Vitamin D, innate immunity and outcomes in community acquired pneumonia

We investigated the associations between vitamin D status, the antimicrobial peptides cathelicidin and beta defensin-2 and outcomes in community acquired pneumonia. In hospitalised patients with community acquired pneumonia, vitamin D deficiency but not antimicrobial peptide levels were associated with increased 30-day mortality. Vitamin D was not associated with levels of the antimicrobial peptide cathelicidin or beta defensin-2

Whole genome sequencing of Mycobacterium tuberculosis reveals slow growth and low mutation rates during latent infections in humans

Very little is known about the growth and mutation rates of Mycobacterium tuberculosis during latent infection in humans. However, studies in rhesus macaques have suggested that latent infections have mutation rates that are higher than that observed during active tuberculosis disease. Elevated mutation rates are presumed risk factors for the development of drug resistance. Therefore, the investigation of mutation rates during human latency is of high importance. We performed whole genome mutation analysis of M. tuberculosis isolates from a multi-decade tuberculosis outbreak of the New Zealand Rangipo strain. We used epidemiological and phylogenetic analysis to identify four cases of tuberculosis acquired from the same index case. Two of the tuberculosis cases occurred within two years of exposure and were classified as recently transmitted tuberculosis. Two other cases occurred more than 20 years after exposure and were classified as reactivation of latent M. tuberculosis infections. Mutation rates were compared between the two recently transmitted pairs versus the two latent pairs. Mean mutation rates assuming 20 hour generation times were 5.5X10⁻¹⁰ mutations/bp/generation for recently transmitted tuberculosis and 7.3X10⁻¹¹ mutations/bp/generation for latent tuberculosis. Generation time versus mutation rate curves were also significantly higher for recently transmitted tuberculosis across all replication rates (p = 0.006). Assuming identical replication and mutation rates among all isolates in the final two years before disease reactivation, the u20hr mutation rate attributable to the remaining latent period was 1.6×10⁻¹¹ mutations/bp/generation, or approximately 30 fold less than that calculated during the two years immediately before disease. Mutations attributable to oxidative stress as might be caused by bacterial exposure to the host immune system were not increased in latent infections. In conclusion, we did not find any evidence to suggest elevated mutation rates during tuberculosis latency in humans, unlike the situation in rhesus macaques

Connexin36 knockout mice display increased sensitivity to pentylenetetrazol-induced seizure-like behaviors

Large-scale synchronous firing of neurons during seizures is modulated by electrotonic coupling between neurons via gap junctions. To explore roles for connexin36 (Cx36) gap junctions in seizures, we examined the seizure threshold of connexin36 knockout (Cx36KO) mice using a pentylenetetrazol (PTZ) model

The utility of single nucleotide DNA variations as predictors of postoperative pain

Objectives: Genetic variation is an important contributor to postsurgical pain and thereby analgesia requirements. A description of the potential predictive power of genetic variants in pain should instruct improvements in pain management postoperatively. We set out to examine whether a set of genetic variants in pain related genes would show any association with actual pain outcomes in a typical surgical population. Methods: A candidate gene study was carried out in 135 surgical patients with 12 DNA variants (single nucleotide polymorphisms or ‘SNPs’) in known or putative pain pathway genes to detect associations with postoperative pain - measured by a verbal rating score (VRS) and patient-controlled analgesia (PCA) usage rate. Standard PCR based molecular biology approaches were used. Results: At 20-24h after surgery, patients with the 1032G/1032G variant pair for the A1032G variant of the potassium channel KCNJ6 gene had a slightly higher median VRS than those with 1032A/1032A or 1032A/1032G pairs (p=0.04; dominant genetic model). This small difference was most apparent in the orthopaedic surgery patients where the 1032G/1032G pair associated with VRS (median(interquartile range)) of 5(4-6) vs. 3(0.5-4) in 1032A/1032A or 1032A/1032G groups. For PCA, patients with 3435C/3435C or 3435C/3435T pairs for ATPdependent efflux pump gene ABCB1 variant C3435T used PCA at a considerably higher rate of 0.89(0.07-1.66) mg.h-1 compared with just 0.11 (0-0.52) mg.h-1 for the 3435T/3435T pair (p=0.03; dominant model). A significantly higher usage rate was also detected for opioid receptor OPRM1 variant IVS2-691 with usage of 0.77(0.01-1.56) mg.h-1 for the IVS2C/IVS2C or IVS2C/IVS2G group vs. 0.24(0-1.26) mg.h-1 in the IVS2G/IVS2G group (p=0.04; recessive model). Conclusion: While this study has identified some significant statistical associations the potential utility of the studied DNA variants in prediction of postoperative pain and patient-controlled opioid analgesia requirements appears to be quite limited at present

