Mise au point d'un test ELISA pour détecter les anticorps anti-Chlamydia pneumoniae

Lorsque nous avons entrepris de mettre au point un test ELISA basé sur la protéine MOMP pour détecter les anticorps anti-Chlamydia pneumoniae, nous espérions entre autre montrer qu'il s'agissait de la protéine immunodominante de cette bactérie mais qu'on [n']a pas pu l'identifier puisqu'il fallait la dénaturer pour l'isoler. Nous voulions aussi se doter d'un test de diagnostic efficace pour retracer les anticorps anti-C. pneumoniae et ainsi être capable d'identifier un facteur de risque de l'athérosclérose quand l'implication de C. pneumoniae dans le développement de cette maladie serait reconnue officiellement. L'identité de la protéine immunodominante de C. pneumoniae va demeurer encore mystérieuse car même si on a été capable d'exprimer la protéine MOMP dans E. coli grâce aux vecteurs pMAL, il nous a été impossible de la produire dans sa conformation native. Cet échec en a entrainé un autre c'est-à-dire la production d'un test ELISA sensible et efficace pour détecter les anticorps produits suite à une infection à C. pneumoniae et par conséquent de faire une corrélation entre leur présence dans l'organisme et le développement de l'athérosclérose. Cependant on a montré qu'un test ELISA est envisageable à condition d'avoir la protéine MOMP native ou une toute autre protéine de C. pneumoniae facile à produire ou insensible à la dénaturation, puisqu'on à [i.e. a] mis au point un test efficace pour détecter les anticorps dirigés contre la protéine MOMP de C. trachomatis

TLN-4601 suppresses growth and induces apoptosis of pancreatic carcinoma cells through inhibition of Ras-ERK MAPK signaling

Abstract Background TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered using Thallion's proprietary DECIPHER® technology, a genomics and bioinformatics platform that predicts the chemical structures of secondary metabolites based on gene sequences obtained by scanning bacterial genomes. Our recent studies suggest that TLN-4601 inhibits the Ras-ERK MAPK pathway post Ras prenylation and prior to MEK activation. The Ras-ERK MAPK signaling pathway is a well-validated oncogenic cascade based on its central role in regulating the growth and survival of cells from a broad spectrum of human tumors. Furthermore, RAS isoforms are the most frequently mutated oncogenes, occurring in approximately 30% of all human cancers, and KRAS is the most commonly mutated RAS gene, with a greater than 90% incidence of mutation in pancreatic cancer. Results To evaluate whether TLN-4601 interferes with K-Ras signaling, we utilized human pancreatic epithelial cells and demonstrate that TLN-4601 treatment resulted in a dose- and time-dependent inhibition of Ras-ERK MAPK signaling. The compound also reduced Ras-GTP levels and induced apoptosis. Finally, treatment of MIA PaCa-2 tumor-bearing mice with TLN-4601 resulted in antitumor activity and decreased tumor Raf-1 protein levels. Conclusion These data, together with phase I/II clinical data showing tolerability of TLN-4601, support conducting a clinical trial in advanced pancreatic cancer patients

Identification of genes expressed in a mesenchymal subset regulating prostate organogenesis using tissue and single cell transcriptomics

Abstract Prostate organogenesis involves epithelial growth controlled by inductive signalling from specialised mesenchymal subsets. To identify pathways active in mesenchyme we used tissue and single cell transcriptomics to define mesenchymal subsets and subset-specific transcript expression. We documented transcript expression using Tag-seq and RNA-seq in female rat Ventral Mesenchymal Pad (VMP) as well as adjacent urethra comprised of smooth muscle and peri-urethral mesenchyme. Transcripts enriched in female VMP were identified with Tag-seq of microdissected tissue, RNA-seq of cell populations, and single cells. We identified 400 transcripts as enriched in the VMP using bio-informatic comparisons of Tag-seq and RNA-seq data, and 44 were confirmed by single cell RNA-seq. Cell subset analysis showed that VMP and adjacent mesenchyme were composed of distinct cell types and that each tissue contained two subgroups. Markers for these subgroups were highly subset specific. Thirteen transcripts were validated by qPCR to confirm cell specific expression in microdissected tissues, as well as expression in neonatal prostate. Immunohistochemical staining demonstrated that Ebf3 and Meis2 showed a restricted expression pattern in female VMP and prostate mesenchyme. We conclude that prostate inductive mesenchyme shows limited cellular heterogeneity and that transcriptomic analysis identified new mesenchymal subset transcripts associated with prostate organogenesis

