Project description:Parkinson’s disease (PD) as a progressive neurodegenerative disorder arises from multiple genetic and environmental factors. However, underlying pathological mechanisms remain poorly understood. Using multiplexed single-cell transcriptomics, we analyze human neural precursor cells (hNPCs) from sporadic PD (sPD) patients. Alterations in gene expression appear in pathways related to primary cilia (PC). Accordingly, in these hiPSC-derived hNPCs and neurons, we observe a shortening of PC. Additionally, we detect a shortening of PC in PINK1-deficient human cellular and mouse models of familial PD. Furthermore, in sPD models, the shortening of PC is accompanied by an increased SHH signal transduction. Inhibition of this pathway rescues the alterations in PC morphology and mitochondrial dysfunction. Thus, increased SHH activity due to ciliary dysfunction is needed for the development of pathoetiological phenotypes observed in sPD, like mitochondrial dysfunction. In sum, altered PC function is part of early PD pathoetiology and inhibiting the overactive SHH signaling is a potential neuroprotective therapy.
Project description:Platinum and palladium are highly sought-after noble metals that due to their low abundance have high value and because of their stability and their roles in catalytic processes are very desirable for industrial purposes. Bacteria are able to produce nanoparticles of platinum and palladium at low temperatures and from low concentration feedstocks contrary to chemical methods and so pose a potentially untapped ‘green’ resource for nanoparticle synthesis. We have used the bacterium Desulfovibrio alaskensis G20 to reduce Pt and Pd ions to zero-valent nanoparticle forms and used differential shotgun proteomics to identify proteins responsible for this reduction . There was found to be a core set of 13 proteins common to both datasets as well as 7 proteins specific to Pt and Pd individually. Over expression of one of Pt-specific genes; the NiFe hydrogenase small subunit, resulted in the formation of larger nanoparticles. For the first time the proteins involved in the metal reduction pathway have been pinpointed and it is our hope that these target genes can then be used for nanoparticle production to tailor specific properties for industrial purposes at the genetic level rather than post-production.
Project description:Purpose:The goals of this study is to analyze the candidate genes motified by CID755674 to promote the mouse embryonic stem cell (ESC) self-renewal through RNA-seq approach. Previously, we found that MEK inhbitior PD0323901 (PD) coporates with protein kinase D inhibitor CID755673 (CID) is able to maitain mouse embryonic stem cell (mESC) self-renewal, we named this conditon is "PC". In order to find out why CID755673 can promote mouse embryonic stem cell self-renewal, we performed RNA-sequence in mESCs treated with PD or PC, and tried to identify the key mechanisms under CID
Project description:We used 500 melanized dopamine neurons isolated from post-mortem human substantia nigra pars compacta of each sample with laser capture microdissection. Total RNA was extracted with Arcturus PicoPure RNA extraction kit and amplified two times with Arcturus RiboAmp RNA amplification kit. the samples were then sent to the Partners Center for Genetics and Genomics (HPCGG.org) for labeling, hybridization and scanning according to standard Affymetrix protocols. Samples from a total of 16 human brains, 8 PDs (4 male and 4 female) and 8 controls (4 male and 4 female) were studied. These were selected from available material after screening total RNA extracted from a small region of the SN for integrity of 18S and 28S species using an Agilent bioanalyzer. We also matched the cases for age and post mortem interval (PMI) (PD Female age, 75 ± 4.4, PMI, 20.5 ± 2.5; PD Male age 77 ± 6.3, PMI 13.85 ± 1.7; Con Female age 68 ± 3.3, PMI 21.8 ± 0.8; Con Male age 70.2 ± 6.3, PMI 18.11 ± 3.6). Vital Statistics of the cases used in this study. PD= Parkinson disease, C= Control, F=Female, M=Male: Case Age Sex PMI Cel file Pdf1 81 F 17 459F cf1 61 f <24 515H Pdf2 84 F 24.08 515K cf2 n/a f >48 515I Pdf3 69 F <24 515J cf3 74 f 23.00 459D Pdf4 66 F <24 515F cf4 69 f 20.70 459A pdm1 68 m 17.16 515E Cm1 67 m 22.33 459C pdm2 67 m 14.5 515D Cm2 63 m 21.50 459B pdm3 94 m 9.25 515G Cm3 62 m 21.20 515L pdm4 78 m 14.50 515B Cm4 89 m 7.42 515C
Project description:Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) can provide choline-based imaging approaches as powerful tools to improve diagnosis and identify new therapeutic targets. Biochemical, protein and mRNA expression analyses demonstrated that the increase in the major choline-containing metabolite phosphocholine (PCho) in EOC compared with normal and non-tumoral immortalized counterparts (EONT) mainly rely upon: 1) ChoK activation, consistent with higher protein content and increased ChoK? mRNA expression levels; 2) PC-plc activation, consistent with higher, previously reported, protein expression. More limited and variable sources of PCho could derive, in some EOC cells, from activation of Phospholipase D or GPC-pd. Phospholipase A2 activity and isoforms’ expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoK? mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgical specimens isolated from EOC patients. Overall, we demonstrated that the elevated PCho pool detected in EOC cells primarily resulted from the upregulation/activation of ChoK and PC-plc involved in the biosynthetic and in a degradative pathway of the PC-cycle, respectively. 20 EOC frozen surgical specimens, 8 EOC cell lines and Ovarian Surface Epithelial (OSE) cells (4 different preparations, 3 of them pooled in one sample).
