Comparative evaluation of 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the log(2) dilution MIC results revealed overall agreement, within the accuracy limits of the standard test (+/-1 log(2) dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test.     

Radiotoxicity of Auger electron-emitting estrogens in MCF-7 spheroids: a potential treatment for estrogen receptor-positive tumors.

To approach treatment of micrometastases of steroid receptor-rich cancers using estrogen receptor-directed therapy with Auger electrons, multicellular spheroids of the estrogen receptor-rich human breast cancer cell line, MCF-7, were prepared and exposed to a range of concentrations of an Auger electron-emitting estrogen, E-17alpha-[123I]-iodovinyl-11beta-methoxyestradiol, [123I]IVME2, in vitro. After washing, the treated spheroids were dissociated to single cells and plated for assay of colony survival, whereby we observed a dose-dependent reduction in survival that was inhibited by inclusion of an excess of unlabeled estradiol in the initial incubation with [123I]IVME2. Spheroids of a range of sizes from 40 to 280 microm showed similar sensitivity to the Auger electron-emitting estrogen. The mean lethal dose was approximately 700 decays per cell and corresponded to an initial [123I]IVME2 concentration of less than 0.5 nM. If the control and treated spheroids were not trypsinized but rather were allowed to grow intact, there was not only a significant reduction in the growth of the treated spheroids, but in 18 days nearly half became necrotic, while few control spheroids were necrotic. Considering the low concentrations of Auger electron-emitting estrogen required for a significant reduction in survival, we believe this approach has merit to pursue in vivo, especially in cases where it may be possible to target the steroid receptor-rich micrometastases directly, such as ovarian cancer.     

Genome-wide association study for calving performance using high-density genotypes in dairy and beef cattle

Background

Calving difficulty and perinatal mortality are prevalent in modern-day cattle production systems. It is well-established that there is a genetic component to both traits, yet little is known about their underlying genomic architecture, particularly in beef breeds. Therefore, we performed a genome-wide association study using high-density genotypes to elucidate the genomic architecture of these traits and to identify regions of the bovine genome associated with them.

Results

Genomic regions associated with calving difficulty (direct and maternal) and perinatal mortality were detected using two statistical approaches: (1) single-SNP (single nucleotide polymorphism) regression and (2) a Bayesian approach. Data included high-density genotypes on 770 Holstein-Friesian, 927 Charolais and 963 Limousin bulls. Several novel or previously identified genomic regions were detected but associations differed by breed. For example, two genomic associations, one each on chromosomes 18 and 2 explained 2.49 % and 3.13 % of the genetic variance in direct calving difficulty in the Holstein-Friesian and Charolais populations, respectively. Imputed Holstein-Friesian sequence data was used to refine the genomic regions responsible for significant associations. Several candidate genes on chromosome 18 were identified and four highly significant missense variants were detected within three of these genes (SIGLEC12, CTU1, and ZNF615). Nevertheless, only CTU1 contained a missense variant with a putative impact on direct calving difficulty based on SIFT (0.06) and Polyphen (0.95) scores. Using imputed sequence data, we refined a genomic region on chromosome 4 associated with maternal calving difficulty in the Holstein-Friesian population and found the strongest association with an intronic variant in the PCLO gene. A meta-analysis was performed across the three breeds for each calving performance trait to identify common variants associated with these traits in the three breeds. Our results suggest that a portion of the genetic variation in calving performance is common to all three breeds.

Conclusion

The genomic architecture of calving performance is complex and mainly influenced by many polymorphisms of small effect. We identified several associations of moderate effect size but the majority were breed-specific, indicating that breed-specific alleles exist for calving performance or that the linkage phase between genotyped allele and causal mutation varies between breeds.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0126-4) contains supplementary material, which is available to authorized users.     

Predicting the fate of a living fossil: how will global warming affect sex determination and hatching phenology in tuatara?

How will climate change affect species'' reproduction and subsequent survival? In many egg-laying reptiles, the sex of offspring is determined by the temperature experienced during a critical period of embryonic development (temperature-dependent sex determination, TSD). Increasing air temperatures are likely to skew offspring sex ratios in the absence of evolutionary or plastic adaptation, hence we urgently require means for predicting the future distributions of species with TSD. Here we develop a mechanistic model that demonstrates how climate, soil and topography interact with physiology and nesting behaviour to determine sex ratios of tuatara, cold-climate reptiles from New Zealand with an unusual developmental biology. Under extreme regional climate change, all-male clutches would hatch at 100% of current nest sites of the rarest species, Sphenodon guntheri, by the mid-2080s. We show that tuatara could behaviourally compensate for the male-biasing effects of warmer air temperatures by nesting later in the season or selecting shaded nest sites. Later nesting is, however, an unlikely response to global warming, as many oviparous species are nesting earlier as the climate warms. Our approach allows the assessment of the thermal suitability of current reserves and future translocation sites for tuatara, and can be readily modified to predict climatic impacts on any species with TSD.     

Mechanism of the reductive activation of succinate dehydrogenase.

When succinate dehydrogenase contains oxalacetate in firmly bound form, activity cannof the enzyme results in dissociation of oxalacetate and activation of the enzyme. The course of reductive titrations appears the same whether or not the enzyme contains oxalacetate, and complete reduction as monitored by bleaching of chromophoric groups requires the incorporation of 6 to 7 reducing equivalents in either case. The stoichiometry is that expected from the non-heme iron and flavin content of the enzyme. Activation of the enzyme during reductive titrations occurs predominantly with the incorporation of the second pair of electrons, while determination of activation levels at various poised potentials shows that the group involved is reduced with the uptake of 2 H+ and 2 e-. These characteristics are consistent with titration of the flavin moiety rather than non-heme iron groups. Thus it appears that activation is concurrent with the reduction of flavin to the hydroquinone form. From the measured half-reduction potential for activation, that of the flavin in an oxalacetate-free enzyme has been estimated at -90 to -60 mv at pH 7.     

Localization of the phosphocholine-binding sites on C-reactive protein by immunoelectron microscopy

C-reactive protein (CRP) was reacted with monoclonal IgG antibody or Fab antibody fragments directed against the phosphocholine- (PC) binding site or a second unrelated site. The resulting immune complexes were viewed by a negative stain immunoelectron microscopy technique. Monoclonal anti-PC-binding site antibody bound to a single epitope on each of the five CRP subunits. The orientation of the PC-binding sites was determined to be slightly medial to one of the planar faces (A-face) of the molecule. The second monoclonal antibody, which was not PC-binding site related, bound to epitopes (one per CRP subunit) that were located slightly lateral to the other planar face (B-face) of the CRP molecule, i.e., opposite of the PC-binding site. Thus, the PC-binding site and the non-PC-binding site are oriented nearly perpendicular but on opposite sides with respect to the plane of the CRP molecule. The functional significance of this configuration is discussed.     

Detection of membrane-associated antigens on lymphoid cells by antibody coupled to staphylococcal protein A.

A simple technique is described for the detection of membrane-associated antigens on lymphoid cells. It is based on the observation that the protein A component of staphylococci binds to the Fc pieces of IgG molecules. Lymphocytes from various sources (mouse, rat, and human tissues) were incubated with hyperimmune antisera directed against surface determinants. Subsequent treatment with a suspension of staphylococci containing protein A permitted visualization of both the presence and distribution of determinants on the cell membrane. The method had comparable sensitivity to the fluorescent sandwich technique and could be used to detect a variety of membrane antigens on both T cells and B cells.