A Structural Model for the Core Nup358-BicD2 Interface.

Dynein motors facilitate the majority of minus-end-directed transport events on microtubules. The dynein adaptor Bicaudal D2 (BicD2) recruits the dynein machinery to several cellular cargo for transport, including Nup358, which facilitates a nuclear positioning pathway that is essential for the differentiation of distinct brain progenitor cells. Previously, we showed that Nup358 forms a "cargo recognition α-helix" upon binding to BicD2; however, the specifics of the BicD2-Nup358 interface are still not well understood. Here, we used AlphaFold2, complemented by two additional docking programs (HADDOCK and ClusPro) as well as mutagenesis, to show that the Nup358 cargo-recognition α-helix binds to BicD2 between residues 747 and 774 in an anti-parallel manner, forming a helical bundle. We identified two intermolecular salt bridges that are important to stabilize the interface. In addition, we uncovered a secondary interface mediated by an intrinsically disordered region of Nup358 that is directly N-terminal to the cargo-recognition α-helix and binds to BicD2 between residues 774 and 800. This is the same BicD2 domain that binds to the competing cargo adapter Rab6, which is important for the transport of Golgi-derived and secretory vesicles. Our results establish a structural basis for cargo recognition and selection by the dynein adapter BicD2, which facilitates transport pathways that are important for brain development.

A marginal structural model analysis for the effect modification by education on the association between cancer diagnosis history and major depressive symptoms: Findings from Midlife Development in the U.S. (MIDUS).

BACKGROUND: Limited research has employed a longitudinal approach to investigate the role of education level as an effect modifier on the relationship between cancer diagnosis history and the experience of major depressive disorder (MDD) with a nationally representative sample. METHODS: We harnessed data from three installments of the MIDUS Longitudinal study (n = 7108). A Marginal Structural Model facilitated the investigation of associations between a history of cancer diagnosis, MDD, and potential modifying effects of education level. Inverse probability weighting helped manage confounding factors. RESULTS: Findings indicated that a cancer diagnosis made one year prior was linked with 3.741 times greater odds of experiencing MDD (95 % CI: 1.411-9.918, p < 0.01). This connection was absent for diagnoses made two years earlier. Among individuals with education up to high school, a recent cancer diagnosis significantly increased the likelihood of MDD in the subsequent wave by 3.45 times (95 % CI: 1.31-9.08, p < 0.05). This pattern was not apparent among better-educated individuals. LIMITATIONS: As the exposure variable was dependent on self-reported questionnaires, recall bias could be a potential limitation. Moreover, unaccounted variables like genetic factors could introduce confounding. CONCLUSIONS: A recent cancer diagnosis, particularly among less educated individuals, correlated with an increased probability of MDD, while the impact was not observed for older diagnoses. These findings emphasize that the timing of a cancer diagnosis and education level need consideration in the mental health assessment of cancer survivors.

Structural Model for Factor X Inhibition of IgM and Complement-Mediated Neutralization of Adenovirus.

Adenovirus has strong therapeutic potential as an oncolytic virus and gene therapy vector. However, injecting human species C serotype 5 adenovirus, HAdv-C5, into the bloodstream leads to numerous interactions with plasma proteins that affect viral tropism and biodistribution, and can lead to potent immune responses and viral neutralization. The HAdv/factor X (FX) interaction facilitates highly efficient liver transduction and protects virus particles from complement-mediated neutralization after intravenous delivery. Ablating the FX interaction site on the HAdv-C5 capsid leaves the virus susceptible to neutralization by natural IgM followed by activation of the complement cascade and covalent binding of complement components C4b and C3b to the viral capsid. Here we present structural models for IgM and complement components C1, C4b, and C3b in complex with HAdv-C5. Molecular dynamics simulations indicate that when C3b binds near the vertex, multiple stabilizing interactions can be formed between C3b, penton base, and fiber. These interactions may stabilize the vertex region of the capsid and prevent release of the virally encoded membrane lytic factor, protein VI, which is packaged inside of the viral capsid, thus effectively neutralizing the virus. In a situation where FX and IgM are competing for binding to the capsid, IgM may not be able to form a bent conformation in which most of its Fab arms interact with the capsid. Our structural modeling of the competitive interaction of FX and IgM with HAdv-C5 allows us to propose a mechanistic model for FX inhibition of IgM-mediated virus neutralization. According to this model, although IgM may bind to the capsid, in the presence of FX it will likely retain a planar conformation and thus be unable to promote activation of the complement cascade at the virus surface.

