Biodesigns (United States)

Abstract Most eukaryotic membrane proteins are inserted into the membrane at the endoplasmic reticulum (ER). This essential but error-prone process relies on molecular quality control machineries to prevent mistargeting and incorrect structure formation. Here we show that the ER membrane protein complex (EMC) forms an evolutionarily conserved supercomplex with the P5A-ATPase Spf1/ATP13A1. This supercomplex combines the transmembrane domain (TMD) insertase function of the EMC and the TMD dislocase activity of Spf1 in a single entity. Our cryo-EM structure of this supercomplex shows that EMC and Spf1 form a shared intramembrane cavity for substrate engagement and reveals that the ATPase cycle of Spf1 regulates access to this cavity. Together, our study suggests that proteins with opposing biochemical activities in membrane protein biogenesis – insertion versus dislocation – form an integrated molecular machine in eukaryotes to proofread membrane protein insertion and topogenesis.

Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

Photoinduced proton-coupled electron transfer and long-range two-proton transport via a Grotthuss-type mechanism are investigated in a biomimetic construct. The ultrafast, nonequilibrium dynamics are assessed via two-dimensional electronic vibrational spectroscopy, in concert with electrochemical and computational techniques. A low-frequency mode is identified experimentally and found to promote double proton and electron transfer, supported by recent theoretical simulations of a similar but abbreviated (non-photoactive) system. Excitation frequency peak evolution and center line slope dynamics show direct evidence of strongly coupled nuclear and electronic degrees of freedom, from which we can conclude that the double proton and electron transfer processes are concerted (up to an uncertainty of 24 fs). The nonequilibrium pathway from the photoexcited Franck–Condon region to the E2PT state is characterized by an ∼110 fs time scale. This study and the tools presented herein constitute a new window into hot charge transfer processes involving an electron and multiple protons.

Gulf War Illness (GWI) is a chronic multi-symptom disorder that affected almost one-third of the deployed veterans during the first Persian Gulf War (1990-91). Symptoms include gastrointestinal disturbances, chronic fatigue, cognitive deficiencies, and neuroinflammation. Our group has recently shown a series of different therapeutic strategies that span oral butyrate, anti-viral drug Ribavirin, and TLR4 antagonist Sparstolonin B effectively improving outcomes to murine GWI-linked symptoms. Andrographolide (AG), a labdane diterpenoid produced by the plant Andrographis paniculata, exerts numerous beneficial effects in various cell types. Adult C57BL/6J mice were exposed to the GWI chemicals (GWC) pyridostigmine bromide and permethrin for a week (GWT group) and simultaneously treated with AG (GWT+AG group). In our previous report, virome dysbiosis showed a strong association with GWI inflammation. Next-Gen Sequencing data showed both virome richness and Shannon diversity were markedly altered in GWT+AG mice than GWT mice. The observed increased relative abundance of Siphoviridae, Myoviridae, and decreased Microviridae abundance in the GWT group was reversed by AG treatment, referring to a restored gut environment. Gut leaching marked by decreased Occludin expression and increased Claudin2 expression was reversed by AG treatment, a likely consequence of AG induced decrease in TLR4 and TLR7 expression. AG administration restored gut barrier integrity, decreased TLR4 activation, NFκb phosphorylation, and gut inflammation. Furthermore, systemic inflammation in GWT mice, evidenced by the high level of serum IL-1β and IL-6, was found to be decreased in GWT+AG mice. Blood-brain barrier (BBB) integrity loss and resulting microglial activation, a common feature of GWI murine pathology were reversed by AG treatment. AG restored dysfunctional BBB by enhancing tight junction protein Claudin5 expression as well as decreased microglial activation. Also, AG administration led to high brain-derived neurotrophic factor (BDNF) expression in the frontal cortex, a molecule with a pronounced role in synaptic plasticity and neuronal regeneration. In summary, these results explicitly imply that AG played a key role in decreasing GWI-associated intestinal, systemic, and neuroinflammation whereas it also improved intestinal barrier and BBB integrity in the GWI mouse model.

