Antibody RING-Mediated Destruction of Endogenous Proteins

International audienc

Analysis of the potential of cancer cell lines to release tissue factor-containing microvesicles: correlation with tissue factor and PAR2 expression

BackgroundDespite the association of cancer-derived circulating tissue factor (TF)-containing microvesicles and hypercoagulable state, correlations with the incidence of thrombosis remain unclear.MethodsIn this study the upregulation of TF release upon activation of various cancer cell lines, and the correlation with TF and PAR2 expression and/or activity was examined. Microvesicle release was induced by PAR2 activation in seventeen cell lines and released microvesicle density, microvesicle-associated TF activity, and phoshpatidylserine-mediated activity were measured. The time-course for TF release was monitored over 90 min in each cell line. In addition, TF mRNA expression, cellular TF protein and cell-surface TF activities were quantified. Moreover, the relative expression of PAR2 mRNA and cellular protein were analysed. Any correlations between the above parameters were examined by determining the Pearson’s correlation coefficients.ResultsTF release as microvesicles peaked between 30–60 min post-activation in the majority of cell lines tested. The magnitude of the maximal TF release positively correlated with TF mRNA (c = 0.717; p

Chemical composition, antimicrobial, antioxidant and anticancer activities of essential oil from Ammodaucus leucotrichus Cosson & Durieu (Apiaceae) growing in South Algeria

ABSTRACT. The chemical composition of the essential oil obtained by hydrodistillation from aerial parts of A. leucotrichus Cosson & Durieu (Apiaceae) grown in the south of Algeria (El-Oued) was determined by GC-MS analysis. The oil was found to be rich in perilladehyde 64.66% and D-Limonene 26.99%. The biological activity of A. leucotrichus Cosson & Durieu essential oil has been investigated. The in vitro antimicrobial activity of the essential oil sample was tested on eight strains, one yeast and one fungi. The test showed interesting antimicrobial properties, especially on Salmonella enterica and E. coli, the antioxidant capacity of the oil was measured using the cyclic voltammetry, and the AAT value of A. leucotrichus essential oil was evaluated 47.84 mg α-TE/L. In addition, the antitumor activity showed that the oil of A. leucotrichus was very significant against the HCT116 colon cancer cell line.               KEY WORDS: Ammodaucus leucotrichus, Antioxidant activity, Anticancer activity, Cyclic voltammetry Bull. Chem. Soc. Ethiop. 2019, 33(3), 541-549. DOI: https://dx.doi.org/10.4314/bcse.v33i3.1

Exploring the Social Validity and Diffusion Potential of Common Naturalistic Developmental Behavioral Intervention Strategies Implemented in Community Preschools

It is imperative that researchers include the perspectives from key voices regarding early support practices, yet very few studies have included direct assessment of autistic individuals and parents of young autistic children. Despite emerging evidence of effectiveness of naturalistic developmental behavioral intervention (NDBI) strategies, it is currently unknown whether autistic adults and parents of autistic individuals know about NDBI and if they view NDBI strategies as socially valid practice. We aimed to explore the perceptions of autistic adults and parents of young autistic children regarding the social validity of NDBI strategies implemented in community preschool classrooms and their dissemination potential. We conducted a convergent mixed methods research design to collect quantitative survey data and qualitative semi-structured interview data. We received survey responses from 33 autistic adults and 37 parents of young autistic children and interviewed 12 autistic adults and 12 parents of young autistic children. We conducted a series of paired samples and independent samples t-tests to compare perceptions between groups and thematic analysis to analyze qualitative data. Results indicated high levels of social validity for NDBI across both groups of participants and the need for dissemination of NDBI. Agreement between autistic adults and parents of young autistic children on the social validity of NDBI and recommendations for dissemination are promising preliminary findings that NDBI researchers and practitioners may draw upon when engaging in collaborative support planning and participatory research efforts

Replacing the wild type loxP site in BACs from the public domain with lox66 using a lox66 transposon

<p>Abstract</p> <p>Background</p> <p>Chromatin adjoining the site of integration of a transgene affects expression and renders comparisons of closely related transgenes, such as those derived from a BAC deletion series retrofitted with enhancer-traps, unreliable. Gene targeting to a pre-determined site on the chromosome is likely to alleviate the problem.</p> <p>Findings</p> <p>A general procedure to replace the <it>loxP </it>site located at one end of genomic DNA inserts in BACs with <it>lox66 </it>is described. Truncating insert DNA from the <it>loxP </it>end with a Tn10 transposon carrying a <it>lox66 </it>site simultaneously substitutes the <it>loxP </it>with a <it>lox66 </it>sequence. The replacement occurs with high stringency, and the procedure should be applicable to all BACs in the public domain. Cre recombination of <it>loxP </it>with <it>lox66 </it>or <it>lox71 </it>was found to be as efficient as another <it>loxP </it>site during phage P1 transduction of small plasmids containing those sites. However the end-deletion of insert DNA in BACs using a <it>lox66 </it>transposon occurred at no more than 20% the efficiency observed with a <it>loxP </it>transposon. Differences in the ability of Cre protein available at different stages of the P1 life cycle to recombine identical versus non-identical <it>lox</it>-sites is likely responsible for this discrepancy. A possible mechanism to explain these findings is discussed.</p> <p>Conclusions</p> <p>The <it>loxP/lox66 </it>replacement procedure should allow targeting BACs to a pre-positioned <it>lox71 </it>site in zebrafish chromosomes; a system where homologous recombination-mediated "knock-in" technology is unavailable.</p