Mise au point d'un test ELISA pour détecter les anticorps anti-Chlamydia pneumoniae

Lorsque nous avons entrepris de mettre au point un test ELISA basé sur la protéine MOMP pour détecter les anticorps anti-Chlamydia pneumoniae, nous espérions entre autre montrer qu'il s'agissait de la protéine immunodominante de cette bactérie mais qu'on [n']a pas pu l'identifier puisqu'il fallait la dénaturer pour l'isoler. Nous voulions aussi se doter d'un test de diagnostic efficace pour retracer les anticorps anti-C. pneumoniae et ainsi être capable d'identifier un facteur de risque de l'athérosclérose quand l'implication de C. pneumoniae dans le développement de cette maladie serait reconnue officiellement. L'identité de la protéine immunodominante de C. pneumoniae va demeurer encore mystérieuse car même si on a été capable d'exprimer la protéine MOMP dans E. coli grâce aux vecteurs pMAL, il nous a été impossible de la produire dans sa conformation native. Cet échec en a entrainé un autre c'est-à-dire la production d'un test ELISA sensible et efficace pour détecter les anticorps produits suite à une infection à C. pneumoniae et par conséquent de faire une corrélation entre leur présence dans l'organisme et le développement de l'athérosclérose. Cependant on a montré qu'un test ELISA est envisageable à condition d'avoir la protéine MOMP native ou une toute autre protéine de C. pneumoniae facile à produire ou insensible à la dénaturation, puisqu'on à [i.e. a] mis au point un test efficace pour détecter les anticorps dirigés contre la protéine MOMP de C. trachomatis

Genome-wide analysis of AR binding and comparison with transcript expression in primary human fetal prostate fibroblasts and cancer associated fibroblasts

The androgen receptor (AR) is a transcription factor, and key regulator of prostate development and cancer, which has discrete functions in stromal versus epithelial cells. AR expressed in mesenchyme is necessary and sufficient for prostate development while loss of stromal AR is predictive of prostate cancer progression. Many studies have characterized genome-wide binding of AR in prostate tumour cells but none have used primary mesenchyme or stroma. We applied ChIPseq to identify genomic AR binding sites in primary human fetal prostate fibroblasts and patient derived cancer associated fibroblasts, as well as the WPMY1 cell line overexpressing AR. We identified AR binding sites that were specific to fetal prostate fibroblasts (7534), cancer fibroblasts (629), WPMY1-AR (2561) as well as those common among all (783). Primary fibroblasts had a distinct AR binding profile versus prostate cancer cell lines and tissue, and showed a localisation to gene promoter binding sites 1 kb upstream of the transcriptional start site, as well as non-classical AR binding sequence motifs. We used RNAseq to define transcribed genes associated with AR binding sites and derived cistromes for embryonic and cancer fibroblasts as well as a cistrome common to both. These were compared to several in vivo ChIPseq and transcript expression datasets; which identified subsets of AR targets that were expressed in vivo and regulated by androgens. This analysis enabled us to deconvolute stromal AR targets active in stroma within tumour samples. Taken together, our data suggest that the AR shows significantly different genomic binding site locations in primary prostate fibroblasts compared to that observed in tumour cells. Validation of our AR binding site data with transcript expression in vitro and in vivo suggests that the AR target genes we have identified in primary fibroblasts may contribute to clinically significant and biologically important AR-regulated changes in prostate tissue.</p

