MLH1 mediates PARP-dependent cell death in response to the methylating agent N-methyl-N-nitrosourea

Background:Methylating agents such as N-methyl-N-nitrosourea (MNU) can cause cell cycle arrest and death either via caspase-dependent apoptosis or via a poly(ADP-ribose) polymerase (PARP)-dependent form of apoptosis. We wished to investigate the possible role of MLH1 in signalling cell death through PARP.Methods:Fibroblasts are particularly dependent on a PARP-mediated cell death response to methylating agents. We used hTERT-immortalised normal human fibroblasts (WT) to generate isogenic MLH1-depleted cells, confirmed by quantitative PCR and western blotting. Drug resistance was measured by clonogenic and cell viability assays and effects on the cell cycle by cell sorting. Damage signalling was additionally investigated using immunostaining.Results:MLH1-depleted cells were more resistant to MNU, as expected. Despite having an intact G2/M checkpoint, the WT cells did not initially undergo cell cycle arrest but instead triggered cell death directly by PARP overactivation and nuclear translocation of apoptosis-inducing factor (AIF). The MLH1-depleted cells showed defects in this pathway, with decreased staining for phosphorylated H2AX, altered PARP activity and reduced AIF translocation. Inhibitors of PARP, but not of caspases, blocked AIF translocation and greatly decreased short-term cell death in both WT and MLH1-depleted cells. This MLH1-dependent response to MNU was not blocked by inhibitors of ATM/ATR or p53.Conclusion:These novel data indicate an important role for MLH1 in signalling PARP-dependent cell death in response to the methylating agent MNU

Inactivation of the dnaK gene in Clostridium difficile 630 Δerm yields a temperature-sensitive phenotype and increases biofilm-forming ability

Abstract Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood. We previously reported that clinically relevant heat-stress (37–41 °C) resulted in a classical heat-stress response with up-regulation of cellular chaperones. We used ClosTron to construct an insertional mutation in the dnaK gene of C. difficile 630 Δerm. The dnaK mutant exhibited temperature sensitivity, grew more slowly than C. difficile 630 Δerm and was less thermotolerant. Furthermore, the mutant was non-motile, had 4-fold lower expression of the fliC gene and lacked flagella on the cell surface. Mutant cells were some 50% longer than parental strain cells, and at optimal growth temperatures, they exhibited a 4-fold increase in the expression of class I chaperone genes including GroEL and GroES. Increased chaperone expression, in addition to the non-flagellated phenotype of the mutant, may account for the increased biofilm formation observed. Overall, the phenotype resulting from dnaK disruption is more akin to that observed in Escherichia coli dnaK mutants, rather than those in the Gram-positive model organism Bacillus subtilis

Experimental Incubations Elicit Profound Changes in Community Transcription in OMZ Bacterioplankton

Sequencing of microbial community RNA (metatranscriptome) is a useful approach for assessing gene expression in microorganisms from the natural environment. This method has revealed transcriptional patterns in situ, but can also be used to detect transcriptional cascades in microcosms following experimental perturbation. Unambiguously identifying differential transcription between control and experimental treatments requires constraining effects that are simply due to sampling and bottle enclosure. These effects remain largely uncharacterized for “challenging” microbial samples, such as those from anoxic regions that require special handling to maintain in situ conditions. Here, we demonstrate substantial changes in microbial transcription induced by sample collection and incubation in experimental bioreactors. Microbial communities were sampled from the water column of a marine oxygen minimum zone by a pump system that introduced minimal oxygen contamination and subsequently incubated in bioreactors under near in situ oxygen and temperature conditions. Relative to the source water, experimental samples became dominated by transcripts suggestive of cell stress, including chaperone, protease, and RNA degradation genes from diverse taxa, with strong representation from SAR11-like alphaproteobacteria. In tandem, transcripts matching facultative anaerobic gammaproteobacteria of the Alteromonadales (e.g., Colwellia) increased 4–13 fold up to 43% of coding transcripts, and encoded a diverse gene set suggestive of protein synthesis and cell growth. We interpret these patterns as taxon-specific responses to combined environmental changes in the bioreactors, including shifts in substrate or oxygen availability, and minor temperature and pressure changes during sampling with the pump system. Whether such changes confound analysis of transcriptional patterns may vary based on the design of the experiment, the taxonomic composition of the source community, and on the metabolic linkages between community members. These data highlight the impressive capacity for transcriptional changes within complex microbial communities, underscoring the need for caution when inferring in situ metabolism based on transcript abundances in experimental incubations

