and metastasis. The findings strongly suggest that interactions of cancer cells with normal ECM play an important role in recurrence and metastasis and further suggest that targeting suppressed cells could represent a heretofore unexploited point of vulnerability in cancer therapy.
Project description:Background: Molecular profiling of diffuse gliomas has provided significant insights into the pathogenesis, classification and prognostication of these malignancies. However, previous molecular studies of glioma have largely focused on genomic readouts and targeted proteomic profiling technologies. Consequently, proteomic and downstream functional landscape of gliomas in general, and molecular subgroups in particular, remains largely unexplored. Here, we utilize liquid chromatography tandem mass spectrometry to profile genomically-defined cohorts of gliomas spanning the full range of World Health Organization (WHO) grades. Methods: Bulk frozen tissue and purified micro-dissected regions from formalin-fixed paraffin-embedded (FFPE) tissues were assembled and utilized to define robust proteomic signatures of both low grade, infiltrative and high-grade tumors. As a final analysis, primary tumor tissue was compared with both IDH-mutated and IDH-wildtype glioblastoma stem cell (GSC) lines to further overcome tissue heterogeneity and pinpoint proteins differences likely arising in the relevant glial cellular drivers of tumor development. Results: In aggregate, 5,496 unique proteins over 3 glioma cohorts were identified, and span common molecular subclasses based on IDH and 1p19q co-deletion status and all four WHO grades. Supervised clustering highlights substantial proteome and systems-level pathway differences between different genetically defined glioma subtypes and WHO grades. By using bulk tumor statistical analysis, 833 proteins distinguish different WHO grade tumors, while FFPE tumor dissection reveals 287 proteins in GBMs with abundance changes according to IDH mutation status. Using our integrative approach, calcium signaling, proteins of the endoplasmic reticulum and extracellular integrin proteins are most conserved proteomic markers that distinguish aggressive, IDH-wt, from IDH-mut GBM tumors in primary and tissue culture models gliomagenesis. Conclusions: This proteomic survey provides the largest and most diverse unbiased protein-based brain tumor resource to date. Current treatments for glial tumors are largely non-specific and overlap between genomic subtypes and WHO grades. Our analysis provides early insight into the vast downstream and epigenetic protein-level differences within this molecular framework. Given the central position proteins occupy in driving biology and phenotype, further characterization of the substantial proteomic diversity that exist between the molecular subtypes and grades of gliomas, proteomics may help define more personalized prognostic and predictive biomarkers for precision care.
Project description:Phosphorylation, as one of the most important and well-studied post-translational modifications, is tightly associated with protein activity and protein functional regulation. Here in this study, we generated a global protein phosphorylation atlas within the pathological site of human RCT patients by using Tandem Mass Tag (TMT) labeling combining with mass spectrometry. GO enrichment analyses and KEGG pathway analyses were performed. At last, a weighted kinase-site phosphorylation network was built to identify potentially core kinase.
Project description:Tyrosine kinase inhibitors (TKI) are highly effective in treatment of chronic myeloid leukemia (CML) but do not eliminate leukemia stem cells (LSC), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSC, suggesting that additional kinase-independent mechanisms contribute to LSC preservation. We investigated whether signals from the bone marrow (BM) microenvironment protect CML LSC from TKI treatment. Coculture with human BM mesenchymal stromal cells (MSC) significantly inhibited apoptosis and preserved CML stem/progenitor cells following TKI exposure, maintaining colony forming ability and engraftment potential in immunodeficient mice. We found that the N-Cadherin receptor plays an important role in MSC-mediated protection of CML progenitors from TKI. N-Cadherin-mediated adhesion to MSC was associated with increased cytoplasmic N-Cadherin-M-NM-2-catenin complex formation, as well as enhanced M-NM-2-catenin nuclear translocation and transcriptional activity. Increased exogenous Wnt-mediated M-NM-2-catenin signaling played an important role in MSC-mediated protection of CML progenitors from TKI treatment. Our results reveal a close interplay between N-Cadherin and the Wnt-M-NM-2-catenin pathway in protecting CML LSC during TKI treatment. Importantly, these results reveal novel mechanisms of resistance of CML LSC to TKI treatment, and suggest new targets for treatment designed to eradicate residual LSC in CML patients. RNA was obtained from CML CD34+ cells treated with or without IM (5M-NM-<M) and MSC for 96 hours, amplified, labeled and hybridized to GeneChip 1.0 arrays (Affymetrix, Santa Clara, CA). Microarray data analysis was performed using R (version 2.9) with genomic analysis packages from Bioconductor (version 2.4). The 33297 probes represented on the microarray were filtered by cross-sample mean, and for standard deviation of greater than the 25% quantile, yielding 18624 probes representing 12553 genes. Linear regression was used to model the gene expression with the consideration of a 2x2 factorial design and matched samples. Differentially expressed genes were identified by calculating empirical Bayes moderated t-statistic, and p-values were adjusted by FDR using the M-bM-^@M-^\LIMMAM-bM-^@M-^] package. Gene Set Enrichment Analysis (GSEA) was performed using GSEA software version 2.04 to detect enrichment of predetermined gene sets using t-scores from all genes for 1263 gene sets in the C2 (curated gene sets) category from the Molecular Signature Database (MsigDB).