A structural model of the iRhom-ADAM17 sheddase complex reveals functional insights into its trafficking and activity.

Several membrane-anchored signal mediators such as cytokines (e.g. TNFα) and growth factors are proteolytically shed from the cell surface by the metalloproteinase ADAM17, which, thus, has an essential role in inflammatory and developmental processes. The membrane proteins iRhom1 and iRhom2 are instrumental for the transport of ADAM17 to the cell surface and its regulation. However, the structure-function determinants of the iRhom-ADAM17 complex are poorly understood. We used AI-based modelling to gain insights into the structure-function relationship of this complex. We identified different regions in the iRhom homology domain (IRHD) that are differentially responsible for iRhom functions. We have supported the validity of the predicted structure-function determinants with several in vitro, ex vivo and in vivo approaches and demonstrated the regulatory role of the IRHD for iRhom-ADAM17 complex cohesion and forward trafficking. Overall, we provide mechanistic insights into the iRhom-ADAM17-mediated shedding event, which is at the centre of several important cytokine and growth factor pathways.

Causal inference in survival analysis using longitudinal observational data: Sequential trials and marginal structural models.

Longitudinal observational data on patients can be used to investigate causal effects of time-varying treatments on time-to-event outcomes. Several methods have been developed for estimating such effects by controlling for the time-dependent confounding that typically occurs. The most commonly used is marginal structural models (MSM) estimated using inverse probability of treatment weights (IPTW) (MSM-IPTW). An alternative, the sequential trials approach, is increasingly popular, and involves creating a sequence of "trials" from new time origins and comparing treatment initiators and non-initiators. Individuals are censored when they deviate from their treatment assignment at the start of each "trial" (initiator or noninitiator), which is accounted for using inverse probability of censoring weights. The analysis uses data combined across trials. We show that the sequential trials approach can estimate the parameters of a particular MSM. The causal estimand that we focus on is the marginal risk difference between the sustained treatment strategies of "always treat" vs "never treat." We compare how the sequential trials approach and MSM-IPTW estimate this estimand, and discuss their assumptions and how data are used differently. The performance of the two approaches is compared in a simulation study. The sequential trials approach, which tends to involve less extreme weights than MSM-IPTW, results in greater efficiency for estimating the marginal risk difference at most follow-up times, but this can, in certain scenarios, be reversed at later time points and relies on modelling assumptions. We apply the methods to longitudinal observational data from the UK Cystic Fibrosis Registry to estimate the effect of dornase alfa on survival.

A thrombophilic allele of clotting Factor VII/VIIa promoting recurrent pulmonary emboli, clinical details, and a structural model of the altered protein: a case report.

BACKGROUND: The clotting or hemostasis system is a meticulously regulated set of enzymatic reactions that occur in the blood and culminate in formation of a fibrin clot. The precisely calibrated signaling system that prevents or initiates clotting originates with the activated Factor Seven (FVIIa) complexed with tissue factor (TF) formed in the endothelium. Here we describe a rare inherited mutation in the FVII gene which is associated with pathological clotting. CASE PRESENTATION: The 52-year-old patient, with European, Cherokee and African American origins, FS was identified as having low FVII (10%) prior to elective surgery for an umbilical hernia. He was given low doses of NovoSeven (therapeutic Factor VIIa) and had no unusual bleeding or clotting during the surgery. In fact, during his entire clinical course he had no unprovoked bleeding. Bleeding instances occurred with hemostatic stresses such as gastritis, kidney calculus, orthopedic surgery, or tooth extraction, and these were handled without factor replacement. On the other hand, FS sustained two unprovoked and life-threatening instances of pulmonary emboli, although he was not treated with NovoSeven at any time close to the events. Since 2020, he has been placed on a DOAC (Direct Oral Anticoagulant, producing Factor Xa inhibition) and has sustained no further clots. POSSIBLE MECHANISM OF (UNAUTHORIZED) FVII ACTIVATION: FS has a congenitally mutated FVII/FVIIa gene, which carries a R315W missense mutation in one allele and a mutated start codon (ATG to ACG) in the other allele, thus rendering the patient effectively homozygous for the missense FVII. Structure based comparisons with known crystal structures of TF-VIIa indicate that the patient's missense mutation is predicted to induce a conformational shift of the C170's loop due to crowding of the bulky tryptophan to a distorted "out" position (Fig. 1). This mobile loop likely forms new interactions with activation loop 3, stabilizing a more active conformation of the FVII and FVIIa protein. The mutant form of FVIIa may be better able to interact with TF, displaying a modified serine protease active site with enhanced activity for downstream substrates such as Factor X. CONCLUSIONS: Factor VII can be considered the gatekeeper of the coagulation system. Here we describe an inherited mutation in which the gatekeeper function is altered. Instead of the expected bleeding manifestations resulting from a clotting factor deficiency, the patient FS suffered clotting episodes. The efficacy of the DOAC in treating and preventing clots in this unusual situation is due to its target site of inhibition (anti-Xa), which lies downstream of the site of action of FVIIa/TF.

