Comparison of genomic profiles of Escherichia coli isolates from urinary tract infections

Formally included in the larger category of extraintestinal pathogenic Escherichia coli (ExPEC), the uropathogenic E. coli remains the most frequent cause of urinary tract infection (UTI), an important endemic health problem. The genomic DNA of E. coli urinary isolates from adults diagnosed with urinary tract infections and of E. coli fecal isolates from healthy subjects was analysed by PCR for the presence of virulence factor encoding genes pap, sfa/foc, afa, hly and cnf and by field inversion gel electrophoresis (FIGE) fingerprinting of XbaI DNA macrorestriction fragments. The aim was to obtain more detailed microbiological data regarding the community circulating strains in respect of their virulence potential and genetic relatedness. Almost 70% of the urinary strains carried at least one of the target virulence genes, and only 35.5% of the fecal E. coli strains were positive in the PCR screening. Taking into account the virulence genotypes exhibited, a part of the strains isolated from the urinary tract could be defined as belonging to the ExPEC pathotype. A unique FIGE profile was obtained for each of the selected isolates and the dendrogram generated by Taxotron software package analysis suggested a polyclonal population of potential uropathogenic strains clustered into 14 groups of only 60% similarity. For better understanding the epidemiology of UTIs, diseases commonly caused by such a heterogeneous species like E. coli, molecular analysis methods could be essential due to their increased power of identification and fingerprinting.     

Incidence of virulence-encoding genes among enteric Escherichia coli strains isolated from healthy subjects

Escherichia coli, heterogeneous species consisting of commensal and pathogenic strains, is causing a broad spectrum of intestinal and extra intestinal diseases, ranging from asymptomatic infections to septicaemia, according to its capacity to produce different virulence factors. The incidence of different virulence-associated genes among the strains isolated from healthy subjects, taking into account that the human gastrointestinal tract is considered an important source for spreading E. coli strains, was evaluated. A total of 241 E. coli strains isolated from 41 healthy subjects, working in the food chain and coming to the laboratory for periodical medical control, were investigated for harbouring patogenicity factors--encoding genes. Extra intestinal virulence-associated genes, pap, sfa/foc, afa, hly, cnf and intestinal ones eaea, bfp, agg, It, st, vtx1 (stx1), vtx2 (stx2) and ipaH, were targeted by PCR using cellular lysate for total DNA. Genes encoding for adherence were the most prevalent. A number of 67 strains (27.80%) were positive for pap genes and 34 strains (14.11%) presented PCR positive results when afa genes were targeted, but sfa/foc genes were identified in only 10 strains (4.15%). Genes encoding for toxigenesis were less prevalent. A total of 9 strains amplified hly genes, 2.49% were positive for cnf genes and only 2 strains presented vtx1(stx1) gene. The results are in concordance with those which demonstrate that healthy subjects carrying strains possessing virulence-encoding genes could represent a reservoir for environmental circulation of such strains, considered life-threatening when a receptive host is encountered.     

Processing of DNA structures via DNA unwinding and branch migration by the S. cerevisiae Mph1 protein

The budding yeast Mph1 protein, the putative ortholog of human FANCM, possesses a 3' to 5' DNA helicase activity and is capable of disrupting the D-loop structure to suppress chromosome arm crossovers in mitotic homologous recombination. Similar to FANCM, genetic studies have implicated Mph1 in DNA replication fork repair. Consistent with this genetic finding, we show here that Mph1 is able to mediate replication fork reversal, and to process the Holliday junction via DNA branch migration. Moreover, Mph1 unwinds 3' and 5' DNA Flap structures that bear key features of the D-loop. These biochemical results not only provide validation for a role of Mph1 in the repair of damaged replication forks, but they also offer mechanistic insights as to its ability to efficiently disrupt the D-loop intermediate.     

Overcoming co‐product inhibition in the nicotinamide independent asymmetric bioreduction of activated CC‐bonds using flavin‐dependent ene‐reductases

Eleven flavoproteins from the old yellow enzyme family were found to catalyze the disproportionation (“dismutation”) of conjugated enones. Incomplete conversions, which were attributed to enzyme inhibition by the co‐product phenol could be circumvented via in situ co‐product removal by scavenging the phenol using the polymeric adsorbent MP‐carbonate. The optimized system allowed to reduce an alkene activated by ester groups in a “coupled‐substrate” approach via nicotinamide‐free hydrogen transfer with >90% conversion and complete stereoselectivity. Biotechnol. Bioeng. 2013;110: 3085–3092. © 2013 The Authors. Biotechnology and Bioengineering Published by Willey Periodicals, Inc.     

Expression of chemokine-like receptor 1 (CMKLR1) on J744A.1 macrophages co-cultured with fibroblast and/or tumor cells: modeling the influence of microenvironment

Maturation of macrophages is influenced by the composition of surrounding microenvironment. Expression of CMKLR1, the receptor for chemerin, is potentially associated with the differentiation status of macrophages. In this study, CMKLR1 was determined on peritoneal and tumor-infiltrating macrophages. CMKLR1 expression was found to be associated with the fibroblast-assisted maturation of J744A.1 monocyte/macrophage cells in the co-cultures established to model tumor microenvironment, whereas the presence of tumor cells was able to upregulate CMKLR1 expression independent of macrophage maturation. In addition, macrophages cultured with tumor cells or in tumor cell-conditioned media responded to recombinant chemerin(17-156) peptide and increased the expression of proinflammatory IL-1β, TNF-α and IL-12 p40 cytokines. The native form of chemerin (prochemerin) supplied by fibroblasts did not induce a functional response. These observations may indicate a potential role for chemerin and CMKLR1 in the regulation of inflammatory responses in the tumor microenvironment.     

