An electrostatic mechanism for Ca(2+)-mediated regulation of gap junction channels.

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca(2+) blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca(2+). The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca(2+) coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca(2+)chelation. Computational analysis revealed that Ca(2+)-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K(+) into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore

Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins

Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins

Metabolic engineering of Clostridium autoethanogenum for selective alcohol production

Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols

Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo

The unique properties of the tumour microenvironment can be exploited by using recombinant anaerobic clostridial spores as highly selective gene delivery vectors. Although several recombinant Clostridium species have been generated during the past decade, their efficacy has been limited. Our goal was to substantially improve the prospects of clostridia as a gene delivery vector. Therefore, we have assessed a series of nitroreductase (NTR) enzymes for their capacity to convert the innocuous CB1954 prodrug to its toxic derivative. Among the enzymes tested, one showed superior prodrug turnover characteristics. In addition, we established an efficient gene transfer procedure, based on conjugation, which allows for the first time genetic engineering of Clostridium strains with superior tumour colonisation properties with high success rates. This conjugation procedure was subsequently used to create a recombinant C. sporogenes overexpressing the isolated NTR enzyme. Finally, analogous to a clinical setting situation, we have tested the effect of multiple consecutive treatment cycles, with antibiotic bacterial clearance between cycles. Importantly, this regimen demonstrated that intravenously administered spores of NTR-recombinant C. sporogenes produced significant antitumour efficacy when combined with prodrug administration

The Hepatitis E Virus Polyproline Region Is Involved in Viral Adaptation

Genomes of hepatitis E virus (HEV), rubivirus and cutthroat virus (CTV) contain a region of high proline density and low amino acid (aa) complexity, named the polyproline region (PPR). In HEV genotypes 1, 3 and 4, it is the only region within the non-structural open reading frame (ORF1) with positive selection (4–10 codons with dN/dS>1). This region has the highest density of sites with homoplasy values >0.5. Genotypes 3 and 4 show ∼3-fold increase in homoplastic density (HD) in the PPR compared to any other region in ORF1, genotype 1 does not exhibit significant HD (p<0.0001). PPR sequence divergence was found to be 2-fold greater for HEV genotypes 3 and 4 than for genotype 1. The data suggest the PPR plays an important role in host-range adaptation. Although the PPR appears to be hypervariable and homoplastic, it retains as much phylogenetic signal as any other similar sized region in the ORF1, indicating that convergent evolution operates within the major HEV phylogenetic lineages. Analyses of sequence-based secondary structure and the tertiary structure identify PPR as an intrinsically disordered region (IDR), implicating its role in regulation of replication. The identified propensity for the disorder-to-order state transitions indicates the PPR is involved in protein-protein interactions. Furthermore, the PPR of all four HEV genotypes contains seven putative linear binding motifs for ligands involved in the regulation of a wide number of cellular signaling processes. Structure-based analysis of possible molecular functions of these motifs showed the PPR is prone to bind a wide variety of ligands. Collectively, these data suggest a role for the PPR in HEV adaptation. Particularly as an IDR, the PPR likely contributes to fine tuning of viral replication through protein-protein interactions and should be considered as a target for development of novel anti-viral drugs

ECOSTRESS: NASA's next generation mission to measure evapotranspiration from the International Space Station

The ECOsystem Spaceborne Thermal Radiometer Experiment on Space Station ECOSTRESS) was launched to the International Space Station on June 29, 2018. The primary science focus of ECOSTRESS is centered on evapotranspiration (ET), which is produced as level‐3 (L3) latent heat flux (LE) data products. These data are generated from the level‐2 land surface temperature and emissivity product (L2_LSTE), in conjunction with ancillary surface and atmospheric data. Here, we provide the first validation (Stage 1, preliminary) of the global ECOSTRESS clear‐sky ET product (L3_ET_PT‐JPL, version 6.0) against LE measurements at 82 eddy covariance sites around the world. Overall, the ECOSTRESS ET product performs well against the site measurements (clear‐sky instantaneous/time of overpass: r2 = 0.88; overall bias = 8%; normalized RMSE = 6%). ET uncertainty was generally consistent across climate zones, biome types, and times of day (ECOSTRESS samples the diurnal cycle), though temperate sites are over‐represented. The 70 m high spatial resolution of ECOSTRESS improved correlations by 85%, and RMSE by 62%, relative to 1 km pixels. This paper serves as a reference for the ECOSTRESS L3 ET accuracy and Stage 1 validation status for subsequent science that follows using these data

Epidemic History and Evolutionary Dynamics of Hepatitis B Virus Infection in Two Remote Communities in Rural Nigeria

