Two novel loci underlie natural differences in Caenorhabditis elegans abamectin responses

Parasitic nematodes cause a massive worldwide burden on human health along with a loss of livestock and agriculture productivity. Anthelmintics have been widely successful in treating parasitic nematodes. However, resistance is increasing, and little is known about the molecular and genetic causes of resistance for most of these drugs. The free-living roundworm Caenorhabditis elegans provides a tractable model to identify genes that underlie resistance. Unlike parasitic nematodes, C. elegans is easy to maintain in the laboratory, has a complete and well annotated genome, and has many genetic tools. Using a combination of wild isolates and a panel of recombinant inbred lines constructed from crosses of two genetically and phenotypically divergent strains, we identified three genomic regions on chromosome V that underlie natural differences in response to the macrocyclic lactone (ML) abamectin. One locus was identified previously and encodes an alpha subunit of a glutamate-gated chloride channel (glc-1). Here, we validate and narrow two novel loci using near-isogenic lines. Additionally, we generate a list of prioritized candidate genes identified in C. elegans and in the parasite Haemonchus contortus by comparison of ML resistance loci. These genes could represent previously unidentified resistance genes shared across nematode species and should be evaluated in the future. Our work highlights the advantages of using C. elegans as a model to better understand ML resistance in parasitic nematodes.     

Improved Filtration Technique for Concentrating and Harvesting Bacteria

An improved technique is described for the filtrative concentration and harvesting of bacterial cultures. A pleated tangential flow filtration unit containing 1,000 cm² of 0.2-μm-pore-size microporous membrane was used to rapidly (30 to 50 min) reduce the volume of 5 liters of bacterial culture of approximately 10⁹ cells per ml to 0.2 to 0.5 liters of concentrated bacterial suspension. The effects of cell concentration, filtration pressure, and tangential flow rate were examined with respect to the rate of concentration and cell viability. Recovery efficiencies were between 60 and 75%, with no apparent impairment of organism viability. Cell concentration exerted the predominant effect on the filtration rate.     

A multiplex set of microsatellite markers for the scarlet rosefinch (Carpodacus erythrinus)

Cardueline finches have become important models in studies of sexual selection and evolution of carotenoid‐based ornamentation. Here, we describe eight new polymorphic microsatellites isolated from the Scarlet rosefinch (Carpodacus erythrinus) and four from the House finch (Carpodacus mexicanus). Together with the cross‐species amplification of additional loci, originally published for two species of songbirds, we optimized a multiplex panel for C. erythrinus allowing genotyping of 22 polymorphic loci. Number of alleles and heterozygosity per locus in a sample of 34 individuals ranged from three to 38 and from 0.27 to 0.94, respectively.     

Budding yeast silencing complexes and regulation of Sir2 activity by protein-protein interactions

Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo.     

Urinary Proteins,Vitamin D and Genetic Polymorphisms as Risk Factors for Febrile Urinary Tract Infection and Relation with Bacteremia: A Case Control Study

Objective/Purpose

Febrile urinary tract infection (UTI) is a common bacterial disease that may lead to substantial morbidity and mortality especially among the elderly. Little is known about biomarkers that predict a complicated course. Our aim was to determine the role of certain urinary cytokines or antimicrobial proteins, plasma vitamin D level, and genetic variation in host defense of febrile UTI and its relation with bacteremia.

Methods

A case-control study. Out of a cohort of consecutive adults with febrile UTI (n = 787) included in a multi-center observational cohort study, 46 cases with bacteremic E.coli UTI and 45 cases with non-bacteremic E.coli UTI were randomly selected and compared to 46 controls. Urinary IL-6, IL-8, LL37, β-defensin 2 and uromodulin as well as plasma 25-hydroxyvitamin D were measured. In 440 controls and 707 UTI patients polymorphisms were genotyped in the genes CXCR1, DEFA4, DEFB1, IL6, IL8, MYD88, UMOD, TIRAP, TLR1, TLR2, TLR5 and TNF.

Results

IL-6, IL-8, and LL37 are different between controls and UTI patients, although these proteins do not distinguish between patients with and without bacteremia. While uromodulin did not differ between groups, inability to produce uromodulin is more common in patients with bacteremia. Most participants in the study, including the controls, had insufficient vitamin D and, at least in winter, UTI patients have lower vitamin D than controls. Associations were found between the CC genotype of IL6 SNP rs1800795 and occurrence of bacteremia and between TLR5 SNP rs5744168 and protection from UTI. The rare GG genotype of IL6 SNP rs1800795 was associated with higher β-defensin 2 production.

Conclusion

Although no biomarker was able to distinguish between UTI with or without bacteremia, two risk factors for bacteremia were identified. These were inability to produce uromodulin and an IL6 rs1800795 genotype.     

