E. coli Ada regulatory protein repairs the SP diastereoisomer of alkylated DNA

Using HPLC and 31P NMR spectroscopy on a chemically synthesized asymmetric mixture of the diastereoisomers of thymidyl(3'----5')thymidyl-O-methyl phosphate absolute configuration has been correlated with chromatographic mobility. The methyl phosphotriester system in alkylated DNA which is repaired by the Ada regulatory protein of E. coli has consequently been established to possess the Sp configuration.     

A comparison of the susceptibility of Culicoides imicola and C. bolitinos to oral infection with eight serotypes of epizootic haemorrhagic disease virus

Abstract. The mechanisms involved in introduction, maintenance and perpetuation of epizootic haemorrhagic disease virus (EHDV) in South Africa are not fully understood. This paper reports on the susceptibility of South African livestock associated Culicoides (Diptera: Ceratopogonidae) species to oral infection with eight EHDV serotypes. Virus was recovered from eight of 17 field-collected Culicoides species 10 days after oral feeding on blood/virus mixtures. Six EHDV serotypes were recovered from C. (Avaritia) imicola Kieffer, and seven serotypes were recovered from C. (A.) bolitinos Meiswinkel. Virus recovery rates in C. imicola ranged from 0.4% for EHDV 2 to 14.4% for EHDV 7, and in C. bolitinos from 0.6% for EHDV 6 to 12.3% for EHDV 2. There was a significant difference in virus recovery rates between serotypes in both species. Other Culicoides species that yielded EHDV after 10 days extrinsic incubation included C. (Meijerehelea) leucostictus Kieffer, C. (Culicoides) magnus Colaço, C. (Beltranmyia) nivosus de Meillon, C. (A.) gulbenkiani Caeiro, C. (Hoffmania) zuluensis de Meillon and C. onderstepoortensis Fiedler. Culicoides midges shown in this study to be susceptible to oral infection with EHDV are widely distributed in South Africa but differ considerably in their abundance, host preference and breeding sites.     

Molecular evolution of the phytochrome gene family in sorghum: changing rates of synonymous and replacement evolution

The photoreceptor phytochromes, encoded by a small gene family, are responsible for controlling the expression of a number of light-responsive genes and photomorphogenic events, including agronomically important phenotypes such as flowering time and shade-avoidance behavior. The understanding and control of flowering time are particularly important goals in sorghum cultivar development for diverse environments, and naturally occurring variation in the phytochrome genes might prove useful in breeding programs. Also of interest is whether variation observed at the phytochrome loci in domesticated sorghum, or in particular races, is a result of human selection. Population genetic studies can reveal evidence of such selection in patterns of polymorphism and divergence. In this study we report a population genetic analysis of the PHY gene family in Sorghum bicolor (L.) Moench in a diverse panel including both cultivated and wild accessions. We show that the level of nucleotide variation in all gene family members is about half the average for this species, consistent with purifying selection acting on these loci. However, the rate of amino acid substitution is accelerated at PHYC compared to the other two loci. In comparisons to a closely related sorghum species, PHYC shows a pattern of intermediate frequency amino acid changes that differ from the patterns observed in comparisons across longer evolutionary distances. There is also a departure from expected patterns of polymorphism and divergence at synonymous sites in PHYC, although the data do not fit a simple model of directional or diversifying selection. Cultivated sorghum has a level of variation similar to that of wild relatives (ssp. verticilliflorum), but many polymorphisms are subspecies-specific, including several amino acid variants.     

Attaching NorA efflux pump inhibitors to methylene blue enhances antimicrobial photodynamic inactivation of Escherichia coli and Acinetobacter baumannii in vitro and in vivo

