Genome Sequences of Listeria monocytogenes Strains Responsible for Cheese- and Cooked Ham Product-Associated Swiss Listeriosis Outbreaks in 2005 and 2011.

The complete genome sequences of three Listeria monocytogenes serotype 1/2a strains, Lm 3136, Lm 3163, and Lm N1546, which were responsible for listeriosis outbreaks in 2005 and 2011 in Switzerland, are presented here

A comparison of faecal microbial populations of South African Windsnyer-type indigenous pigs (SAWIPs) and Large White × Landrace (LW × LR) crosses fed diets containing ensiled maize cobs

Faecal microbial communities in South African Windsnyer-type indigenous pigs (SAWIPs) and Large White × Landrace (LW × LR) crosses were investigated using high-throughput sequencing of the 16S rDNA genes. The faecal microbial communities in LW × LR crosses and SAWIPs fed control (CON) and high maize cob (HMC) diets were evaluated through parallel sequencing of 16S rDNA genes. Butrivibrio, Faecalibacterium and Desulfovibrio, although present in LW × LR pigs, were absent from the SAWIP microbial community. Bacteroides, Succiniclasticum, Peptococcus and Akkermansia were found in SAWIPs but not in LW × LR crosses. The ratios of Bacteroidia to Clostridia on the CON and HMC diets were similar (0.37 versus 0.39) in SAWIPs but different (0.24 versus 0.1) in LW × LR crosses. The faecal microbial profiles determined were different between the LW × LR and SAWIP breeds but not between pigs fed the CON and HMC diets. The composition of faecal bacterial communities in SAWIPs was determined for the first time. The differences in microbial communities detected may explain the enhanced ability of SAWIPs to digest fibrous diets compared with the LW × LR crosse

Enumeration of Mycobacterium avium subsp. paratuberculosis by quantitative real-time PCR, culture on solid media and optical densitometry

<p>Abstract</p> <p>Background</p> <p>Different approaches are used for determining the number of <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) cells in a suspension. The majority of them are based upon culture (determination of CFU) or visual/instrumental direct counting of MAP cells. In this study, we have compared the culture method with a previously published F57 based quantitative real-time PCR (F57qPCR) method, to determine their relative abilities to count the number of three different MAP isolates in suspensions with the same optical densities (OD). McFarland turbidity standards were also compared with F57qPCR and culture, due to its frequent inclusion and use in MAP studies.</p> <p>Findings</p> <p>The numbers of MAP in two-fold serial dilutions of isolates with respective OD measurements were determined by F57qPCR and culture. It was found that culture provided lower MAP CFU counts by approximately two log₁₀, compared to F57qPCR. The McFarland standards (as defined for <it>E. coli</it>) showed an almost perfect fit with the enumeration of MAP performed by F57qPCR.</p> <p>Conclusions</p> <p>It is recommended to use culture and/or qPCR estimations of MAP numbers in experiments where all subsequent counts are performed using the same method. It is certainly not recommended the use of culture as the standard for qPCR experiments and <it>vice versa</it>.</p

Down-Regulation of Replication Factor C-40 (RFC40) Causes Chromosomal Missegregation in Neonatal and Hypertrophic Adult Rat Cardiac Myocytes

BACKGROUND: Adult mammalian cardiac myocytes are generally assumed to be terminally differentiated; nonetheless, a small fraction of cardiac myocytes have been shown to replicate during ventricular remodeling. However, the expression of Replication Factor C (RFC; RFC140/40/38/37/36) and DNA polymerase δ (Pol δ) proteins, which are required for DNA synthesis and cell proliferation, in the adult normal and hypertrophied hearts has been rarely studied. METHODS: We performed qRT-PCR and Western blot analysis to determine the levels of RFC and Pol δ message and proteins in the adult normal cardiac myocytes and cardiac fibroblasts, as well as in adult normal and pulmonary arterial hypertension induced right ventricular hypertrophied hearts. Immunohistochemical analyses were performed to determine the localization of the re-expressed DNA replication and cell cycle proteins in adult normal (control) and hypertrophied right ventricle. We determined right ventricular cardiac myocyte polyploidy and chromosomal missegregation/aneuploidy using Fluorescent in situ hybridization (FISH) for rat chromosome 12. RESULTS: RFC40-mRNA and protein was undetectable, whereas Pol δ message was detectable in the cardiac myocytes isolated from control adult hearts. Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts. Immunohistochemical analyses revealed that in addition to RFC40, proliferative and mitotic markers such as cyclin A, phospho-Aurora A/B/C kinase and phospho-histone 3 were also re-expressed/up-regulated simultaneously in the cardiac myocytes. Interestingly, FISH analyses demonstrated cardiac myocytes polyploidy and chromosomal missegregation/aneuploidy in these hearts. Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers. CONCLUSION: Our novel findings suggest that transcription of RFC40 is suppressed in the normal adult cardiac myocytes and its insufficient re-expression may be responsible for causing chromosomal missegregation/aneuploidy and in cardiac myocytes during right ventricular hypertrophy

In vitro nuclear interactome of the HIV-1 Tat protein

<p>Abstract</p> <p>Background</p> <p>One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry.</p> <p>Results</p> <p>Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied <it>in silico </it>analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture.</p> <p>Conclusion</p> <p>We have completed the <it>in vitro </it>Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.</p

Reduced host cell invasiveness and oxidative stress tolerance in double and triple csp gene family deletion mutants of L. monocytogenes

The cold shock protein (Csp) family comprises small, highly conserved proteins that bind nucleic acids to modulate various bacterial gene expressions. In addition to cold adaptation functions, this group of proteins is thought to facilitate various cellular processes to promote normal growth and stress adaptation responses. Three proteins making up the Listeria monocytogenes Csp family (CspA, CspB, and CspD) promote both cold and osmotic stress adaptation functions in this bacterium. The contribution of these three Csps in the host cell invasion processes of L. monocytogenes was investigated based on human Caco-2 and murine macrophage in vitro cell infection models. The DeltacspB, DeltacspD, DeltacspAB, DeltacspAD, DeltacspBD, and DeltacspABD strains were all significantly impaired in Caco-2 cell invasion compared with the wild-type strain, whereas in the murine macrophage infection assay only, the double (DeltacspBD) and triple (DeltacspABD) csp mutants were also significantly impaired in cell invasion compared with the wild-type strain. The DeltacspBD and DeltacspABD mutants displayed the most severely impaired invasion phenotypes. The invasion ability of these two mutant strains was also further analyzed using cold-stress-exposed organisms. In both cell infection models a significant reduction in invasiveness was observed after cold stress exposure of Listeria organisms. The negative impact of cold stress on subsequent cell invasion ability was, however, more severe in cold-sensitive csp mutants (DeltacspBD and DeltacspABD) compared with the wild type. The impaired macrophage invasion and intracellular growth of DeltacspBD and DeltacspABD also led us to examine oxidative stress resistance capacity in these two mutant strains. Both strains also displayed higher oxidative stress sensitivity relative to the wild-type strain. Our data indicate that besides cold and osmotic stress adaptation roles, Csp family proteins also promote efficient host cell invasion and oxidative stress adaptation processes in L. monocytogenes