Antibiotic resistance and pathogenicity factors in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from mastitic Sahiwal cattle

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious problem in dairy animals suffering from mastitis. In the present study, the distribution of mastitic MRSA and antibiotic resistance was studied in 107 strains of S. aureus isolated from milk samples from 195 infected udders. The characterizations pathogenic factors (adhesin and toxin genes) and antibiotic susceptibility of isolates were carried out using gene amplification and disc diffusion assays, respectively. A high prevalence of MRSA was observed in the tested isolates (13.1%). The isolates were also highly resistant to antibiotics, i.e. 36.4% were resistant to streptomycin, 33.6% to oxytetracycline, 29.9% to gentamicin and 26.2% each to chloramphenicol, pristinomycin and ciprofloxacin. A significant variation in the expression of pathogenic factors (Ig, coa and clf) was observed in these isolates. The overall distribution of adhesin genes ebp, fib, bbp, fnbB, cap5, cap8, map and cna in the isolates was found to be 69.1, 67.2, 6.5, 20.5, 60.7, 26.1, 81.3 and 8.4%, respectively. The presence of fib, fnbB, bbp and map genes was considerably greater in MRSA than in methicillin-susceptible S. aureus (MSSA) isolates. The proportions of toxin genes, namely, hlb, seb, sec, sed, seg and sei, in the isolates were found to be 94.3, 0.9, 8.4, 0.9, 10.2 and 49.5%, respectively. The proportions of agr genes I, II, III and IV were found to be 39.2, 27.1, 21.5 and 12.1%, respectively. A few isolates showed similar antibiotic-resistance patterns, which could be due to identical strains or the dissemination of the same strains among animals. These findings can be utilized in mastitis treatment programmes and antimicrobials strategies in organized herds.     

Annual methane uptake by temperate semiarid steppes as regulated by stocking rates,aboveground plant biomass and topsoil air permeability

Overgrazing‐induced degradation of temperate semiarid steppes may affect the soil sink for atmospheric methane (CH₄). However, previous studies have primarily focused on the growing season and on single grazing patterns. Thus, the response of annual CH₄ uptake by steppes compared with various grazing practices is uncertain. In this study, we investigated the effects of grazing on the annual CH₄ uptake by two typical Eurasian semiarid steppes (the Stipa grandis steppe and the Leymus chinensis steppe) located in Inner Mongolia, China. The CH₄ fluxes were measured year‐round using static chambers and gas chromatography at 12 field sites that differed primarily in grazing intensities. Our results indicated that steppe soils were CH₄ sinks throughout the year. The annual CH₄ uptake correlated with stocking rates, whereas the seasonality of CH₄ uptake was primarily dominated by temperature. The annual CH₄ uptake at all sites averaged 3.7±0.7 kg C ha^?1 yr^?1 (range: 2.3–4.5), where approximately 35% (range: 23–40%) occurred during the nongrowing season. Light‐to‐moderate grazing (stocking rate≤1 sheep ha^?1 yr^?1) did not significantly change the annual CH₄ uptake compared with ungrazed steppes, but heavy grazing reduced annual CH₄ uptake significantly (by 24–31%, P<0.05). These findings imply that easing the pressure of heavily grazed steppes (e.g. moving to light or moderate stocking rates) would help restore steppe soil sinks for atmospheric CH₄. The empirical equations based on the significant relationships between annual CH₄ uptake and stocking rates, aboveground plant biomass and topsoil air permeability (P<0.01) could provide simple approaches for the estimation of regional CH₄ uptake by temperate semiarid steppes.     

Comparison of the Safety and Pharmacokinetics of ST-246? after IV Infusion or Oral Administration in Mice,Rabbits and Monkeys

Background

ST-246® is an antiviral, orally bioavailable small molecule in clinical development for treatment of orthopoxvirus infections. An intravenous (IV) formulation may be required for some hospitalized patients who are unable to take oral medication. An IV formulation has been evaluated in three species previously used in evaluation of both efficacy and toxicology of the oral formulation.