DNA adsorption by nanocrystalline allophane spherules and nanoaggregates, and implications for carbon sequestration in Andisols

This study provides fundamental knowledge about the interaction of allophane, deoxyribonucleic acid (DNA), and organic matter in soils, and how allophane sequesters DNA. The adsorption capacities of salmon-sperm DNA on pure synthetic allophane (characterised morphologically and chemically) and on humic-acid-rich synthetic allophane were determined, and the resultant DNA–allophane complexes were characterised using synchrotron-radiation-derived P X-ray absorption near-edge fine structure (XANES) spectroscopy and infrared (IR) spectroscopy. The synthetic allophane adsorbed up to 34 μg mg⁻¹ of salmon-sperm DNA. However, the presence of humic acid significantly lowered the DNA uptake on the synthetic allophane to 3.5 μg mg⁻¹ by occupying the active sites on allophane so that DNA was repulsed. Both allophane and humic acid adsorbed DNA chemically through its phosphate groups. IR spectra for the allophane–DNA complex showed a chemical change of the Si–O–Al stretching of allophane after DNA adsorption, possibly because of the alteration of the steric distance of the allophane outer wall, or because of the precipitation of aluminium phosphate on allophane after DNA adsorption on it, or both. The aluminol groups of synthetic allophane almost completely reacted with additions of small amounts of DNA (~ 2–6 μg mg⁻¹ ), but the chemical adsorption of DNA on allophane simultaneously led to the formation of very porous allophane aggregates up to ~ 500 μm in diameter. The formation of the allophane nano- and microaggregates enabled up to 28 μg mg⁻¹ of DNA to be adsorbed (~ 80% of total) within spaces (pores) between allophane spherules and allophane nanoaggregates (as “physical adsorption”), giving a total of 34 μg mg⁻¹ of DNA adsorbed by the allophane. The stability of the allophane–DNA nano- and microaggregates likely prevents encapsulated DNA from exposure to oxidants, and DNA within small pores between allophane spherules and nanoaggregates may not be accessible to enzymes or microbes, hence enabling DNA protection and preservation in such materials. By implication, substantial organic carbon is therefore likely to be sequestered and protected in allophanic soils (Andisols) in the same way as demonstrated here for DNA, that is, predominantly by encapsulation within a tortuous network of nanopores and submicropores amidst stable nanoaggregates and microaggregates, rather than by chemisorption alone

A new method to extract and purify DNA from allophanic soils and paleosols, and potential for paleoenvironmental reconstruction and other applications