Purpose: GI-4000, a series of recombinant yeast expressing four different mutated RAS proteins, was evaluated in subjects with resected ras-mutated pancreas cancer.Methods: Subjects (n = 176) received GI-4000 or placebo plus gemcitabine. Subjects' tumors were genotyped to identify which matched GI-4000 product to administer. Immune responses were measured by interferon-γ (IFNγ) ELISpot assay and by regulatory T cell (Treg) frequencies on treatment. Pretreatment plasma was retrospectively analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-ToF) mass spectrometry for proteomic signatures predictive of GI-4000 responsiveness.Results: GI-4000 was well tolerated, with comparable safety findings between treatment groups. The GI-4000 group showed a similar pattern of median recurrence-free and overall survival (OS) compared with placebo. For the prospectively defined and stratified R1 resection subgroup, there was a trend in 1 year OS (72% vs. 56%), an improvement in OS (523.5 vs. 443.5 days [hazard ratio (HR) = 1.06 [confidence interval (CI): 0.53–2.13], p = 0.872), and increased frequency of immune responders (40% vs. 8%; p = 0.062) for GI-4000 versus placebo and a 159-day improvement in OS for R1 GI-4000 immune responders versus placebo (p = 0.810). For R0 resection subjects, no increases in IFNγ responses in GI-4000–treated subjects were observed. A higher frequency of R0/R1 subjects with a reduction in Tregs (CD4+/CD45RA+/Foxp3low) was observed in GI-4000–treated subjects versus placebo (p = 0.033). A proteomic signature was identified that predicted response to GI-4000/gemcitabine regardless of resection status.Conclusion: These results justify continued investigation of GI-4000 in studies stratified for likely responders or in combination with immune check-point inhibitors or other immunomodulators, which may provide optimal reactivation of antitumor immunity.ClinicalTrials.gov Number: NCT00300950.

Opening the possibility of sustainable industrial (3R)-acetoin biomanufacturingin vitro.

ABSTRACT Coupling the study of sustainability with geoscience may enable students to explore science in a more sophisticated way by examining the social–technological–ecological relationships that exist between human–nonhuman and flora–fauna–land interactions. Elementary educators are a population capable of making these issues come to life for today's youngest citizens, who will ultimately become tomorrow's changemakers. This study explores Sustainability Science for Teachers, a semester-long hybrid course designed to enable future teachers to engage in sustainability and science concepts while developing their understanding of science from the human perspective and in which an issues-based curriculum underpins social and biosphere responsibility. The course's Water unit is explored as a case study of the melding of sustainability and geoscience to engage teachers in a more nuanced understanding of science education. The unit's curriculum is presented and its design process is explained, followed by a cross-sectional analysis of student outcomes. Data from preservice teachers enrolled in the course, as well as course alumni, were collected over a 4-y period. A mixed methods evaluation of teachers' opinions and products indicate that the Water unit facilitated the development of new understanding and new ways of thinking about teaching their future students. Opportunities and challenges for fusing the geosciences, sustainability concepts, and preservice teacher education in a novel and impactful fashion are discussed.

Helicobacter pylori is one of the most common pathogenic bacteria that causes chronic gastritis, peptic ulcers, and gastric cancer. It is very difficult to treat H. pylori bacterial infections, in part due to its polymorphisms. The objective of this study was to identify critical contributors to the pathogenicity of H. pylori bacteria through bibliometric screening of pathogenic genes. The pathogenic genes of H. pylori strain 26695 were collected from major publication databases using the bibliometric approach. Information about the pathways, operons, and functions of these genes was obtained from the KEGG and DOOR databases. We inferred that HP0067, HP0069, HP1109, HP1036, and HP1037 are key pathogenic genes in H. pylori pathogenicity. These genes provided new targets for the clinical diagnosis and prognosis judgment of H. pylori. Finally, this study may serve as a model for other genomic level studies when more H. pylori genomes become available. These results lay a theoretical foundation for further studies on the detection of novel pathogenic genes and relevant clinical analyses.