BACKGROUND: In Nigeria, hepatitis B virus (HBV) infection has reached hyperendemic levels and its nature and origin have been described as a puzzle. In this study, we investigated the molecular epidemiology and epidemic history of HBV infection in two semi-isolated rural communities in North/Central Nigeria. It was expected that only a few, if any, HBV strains could have been introduced and effectively transmitted among these residents, reflecting limited contacts of these communities with the general population in the country. METHODS AND FINDINGS: Despite remoteness and isolation, approximately 11% of the entire population in these communities was HBV-DNA seropositive. Analyses of the S-gene sequences obtained from 55 HBV-seropositive individuals showed the circulation of 37 distinct HBV variants. These HBV isolates belong predominantly to genotype E (HBV/E) (n=53, 96.4%), with only 2 classified as sub-genotype A3 (HBV/A3). Phylogenetic analysis showed extensive intermixing between HBV/E variants identified in these communities and different countries in Africa. Quasispecies analysis of 22 HBV/E strains using end-point limiting-dilution real-time PCR, sequencing and median joining networks showed extensive intra-host heterogeneity and inter-host variant sharing. To investigate events that resulted in such remarkable HBV/E diversity, HBV full-size genome sequences were obtained from 47 HBV/E infected persons and P gene was subjected to Bayesian coalescent analysis. The time to the most recent common ancestor (tMRCA) for these HBV/E variants was estimated to be year 1952 (95% highest posterior density (95% HPD): 1927-1970). Using additional HBV/E sequences from other African countries, the tMRCA was estimated to be year 1948 (95% HPD: 1924-1966), indicating that HBV/E in these remote communities has a similar time of origin with multiple HBV/E variants broadly circulating in West/Central Africa. Phylogenetic analysis and statistical neutrality tests suggested rapid HBV/E population expansion. Additionally, skyline plot analysis showed an increase in the size of the HBV/E-infected population over the last approximately 30-40 years. CONCLUSIONS: Our data suggest a massive introduction and relatively recent HBV/E expansion in the human population in Africa. Collectively, these data show a significant shift in the HBV/E epidemic dynamics in Africa over the last century

Reflections on the 'History and Historians' of the black woman's role in the community of slaves: enslaved women and intimate partner sexual violence

Taking as points of inspiration Peter Parish’s 1989 book, Slavery: History and Historians, and Angela Davis’s seminal 1971 article, “Reflections on the black woman’s role in the community of slaves,” this probes both historiographically and methodologically some of the challenges faced by historians writing about the lives of enslaved women through a case study of intimate partner violence among enslaved people in the antebellum South. Because rape and sexual assault have been defined in the past as non-consensual sexual acts supported by surviving legal evidence (generally testimony from court trials), it is hard for historians to research rape and sexual violence under slavery (especially marital rape) as there was no legal standing for the rape of enslaved women or the rape of any woman within marriage. This article suggests enslaved women recognized that black men could both be perpetrators of sexual violence and simultaneously be victims of the system of slavery. It also argues women stoically tolerated being forced into intimate relationships, sometimes even staying with “husbands” imposed upon them after emancipation

Meta-analysis of pre-clinical studies of early decompression in acute spinal cord injury:a battle of time and pressure

The use of early decompression in the management of acute spinal cord injury (SCI) remains contentious despite many pre-clinical studies demonstrating benefits and a small number of supportive clinical studies. Although the pre-clinical literature favours the concept of early decompression, translation is hindered by uncertainties regarding overall treatment efficacy and timing of decompression.We performed meta-analysis to examine the pre-clinical literature on acute decompression of the injured spinal cord. Three databases were utilised; PubMed, ISI Web of Science and Embase. Our inclusion criteria consisted of (i) the reporting of efficacy of decompression at various time intervals (ii) number of animals and (iii) the mean outcome and variance in each group. Random effects meta-analysis was used and the impact of study design characteristics assessed with meta-regression.Overall, decompression improved behavioural outcome by 35.1% (95%CI 27.4-42.8; I(2)=94%, p<0.001). Measures to minimise bias were not routinely reported with blinding associated with a smaller but still significant benefit. Publication bias likely also contributed to an overestimation of efficacy. Meta-regression demonstrated a number of factors affecting outcome, notably compressive pressure and duration (adjusted r(2)=0.204, p<0.002), with increased pressure and longer durations of compression associated with smaller treatment effects. Plotting the compressive pressure against the duration of compression resulting in paraplegia in individual studies revealed a power law relationship; high compressive forces quickly resulted in paraplegia, while low compressive forces accompanying canal narrowing resulted in paresis over many hours.These data suggest early decompression improves neurobehavioural deficits in animal models of SCI. Although much of the literature had limited internal validity, benefit was maintained across high quality studies. The close relationship of compressive pressure to the rate of development of severe neurological injury suggests that pressure local to the site of injury might be a useful parameter determining the urgency of decompression