N-1-Alkyl-2-oxo-2-aryl amides as novel antagonists of the TRPA1 receptor

A series of potent antagonists of the ion channel transient receptor potential A1 (TRPA1) was developed by modifying lead structure 16 that was discovered by high-throughput screening. Based on lead compound 16, a SAR was established, showing a narrow region at the nitro-aromatic R(1) moiety and at the warhead, while the R(2) side had a much wider scope including ureas and carbamates. Compound 16 inhibits Ca(2+)-activated TRPA1 currents reversibly in whole cell patch clamp experiments, indicating that under in vivo conditions, it does not react covalently, despite its potentially electrophilic ketone.     

Mammalian Alpha Arrestins Link Activated Seven Transmembrane Receptors to Nedd4 Family E3 Ubiquitin Ligases and Interact with Beta Arrestins

The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination – which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1–5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the implications of the alpha arrestin functions in fungi having evolved into coordinated alpha/beta arrestin functions in animals.     

Rerouting Cellular Electron Flux To Increase the Rate of Biological Methane Production

Methanogens are anaerobic archaea that grow by producing methane, a gas that is both an efficient renewable fuel and a potent greenhouse gas. We observed that overexpression of the cytoplasmic heterodisulfide reductase enzyme HdrABC increased the rate of methane production from methanol by 30% without affecting the growth rate relative to the parent strain. Hdr enzymes are essential in all known methane-producing archaea. They function as the terminal oxidases in the methanogen electron transport system by reducing the coenzyme M (2-mercaptoethane sulfonate) and coenzyme B (7-mercaptoheptanoylthreonine sulfonate) heterodisulfide, CoM-S-S-CoB, to regenerate the thiol-coenzymes for reuse. In Methanosarcina acetivorans, HdrABC expression caused an increased rate of methanogenesis and a decrease in metabolic efficiency on methylotrophic substrates. When acetate was the sole carbon and energy source, neither deletion nor overexpression of HdrABC had an effect on growth or methane production rates. These results suggest that in cells grown on methylated substrates, the cell compensates for energy losses due to expression of HdrABC with an increased rate of substrate turnover and that HdrABC lacks the appropriate electron donor in acetate-grown cells.     

Fate of Escherichia coli O157:H7 in Manure-Amended Soil

Escherichia coli O157:H7 cells survived for up to 77, >226, and 231 days in manure-amended autoclaved soil held at 5, 15, and 21°C, respectively. Pathogen populations declined more rapidly in manure-amended unautoclaved soil under the same conditions, likely due to antagonistic interactions with indigenous soil microorganisms. E. coli O157:H7 cells were inactivated more rapidly in both autoclaved and unautoclaved soils amended with manure at a ratio of 1 part manure to 10 parts soil at 15 and 21°C than in soil samples containing dilute amounts of manure. The manure-to-soil ratio, soil temperature, and indigenous microorganisms of the soil appear to be contributory factors to the pathogen's survival in manure-amended soil.     

Association and Interaction Analyses of GABBR1 and GABBR2 with Nicotine Dependence in European- and African-American Populations

Previous studies have demonstrated that the γ-aminobutyric acid type B (GABA_B) receptor plays an essential role in modulating neurotransmitter release and regulating the activity of ion channels and adenyl cyclase. However, whether the naturally occurring polymorphisms in the two GABA_B receptor subunit genes interact with each other to alter susceptibility to nicotine dependence (ND) remains largely unknown. In this study, we genotyped 5 and 33 single nucleotide polymorphisms (SNPs) for GABA_B receptor subunit 1 and 2 genes (GABBR1, GABBR2), respectively, in a sample of 2037 individuals from 602 nuclear families of African- American (AA) or European-American (EA) origin. We conducted association analyses to determine (1) the association of each subunit gene with ND at both the individual SNP and haplotype levels and (2) the collective effect(s) of SNPs in both GABA_B subunits on the development of ND. Several individual SNPs and haplotypes in GABBR2 were significantly associated with ND in both ethnic samples. Two haplotypes in AAs and one haplotype in EAs showed a protective effect against ND, whilst two other haplotypes in AAs and three haplotypes in EAs showed a risk effect for developing ND. Interestingly, these significant haplotypes were confined to two regions of GABBR2 in the AA and EA samples. Additionally, we found two minor haplotypes in GABBR1 to be positively associated with Heaviness of Smoking Index (HSI) in the EA sample. Finally, we demonstrated the presence of epistasis between GABBR1 and GABBR2 for developing ND. The variants of GABBR1 and GABBR2 are significantly associated with ND, and the involvement of GABBR1 is most likely through its interaction with GABBR2, whereas GABBR2 polymorphisms directly alter susceptibility to ND. Future studies are needed with more dense SNP coverage of GABBR1 and GABBR2 to verify the epistatic effects of the two subunit genes.     