Comprehensive analysis of temporal alterations in cellular proteome of bacillus subtilis under curcumin treatment

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division

Genomic analysis of Acinetobacter baumannii prophages reveals remarkable diversity and suggests significant impact on bacterial virulence and fitness

[Abstract] Bacterial genomics has revealed substantial amounts of prophage DNA in bacterial genomes. This integrated viral DNA has been shown to play important roles in the evolution of bacterial pathogenicity. Acinetobacter baumannii has shown a fast progression as a nosocomial multi-resistant pathogen in recent years, and is now considered one of the most dangerous microorganisms in hospital environments. The role of prophages in the evolution of A. baumannii pathogenicity has not yet been explored. In this context, we aimed at evaluating the impact of prophages on A. baumannii genomic diversity and pathogenicity. [...]info:eu-repo/semantics/publishedVersio

Effect of 180-day water storage on bonding effectiveness of self-adhesive systems to occlusal and proximal dentin

This study aimed to evaluate the microshear bond strength (μSBS) values of adhesive systems to occlusal and proximal dentin submitted to water storage agin

Influência das avós na alimentação de lactentes: o que dizem suas filhas e noras Influencia de las abuelas en la alimentación de lactantes Influence of grandmothers on infant feeding: what they say to their daughters and granddaughters

OBJETIVO: Identificar as práticas de primíparas sobre a alimentação de crianças lactentes aprendidas com a mãe e/ou sogra. MÉTODOS: Trata-se de pesquisa qualitativa, do tipo descritiva. Os sujeitos foram 11 primíparas, usuárias do Sistema Único de Saúde de Palmeira das Missões/RS. Os dados foram coletados por entrevista semiestruturada e analisados por meio da análise temática. RESULTADOS: Emergiram três temas: práticas de alimentação transmitidas pelas avós; influência das avós na alimentação do lactente; sentimentos das primíparas em relação às ações das avós relativas à alimentação do lactente. CONCLUSÕES: As avós são importantes no que diz respeito à transmissão de conhecimentos, saberes e experiências relativas à alimentação do lactente. Para que a assistência de enfermagem priviligie a autonomia dos sujeitos que envolve corresponsabilidade e congruência cultural, o enfermeiro deve considerar as interações do grupo familiar que, em seu movimento dialético, modulam e são moduladas pela cultura.<br>OBJETIVO: Identificar las prácticas de primigestas sobre la alimentación de niños lactantes aprendidas con la madre y/o suegra. MÉTODOS: Se trata de una investigación cualitativa, de tipo descriptiva. Los sujetos fueron 11 primigestas, usuarias del Sistema Único de Salud de Palmeira das Missões/RS. Los datos fueron recolectados por entrevista semiestructurada y analizados por medio del análisis temático. RESULTADOS: Emergieron tres temas: prácticas de alimentación transmitidas por las abuelas; influencia de las abuelas en la alimentación del lactante; sentimientos de las primigestas en relación a las acciones de las abuelas relativas a la alimentación del lactante. CONCLUSIONES: Las abuelas son importantes en lo referente a la transmisión de conocimientos, saberes y experiencias relativas a la alimentación del lactante. Para que la asistencia de enfermería privilegie la autonomía de los sujetos que involucra corresponsabilidad y congruencia cultural, el enfermero debe considerar las interacciones del grupo familiar que, en su movimiento dialéctico, modulan y son moduladas por la cultura.<br>OBJECTIVE: To identify practices of primiparas about infant feeding of infants learned from the mother and/or grandmother. METHODS: This was a qualitative, descriptive research study. The subjects were 11 primiparas, who used the Unified Health System of Palmeira das Missões/ RS. Data were collected through semi-structured interviews and analyzed using thematic analysis. RESULTS: Three themes emerged: practices of infant feeding transmitted by grandmothers; influence of grandmothers on infant feeding; feelings of primiparas in regard to the actions of grandmothers on infant feeding. CONCLUSIONS: The grandmothers are important with regard to the transmission of knowledge, wisdom and experiences related to infant feeding. For nursing care focused on the subjects' autonomy and responsibility that involves cultural congruence, the nurse should consider that the interactions of the family group, in its dialectical movement, modulate and are modulated by their culture