Penman-Monteith approaches for estimating crop evapotranspiration in screenhouses—a case study with table-grape

In arid and semi-arid regions many crops are grown under screens or in screenhouses to protect them from excessive radiation, strong winds, hailstorms and insects, and to reduce crop water requirements. Screens modify the crop microclimate, which means that it is necessary to accurately estimate crop water use under screens in order to improve the irrigation management and thereby increase water-use efficiency. The goal of the present study was to develop a set of calibrated relationships between inside and outside climatic variables, which would enable growers to predict crop water use under screens, based on standard external meteorological measurements and evapotranspiration (ET) models. Experiments were carried out in the Jordan Valley region of eastern Israel in a table-grape vineyard that was covered with a transparent screen providing 10 % shading. An eddy covariance system was deployed in the middle of the vineyard and meteorological variables were measured inside and outside the screenhouse. Two ET models were evaluated: a classical Penman-Monteith model (PM) and a Penman-Monteith model modified for screenhouse conditions by the inclusion of an additional boundary-layer resistance (PMsc). Energy-balance closure analysis, presented as a linear relation between half-hourly values of available and consumed energy (1,344 data points), yielded the regression Y?=?1.05X–9.93 (W m^?2), in which Y = sum of latent and sensible heat fluxes, and X = net radiation minus soil heat flux, with R ²?=?0.81. To compensate for overestimation of the eddy fluxes, ET was corrected by forcing the energy balance closure. Average daily ET under the screen was 5.4?±?0.54 mm day^?1, in general agreement with the model estimates and the applied irrigation. The results showed that measured ET under the screen was, on average, 34 % lower than that estimated outside, indicating significant potential water saving through screening irrigated vineyards. The PM model was somewhat more accurate than the PMsc for estimating ET under the screen. A model sensitivity analysis illustrates how changes in certain climatic conditions or screen properties would affect evapotranspiration.     

Assembly of the SIR complex and its regulation by O-acetyl-ADP-ribose, a product of NAD-dependent histone deacetylation

Assembly of silent chromatin domains in budding yeast involves the deacetylation of histone tails by Sir2 and the association of the Sir3 and Sir4 proteins with hypoacetylated histone tails. Sir2 couples deacetylation to NAD hydrolysis and the synthesis of a metabolite, O-acetyl-ADP-ribose (AAR), but the functional significance of NAD hydrolysis or AAR, if any, is unknown. Here we examine the association of the Sir2, Sir3, and Sir4 proteins with each other and histone tails. Our analysis reveals that deacetylation of histone H4-lysine 16 (K16), which is critical for silencing in vivo, is also critical for the binding of Sir3 and Sir4 to histone H4 peptides in vitro. Moreover, AAR itself promotes the association of multiple copies of Sir3 with Sir2/Sir4 and induces a dramatic structural rearrangement in the SIR complex. These results suggest that Sir2 activity modulates the assembly of the SIR complex through both histone deacetylation and AAR synthesis.     

Rapid epidemiological analysis of Acinetobacter strains by Raman spectroscopy

From earlier publications, we noticed that Raman spectra could potentially be used for subspecies identification of microorganisms. Here we evaluated the technique for its use as a typing tool of Acinetobacter species, using a collection of well-characterised strains from five hospital outbreaks. The strains were previously analysed using molecular techniques as cell envelope protein profiling and ribotyping. In this study, we have typed the strains by AFLP analysis and Raman spectroscopy. We compared the results using hierarchical cluster analysis, which showed highly similar groupings by both techniques. There seemed to be some misclassification between two sets of outbreak strains in the Raman analysis. We ascribe this to the clonal relationship between the strains of both outbreaks, described earlier. This results from a highly similar biochemical composition of the strains involved, and hence a highly similar Raman spectrum. We conclude that Raman spectroscopy could be an easy-to-use alternative in epidemiological studies of Acinetobacter strains and a promising starting point for the development of epidemiological studies in general.     

Genome-wide analysis of re-replication reveals inhibitory controls that target multiple stages of replication initiation

DNA replication must be tightly controlled during each cell cycle to prevent unscheduled replication and ensure proper genome maintenance. The currently known controls that prevent re-replication act redundantly to inhibit pre-replicative complex (pre-RC) assembly outside of the G1-phase of the cell cycle. The yeast Saccharomyces cerevisiae has been a useful model organism to study how eukaryotic cells prevent replication origins from reinitiating during a single cell cycle. Using a re-replication-sensitive strain and DNA microarrays, we map sites across the S. cerevisiae genome that are re-replicated as well as sites of pre-RC formation during re-replication. Only a fraction of the genome is re-replicated by a subset of origins, some of which are capable of multiple reinitiation events. Translocation experiments demonstrate that origin-proximal sequences are sufficient to predispose an origin to re-replication. Origins that reinitiate are largely limited to those that can recruit Mcm2-7 under re-replicating conditions; however, the formation of a pre-RC is not sufficient for reinitiation. Our findings allow us to categorize origins with respect to their propensity to reinitiate and demonstrate that pre-RC formation is not the only target for the mechanisms that prevent genomic re-replication.