Resistance of bacteria to antibiotics is a public health concern worldwide due to the increasing failure of standard antibiotic therapies. Antimicrobial photodynamic inactivation (aPDI) is a promising non-antibiotic alternative for treating localized bacterial infections that uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species and kill microbes. Phenothiazinium photosensitizers like methylene blue (MB) and toluidine blue O are hydrophobic cations that are naturally expelled from bacterial cells by multidrug efflux pumps, which reduces their effectiveness. We recently reported the discovery of a NorA efflux pump inhibitor-methylene blue (EPI-MB) hybrid compound INF55-(Ac)en–MB that shows enhanced photodynamic inactivation of the Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) relative to MB, both in vitro and in vivo. Here, we report the surprising observation that INF55-(Ac)en–MB and two related hybrids bearing the NorA efflux pump inhibitors INF55 and INF271 also show enhanced aPDI activity in vitro (relative to MB) against the Gram-negative bacteria Escherichia coli and Acinetobacter baumannii, despite neither species expressing the NorA pump. Two of the hybrids showed superior effects to MB in murine aPDI infection models. The findings motivate wider exploration of aPDI with EPI-MB hybrids against Gram-negative pathogens and more detailed studies into the molecular mechanisms underpinning their activity.     

Pain management using photobiomodulation: Mechanisms,location, and repeatability quantified by pain threshold and neural biomarkers in mice

Photobiomodulation (PBM) is a simple, efficient and cost‐effective treatment for both acute and chronic pain. We previously showed that PBM applied to the mouse head inhibited nociception in the foot. Nevertheless, the optimum parameters, location for irradiation, duration of the effect and the mechanisms of action remain unclear. In the present study, the pain threshold in the right hind paw of mice was studied, after PBM (810 nm CW laser, spot size 1 or 6 cm², 1.2–36 J/cm²) applied to various anatomical locations. The pain threshold, measured with von Frey filaments, was increased more than 3‐fold by PBM to the lower back (dorsal root ganglion, DRG), as well as to other neural structures along the pathway such as the head, neck and ipsilateral (right) paw. On the other hand, application of PBM to the contralateral (left) paw, abdomen and tail had no effect. The optimal effect occurred 2 to 3 hours post‐PBM and disappeared by 24 hours. Seven daily irradiations showed no development of tolerance. Type 1 metabotropic glutamate receptors decreased, and prostatic acid phosphatase and tubulin‐positive varicosities were increased as shown by immunofluorescence of DRG samples. These findings elucidate the mechanisms of PBM for pain and provide insights for clinical practice.      

Photodynamic therapy for Staphylococcus aureus infected burn wounds in mice.

The rise of multiply antibiotic resistant bacteria has led to searches for novel antimicrobial therapies to treat infections. Photodynamic therapy (PDT) is a potential candidate; it uses the combination of a photosensitizer with visible light to produce reactive oxygen species that lead to cell death. We used PDT mediated by meso-mono-phenyl-tri(N-methyl-4-pyridyl)-porphyrin (PTMPP) to treat burn wounds in mice with established Staphylococcus aureus infections The third degree burn wounds were infected with bioluminescent S. aureus. PDT was applied after one day of bacterial growth by adding a 25% DMSO/500 microM PTMPP solution to the wound followed by illumination with red light and periodic imaging of the mice using a sensitive camera to detect the bioluminescence. More than 98% of the bacteria were eradicated after a light dose of 210 J cm(-2) in the presence of PTMPP. However, bacterial re-growth was observed. Light alone or PDT both delayed the wound healing. These data suggest that PDT has the potential to rapidly reduce the bacterial load in infected burns. The treatment needs to be optimized to reduce wound damage and prevent recurrence.     

Prevalence of neutralizing antibodies to bovid herpesvirus 2 in African wildlife

A total of 3,470 sera, collected between 1963 and 1980 from 45 different species of wildlife in nine African countries, was examined for virus neutralizing (VN) antibodies to bovid herpesvirus 2. Antibodies were demonstrated in 20 species including 15 Bovidae, two Suidae, hippopotamus (Hippopotamus amphibius), giraffe (Giraffa camelopardalis) and a green monkey (Cercopithecus aethiops); 11 of these species had not been previously recorded as sero-positive. Although the significance of neutralizing antibodies in the absence of virus isolation remains in doubt, results suggest that infection is widespread in wildlife. The highest VN titres were recorded in waterbuck (Kobus ellipsiprymnus and K. defassa), reedbuck (Redunca arundinum) and buffalo (Syncerus caffer). Infection appears to be continuous in free-living populations of buffalo and antibodies are present in the majority of animals by the age of 2 yr.