Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis

Background

MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR–92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR–92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR–92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells.

Methodology/Principal Findings

We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR–92 expression by qRT-PCR. Expression of ERβ1, a direct miR–92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR–92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR–92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR–92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR–92 levels in NFs but not CAFs enhanced invasion of both MCF–7 and MDA-MB–231 breast cancer epithelial cells.

Conclusions

miR–92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR–92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERβ1 may not be the most important miR–92 target in breast cancer.     

First Report of OXA-4, an ESBL Isolated from Pseudomonas aeruginosa a South Indian Strain

The OXA-type β-lactamases are so named because of their oxacillin-hydrolyzing abilities. In this study we characterize an extended spectrum β-lactamase, designated OXA-4, produced by a clinical isolate of Pseudomonas aeruginosa. ESBL production was detected by double disk synergy test. The P. aeruginosa isolate was obtained from endotracheal suction tip of 84 years old male patient diagnosed with CVA and hypertension. ESBL producing OXA β-lactamases was detected by PCR with primers specific to the conserved regions of the coding genes. Iso electric focusing was done to confirm the significance, sequencing the amplified product was also done. In the phenotypic identification, the strain was highly resistant to third generation cephalosporins and also to imipenem. The PCR amplified product for OXA β-lactamase was viewed at 919 bp. The pI point for the same was identified at 7.2. With the help of sequencing the amplified OXA β-lactamase was identified as OXA-4 gene. Here we report P. aeruginosa producing OXA-4 ESBL for the first time in the Indian subcontinent.     

S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection

Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting ‘anchoring’ non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.     

Influence of cGMP on Feeding Potential of Predatory Coccinellid,Cryptolaemus montrouzieri Mulsant and Isolation of Partial foraging Gene

The cyclic guanomonophosphate (cGMP) dependent protein kinase (PKG) plays an important role in the food related behaviours of several insect species. Here we report the influence of cGMP dependent PKG on prey consumption of adult predatory coccinellid, Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae). The oral cGMP treatment (which increases PKG activity) enhanced the feeding potential of C. montrouzieri. The good foragers responded more positively to the cGMP treatment compared to the poor foragers. The cGMP levels estimated through ELISA were significantly (P?<?0.001) high in the digestive tissues of unfed as well as cGMP treated C. montrouzieri compared to normal fed beetles. This finding suggests that cGMP is involved in the higher feeding rates of C. montrouzieri and the partial foraging gene (~455 bp) which encodes the cGMP dependent PKG was isolated from genomic DNA of C. montrouzieri using gene specific primers.     

Incorporation of exogenous precursors into uridine and ribonucleic acid. Nucleotide compartmentation in the renal cortex in vivo

The possibility of compartmentation of UTP in vivo was investigated in the renal cortex of unanaesthetized rats. In addition, liver and spleen were studied in order to compare tissues with different utilization of precursors for pyrimidine nucleotide synthesis. After continuous 2h infusions of [(3)H]uridine or [(3)H]orotate, their incorporation into UTP, UDP-sugars and RNA was quantified. Rates of RNA synthesis were calculated by dividing the incorporation of precursor into RNA by the average specific radioactivity of the UTP pool. Although similar RNA-synthesis rates might have been expected with the two precursors, higher rates were found with uridine than with orotate. The relative incorporation into UDP-sugars of these precursors was also different. Similar results were obtained in the liver. In the spleen, equal amounts of both precursors were incorporated into UTP, but [(3)H]orotate incorporation did not lead to labelling of RNA. To evaluate the heterogeneity of cells with respect to the metabolism of pyrimidines, precursor incorporation was studied in isolated glomeruli and by radioautography. Incorporation into glomeruli was qualitatively similar to but quantitatively different from results in the renal cortex. Although there is obvious tissue heterogeneity, compartmentation of UTP pools is the most credible explanation for the results obtained with the renal cortex and liver. Consequently RNA and UDP-sugars may originate from two different UTP pools. Tissue heterogeneity is the likely explanation for the results obtained in the spleen. Studies of synthesis of pyrimidine and RNA, particularly in relation to growth and regeneration, must take into consideration the precursor used, the apparent existence of UTP compartmentation and the degree of cellular heterogeneity.     

PIQET: the design and development of an online ‘streamlined’ LCA tool for sustainable packaging design decision support

Background, aim and scope  

‘Streamlined’ life cycle assessment (LCA) tools hold out the possibility of providing LCA information quickly and easily in order to support a variety of decision-making environments and situations. The utility of such tools is closely related to the accuracy needs and possibilities, and the particular decisions to be supported. In order to facilitate the provision and application of LCA information in decision making during packaging design, development and utilisation, there is a prima facia case for a ‘streamlined’ LCA tool, provided it meets a set of requirements, including functionality, accuracy, validity, reliability and usability.     

Prostasin regulates epithelial monolayer function: cell-specific Gpld1-mediated secretion and functional role for GPI anchor

Prostasin, a trypsinlike serine peptidase, is highly expressed in prostate, kidney, and lung epithelia, where it is bound to the cell surface, secreted, or both. Prostasin activates the epithelial sodium channel (ENaC) and suppresses invasion of prostate and breast cancer cells. The studies reported here establish mechanisms of membrane anchoring and secretion in kidney and lung epithelial cells and demonstrate a critical role for prostasin in regulating epithelial monolayer function. We report that endogenous mouse prostasin is glycosylphosphatidylinositol (GPI) anchored to the cell surface and is constitutively secreted from the apical surface of kidney cortical collecting duct cells. Using site-directed mutagenesis, detergent phase separation, and RNA interference approaches, we show that prostasin secretion depends on GPI anchor cleavage by endogenous GPI-specific phospholipase D1 (Gpld1). Secretion of prostasin by kidney and lung epithelial cells, in contrast to prostate epithelium, does not depend on COOH-terminal processing at conserved Arg³²². Using stably transfected M-1 cells expressing wild-type, catalytically inactive, or chimeric transmembrane (not GPI)-anchored prostasins we establish that prostasin regulates transepithelial resistance, current, and paracellular permeability by GPI anchor- and protease activity-dependent mechanisms. These studies demonstrate a novel role for prostasin in regulating epithelial monolayer resistance and permeability in kidney epithelial cells and, furthermore, show specifically that prostasin is a critical regulator of transepithelial ion transport in M-1 cells. These functions depend on the GPI anchor as well as the peptidase activity of prostasin. These studies suggest that cell-specific Gpld1- or peptidase-dependent pathways for prostasin secretion may control prostasin functions in a tissue-specific manner. serine protease; epithelial sodium channel; glycosylphosphatidylinositol anchor; transepithelial resistance; tight junction