Project description:Expression data from peritoneal biopsies of patients with encapsulating peritoneal sclerosis (EPS), patients undergoing first implantation of a peritoneal dialysis catheter (PD), and patients undergoing abdominal surgery for non-peritoneal conditions (controls) We used microarrays to determine the transcriptional profiles of peritoneal membrane in patients with encapsulating peritoneal sclerosis (EPS), patients undergoing first insertion of a peritoneal dialysis cathetier (PD), and uremic patients without history of PD or EPS, undergoing abdominal surgery for non-peritoneal problems (CON) Encapsulating peritoneal sclerosis (EPS) is a devastating complication of peritoneal dialysis (PD), characterized by marked inflammation and severe fibrosis of the peritoneum, and associated with high morbidity and mortality. EPS can occur years after termination of PD and, in severe cases, leads to intestinal obstruction and ileus requiring surgical intervention. Despite ongoing research, the pathogenesis of EPS remains unclear. We performed a global transcriptome analysis of peritoneal tissue specimens from EPS patients, PD patients without EPS, and uremic patients without history of PD or EPS (Uremic). Unsupervised and supervised bioinformatics analysis revealed distinct transcriptional patterns that discriminated these three clinical groups. The analysis identified a signature of 219 genes expressed differentially in EPS as compared to PD and Uremic groups. Canonical pathway analysis of differentially expressed genes showed enrichment in several pathways, including antigen presentation, dendritic cell maturation, B cell development, chemokine signaling and humoral and cellular immunity (P value <0.05). Further interactive network analysis depicted effects of EPS-associated genes on networks linked to inflammation, immunological response, and cell proliferation. Gene expression changes were confirmed by qRT-PCR for a subset of the differentially expressed genes. EPS patient tissues exhibited elevated expression of genes encoding sulfatase1, thrombospondin 1, fibronectin 1 and alpha smooth muscle actin, among many others, while in EPS and PD tissues mRNAs encoding leptin and retinol-binding protein 4 were markedly down-regulated, compared to Uremic group patients. Immunolocalization of Collagen 1 alpha 1 revealed that Col1a1 protein was predominantly expressed in the submesothelial compact zone of EPS patient peritoneal samples, whereas PD patient peritoneal samples exhibited homogenous Col1a1 staining throughout the tissue samples. The results are compatible with the hypothesis that encapsulating peritoneal sclerosis is a distinct pathological process from the simple peritoneal fibrosis that accompanies all PD treatment. Total RNA was isolated from frozen peritoneal biopsy specimens obtained at time of surgery. RNA was hybridized to Affymetrix arrays, and analyzed. Select transcripts were subjected to validation by rt-pcr and by immunodetection.
Project description:The transcriptional regulation of drug-metabolizing enzymes and transporters (here collectively referred to as DMEs) in the developing proximal tubule is not well understood. As in the liver, DME regulation in the PT may be mediated through nuclear receptors which are thought to “sense” deviations from homeostasis by being activated by ligands, some of which are handled by DMEs, including drug transporters. Systems analysis of transcriptomic data during kidney development predicted a set of upstream transcription factors, including Hnf4a and Hnf1a, as well as Nr3c1 (Gr), Nfe2l2 (Nrf2), Ppara, and Tp53. Motif analysis of cis-regulatory further suggested that Hnf4a and Hnf1a are the main transcriptional regulators in the PT. Available expression data from tissue-specific Hnf4a KO tissues revealed that distinct subsets of DMEs were regulated by Hnf4a in a tissue-specific manner. ChIP-seq was performed to characterize the PT-specific binding sites of Hnf4a in rat kidneys at three developmental stages (prenatal, immature, adult), which further supported a major role for Hnf4a in regulating PT gene expression, including DMEs. In ex vivo kidney organ culture, an antagonist of Hnf4a (but not a similar inactive compound) led to predicted changes in DME expression, including among others Fmo1, Cyp2d2, Cyp2d4, Nqo2, as well as organic cation transporters and organic anion transporters Slc22a1(Oct1), Slc22a2 (Oct2), Slc22a6 (Oat1), Slc22a8(Oat3), and Slc47a1(Mate1). Conversely, overexpression of Hnf1a and Hnf4a in primary mouse embryonic fibroblasts (MEFs), sometimes considered a surrogate for mesenchymal stem cells, induced expression of several of these proximal tubule DMEs, as well as epithelial markers and a PT-specific brush border marker Ggt1. These cells had organic anion transporter function. Taken together, the data strongly supports a critical role for HNF4a and Hnf1a in the tissue-specific regulation of drug handling and differentiation toward a PT cellular identity. Hnf4a binding was examined in rat kidneys at three timepoints (E20, P13 and Adult) and p300 binding was examined in adult rat kidney cortex tissue using ChIP-seq. Four corresponding input DNA samples were used as controls for peak calling.
Project description:Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) can provide choline-based imaging approaches as powerful tools to improve diagnosis and identify new therapeutic targets. Biochemical, protein and mRNA expression analyses demonstrated that the increase in the major choline-containing metabolite phosphocholine (PCho) in EOC compared with normal and non-tumoral immortalized counterparts (EONT) mainly rely upon: 1) ChoK activation, consistent with higher protein content and increased ChoKalpha mRNA expression levels; 2) PC-plc activation, consistent with higher, previously reported, protein expression. More limited and variable sources of PCho could derive, in some EOC cells, from activation of Phospholipase D or GPC-pd. Phospholipase A2 activity and isoforms’ expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoKalpha mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgical specimens isolated from EOC patients. Overall, we demonstrated that the elevated PCho pool detected in EOC cells primarily resulted from the upregulation/activation of ChoK and PC-plc involved in the biosynthetic and in a degradative pathway of the PC-cycle, respectively.