Age at Death of Creutzfeldt-Jakob Disease in Subsequent Family Generation Carrying the E200K Mutation of the Prion Protein Gene

Background

The E200K mutation of the prion protein gene (PRNP) is the most frequent amino acid substitution in genetic Creutzfeldt-Jakob disease and is the only one responsible for the appearance of clustered cases in the world. In the Israel and Slovakian clusters, age of disease onset was reduced in successive generations but the absence of a clear molecular basis raised the possibility that this event was an observational bias. The aim of the present study was to investigate possible selection biases or confounding factors related to anticipation in E200K CJD patients belonging to a cluster in Southern Italy.

Methods

Clinical and demographical data of 41 parent-offspring pairs from 19 pedigrees of the Italian cluster of E200K patients were collected. Age at death of parents was compared with age at death of E200K CJD offspring. Subgroup analyses were performed for controlling possible selection biases, confounding factors, or both.

Results

The mean age at death/last follow-up of the parent generation was 71.4 years while that of CJD offspring was 59.3 years with an estimated anticipation of 12.1 years. When the same analysis was performed including only parents with CJD or carrying the E200K mutation (n = 26), the difference between offspring and parents increased to 14.8 years.

Conclusions

These results show that early age at death occurs in offspring of families carrying the E200K PRNP mutation and that this event is not linked to observational biases. Although molecular or environmental bases for this occurrence remain unsettled, this information is important for improving the accuracy of information to give to mutated carriers.     

Androgen receptor drives cellular senescence

The accepted androgen receptor (AR) role is to promote proliferation and survival of prostate epithelium and thus prostate cancer progression. While growth-inhibitory, tumor-suppressive AR effects have also been documented, the underlying mechanisms are poorly understood. Here, we for the first time link AR anti-cancer action with cell senescence in vitro and in vivo. First, AR-driven senescence was p53-independent. Instead, AR induced p21, which subsequently reduced ΔN isoform of p63. Second, AR activation increased reactive oxygen species (ROS) and thereby suppressed Rb phosphorylation. Both pathways were critical for senescence as was proven by p21 and Rb knock-down and by quenching ROS with N-Acetyl cysteine and p63 silencing also mimicked AR-induced senescence. The two pathways engaged in a cross-talk, likely via PML tumor suppressor, whose localization to senescence-associated chromatin foci was increased by AR activation. All these pathways contributed to growth arrest, which resolved in senescence due to concomitant lack of p53 and high mTOR activity. This is the first demonstration of senescence response caused by a nuclear hormone receptor.     

Vascular endothelial growth factor stimulates organ-specific host matrix metalloproteinase-9 expression and ovarian cancer invasion

Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP) regulate each other, contributing to tumor progression. We have previously reported that MMP9 induces the release of tumor VEGF, promoting ascites formation in human ovarian carcinoma xenografts. The aim of this study was to investigate whether tumor-derived VEGF regulated the expression of gelatinase by the stroma, influencing the invasive properties of ovarian tumors. Tumor variants derived from 1A9 human ovarian carcinoma, stably expressing VEGF(121) in the sense (1A9-VS-1) and antisense orientations (1A9-VAS-3), were used. In vivo, zymographic analysis of tumors from 1A9-VS-1 implanted in the peritoneal cavity of nude mice showed higher levels of gelatinases, particularly murine MMP9, indicating that VEGF stimulates host expression of the matrix-degrading enzyme. Murine MMP9 expression was also high in the ovaries of mice bearing 1A9-VS-1 tumors. The effect on host MMP9 activity was organ-specific. The levels of host pro-MMP9 in ovaries correlated with the plasma levels of tumor VEGF and with the selective invasion of the ovaries. Induction of host MMP9 expression in tumors and ovaries was independent of the site of tumor growth as it was seen in mice carrying both intraperitoneal and subcutaneous tumors. The anti-VEGF antibody bevacizumab (Avastin) inhibited MMP9 expression and tumor invasion in the ovaries of mice bearing 1A9-VS-1 tumors. These findings point to a complex cross-talk between VEGF and MMPs in the progression of ovarian tumor and suggest the possibility of using VEGF inhibitors to affect MMP-dependent tumor invasion.     

Threatened but understudied: supporting conservation by understanding the genetic structure of the flat-headed cat

The flat-headed cat (Prionailurus planiceps) is a wetland specialist, currently facing habitat loss on a serious scale due to massive destruction of lowland forests and wetlands in Southeast Asia. Despite its ‘endangered’ status in the IUCN Red List, there has virtually been no investigation on the population structure nor on the evolutionary history of the flat-headed cat. To fill this gap, we used full mitochondrial genome sequences, obtained from archival samples, covering the historical distribution of the flat-headed cat. Our data revealed a high genetic differentiation (F _st = 0.81, P?<?0.001) between mitochondrial lineages from Borneo and those from Thai-Malay Peninsula/Sumatra, a split that was dated to ~575 Kya. Such a significant differentiation clearly distinguishes the Bornean flat-headed cat population from all other populations and suggests that there should be a reassessment of the flat-headed cat’s intraspecific taxonomy. However, morphological and nuclear data are required to corroborate our mtDNA results. Until such data become available, we recommend that for future conservation efforts and captive breeding programmes the two genetically distinct flat-headed cat populations are managed separately as two lineages to maintain the original genetic diversity of this endangered species.