Role of bolA and rpoS genes in biofilm formation and adherence pattern by Escherichia coli K-12 MG1655 on polypropylene, stainless steel, and silicone surfaces

Escherichia coli has developed sophisticated means to sense, respond, and adapt in stressed environment. It has served as a model organism for studies in molecular genetics and physiology since the 1960s. Stress response genes are induced whenever a cell needs to adapt and survive under unfavorable growth conditions. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma (σ) factor, which controls the expression of a large number of genes, which are involved in responses to various stress factors as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stressed environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. This study has been undertaken to address the adherence pattern and formation of biofilms by E. coli on stainless steel, polypropylene, and silicone surfaces after 24 h of growth at 37 °C. Scanning electron microscopy was used for direct examination of the cell attachment and biofilm formation on various surfaces and it was found that, in the presence of bolA, E. coli cells were able to attach to the stainless steel and silicone very well. By contrast, polypropylene surface was not found to be attractive for E. coli cells. This indicates that bolA responded and can play a major role in the presence and absence of rpoS in cell attachment

MicroRNAs in pulmonary arterial remodeling

Pulmonary arterial remodeling is a presently irreversible pathologic hallmark of pulmonary arterial hypertension (PAH). This complex disease involves pathogenic dysregulation of all cell types within the small pulmonary arteries contributing to vascular remodeling leading to intimal lesions, resulting in elevated pulmonary vascular resistance and right heart dysfunction. Mutations within the bone morphogenetic protein receptor 2 gene, leading to dysregulated proliferation of pulmonary artery smooth muscle cells, have been identified as being responsible for heritable PAH. Indeed, the disease is characterized by excessive cellular proliferation and resistance to apoptosis of smooth muscle and endothelial cells. Significant gene dysregulation at the transcriptional and signaling level has been identified. MicroRNAs are small non-coding RNA molecules that negatively regulate gene expression and have the ability to target numerous genes, therefore potentially controlling a host of gene regulatory and signaling pathways. The major role of miRNAs in pulmonary arterial remodeling is still relatively unknown although research data is emerging apace. Modulation of miRNAs represents a possible therapeutic target for altering the remodeling phenotype in the pulmonary vasculature. This review will focus on the role of miRNAs in regulating smooth muscle and endothelial cell phenotypes and their influence on pulmonary remodeling in the setting of PAH

Impact of multi-metals (Cd, Pb and Zn) exposure on the physiology of the yeast Pichia kudriavzevii

Metal contamination of the environment is frequently associated to the presence of two or more metals. This work aimed to study the impact of a mixture of metals (Cd, Pb and Zn) on the physiology of the non-conventional yeast Pichia kudriavzevii. The incubation of yeast cells with 5 mg/l Cd, 10 mg/l Pb and 5 mg/l Zn, for 6 h, induced a loss of metabolic activity (assessed by FUN-1 staining) and proliferation capacity (evaluated by a clonogenic assay), with a small loss of membrane integrity (measured by trypan blue exclusion assay). The staining of yeast cells with calcofluor white revealed that no modification of chitin deposition pattern occurred during the exposure to metal mixture. Extending for 24 h, the exposure of yeast cells to metal mixture provoked a loss of membrane integrity, which was accompanied by the leakage of intracellular components. A marked loss of the metabolic activity and the loss of proliferation capacity were also observed. The analysis of the impact of a single metal has shown that, under the conditions studied, Pb was the metal responsible for the toxic effect observed in the metal mixture. Intracellular accumulation of Pb seems to be correlated with the metals toxic effects observed.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "BioInd-Biotechnology and Bioengineering for improved Industrial and Agro-Food processes" (NORTE-07-0124-FEDER-000028), Co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. Manuela D. Machado gratefully acknowledges the post-doctoral grant from FCT (SFRH/BPD/72816/2010). Vanessa A. Mesquita gratefully acknowledges the grant from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES). The authors also thank to Doctor Rosane Freitas Schwan to offer the yeast strain and to Doctor Helena M.V.M. Soares, from the Faculty of Engineering of Porto University, for the use of analytical facilities (AAS with flame atomization and AAS with electrothermal atomization)

Evaluation of high-throughput genomic assays for the Fc gamma receptor locus

Cancer immunotherapy has been revolutionised by the use of monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics