A comparison of amplification methods to detect Avian Influenza viruses in California wetlands targeted via remote sensing of waterfowl.

Migratory waterfowl, including geese and ducks, are indicated as the primary reservoir of avian influenza viruses (AIv) which can be subsequently spread to commercial poultry. The US Department of Agriculture's (USDA) surveillance efforts of waterfowl for AIv have been largely discontinued in the contiguous United States. Consequently, the use of technologies to identify areas of high waterfowl density and detect the presence of AIv in habitat such as wetlands has become imperative. Here we identified two high waterfowl density areas in California using processed NEXt generation RADar (NEXRAD) and collected water samples to test the efficacy of two tangential flow ultrafiltration methods and two nucleic acid based AIv detection assays. Whole-segment amplification and long-read sequencing yielded more positive samples than standard M-segment qPCR methods (57.6% versus 3.0%, p < .0001). We determined that this difference in positivity was due to mismatches in published primers to our samples and that these mismatches would result in failing to detect in the vast majority of currently sequenced AIv genomes in public databases. The whole segment sequences were subsequently used to provide subtype and potential host information of the AIv environmental reservoir. There was no statistically significant difference in sequencing reads recovered from the RexeedTM filtration compared to the unfiltered surface water. This overall approach combining remote sensing, filtration and sequencing provides a novel and potentially more effective, surveillance approach for AIv.

Sulforaphane alters the acidification of the yeast vacuole.

Sulforaphane (SFN) is a compound [1-isothiocyanato-4-(methylsulfinyl)-butane] found in broccoli and other cruciferous vegetables that is currently of interest because of its potential as a chemopreventive and a chemotherapeutic drug. Recent studies in a diverse range of cellular and animal models have shown that SFN is involved in multiple intracellular pathways that regulate xenobiotic metabolism, inflammation, cell death, cell cycle progression, and epigenetic regulation. In order to better understand the mechanisms of action behind SFN-induced cell death, we undertook an unbiased genome wide screen with the yeast knockout (YKO) library to identify SFN sensitive (SFNS) mutants. The mutants were enriched with knockouts in genes linked to vacuolar function suggesting a link between this organelle and SFN's mechanism of action in yeast. Our subsequent work revealed that SFN increases the vacuolar pH of yeast cells and that varying the vacuolar pH can alter the sensitivity of yeast cells to the drug. In fact, several mutations that lower the vacuolar pH in yeast actually made the cells resistant to SFN (SFNR). Finally, we show that human lung cancer cells with more acidic compartments are also SFNR suggesting that SFN's mechanism of action identified in yeast may carry over to higher eukaryotic cells.

Next-generation sequencing of dsRNA is greatly improved by treatment with the inexpensive denaturing reagent DMSO.

dsRNA is the genetic material of important viruses and a key component of RNA interference-based immunity in eukaryotes. Previous studies have noted difficulties in determining the sequence of dsRNA molecules that have affected studies of immune function and estimates of viral diversity in nature. DMSO has been used to denature dsRNA prior to the reverse-transcription stage to improve reverse transcriptase PCR and Sanger sequencing. We systematically tested the utility of DMSO to improve the sequencing yield of a dsRNA virus (Φ6) in a short-read next-generation sequencing platform. DMSO treatment improved sequencing read recovery by over two orders of magnitude, even when RNA and cDNA concentrations were below the limit of detection. We also tested the effects of DMSO on a mock eukaryotic viral community and found that dsRNA virus reads increased with DMSO treatment. Furthermore, we provide evidence that DMSO treatment does not adversely affect recovery of reads from a ssRNA viral genome (influenza A/California/07/2009). We suggest that up to 50 % DMSO treatment be used prior to cDNA synthesis when samples of interest are composed of or may contain dsRNA.

Characterization of the definitive classical calpain family of vertebrates using phylogenetic, evolutionary and expression analyses.

The calpains are a superfamily of proteases with extensive relevance to human health and welfare. Vast research attention is given to the vertebrate 'classical' subfamily, making it surprising that the evolutionary origins, distribution and relationships of these genes is poorly characterized. Consequently, there exists uncertainty about the conservation of gene family structure, function and expression that has been principally defined from work with mammals. Here, more than 200 vertebrate classical calpains were incorporated in phylogenetic analyses spanning an unprecedented range of taxa, including jawless and cartilaginous fish. We demonstrate that the common vertebrate ancestor had at least six classical calpains, including a single gene that gave rise to CAPN11, 1, 2 and 8 in the early jawed fish lineage, plus CAPN3, 9, 12, 13 and a novel calpain gene, hereafter named CAPN17. We reveal that while all vertebrate classical calpains have been subject to persistent purifying selection during evolution, the degree and nature of selective pressure has often been lineage-dependent. The tissue expression of the complete classic calpain family was assessed in representative teleost fish, amphibians, reptiles and mammals. This highlighted systematic divergence in expression across vertebrate taxa, with most classic calpain genes from fish and amphibians having more extensive tissue distribution than in amniotes. Our data suggest that classical calpain functions have frequently diverged during vertebrate evolution and challenge the ongoing value of the established system of classifying calpains by expression.

Degradation of the Saccharomyces cerevisiae mating-type regulator alpha1: genetic dissection of cis-determinants and trans-acting pathways.

Mating phenotype in the yeast Saccharomyces cerevisiae is a dynamic trait, and efficient transitions between alternate haploid cell types allow the organism to access the advantageous diploid form. Mating identity is determined by cell type-specific transcriptional regulators, but these factors must be rapidly removed upon mating-type switching to allow the master regulators of the alternate state to establish a new gene expression program. Targeted proteolysis by the ubiquitin-proteasome system is a commonly employed strategy to quickly disassemble regulatory networks, and yeast use this approach to evoke efficient switching from the alpha to the a phenotype by ensuring the rapid removal of the alpha2 transcriptional repressor. Transition to the a cell phenotype, however, also requires the inactivation of the alpha1 transcriptional activator, but the mechanism by which this occurs is currently unknown. Here, we report a central role for the ubiquitin-proteasome system in alpha1 inactivation. The alpha1 protein is constitutively short lived and targeted for rapid turnover by multiple ubiquitin-conjugation pathways. Intriguingly, the alpha-domain, a conserved region of unknown function, acts as a degradation signal for a pathway defined by the SUMO-targeted ligase Slx5-Slx8, which has also been implicated in the rapid destruction of alpha2. Our observations suggest coordinate regulation in the turnover of two master regulatory transcription factors ensures a rapid mating-type switch.

A ubiquitin-selective AAA-ATPase mediates transcriptional switching by remodelling a repressor-promoter DNA complex.

Switches between different phenotypes and their underlying states of gene transcription occur as cells respond to intrinsic developmental cues or adapt to changing environmental conditions. Post-translational modification of the master regulatory transcription factors that define the initial phenotype is a common strategy to direct such transitions. Emerging evidence indicates that the modification of key transcription factors by the small polypeptide ubiquitin has a central role in many of these transitions. However, the molecular mechanisms by which ubiquitylation regulates the switching of promoters between active and inactive states are largely unknown. Ubiquitylation of the yeast transcriptional repressor alpha2 is necessary to evoke the transition between mating-types, and here we dissect the impact of this modification on alpha2 dynamics at its target promoters. Ubiquitylation of alpha2 does not alter DNA occupancy by depleting the existing pool of the transcription factor, despite its well-characterized function in directing repressor turnover. Rather, alpha2 ubiquitylation has a direct role in the rapid removal of the repressor from its DNA targets. This disassembly of alpha2 from DNA depends on the ubiquitin-selective AAA-ATPase Cdc48. Our findings expand the functional targets of Cdc48 to include active transcriptional regulatory complexes in the nucleus. These data reveal an ubiquitin-dependent extraction pathway for dismantling transcription factor-DNA complexes and provide an archetype for the regulation of transcriptional switching events by ubiquitylation.

Y-chromosome microdeletions and recurrent pregnancy loss.

OBJECTIVE: To determine the prevalence of Y-chromosome microdeletions in recurrent pregnancy loss (RPL) couples as compared with couples with male factor infertility and fertile couples. DESIGN: Controlled clinical study. SETTING: Andrology laboratory and RPL clinic. PATIENT(S): Seventeen men from RPL couples, 18 men from couples with a live birth and no history of miscarriages, and 10 men from couples with male factor infertility. INTERVENTION(S): Buccal smears for Y-chromosome microdeletion testing. MAIN OUTCOME MEASURE(S): The DNA was tested for microdeletions in the proximal AZFc region by polymerase chain reaction (PCR). RESULT(S): Fourteen of the 17 men (82%) tested had microdeletions in one or more of the four segments studied. Two of the 10 male factor infertility patients (20%) had microdeletions in 2 different segments. None of the 18 fertile men had any microdeletions in the 4 segments of the proximal AZFc region studied. CONCLUSION(S): The prevalence of the Y-chromosome microdeletions in the proximal AZFc region was much higher in men from RPL couples than from fertile or infertile couples. Although these patients are from a tertiary referral center that may skew the population and findings, one may consider Y-chromosome microdeletion testing particularly of the AZFc region in the evaluation of RPL couples when all other tests fail to reveal the etiology.

Effect of protein structure on mitochondrial import.

Most proteins that are to be imported into the mitochondrial matrix are synthesized as precursors, each composed of an N-terminal targeting sequence followed by a mature domain. Precursors are recognized through their targeting sequences by receptors at the mitochondrial surface and are then threaded through import channels into the matrix. Both the targeting sequence and the mature domain contribute to the efficiency with which proteins are imported into mitochondria. Precursors must be in an unfolded conformation during translocation. Mitochondria can unfold some proteins by changing their unfolding pathways. The effectiveness of this unfolding mechanism depends on the local structure of the mature domain adjacent to the targeting sequence. This local structure determines the extent to which the unfolding pathway can be changed and, therefore, the unfolding rate increased. Atomic force microscopy studies find that the local structures of proteins near their N and C termini also influence their resistance to mechanical unfolding. Thus, protein unfolding during import resembles mechanical unfolding, and the specificity of import is determined by the resistance of the mature domain to unfolding as well as by the properties of the targeting sequence.