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The nuclear pore complex forms a highly crowded selective barrier with intrinsically disordered regions at the nuclear membrane to coordinate nucleocytoplasmic molecular communications. Although oxidative stress is known to alter the barrier function, the molecular mechanism underlying this adaptive control of the nuclear pore complex remains unknown. Here we uncover a systematic control of the crowding barrier within the nuclear pore in response to various redox environments. Direct measurements of the crowding states using a crowding-sensitive FRET (Forster resonance energy transfer) probe reveal specific roles of the nuclear pore subunits that adjust the degree of crowding in response to different redox conditions, by adaptively forming or disrupting redox-sensitive disulfide bonds. Relationships between crowding control and the barrier function of the nuclear pore are investigated by single-molecular fluorescence measurements of nucleartransport. Based on these findings, we propose a proximal control model of molecular crowding in vivo that is dynamically regulated at the molecular level.

Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.

The widely abused prescription opioid oxycodone is a mu-opioid receptor (MOP-r) agonist and addiction to such opioids is a relapsing disorder. The human MOP-r gene (OPRM1) has an important functional single nucleotide polymorphism (SNP), A118G, which affects risk of severe opioid use disorders. A112G (G/G) knock-in mice are models of human A118G carriers. We examined oxycodone self-administration (SA) in male and female G/G versus wild type (A/A) mice in SA sessions and in relapse-like behavior. Adult male and female G/G and A/A mice self-administered oxycodone (0.25 mg/kg/infusion, FR1) for 10 consecutive days. Following 10-day home cage drug free withdrawal, the mice were re-exposed to oxycodone SA for a further 10 days. MOP-r receptor mRNA in various brain regions were examined immediately after the last re-exposure session. We found that G/G mice had greater oxycodone SA than A/A mice in the initial and in re-exposure sessions. Mice of both genotypes had greater oxycodone intake during the re-exposure period than during the initial exposure. We also detected differences in MOP-r gene expression due to genotype, sex and oxycodone SA history in the dorsal striatum, hippocampus, and prefrontal cortex. These studies may improve our understanding of MOP-r-agonist self-exposure and relapse in human carriers of the A118G SNP.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 was recovered from infections in humans exposed to animals, raising public health concerns. However, contact with food producing chain as a means of transmission of LA-MRSA to humans remains poorly understood. We aimed to assess if pork production chain is a source of MRSA ST398 for human colonization and infection. MRSA from live pigs, meat, the environment, and slaughterhouse workers were analyzed by Pulsed-Field Gel Electrophoresis (PFGE), spa, MLST typing, SNPs and for antibiotic resistance and virulence gene profiles. We compared core and accessory genomes of MRSA ST398 isolated from slaughterhouse and hospital. We detected MRSA ST398 (t011, t108, t1451) along the entire pork production chain (live pigs: 60%; equipment: 38%; meat: 23%) and in workers (40%). All MRSA ST398 were multidrug resistant, and the majority carried genes encoding biocide resistance and enterotoxins. We found 23 cross-transmission events between live pigs, meat, and workers (6-55 SNPs). MRSA ST398 from infection and slaughterhouse environment belonged to the same clonal type (ST398, t011, SCCmec V), but differed in 321-378 SNPs. Pork production chain can be a source of MRSA ST398 for colonization of human slaughterhouse workers, which can represent a risk of subsequent meat contamination and human infection.

Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (Mtb). The emergence of Rif-resistant (RifR) Mtb presents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of RifR RNAPs, including the most prevalent clinical RifR RNAP substitution found in Mtb infected patients (S456>L of the beta subunit). Here, we present structural and biochemical data demonstrating that Sor inhibits the wild-type Mtb RNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the RifR S456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suit able starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.

Eating disorders are life-interrupting psychiatric conditions with high morbidity and mortality, yet the basic mechanisms underlying these conditions are understudied compared with other psychiatric disorders. In this opinion, we suggest that recent knowledge gleaned from genomic and neuroimaging investigations of eating disorders in humans presents a rich opportunity to sharpen animal models of eating disorders and to identify neural mechanisms that contribute to the risk and maintenance of these conditions. Our article reflects the state of the science, with a primary focus on anorexia nervosa (AN) and binge-eating behavior, and encourages further study of all conditions categorized under feeding and eating disorders.

Eight structures of human neutralizing antibodies that target the SARS-CoV-2 spike receptor-binding domain are reported and classified into four categories, suggesting combinations for clinical use. The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein(1-5) show promise therapeutically and are being evaluated clinically(6-8). Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies(5) in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to 'up' RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and 'down' RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs(9). Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2.

Stress is a common, if often unpredictable life event. It can be defined from an evolutionary perspective as a force an organism perceives it must adapt to. Thus stress is a useful tool to study adaptation and the adaptive capacity of organisms. The deep genome, long neglected as a pile of "junk" has emerged as a source of regulatory DNA and RNA as well as a potential stockpile of adaptive capacity at the organismal and species levels. Recent work on the regulation of transposable elements (TEs), the principle constituents of the deep genome, by stress has shown that these elements are responsive to host stress and other environmental cues. Further, we have shown that some are likely directly regulated by the glucocorticoid receptor (GR), one of the two major vertebrate stress steroid receptors in a fashion that appears adaptive. On the basis of this and other emerging evidence I argue that the deep genome may represent an adaptive toolkit for organisms to respond to their environments at both individual and evolutionary scales. This argues that genomes may be adapted for what Waddington called "trait adaptability" rather than being purely passive objects of natural selection and single nucleotide level mutation.