Biochemically validated structural model of the 15-subunit intraflagellar transport complex IFT-B.

Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT-A and IFT-B that are transported by molecular motors. The IFT-B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15-subunit IFT-B complex. The model was validated using cross-linking/mass-spectrometry data on reconstituted IFT-B complexes, X-ray scattering in solution, diffraction from crystals as well as site-directed mutagenesis and protein-binding assays. The IFT-B structure reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The IFT-B complex organizes into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.

A New Structural Model of Apolipoprotein B100 Based on Computational Modeling and Cross Linking.

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the "divide and conquer" algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions.

Childhood stunting and subsequent educational outcomes: a marginal structural model analysis from a South African longitudinal study.

OBJECTIVE: To examine the association between childhood stunting and grade completion (as educational outcome) in South Africa. DESIGN: Longitudinal study. Data were obtained using the National Income Dynamics Study over five waves (2008 to 2017). Children were tracked at wave 1 in 2008 until wave 5 in 2017 to determine their total years of schooling. We controlled for time-variant and time-varying confounding with a marginal structural model to estimate the associations between childhood stunting and subsequent grade completion. SETTING: Nationally representative study of South African households. PARTICIPANTS: A total of 2629 children aged 2 and 3 years in 2008. RESULTS: We observed a substantial decrease in the prevalence of stunting between wave 1 (28·2 %) and wave 4 (8·6 %). Our marginal structural model results suggest that childhood stunting was significantly associated with decreased odds (22 % less likely) of grade completion (OR = 0·78; 95 % CI: 0·40, 0·86; P = 0·015), while those who were only stunted during early childhood had a 29 % reduction in the odds of grade completion (OR = 0·71; 95 % CI: 0·51, 0·82; P = 0·020). CONCLUSION: These findings underscore the fact that stunting is a significant predictor of academic achievement, whose effects might be long-lasting.

Bias and Type I error Control in Correcting Treatment Effect for Treatment Switching Using Marginal Structural Models in Phase III Oncology Trials.

This research focuses on the bias and type I error control issues when the marginal structural models (MSMs) are applied to evaluate the causal survival benefits of active intervention versus control in randomized clinical trials (RCTs) with treatment switching after disease progression. When MSMs are applied in the RCT setting, the question of interest, model specifications, strategies for type I error control, bias reduction, etc. differ somewhat from those for observational studies. This manuscript discusses the approaches used to accommodate these differences. Through Monte Carlo simulations and a case study, our research demonstrates that, with sufficient attention paid to issues applicable to RCTs in particular, MSMs may perform better than the inverse probability of censoring weighting (IPCW) method in analyzing the survival endpoint in RCTs with treatment switching because more information is used by the MSM.

Virtual Quasi-2D Intermediates as Building Blocks for Plausible Structural Models of Amyloid Fibrils from Proteins with Complex Topologies: A Case Study of Insulin.

