The epithelial anion transporter SLC26A9 contributes to airway surface hydration and gastric acid production. Colocalizing with CFTR, SLC26A9 has been proposed as a target for the treatment of cystic ...fibrosis. To provide molecular details of its transport mechanism, we present cryo-EM structures and a functional characterization of murine Slc26a9. These structures define the general architecture of eukaryotic SLC26 family members and reveal an unusual mode of oligomerization which relies predominantly on the cytosolic STAS domain. Our data illustrates conformational transitions of Slc26a9, supporting a rapid alternate-access mechanism which mediates uncoupled chloride transport with negligible bicarbonate or sulfate permeability. The characterization of structure-guided mutants illuminates the properties of the ion transport path, including a selective anion binding site located in the center of a mobile module within the transmembrane domain. This study thus provides a structural foundation for the understanding of the entire SLC26 family and potentially facilitates their therapeutic exploitation.
Although vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) is traditionally regarded as an endothelial cell protein, evidence suggests that VEGFRs may be expressed by cancer cells. ...Glioblastoma multiforme (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with the humanized VEGF antibody bevacizumab reduces GBM tumor growth; however, the clinical benefits are transient and invariably followed by tumor recurrence. In this study, we show that VEGFR2 is preferentially expressed on the cell surface of the CD133(+) human glioma stem-like cells (GSCs), whose viability, self-renewal, and tumorigenicity rely, at least in part, on signaling through the VEGF-VEGFR2-Neuropilin-1 (NRP1) axis. We find that the limited impact of bevacizumab-mediated VEGF blockage may reflect ongoing autocrine signaling through VEGF-VEGFR2-NRP1, which is associated with VEGFR2-NRP1 recycling and a pool of active VEGFR2 within a cytosolic compartment of a subset of human GBM cells. Whereas bevacizumab failed to inhibit prosurvival effects of VEGFR2-mediated signaling, GSC viability under unperturbed or radiation-evoked stress conditions was attenuated by direct inhibition of VEGFR2 tyrosine kinase activity and/or shRNA-mediated knockdown of VEGFR2 or NRP1. We propose that direct inhibition of VEGFR2 kinase may block the highly dynamic VEGF-VEGFR2-NRP1 pathway and inspire a GBM treatment strategy to complement the currently prevalent ligand neutralization approach.
Since the Fermi discovery of γ-rays from novae, one of the biggest questions in the field has been how novae generate such high-energy emission. Shocks must be a fundamental ingredient. Six months of ...radio observations of the 2012 Nova V5589 Sgr with the VLA and 15 weeks of X-ray observations with Swift/XRT show that the radio emission consisted of: (1) a shock-powered, non-thermal flare; and (2) weak thermal emission from 10−5 M⊙ of freely expanding, photoionized ejecta. Absorption features in the optical spectrum and the peak optical brightness suggest that V5589 Sgr lies 4 kpc away (3.2–4.6 kpc). The shock-powered flare dominated the radio light curve at low frequencies before day 100. The spectral evolution of the radio flare, its high radio brightness temperature, the presence of unusually hard (kT
x
> 33 keV) X-rays, and the ratio of radio to X-ray flux near radio maximum all support the conclusions that the flare was shock-powered and non-thermal. Unlike most other novae with strong shock-powered radio emission, V5589 Sgr is not embedded in the wind of a red-giant companion. Based on the similar inclinations and optical line profiles of V5589 Sgr and V959 Mon, we propose that shocks in V5589 Sgr formed from collisions between a slow flow with an equatorial density enhancement and a subsequent faster flow. We speculate that the relatively high speed and low mass of the ejecta led to the unusual radio emission from V5589 Sgr, and perhaps also to the non-detection of γ-rays.
