Project description:microRNA array of 4 cell lines: WI-38 primary fibroblasts (â??Controlâ??), slow growers (early passage after immortalization, Slow), fast growers (extensive passaging after immortalization) and fast growers transformed by constitutively activated mutant H-RasV12 (â??Rasâ??) WI-38 cells were grown in 37° in MEM supplemented with 10% non-heat-inactivated fetal bovine serum (Sigma), pen-strep, sodium pyruvate, L-glutamine solution (Beit HaEmek). RNA was extracted using Nucleospin miRNA kit (Macherey-Nagel), according to manufacturerâ??s instructions. microRNA array analysis was done in duplicates, using the miRNA Complete Labeling and Hyb Kit (Agilent, 5190-0456) according to the manufacturer's instructions. Briefly, for each sample 100ng RNA was dephosphorylated, denatured, labeled with Cyanine 3-pCp and purified using Micro Bio-Spin 6 Columns. Hybridization was done for 20h with Agilent SurePrint G3 Unrestricted miRNA 8x60K (Release 19.0) arrays. Arrays were scanned using an Agilent DNA microarray scanner, and analyzed using the AgiMicroRna package in R with the RMA algorithm. The function filterMicroRna was applied with the following parameters: control = TRUE, IsGeneDetected = TRUE, wellaboveNEG = TRUE, limIsGeneDetected = 50 and limNEG = 25.
Project description:Several features differentiate aged cells from young cells, many of which are due to changes in gene expression during the aging process. The mechanisms of altered gene expression in aging cells remain incompletely understood, and we hypothesized that long non-coding (lnc) RNAs mediate at least some of these changes. To determine candidate lncRNAs which are associated with aging process, we screened for alterations in lncRNA expression with aging in skin fibroblasts. Twenty-eight lncRNAs were found to be expressed more than 2-fold in aged fibroblasts, and thirty-five lncRNAs expressed less than 0.5-fold.
Project description:In an attempt to improve the antitumor activity and reduce the side effects of irinotecan (2), novel prodrugs of SN-38 (3) were prepared by conjugating amino acids or dipeptides to the 10-hydroxyl group of SN-38 via a carbamate linkage. The synthesized compounds completely generated SN-38 in pH 7.4 buffer or in human plasma, while remaining stable under acidic conditions. All prodrug compounds demonstrated much greater in vitro antitumor activities against HeLa cells and SGC-7901 cells than irinotecan. The most active compounds, 5h, 7c, 7d, and 7f, exhibited IC50 values that were 1000 times lower against HeLa cells and 30 times lower against SGC-7901 cells than those of irinotecan, and the inhibitory activities of these prodrugs against acetylcholinesterase (AchE) were significantly reduced, with IC50 values more than 6.8 times greater than that of irinotecan. In addition, compound 5e exhibited the same level of tumor growth inhibitory activity as irinotecan (CPT-11) in a human colon xenograft model in vivo.
Project description:Background and purposeThe opioid system plays a crucial role in several physiological processes in the CNS and in the periphery. It has also been shown that selective opioid receptor agonists exert potent inhibitory action on pruritus and pain. In this study we examined whether two analogues of Salvinorin A, PR-37 and PR-38, exhibit antipruritic properties in mice.Experimental approachTo examine the antiscratch effect of PR-37 and PR-38 we used a mouse model of compound 48/80-induced pruritus. In order to elucidate the mechanism of action of tested compounds, specific antagonists of opioid and cannabinoid receptors were used. The effect of PR-37 on the CNS was assessed by measuring motor parameters and exploratory behaviours in mice.Key resultsPR-37 and PR-38, jnjected s.c., significantly reduced the number of compound 48/80-induced scratching behaviours in mice in a dose- and time-dependent manner. PR-38 was also active when orally administered. The antiscratch activity of PR-37 was blocked by the selective κ opioid receptor antagonist, nor-binaltorphimine, and that of PR-38 by the selective μ opioid receptor antagonist, β-funaltrexamine.Conclusion and implicationsIn conclusion, a novel framework for the development of new antipruritic drugs derived from salvinorin A has been validated.
Project description:CPT-11 is a clinically used cancer drug, and it is a prodrug of the potent topoisomerase I inhibitor, SN-38 (7-ethyl-10-hydroxycamptothecin). To bypass the need for the in vivo conversion of CPT-11 and increase the therapeutic index, bifunctional derivatives of SN-38 were prepared for use in antibody-based targeted therapy of cancer. The general synthetic scheme incorporated an acetylene-azide click cycloaddition step in the design, a short polyethylene glycol spacer for aqueous solubility, and a maleimide group for conjugation. Conjugates of a humanized anti-CEACAM5 monoclonal antibody, hMN-14, prepared using these SN-38 derivatives were evaluated in vitro for stability in buffer and human serum and for antigen-binding and cytotoxicity in a human colon adenocarcinoma cell line. Conjugates of hMN-14 and SN-38 derivatives 16 and 17 were found promising for further development.