Building proteomic tool boxes to monitor MHC class I and class II peptides

Major histocompatibility complex Class I (MHCI) and Class II (MHCII) presented peptides powerfully modulate T cell immunity and play a vital role in generating effective anti‐tumor and anti‐viral immune responses in mammals. Characterizing these MHCI or MHCII presented peptides can help generate therapeutic treatments, afford information on T cell mediated biomarkers, provide insight into disease progression, and reduce adverse anti‐drug side effects from engineered biotherapeutics. Here, we explore the tools and techniques commonly employed to discover both MHCI‐ and MHCII‐presented peptides. We describe complementary strategies that enhance the characterization of these peptides and the informatics tools employed for both predicting and characterizing MHCI‐ and MHCII‐presented epitopes. The evolution of methodologies for isolating MHC‐presented peptides is discussed, as are the mass spectrometric workflows that can be employed for their characterization. We provide a perspective on where this field is headed, and how these tools may be applicable to the discovery and monitoring of epitopes in a variety of scenarios.     

Long‐term continental changes in wing length,but not bill length,of a long‐distance migratory shorebird

We compiled a >50‐year record of morphometrics for semipalmated sandpipers (Calidris pusilla), a shorebird species with a Nearctic breeding distribution and intercontinental migration to South America. Our data included >57,000 individuals captured 1972–2015 at five breeding locations and three major stopover sites, plus 139 museum specimens collected in earlier decades. Wing length increased by ca. 1.5 mm (>1%) prior to 1980, followed by a decrease of 3.85 mm (nearly 4%) over the subsequent 35 years. This can account for previously reported changes in metrics at a migratory stopover site from 1985 to 2006. Wing length decreased at a rate of 1,098 darwins, or 0.176 haldanes, within the ranges of other field studies of phenotypic change. Bill length, in contrast, showed no consistent change over the full period of our study. Decreased body size as a universal response of animal populations to climate warming, and several other potential mechanisms, are unable to account for the increasing and decreasing wing length pattern observed. We propose that the post‐WWII near‐extirpation of falcon populations and their post‐1973 recovery driven by the widespread use and subsequent limitation on DDT in North America selected initially for greater flight efficiency and latterly for greater agility. This predation danger hypothesis accounts for many features of the morphometric data and deserves further investigation in this and other species.     

Asynchrony among local communities stabilises ecosystem function of metacommunities

Temporal stability of ecosystem functioning increases the predictability and reliability of ecosystem services, and understanding the drivers of stability across spatial scales is important for land management and policy decisions. We used species‐level abundance data from 62 plant communities across five continents to assess mechanisms of temporal stability across spatial scales. We assessed how asynchrony (i.e. different units responding dissimilarly through time) of species and local communities stabilised metacommunity ecosystem function. Asynchrony of species increased stability of local communities, and asynchrony among local communities enhanced metacommunity stability by a wide range of magnitudes (1–315%); this range was positively correlated with the size of the metacommunity. Additionally, asynchronous responses among local communities were linked with species’ populations fluctuating asynchronously across space, perhaps stemming from physical and/or competitive differences among local communities. Accordingly, we suggest spatial heterogeneity should be a major focus for maintaining the stability of ecosystem services at larger spatial scales.