Methodology/Principal Findings

The pharmacokinetics of ST-246 after IV infusions in mice, rabbits and nonhuman primates (NHP) were compared to those obtained after oral administration. Ten minute IV infusions of ST-246 at doses of 3, 10, 30, and 75 mg/kg in mice produced peak plasma concentrations ranging from 16.9 to 238 µg/mL. Elimination appeared predominately first-order and exposure dose-proportional up to 30 mg/kg. Short IV infusions (5 to 15 minutes) in rabbits resulted in rapid distribution followed by slower elimination. Intravenous infusions in NHP were conducted at doses of 1 to 30 mg/kg. The length of single infusions in NHP ranged from 4 to 6 hours. The pharmacokinetics and tolerability for the two highest doses were evaluated when administered as two equivalent 4 hour infusions initiated 12 hours apart. Terminal elimination half-lives in all species for oral and IV infusions were similar. Dose-limiting central nervous system effects were identified in all three species and appeared related to high C_max plasma concentrations. These effects were eliminated using slower IV infusions.

Conclusions/Significance

Pharmacokinetic profiles after IV infusion compared to those observed after oral administration demonstrated the necessity of longer IV infusions to (1) mimic the plasma exposure observed after oral administration and (2) avoid C_max associated toxicity. Shorter infusions at higher doses in NHP resulted in decreased clearance, suggesting saturated distribution or elimination. Elimination half-lives in all species were similar between oral and IV administration. The administration of ST-246 was well tolerated as a slow IV infusion.     

Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting

Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.     

Early Triassic conodont clusters from South China: revision of the architecture of the 15 element apparatuses of the superfamily Gondolelloidea

Abstract: Several fused clusters of conodont elements of the genera Neospathodus and Novispathodus were recovered from limestone beds at the Dienerian–Smithian and Smithian–Spathian boundaries, respectively, from several localities in Guangxi province, South China. Conodont clusters are otherwise extremely rare in the Triassic, and these are first described for the Early Triassic. The exceptional specimens partially preserve the relative three‐dimensional position and orientation of ramiform elements and are therefore extremely important for testing hypotheses on the architecture of apparatuses. These specimens partly confirm the previous reconstruction of the Novispathodus apparatus by Orchard. Within apparatuses of members of superfamily Gondolelloidea, elements previously identified as occupying the S₁ and S₂ positions instead occupy the S₂ and S₁ positions. Similarly, within apparatuses of members of the subfamily Novispathodinae, elements previously referred to S₃ and S₄ positions are reinterpreted to have occupied S₄ and S₃ positions, respectively.     

The 'Green Revolution' dwarfing genes play a role in disease resistance in Triticum aestivum and Hordeum vulgare

The Green Revolution dwarfing genes, Rht-B1b and Rht-D1b, encode mutant forms of DELLA proteins and are present in most modern wheat varieties. DELLA proteins have been implicated in the response to biotic stress in the model plant, Arabidopsis thaliana. Using defined wheat Rht near-isogenic lines and barley Sln1 gain of function (GoF) and loss of function (LoF) lines, the role of DELLA in response to biotic stress was investigated in pathosystems representing contrasting trophic styles (biotrophic, hemibiotrophic, and necrotrophic). GoF mutant alleles in wheat and barley confer a resistance trade-off with increased susceptibility to biotrophic pathogens and increased resistance to necrotrophic pathogens whilst the converse was conferred by a LoF mutant allele. The polyploid nature of the wheat genome buffered the effect of single Rht GoF mutations relative to barley (diploid), particularly in respect of increased susceptibility to biotrophic pathogens. A role for DELLA in controlling cell death responses is proposed. Similar to Arabidopsis, a resistance trade-off to pathogens with contrasting pathogenic lifestyles has been identified in monocotyledonous cereal species. Appreciation of the pleiotropic role of DELLA in biotic stress responses in cereals has implications for plant breeding.     

Does transmission of the rust pathogen,Puccinia punctiformis,require stem mining vectors?

Recent research in Europe has suggested that stem mining insects may be important for vectoring the pathogen Puccinia punctiformis, and largely responsible for its systemic infection in the weed, Cirsium arvense. However, here we present comparative survey data showing that the level of systemic disease is the same in Europe and New Zealand, with and without stem miners, respectively, casting doubt on the idea that these insects are necessary for transmission of the fungus.     

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages of their life cycle in opossums.