Project description:We performed a microarray experiment to test the hypothesis that pre-labour premature rupture of fetal membranes (PPROM) has a different underlying pathology from spontaneous premature labour with intact membranes (PTL). We thought that the different genetic signature would be detected in the cervical tissue. Our study included cervical biopsies obtained as previously described from women undergoing caesarean section (CS) following PPROM, PTL, term labour (TL) or delivering at term prior to the presentation of labour (Term no labour âTNL). After quality control of the expression arrays, any association between sample covariates was evaluated and no evidence was found of unexpected associations. The differences in expression levels for each statistical comparison were then computed. We identified genes that were differentially expressed with an adjusted pvalue < 0.01 from hypothesis testing together with a fold change >= 2. The strongest effect, i.e. the contrast with the highest number of DEGs, was observed for âTerm labour vs Term not labourâ (1,285 genes) while the contrast with the fewest DEGs was âTerm labour vs Preterm premature rupture of membranesâ (16 genes). A table showing the number of differentially expressed genes, for each comparison, can be found in the Results section of this report. We then examined the number of genes overlapping between the statistical comparisons performed (see section 2.5 of the Results) and found that the contrast âTerm labour vs Term not labourâ had a large number of DEGs in common with the five other contrasts.
Project description:Dupuytren's contracture (DC) is the most common inherited connective tissue disease of humans and is hypothesized to be associated with aberrant wound healing of the palmar fascia. Fibroblasts and myofibroblasts are believed to play an important role in the genesis of DC and the fibroproliferation and contraction that are hallmarks of this disease. This study compares the gene expression profiles of fibroblasts isolated from DC patients and controls in an attempt to identify key genes whose regulation might be significantly altered in fibroblasts found within the palmar fascia of Dupuytren's patients. Total RNA isolated from diseased palmar fascia (DC) and normal palmar fascia (obtained during carpal tunnel release; 6 samples per group) was subjected to quantitative analyses using two different microarray platforms (GE Code Link™ and Illumina™) to identify and validate differentially expressed genes. The data obtained was analyzed using The Significance Analysis of Microarrays (SAM) software through which we identified 69 and 40 differentially regulated gene transcripts using the CodeLink™ and Illumina™ platforms, respectively. The CodeLink™ platform identified 18 upregulated and 51 downregulated genes. Using the Illumina™ platform, 40 genes were identified as downregulated, eleven of which were identified by both platforms. Quantitative RT-PCR confirmed the downregulation of three high-interest candidate genes which are all components of the extracellular matrix: proteoglycan 4 (PRG4), fibulin-1 (FBLN-1) transcript variant D, and type XV collagen alpha 1 chain. Overall, our study has identified a variety of candidate genes that may be involved in the pathophysiology of Dupuytren's contracture and may ultimately serve as attractive molecular targets for alternative therapies. Dupuytren's contracture and may ultimately serve as attractive molecular targets for alternative therapies. 6 Samples from carpal-tunnel derived fibroblasts and 6 Samples from Dupuytren's contracture-derived fibroblasts were hybridized to Illumina's Sentrix Human-6 Expression Beadchip and GE Code Link arrays.
Project description:Interleukin (IL)-10 plays an important role in immune regulation in the intestine. Immune deregulation is suggested in the pathogenesis of inflammatory bowel disease (IBD). This study aims to elucidate the role of IL-23 in the suppression of IL-10 in the IBD intestinal mucosa. Surgically removed colon specimens were obtained from 16 IBD patients. The expressions of IL-10, IL-23, and IgA in the specimens were examined at the protein and gene transcriptional levels. The gene transcription of IL-10 was assessed by chromatin immunoprecipitation assay and promoter accessibility assay. The levels of IgA and IL-10 were significantly lower, whereas the levels of IL-23 were higher, in IBD specimens than in normal controls. The levels of IgA and IL-10 were negatively correlated with the infiltration of inflammatory cells in the IBD mucosa. The production of IL-10 by lamina propria mononuclear cells was lower in the IBD group than in the control group, and these levels could be enhanced by blocking IL-23. The gene transcription of IL-10 was significantly suppressed in CD4(+) T cells of IBD mucosa; this phenomenon could be replicated in vitro by adding IL-23 in the culture of polarized Th2 cells. Overexpression of IL-23 in the intestinal mucosa suppresses the production of IL-10, which weakens the defensive barrier by reducing the production of IgA in the gut.