Conformational transitions of globular proteins into amyloid fibrils are complex multistage processes exceedingly challenging to simulate using molecular dynamics (MD). Slow monomer diffusion rates and rugged free energy landscapes disfavor swift self-assembly of orderly amyloid architectures within timescales accessible to all-atom MD. Here, we conduct a multiscale MD study of the amyloidogenic self-assembly of insulin: a small protein with a complex topology defined by two polypeptide chains interlinked by three disulfide bonds. To avoid kinetic traps, unconventional preplanarized insulin conformations are used as amyloid building blocks. These starting conformers generated through uniaxial compression of the native monomer in various spatial directions represent 6 distinct (out of 16 conceivable) two-dimensional (2D) topological classes varying in N-/C-terminal segments of insulin's A- and B-chains being placed inside or outside of the central loop constituted by the middle sections of both chains and Cys7A-Cys7B/Cys19B-Cys20A disulfide bonds. Simulations of the fibrillar self-assembly are initiated through a biased in-register alignment of two, three, or four layers of flat conformers belonging to a single topological class. The various starting topologies are conserved throughout the self-assembly process resulting in polymorphic amyloid fibrils varying in structural features such as helical twist, presence of cavities, and overall stability. Some of the protofilament structures obtained in this work are highly compatible with the earlier biophysical studies on insulin amyloid and high-resolution studies on insulin-derived amyloidogenic peptide models postulating the presence of steric zippers. Our approach provides in silico means to study amyloidogenic tendencies and viable amyloid architectures of larger disulfide-constrained proteins with complex topologies.

Physicochemical Heuristics for Identifying High Fidelity, Near-Native Structural Models of Peptide/MHC Complexes.

There is long-standing interest in accurately modeling the structural features of peptides bound and presented by class I MHC proteins. This interest has grown with the advent of rapid genome sequencing and the prospect of personalized, peptide-based cancer vaccines, as well as the development of molecular and cellular therapeutics based on T cell receptor recognition of peptide-MHC. However, while the speed and accessibility of peptide-MHC modeling has improved substantially over the years, improvements in accuracy have been modest. Accuracy is crucial in peptide-MHC modeling, as T cell receptors are highly sensitive to peptide conformation and capturing fine details is therefore necessary for useful models. Studying nonameric peptides presented by the common class I MHC protein HLA-A*02:01, here we addressed a key question common to modern modeling efforts: from a set of models (or decoys) generated through conformational sampling, which is best? We found that the common strategy of decoy selection by lowest energy can lead to substantial errors in predicted structures. We therefore adopted a data-driven approach and trained functions capable of predicting near native decoys with exceptionally high accuracy. Although our implementation is limited to nonamer/HLA-A*02:01 complexes, our results serve as an important proof of concept from which improvements can be made and, given the significance of HLA-A*02:01 and its preference for nonameric peptides, should have immediate utility in select immunotherapeutic and other efforts for which structural information would be advantageous.

Structural Model of the Human BTG2-PABPC1 Complex by Combining Mutagenesis, NMR Chemical Shift Perturbation Data and Molecular Docking.

Degradation of cytoplasmic mRNA in eukaryotes involves the shortening and removal of the mRNA poly(A) tail by poly(A)-selective ribonuclease (deadenylase) enzymes. In human cells, BTG2 can stimulate deadenylation of poly(A) bound by cytoplasmic poly(A)-binding protein PABPC1. This involves the concurrent binding by BTG2 of PABPC1 and the Caf1/CNOT7 nuclease subunit of the Ccr4-Not deadenylase complex. To understand in molecular detail how PABPC1 and BTG2 interact, we set out to identify amino acid residues of PABPC1 and BTG2 contributing to the interaction. To this end, we first used algorithms to predict PABPC1 interaction surfaces. Comparison of the predicted interaction surface with known residues involved in the binding to poly(A) resulted in the identification of a putative interaction surface for BTG2. Subsequently, we used pulldown assays to confirm the requirement of PABPC1 residues for the interaction with BTG2. Analysis of RNA-binding by PABPC1 variants indicated that PABPC1 residues required for interaction with BTG2 do not interfere with poly(A) binding. After further defining residues of BTG2 that are required for the interaction with PABPC1, we used information from published NMR chemical shift perturbation experiments to guide docking and generate a structural model of the BTG2-PABPC1 complex. A quaternary poly(A)-PABPC1-BTG2-Caf1/CNOT7 model showed that the 3' end of poly(A) RNA is directed towards the catalytic centre of Caf1/CNOT7, thereby providing a rationale for enhanced deadenylation by Caf1/CNOT7 in the presence of BTG2 and PABPC1.