BRCA1 can stimulate gene transcription by a unique mechanism

Most familial breast and ovarian cancers have been linked to mutations in the BRCA1 gene. BRCA1 has been shown to affect gene transcription but how it does so remains elusive. Here we show that BRCA1 can stimulate transcription without the requirement for a DNA-tethering function in mammalian and yeast cells. Furthermore, the BRCA1 C-terminal region can stimulate transcription of the p53-responsive promoter, MDM2. Unlike many enhancer-specific activators, non-tethered BRCA1 does not require a functional TATA element to stimulate transcription. Our results suggest that BRCA1 can enhance transcription by a function additional to recruiting the transcriptional machinery to a targeted gene

SOCS1 Deficiency Promotes Hepatocellular Carcinoma via SOCS3-Dependent CDKN1A Induction and NRF2 Activation

SOCS1 deficiency, which increases susceptibility to hepatocellular carcinoma (HCC), promotes CDKN1A expression in the liver. High CDKN1A expression correlates with disease severity in many cancers. Here, we demonstrate a crucial pathogenic role of CDKN1A in diethyl nitrosamine (DEN)-induced HCC in SOCS1-deficient mice. Mechanistic studies on DEN-induced genotoxic response revealed that SOCS1-deficient hepatocytes upregulate SOCS3 expression, SOCS3 promotes p53 activation, and Cdkn1a induction that were abolished by deleting either Socs3 or Tp53. Previous reports implicate CDKN1A in promoting oxidative stress response mediated by NRF2, which is required for DEN-induced hepatocarcinogenesis. We show increased induction of NRF2 and its target genes in SOCS1-deficient livers following DEN treatment that was abrogated by the deletion of either Cdkn1a or Socs3. Loss of SOCS3 in SOCS1-deficient mice reduced the growth of DEN-induced HCC without affecting tumor incidence. In the TCGA-LIHC dataset, the SOCS1-low/SOCS3-high subgroup displayed increased CDKN1A expression, enrichment of NRF2 transcriptional signature, faster disease progression, and poor prognosis. Overall, our findings show that SOCS1 deficiency in hepatocytes promotes compensatory SOCS3 expression, p53 activation, CDKN1A induction, and NRF2 activation, which can facilitate cellular adaptation to oxidative stress and promote neoplastic growth. Thus, the NRF2 pathway represents a potential therapeutic target in SOCS1-low/SOCS3-high HCC cases

SOCS1 Deficiency Promotes Hepatocellular Carcinoma via SOCS3-Dependent CDKN1A Induction and NRF2 Activation

SOCS1 deficiency, which increases susceptibility to hepatocellular carcinoma (HCC), promotes CDKN1A expression in the liver. High CDKN1A expression correlates with disease severity in many cancers. Here, we demonstrate a crucial pathogenic role of CDKN1A in diethyl nitrosamine (DEN)-induced HCC in SOCS1-deficient mice. Mechanistic studies on DEN-induced genotoxic response revealed that SOCS1-deficient hepatocytes upregulate SOCS3 expression, SOCS3 promotes p53 activation, and Cdkn1a induction that were abolished by deleting either Socs3 or Tp53. Previous reports implicate CDKN1A in promoting oxidative stress response mediated by NRF2, which is required for DEN-induced hepatocarcinogenesis. We show increased induction of NRF2 and its target genes in SOCS1-deficient livers following DEN treatment that was abrogated by the deletion of either Cdkn1a or Socs3. Loss of SOCS3 in SOCS1-deficient mice reduced the growth of DEN-induced HCC without affecting tumor incidence. In the TCGA-LIHC dataset, the SOCS1-low/SOCS3-high subgroup displayed increased CDKN1A expression, enrichment of NRF2 transcriptional signature, faster disease progression, and poor prognosis. Overall, our findings show that SOCS1 deficiency in hepatocytes promotes compensatory SOCS3 expression, p53 activation, CDKN1A induction, and NRF2 activation, which can facilitate cellular adaptation to oxidative stress and promote neoplastic growth. Thus, the NRF2 pathway represents a potential therapeutic target in SOCS1-low/SOCS3-high HCC cases