Using diagnostic data and contemporary sampling efforts, we conducted surveillance for a diversity of pathogens, toxicants, and diseases of muskrats (Ondatra zibethicus). Between 1977 and 2019, 26 ...diagnostic cases were examined from Kansas and throughout the Southeast and Mid-Atlantic, USA. We identified multiple causes of mortality in muskrats, but trauma (8/26), Tyzzer's disease (5/6), and cysticercosis (5/26) were the most common. We also conducted necropsies, during November 2018-January 2019 Pennsylvania muskrat trapping season, on 380 trapper-harvested muskrat carcasses after the pelt was removed. Tissue samples and exudate were tested for presence of or exposure to a suite of pathogens and contaminants. Gastrointestinal tracts were examined for helminths. Intestinal helminths were present in 39.2% of necropsied muskrats, with Hymenolepis spp. (62%) and echinostome spp. (44%) being the most common Molecular testing identified a low prevalence of infection with Clostridium piliforme in the feces and Sarcocystis spp. in the heart. We detected a low seroprevalence to Toxoplasma gondii (1/380). No muskrats were positive for Francisella tularensis or Babesia spp. Cysticercosis was detected in 20% (5/26) of diagnostic cases and 15% (57/380) of our trapper-harvested muskrats. Toxic concentrations of arsenic, cadmium, lead, or mercury were not detected in tested liver samples. Copper, molybdenum, and zinc concentrations were detected at acceptable levels comparative to previous studies. Parasite intensity and abundance were typical of historic reports; however, younger muskrats had higher intensity of infection than older muskrats which is contradictory to what has been previously reported. A diversity of pathogens and contaminants have been reported from muskrats, but the associated disease impacts are poorly understood. Our data are consistent with historic reports and highlight the wide range of parasites, pathogens and contaminants harbored by muskrats in Pennsylvania. The data collected are a critical component in assessing overall muskrat health and serve as a basis for understanding the impacts of disease on recent muskrat population declines.
The ecosystem performance approach, used in a previously published case study focusing on the Nebraska Sandhills, proved to minimize impacts of non-climatic factors (e.g., overgrazing, fire, pests) ...on the remotely-sensed signal of seasonal vegetation greenness resulting in a better attribution of its changes to climate variability. The current study validates the applicability of this approach for assessment of seasonal and interannual climate impacts on forage production in the western United States semi-arid grasslands. Using a piecewise regression tree model, we developed the Expected Ecosystem Performance (EEP), a proxy for annual forage production that reflects climatic influences while minimizing impacts of management and disturbances. The EEP model establishes relations between seasonal climate, site-specific growth potential, and long-term growth variability to capture changes in the growing season greenness measured via a time-integrated Normalized Difference Vegetation Index (NDVI) observed using a Moderate Resolution Imaging Spectroradiometer (MODIS). The resulting 19 years of EEP were converted to expected biomass (EB, kg ha−1 year−1) using a newly-developed relation with the Soil Survey Geographic Database range production data (R2 = 0.7). Results were compared to ground-observed biomass datasets collected by the U.S. Department of Agriculture and University of Nebraska-Lincoln (R2 = 0.67). This study illustrated that this approach is transferable to other semi-arid and arid grasslands and can be used for creating timely, post-season forage production assessments. When combined with seasonal climate predictions, it can provide within-season estimates of annual forage production that can serve as a basis for more informed adaptive decision making by livestock producers and land managers.
Productivity throughout the North American Great Plains grasslands is generally considered to be water limited, with the strength of this limitation increasing as precipitation decreases. We ...hypothesize that cumulative actual evapotranspiration water loss (AET) from April to July is the precipitation-related variable most correlated to aboveground net primary production (ANPP) in the U.S. Great Plains (GP). We tested this by evaluating the relationship of ANPP to AET, precipitation, and plant transpiration (Tr). We used multi-year ANPP data from five sites ranging from semiarid grasslands in Colorado and Wyoming to mesic grasslands in Nebraska and Kansas, mean annual NRCS ANPP, and satellite-derived normalized difference vegetation index (NDVI) data. Results from the five sites showed that cumulative April-to-July AET, precipitation, and Tr were well correlated (R2: 0.54–0.70) to annual changes in ANPP for all but the wettest site. AET and Tr were better correlated to annual changes in ANPP compared to precipitation for the drier sites, and precipitation in August and September had little impact on productivity in drier sites. April-to-July cumulative precipitation was best correlated (R2 = 0.63) with interannual variability in ANPP in the most mesic site, while AET and Tr were poorly correlated with ANPP at this site. Cumulative growing season (May-to-September) NDVI (iNDVI) was strongly correlated with annual ANPP at the five sites (R2 = 0.90). Using iNDVI as a surrogate for ANPP, we found that county-level cumulative April–July AET was more strongly correlated to ANPP than precipitation for more than 80% of the GP counties, with precipitation tending to perform better in the eastern more mesic portion of the GP. Including the ratio of AET to potential evapotranspiration (PET) improved the correlation of AET to both iNDVI and mean county-level NRCS ANPP. Accounting for how different precipitation-related variables control ANPP (AET in drier portion, precipitation in wetter portion) provides opportunity to develop spatially explicit forecasting of ANPP across the GP for enhancing decision-making by land managers and use of grassland ANPP for biofuels.