A Structural Model of Quality of Life in Patients after Colorectal Cancer Surgery.

BACKGROUND: This study was conducted to determine a hypothetical model for the factors affecting the quality of life of postoperative colorectal cancer patients. METHODS: We collected data from 209 patients that was analyzed using SPSS 22.0 and AMOS 25. RESULTS: Predictive variables of the hypothesis model included an exogenous variable, social support, as well as endogenous variables self-efficacy, symptoms, health promotion behavior, and quality of life. Testing of the correction model showed that significant paths directly affecting quality of life of patients after colorectal cancer surgery included health promotion behavior, symptoms, and self-efficacy and also showed an explanation power of 58.7%. Social support was found to have a significant impact on the quality of life indirectly through self-efficiency. CONCLUSIONS: To improve the quality of life of colorectal cancer patients, it is necessary to develop a nursing intervention program that strengthens patients' health promotion behaviors to alleviate their symptoms and improve their social support and self-efficacy.

Structural Comparative Modeling of Multi-Domain F508del CFTR.

Cystic fibrosis (CF) is a rare genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs-which increases the risk of infection and eventually causes death. CFTR is an ATP-binding cassette (ABC) transporter family protein composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 (F508del), making F508del CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain F508del CFTR structure has been determined and few studies have modeled F508del using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a F508del model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and F508del CFTR structural models. These models demonstrate the destabilizing effects of F508del on NBD1 and the NBD1/TMD interface in both the inactive and active conformation of CFTR. Furthermore, we modeled F508del/R1070W and F508del bound to the CFTR corrector VX-809. Our models reveal the stabilizing effects of VX-809 on multi-domain models of F508del CFTR and pave the way for rational design of additional drugs that target F508del CFTR for treatment of CF.

The Viral Class II Membrane Fusion Machinery: Divergent Evolution from an Ancestral Heterodimer.

A key step during the entry of enveloped viruses into cells is the merger of viral and cell lipid bilayers. This process is driven by a dedicated membrane fusion protein (MFP) present at the virion surface, which undergoes a membrane-fusogenic conformational change triggered by interactions with the target cell. Viral MFPs have been extensively studied structurally, and are divided into three classes depending on their three-dimensional fold. Because MFPs of the same class are found in otherwise unrelated viruses, their intra-class structural homology indicates horizontal gene exchange. We focus this review on the class II fusion machinery, which is composed of two glycoproteins that associate as heterodimers. They fold together in the ER of infected cells such that the MFP adopts a conformation primed to react to specific clues only upon contact with a target cell, avoiding premature fusion in the producer cell. We show that, despite having diverged in their 3D fold during evolution much more than the actual MFP, the class II accompanying proteins (AP) also derive from a distant common ancestor, displaying an invariant core formed by a ß-ribbon and a C-terminal immunoglobulin-like domain playing different functional roles-heterotypic interactions with the MFP, and homotypic AP/AP contacts to form spikes, respectively. Our analysis shows that class II APs are easily identifiable with modern structural prediction algorithms, providing useful information in devising immunogens for vaccine design.

Structural model of human PORCN illuminates disease-associated variants and drug-binding sites.

Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Based on homology to mammalian MBOAT proteins, we developed and validated a structural model of human PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model enhances our mechanistic understanding of PORCN substrate recognition and catalysis, as well as the inhibition of its enzymatic activity, and can facilitate the development of improved inhibitors and the understanding of disease-relevant PORCN mutants. This article has an associated First Person interview with the joint first authors of the paper.

Maternal Complications and Prescription Opioid Exposure During Pregnancy: Using Marginal Structural Models.

