Keratin and S100 calcium-binding proteins are major constituents of the bovine teat canal lining

The bovine teat canal provides the first-line of defence against pathogenic bacteria infecting the mammary gland, yet the protein composition and host-defence functionality of the teat canal lining (TCL) are not well characterised. In this study, TCL collected from six healthy lactating dairy cows was subjected to two-dimensional electrophoresis (2-DE) and mass spectrometry. The abundance and location of selected identified proteins were determined by western blotting and fluorescence immunohistochemistry. The variability of abundance among individual cows was also investigated. Two dominant clusters of proteins were detected in the TCL, comprising members of the keratin and S100 families of proteins. The S100 proteins were localised to the teat canal keratinocytes and were particularly predominant in the cornified outermost layer of the teat canal epithelium. Significant between-animal variation in the abundance of the S100 proteins in the TCL was demonstrated. Four of the six identified S100 proteins have been reported to have antimicrobial activity, suggesting that the TCL has additional functionality beyond being a physical barrier to invading microorganisms. These findings provide new insights into understanding host-defence of the teat canal and resistance of cows to mastitis

The Interaction of Selenium with Chemotherapy and Radiation on Normal and Malignant Human Mononuclear Blood Cells

Selenium, a trace element with anticancer properties, can reduce harmful toxicities of chemotherapy and radiotherapy without compromising efficacy. However, the dose-response relationship in normal versus malignant human cells is unclear. We evaluated how methylseleninic acid (MSA) modulates the toxicity and efficacy of chemotherapy and radiation on malignant and non-malignant human mononuclear blood cells in vitro. We specifically investigated its effects on endoplasmic reticulum stress induction, intracellular glutathione concentration, DNA damage and viability of peripheral blood mononuclear cells and THP1 monocytic leukaemia cells in response to radiation, cytosine arabinoside or doxorubicin chemotherapy. MSA, at lower concentrations, induced protective responses in normal cells but cytotoxic effects in malignant cells, alone and in conjunction with chemotherapy or radiation. However, in normal cells higher concentrations of MSA were directly toxic and increased the cytotoxicity of radiation but not chemotherapy. In malignant cells higher MSA concentrations were generally more effective in combination with cancer treatments. Thus, optimal MSA concentrations differed between normal and malignant cells and treatments. This work supports clinical reports that selenium can significantly reduce dose-limiting toxicities of anticancer therapies and potentially improve efficacy of anticancer treatments. The optimal selenium compound and dose is not yet determined

Development of a qPCR Method to Measure Mitochondrial and Genomic DNA Damage with Application to Chemotherapy-Induced DNA Damage and Cryopreserved Cells

DNA damage quantitation assays such as the comet assay have focused on the measurement of total nuclear damage per cell. The adoption of PCR-based techniques to quantify DNA damage has enabled sequence- and organelle-specific assessment of DNA lesions. Here we report on an adaptation of a qPCR technique to assess DNA damage in nuclear and mitochondrial targets relative to control. Novel aspects of this assay include application of the assay to the Rotor-Gene platform with optimized DNA polymerase/fluorophore/primer set combination in a touchdown PCR protocol. Assay validation was performed using ultraviolet C radiation in A549 and THP1 cancer cell lines. A comparison was made to the comet assay applied to peripheral blood mononuclear cells, and an estimation of the effects of cryopreservation on ultraviolet C-induced DNA damage was carried out. Finally, dose responses for DNA damage were measured in peripheral blood mononuclear cells following exposure to the cytotoxic agents bleomycin and cisplatin. We show reproducible experimental outputs across the tested conditions and concordance with published findings with respect to mitochondrial and nuclear genotoxic susceptibilities. The application of this DNA damage assay to a wide range of clinical and laboratory-derived samples is both feasible and resource-efficient

Application of Streptococcus uberis Multilocus Sequence Typing: Analysis of the Population Structure Detected among Environmental and Bovine Isolates from New Zealand and the United Kingdom

We recently developed a multilocus sequence typing (MLST) scheme to differentiate S. uberis isolates and facilitate an understanding of the population biology of this pathogen. The scheme was initially used to study a collection of 160 bovine milk isolates from the United Kingdom and showed that the majority of isolates were from one clonal complex (designated the ST-5 complex). Here we describe the MLST analysis of a collection of New Zealand isolates. These were obtained from diverse sources, including bovine milk, other bovine anatomical sites, and environmental sources. The complete allelic profiles of 253 isolates were determined. The collection was highly diverse and included 131 different sequence types (STs). The New Zealand and United Kingdom populations were distinct, since none of the 131 STs were represented within the previously studied collection of 160 United Kingdom S. uberis isolates. However, seven of the STs were members of the ST-5 clonal complex, the major complex within the United Kingdom collection. Two new clonal complexes were identified: ST-143 and ST-86. All three major complexes were isolated from milk, other bovine sites, and the environment. Carriage of the hasA gene, which is necessary for capsule formation, correlated with clonal complex and isolation from clinical cases of mastitis

Evolutionary history of <i>M. tuberculosis</i> strains isolated from a cluster of cases in New Zealand.

<p>Relationships were inferred by examining parsimony and nonparsimony informative single nucleotide polymorphisms (SNPs). <b>Panel A</b>: parallelogram of network analysis using 34 SNPs including five homoplastic SNPs. <b>Panel B</b>: Evolutionary tree using the same 34 SNPs as in A rooted on <i>M. tuberculosis</i> H₃₇Rv analyzed using Neighbor-joining method. Panel C. Evolutionary tree examining 29 nonparsimony SNPs using the Neighbor-joining method. The evolutionary distances represent the number of SNP differences per strain.</p

Single nucleotide polymorphisms identified in sequenced isolates.

1<p>reference genome.</p

In vivo mutation rates in <i>M. tuberculosis</i> strains for generation times ranging from 18 to 240 hours.

<p>The mutation rate for each of the <i>M. tuberculosis</i> isolates was estimated using equation (1) in Ford <i>et al</i>, and calculated as untis of mutation/bp/generation <b>Panel A</b>: The mutation rate for each <i>M. tuberculosis</i> isolate in the study. Isolate C1 and E are from recent infection while strains O and S from reactivation after a prolonged period of latency. The yellow areas represent 95% confidence intervals. <b>Panel B</b>: Isolates with recent infection (E and C1) and reactivation after prolonged latency (O and S) were combined to obtain overall estimates of mutation rates of recent and latent-reactivated disease. The yellow areas represent 95% confidence intervals.</p

Epidemiological relationships among 11 New Zealand <i>M. tuberculosis</i> cases.

<p>Chronological representation of different subjects in a New Zealand TB outbreak. Each square represent a subject at the time of the TB diagnosis. Broken lines represent known close direct contact with the initial index case “X” during X's period of infectiousness. Solid lines show assumed connections between a case of presumed reactivation (C1 to C2) and a case of potential child-parent transmission (A to T).</p

Number of single nucleotide polymorphisms differences between each strain.

<p>Number of single nucleotide polymorphisms differences between each strain.</p