The ongoing COVID‐19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously ...to the SARS‐CoV‐2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo‐EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri‐bispecific fusion constructs that exhibit up to 100‐ and 1,000‐fold increase in neutralization potency, respectively. Cryo‐EM of the sybody‐spike complex additionally reveals a novel up‐out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS‐CoV‐2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS‐CoV‐2 escape mutants.
Synopsis
Sybodies Sb#15 and Sb#68 inhibit SARS‐CoV‐2 infectivity by targeting non‐overlapping epitopes on the spike glycoprotein. Covalent sybody fusion and valency engineering enhances neutralization potency against variants and impedes emergence of escape mutants.
Two synthetic nanobodies were in vitro selected against the SARS‐CoV‐2 receptor‐binding domain (RBD).
Sb#15 and Sb#68 neutralize viral infection by blocking ACE2 association with the SARS‐CoV‐2 spike protein.
Sb#15 and Sb#68 bind to distinct epitopes and promote a unique up/out conformation of the RBD.
Sybody fusions increase neutralization potency and attenuate the emergence of novel escape mutants.
Sybodies Sb#15 and Sb#68 inhibit SARS‐CoV‐2 infectivity by targeting non‐overlapping epitopes on the spike glycoprotein. Covalent sybody fusion and valency engineering enhances neutralization potency against variants and impedes emergence of escape mutants.
It has recently been discovered that some, if not all, classical novae emit GeV gamma-rays during outburst, but the mechanisms involved in the production ofgamma-rays are still not well understood. ...We present here a comprehensive multiwavelength data set-from radio to X-rays-for the most gamma-ray-luminous classical nova to date, V1324 Sco. Using this data set, we show that V1324 Sco is a canonical dusty Fe ii-type nova, with a maximum ejecta velocity of 2600 km s−1 and an ejecta mass of a few . There is also evidence for complex shock interactions, including a double-peaked radio light curve which shows high brightness temperatures at early times. To explore why V1324 Sco was so gamma-ray luminous, we present a model of the nova ejecta featuring strong internal shocks and find that higher gamma-ray luminosities result from higher ejecta velocities and/or mass-loss rates. Comparison of V1324 Sco with other gamma-ray-detected novae does not show clear signatures of either, and we conclude that a larger sample of similarly well-observed novae is needed to understand the origin and variation of gamma-rays in novae.
Thiostrepton, a macrocyclic thiopeptide antibiotic, inhibits prokaryotic translation by interfering with the function of elongation factor G (EF-G). Here, we have used 70S ribosome binding and GTP ...hydrolysis assays to study the effects of thiostrepton on EF-G and a newly described translation factor, elongation factor 4 (EF4). In the presence of thiostrepton, ribosome-dependent GTP hydrolysis is inhibited for both EF-G and EF4, with IC(50) values equivalent to the 70S ribosome concentration (0.15 µM). Further studies indicate the mode of thiostrepton inhibition is to abrogate the stable binding of EF-G and EF4 to the 70S ribosome. In support of this model, an EF-G truncation variant that does not possess domains IV and V was shown to possess ribosome-dependent GTP hydrolysis activity that was not affected by the presence of thiostrepton (>100 µM). Lastly, chemical footprinting was employed to examine the nature of ribosome interaction and tRNA movements associated with EF4. In the presence of non-hydrolyzable GTP, EF4 showed chemical protections similar to EF-G and stabilized a ratcheted state of the 70S ribosome. These data support the model that thiostrepton inhibits stable GTPase binding to 70S ribosomal complexes, and a model for the first step of EF4-catalyzed reverse-translocation is presented.
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