INTRODUCTION: Prescription opioids are frequently used for pain management in pregnancy. Studies examining perinatal complications in mothers who received prescription opioids during pregnancy are still limited. OBJECTIVES: The aim of this study was to assess the association of prescription opioid use and maternal pregnancy and obstetric complications. METHODS: This retrospective cohort study with the Rhode Island (RI) Medicaid claims data linked to vital statistics throughout 2008-2015 included pregnant women aged 12-55 years with one or multiple live births. Women were excluded if they had cancer, opioid use disorder, or opioid dispensing prior to but not during pregnancy. Main outcomes included adverse pregnancy and obstetric complications. Marginal Structural Cox Models with time-varying exposure and covariates were applied to control for baseline and time-varying covariates. Analyses were conducted for outcomes that occurred 1 week after opioid exposure (primary) or within the same week as exposure (secondary). Sensitivity studies were conducted to assess the effects of different doses and individual opioids. RESULTS: Of 9823 eligible mothers, 545 (5.5%) filled one or more prescription opioid during pregnancy. Compared with those unexposed, no significant risk was observed in primary analyses, while in secondary analyses opioid-exposed mothers were associated with an increased risk of cesarean antepartum depression (HR 3.19; 95% CI 1.22-8.33), and cardiac events (HR 9.44; 95% CI 1.19-74.83). In sensitivity analyses, results are more prominent in high dose exposure and are consistent for individual opioids. CONCLUSIONS: Prescription opioid use during pregnancy is associated with an increased risk of maternal complications.

The cellular environment shapes the nuclear pore complex architecture.

Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE)1-3. These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel4-6. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that-compared with previous human NPC models obtained from purified NEs-the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture.

Structural Modeling of the Treponema pallidum Outer Membrane Protein Repertoire: a Road Map for Deconvolution of Syphilis Pathogenesis and Development of a Syphilis Vaccine.

Treponema pallidum, an obligate human pathogen, has an outer membrane (OM) whose physical properties, ultrastructure, and composition differ markedly from those of phylogenetically distant Gram-negative bacteria. We developed structural models for the outer membrane protein (OMP) repertoire (OMPeome) of T. pallidum Nichols using solved Gram-negative structures, computational tools, and small-angle X-ray scattering (SAXS) of selected recombinant periplasmic domains. The T. pallidum "OMPeome" harbors two "stand-alone" proteins (BamA and LptD) involved in OM biogenesis and four paralogous families involved in the influx/efflux of small molecules: 8-stranded ß-barrels, long-chain-fatty-acid transporters (FadLs), OM factors (OMFs) for efflux pumps, and T. pallidum repeat proteins (Tprs). BamA (TP0326), the central component of a ß-barrel assembly machine (BAM)/translocation and assembly module (TAM) hybrid, possesses a highly flexible polypeptide-transport-associated (POTRA) 1-5 arm predicted to interact with TamB (TP0325). TP0515, an LptD ortholog, contains a novel, unstructured C-terminal domain that models inside the ß-barrel. T. pallidum has four 8-stranded ß-barrels, each containing positively charged extracellular loops that could contribute to pathogenesis. Three of five FadL-like orthologs have a novel α-helical, presumptively periplasmic C-terminal extension. SAXS and structural modeling further supported the bipartite membrane topology and tridomain architecture of full-length members of the Tpr family. T. pallidum's two efflux pumps presumably extrude noxious small molecules via four coexpressed OMFs with variably charged tunnels. For BamA, LptD, and OMFs, we modeled the molecular machines that deliver their substrates into the OM or external milieu. The spirochete's extended families of OM transporters collectively confer a broad capacity for nutrient uptake. The models also furnish a structural road map for vaccine development. IMPORTANCE The unusual outer membrane (OM) of T. pallidum, the syphilis spirochete, is the ultrastructural basis for its well-recognized capacity for invasiveness, immune evasion, and persistence. In recent years, we have made considerable progress in identifying T. pallidum's repertoire of OMPs. Here, we developed three-dimensional (3D) models for the T. pallidum Nichols OMPeome using structural modeling, bioinformatics, and solution scattering. The OM contains three families of OMP transporters, an OMP family involved in the extrusion of noxious molecules, and two "stand-alone" proteins involved in OM biogenesis. This work represents a major advance toward elucidating host-pathogen interactions during syphilis; understanding how T. pallidum, an extreme auxotroph, obtains a wide array of biomolecules from its obligate human host; and developing a vaccine with global efficacy.