Andisols, developed from late-Quaternary tephra (volcanic ash) deposits and dominated by the nanocrystalline aluminosilicate, allophane, contain large stores of organic matter and are potential reservoirs for DNA. However, DNA recovery from Andisols and other allophane-bearing soils has been difficult and inefficient because of strong chemical bonding between DNA and both allophane and organic matter, and also because much DNA can be encased and physically protected in nanopores in allophane nano/microaggregates. We have therefore developed a new two-step DNA isolation method for allophanic soils and buried paleosols, including those low in clay, which circumvents these problems. The method centres on (1) releasing mainly microbial DNA, and extracellular (unbound) DNA, using an alkaline phosphate buffer (“Rai’s lysis buffer”) that blocks re-adsorption sites on the allophanic materials, and (2) the novel application of acidified ammonium oxalate (Tamm’s reagent) to dissolve the allophane and to release DNA which had been chemically-bound and also which had been protected within nanopores. Ammonium oxalate has not previously been applied to soil DNA extraction. DNA yields up to 44.5 µg g-1 soil (oven-dry basis) were obtained from three field-moist natural allophanic soil samples from northern New Zealand using this two-step method. Following extraction, we evaluated different DNA purification methods. Gel electrophoresis of the extracted DNA followed by gel purification of the DNA from the agarose gel, despite some DNA loss, was the only purification method that removed sufficient humic material for successful DNA amplification using the polymerase chain reaction (PCR) of multiple gene regions. Sequencing of PCR products obtained from a buried allophanic paleosol at 2.2-m depth on a sandy Holocene tephra yielded endemic and exotic plants that differed from the European grasses growing currently on the soil’s surface. This difference suggests that the DNA extraction method is able to access (paleo)environmental DNA derived from previous vegetation cover. Our DNA extraction and purification method hence may be applied to Andisols and allophane-bearing paleosols, potentially offering a means to isolate paleoenvironmental DNA and thus facilitate reconstruction of past environments in volcanic landscapes, datable using tephrochronology, and also aid biodiversity understanding of andic soils and paleosols

Complete genome sequence of a New Zealand Mycobacterium tuberculosis strain responsible for ongoing transmission over the past 30 years.

We report here the complete genome sequence of Mycobacterium tuberculosis strain Colonial S-type 1 (CS1), which has been responsible for ongoing outbreaks of tuberculosis in New Zealand over the past 30 years. CS1 appears to be highly transmissible, with greater rates of progression to active disease, compared to other circulating M. tuberculosis strains; therefore, comparison of its genomic content is of interest

Strangles-The disease which will not go away

This was a series of talks organised by the New Zealand Equine Research Foundation and sponsered by Baye

Genetic diversity and population structure of the pot-belly seahorse Hippocampus abdominalis in New Zealand

Genetic diversity and population structure of New Zealand populations of the pot-belly seahorse Hippocampus abdominalis were explored via three mitochondrial DNA markers (814 bp of cytochrome b, 624 bp of cytochrome oxidase 1 and 404 bp of the control region) and four microsatellite loci (Habd3, Habd6, Habd7 and Habd9). These markers revealed between 0.7–2.2% sequence divergence and 32–46 alleles with low differentiation among populations around the coastline. In total, 169 seahorses (79 females and 90 males) were sampled with 36 sequences generated for each marker and 166 genotypes attained. It is hoped that this new knowledge will aid in the development of future conservation management

The molecular identification of Streptococcus equi subsp. equi strains isolated within New Zealand

AIMS: To identify Streptococcus equi subsp. equi (S. equi) by PCR analysis and obtain isolates by culture, in order to investigate the strains of S. equi infecting horses within New Zealand. METHODS: A diagnostic PCR, based on the amplification of the seeI gene for S. equi, was used on 168 samples submitted from horses with and without clinical signs of strangles. Samples were also processed and cultured on selective media for the isolation of β-haemolytic colonies. In addition, the hypervariable region of the seM gene of S. equi was amplified and then sequenced for strain typing purposes. RESULTS: Of the 168 samples, 35 tested positive for S. equi using PCR. Thirty-two confirmed samples were from horses with a clinical diagnosis of strangles and three were from horses where clinical information was unavailable. Only 22/35 (63%) confirmed S. equi samples were successfully isolated following culture. Strain typing demonstrated that two novel seM alleles of S. equi were found in New Zealand with SeM-99 strains being restricted to the North Island while SeM-100 strains were found in both North and South Islands. CONCLUSIONS: The application of PCR for the laboratory confirmation of strangles allowed for a rapid and sensitive identification of S. equi. Moreover, seM typing revealed that within the samples examined two strains of S. equi co-circulated within the North Island of New Zealand but only one strain in the South Island. CLINICAL RELEVANCE: PCR reduces the time required to obtain laboratory confirmation of strangles compared with culture methods. It also has greater sensitivity in detecting S. equi infections, which is of particular importance in the detection of carrier animals which normally shed low numbers of bacteria. Additionally, seM molecular typing can differentiate between bacterial strains, assisting in the monitoring of local strains of S. equi subsp. equi causing disease