The rockefeller university

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.

Conventional cancer cell lines and animal models have been mainstays of cancer research. More recently, human pluripotent stem cells (hPSCs) and hPSC-derived organoid technologies, together with genome engineering approaches, have provided a complementary platform to model cancer progression. Here, we review the application of these technologies in cancer modeling with respect to the cell-of-origin, cancer propagation, and metastasis. We further discuss the benefits and challenges accompanying the use of hPSC models for cancer research and discuss their broad applicability in drug discovery, biomarker identification, decoding molecular mechanisms, and the deconstruction of clonal and intra-tumoral heterogeneity. In summary, hPSC-derived organoids provide powerful models to recapitulate the pathogenic states in cancer and to perform drug discovery.

Telomere shortening is a presumed tumor suppressor pathway that imposes a proliferative barrier (the Hayflick limit) during tumorigenesis. This model predicts that excessively long somatic telomeres predispose to cancer. Here, we describe cancer-prone families with two unique TINF2 mutations that truncate TIN2, a shelterin subunit that controls telomere length. Patient lymphocyte telomeres were unusually long. We show that the truncated TIN2 proteins do not localize to telomeres, suggesting that the mutations create loss-of-function alleles. Heterozygous knock-in of the mutations or deletion of one copy of TINF2 resulted in excessive telomere elongation in clonal lines, indicating that TINF2 is haploinsufficient for telomere length control. In contrast, telomere protection and genome stability were maintained in all heterozygous clones. The data establish that the TINF2 truncations predispose to a tumor syndrome. We conclude that TINF2 acts as a haploinsufficient tumor suppressor that limits telomere length to ensure a timely Hayflick limit.

Background Embryonic exposure to ethanol (EtOH) produces marked disturbances in neuronal development and alcohol-related behaviors, with low-moderate EtOH doses stimulating neurogenesis without producing apoptosis and high doses having major cytotoxic effects while causing gross morphological abnormalities. With the pro-inflammatory chemokine system, Cxcl12, and its main receptor Cxcr4, known to promote processes of neurogenesis, we examined here this neuroimmune system in the embryonic hypothalamus to test directly if it mediates the stimulatory effects low-moderate EtOH doses have on neuronal development. Methods We used the zebrafish (Danio rerio) model, which develops externally and allows one to investigate the developing brain in vivo with precise control of dose and timing of EtOH delivery in the absence of maternal influence. Zebrafish were exposed to low-moderate EtOH doses (0.1, 0.25, 0.5% v/v), specifically during a period of peak hypothalamic development from 22 to 24 hours postfertilization, and in some tests were pretreated from 2 to 22 hpf with the Cxcr4 receptor antagonist, AMD3100. Measurements in the hypothalamus at 26 hpf were taken of cxcl12a and cxcr4b transcription, signaling, and neuronal density using qRT-PCR, RNAscope, and live imaging of transgenic zebrafish. Results Embryonic EtOH exposure, particularly at the 0.5% dose, significantly increased levels of cxcl12a and cxcr4b mRNA in whole embryos, number of cxcl12a and cxcr4b transcripts in developing hypothalamus, and internalization of Cxcr4b receptors in hypothalamic cells. Embryonic EtOH also caused an increase in the number of hypothalamic neurons and coexpression of cxcl12a and cxcr4b transcripts within these neurons. Each of these stimulatory effects of EtOH in the embryo was blocked by pretreatment with the Cxcr4 antagonist AMD3100. Conclusions These results provide clear evidence that EtOH's stimulatory effects at low-moderate doses on the number of hypothalamic neurons early in development are mediated, in part, by increased transcription and intracellular activation of this chemokine system, likely due to autocrine signaling of Cxcl12a at its Cxcr4b receptor within the neurons.

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.

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.

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RII beta, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RII beta:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RII beta C-2(2) but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RII beta holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RII beta holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

The Aedesaegypti mosquito shows extreme sexual dimorphism in feeding. Only females are attracted to and obtain a blood-meal from humans, which they use to stimulate egg production. The fruitless gene is sex-specifically spliced and encodes a BTB zinc-finger transcription factor proposed to be a master regulator of male courtship and mating behavior across insects. We generated fruitless mutant mosquitoes and showed that males failed to mate, confirming the ancestral function of this gene in male sexual behavior. Remarkably, fruitless males also gain strong attraction to a live human host, a behavior that wild-type males never display, suggesting that male mosquitoes possess the central or peripheral neural circuits required to host-seek and that removing fruitless reveals this latent behavior in males. Our results highlight an unexpected repurposing of a master regulator of male-specific sexual behavior to control one module of female-specific blood-feeding behavior in a deadly vector of infectious diseases.

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.