Leptin differentially regulate STAT3 activation in ob/ob mouse adipose mesenchymal stem cells

Background Leptin-deficient ob/ob mice exhibit adipocyte hypertrophy and hyperplasia as well as elevated adipose tissue and systemic inflammation. Multipotent stem cells isolated from adult adipose tissue can differentiate into adipocytes ex vivo and thereby contribute toward increased adipocyte cell numbers, obesity, and inflamm ation. Currently, information is lacking regarding regulation of adipose stem cell numbers as well as leptin-induced inflammation and its signaling pathway in ob/ob mice. Methods Using leptin deficient ob/ob mice, we investigated whether leptin injection into ob/ob mice increases adipose stem cell numbers and adipose tissue inflammatory marker MCP-1 mRNA and secretion levels. We also determined leptin mediated signaling pathways in the adipose stem cells. Results We report here that adipose stem cell number is significantly increased following leptin injection in ob/ob mice and with treatment of isolated stem cells with leptin in vitro. Leptin also up-regulated MCP-1 secretion in a dose- and time-dependent manner. We further showed that increased MCP-1 mRNA levels were due to increased phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) Ser727 but not STAT3 Tyr705 phosphorylation, suggesting differential regulation of MCP-1 gene expression under basal and leptin-stimulated conditions in adipose stem cells. Conclusions Taken together, these studies demonstrate that leptin increases adipose stem cell number and differentially activates STAT3 protein resulting in up-regulation of MCP-1 gene expression. Further studies of mechanisms mediating adipose stem cell hyperplasia and leptin signaling in obesity are warranted and may help identify novel anti-obesity target strategies

Genetic Deletion of DNAJB3 Using CRISPR-Cas9, Produced Discordant Phenotypes

Several pathways and/or genes have been shown to be dysregulated in obesity-induced insulin resistance (IR) and type 2 diabetes (T2D). We previously showed, for the first time, impaired expression of DNAJB3 mRNA and protein in subjects with obesity, which was concomitant with increased metabolic stress. Restoring the normal expression of DNAJB3 attenuated metabolic stress and improved insulin signaling both in vivo and in vitro, suggesting a protective role of DNAJB3 against obesity and T2D. The precise underlying mechanisms remained, however, unclear. This study was designed to confirm the human studies in a mouse model of dietary obesity-induced insulin resistance, and, if validated, to understand the underlying mechanisms. We hypothesized that mice lacking DNAJB3 would be more prone to high-fat (HF)-diet-induced increase in body weight and body fat, inflammation, glucose intolerance and insulin resistance as compared with wild-type (WT) littermates. Three DNAJB3 knockout (KO) lines were generated (KO 30, 44 and 47), using CRISPR-Cas9. Male and female KO and WT mice were fed a HF diet (45% kcal fat) for 16 weeks. Body weight was measured biweekly, and a glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted at week 13 and 14, respectively. Body composition was determined monthly by nuclear magnetic resonance (NMR). Following euthanasia, white adipose tissue (WAT) and skeletal muscle were harvested for further analyses. Compared with WT mice, male and female KO 47 mice demonstrated higher body weight and fat mass. Similarly, KO 47 mice also showed a slower rate of glucose clearance in GTT that was consistent with decreased mRNA expression of the GLUT4 gene in WAT but not in the muscle. Both male and female KO 47 mice exhibited higher mRNA levels of the pro-inflammatory marker TNF-a in WAT only, whereas increased mRNA levels of MCP1 chemokine and the ER stress marker BiP/Grp78 were observed in male but not in female KO 47 mice. However, we did not observe the same changes in the other KO lines. Taken together, the phenotype of the DNAJB3 KO 47 mice was consistent with the metabolic changes and low levels of DNAJB3 reported in human subjects. These findings suggest that DNAJB3 may play an important role in metabolic functions and glucose homeostasis, which warrants further phenotyping and intervention studies in other KO 47 and other KO mice, as well as investigating this protein as a potential therapeutic target for obesity and T2D

Integrated genomic characterization of oesophageal carcinoma

Oesophageal cancers are prominent worldwide; however, there are few targeted therapies and survival rates for these cancers remain dismal. Here we performed a comprehensive molecular analysis of 164 carcinomas of the oesophagus derived from Western and Eastern populations. Beyond known histopathological and epidemiologic distinctions, molecular features differentiated oesophageal squamous cell carcinomas from oesophageal adenocarcinomas. Oesophageal squamous cell carcinomas resembled squamous carcinomas of other organs more than they did oesophageal adenocarcinomas. Our analyses identified three molecular subclasses of oesophageal squamous cell carcinomas, but none showed evidence for an aetiological role of human papillomavirus. Squamous cell carcinomas showed frequent genomic amplifications of CCND1 and SOX2 and/or TP63, whereas ERBB2, VEGFA and GATA4 and GATA6 were more commonly amplified in adenocarcinomas. Oesophageal adenocarcinomas strongly resembled the chromosomally unstable variant of gastric adenocarcinoma, suggesting that these cancers could be considered a single disease entity. However, some molecular features, including DNA hypermethylation, occurred disproportionally in oesophageal adenocarcinomas. These data provide a framework to facilitate more rational categorization of these tumours and a foundation for new therapies.ope