The rockefeller university

Multidrug resistance-associated protein 2 (MRP2) is an ATP-powered exporter important for maintaining liver homeostasis and a potential contributor to chemotherapeutic resistance. Using cryogenic electron microscopy (cryo-EM), we determine the structures of human MRP2 in three conformational states: an autoinhibited state, a substrate-bound pre-translocation state, and an ATP-bound post-translocation state. In the autoinhibited state, the cytosolic regulatory (R) domain plugs into the transmembrane substrate-binding site and extends into the cytosol to form a composite ATP-binding site at the surface of nucleotide-binding domain 2. Substrate displaces the R domain, permitting conformational changes necessary for transport. These observations suggest that the R domain functions as a selectivity gauge, where only at sufficiently high concentrations can the substrate effectively initiate transport. Comparative structural analyzes of MRP2 bound to various substrates, as determined in this study and others, reveal how MRP2 recognizes a diverse array of compounds, supporting its role in multidrug resistance.

Fascin cross-links actin filaments (F-actin) into bundles that support tubular membrane protrusions including filopodia and stereocilia. Fascin dysregulation drives aberrant cell migration during metastasis, and fascin inhibitors are under development as cancer therapeutics. Here, we use cryo-EM, cryo-electron tomography coupled with custom denoising and computational modeling to probe human fascin-1's F-actin cross-linking mechanisms across spatial scales. Our fascin cross-bridge structure reveals an asymmetric F-actin binding conformation that is allosterically blocked by the inhibitor G2. Reconstructions of seven-filament hexagonal bundle elements, variability analysis and simulations show how structural plasticity enables fascin to bridge varied interfilament orientations, accommodating mismatches between F-actin's helical symmetry and bundle hexagonal packing. Tomography of many-filament bundles and modeling uncover geometric rules underlying emergent fascin binding patterns, as well as the accumulation of unfavorable cross-links that limit bundle size. Collectively, this work shows how fascin harnesses fine-tuned nanoscale structural dynamics to build and regulate micron-scale F-actin bundles.

Approximately 3.4 million patients worldwide are diagnosed each year with cancers that have pathogenic mutations in one of three RAS proto-oncogenes (KRAS, NRAS and HRAS)1,2. These mutations impair the GTPase activity of RAS, leading to activation of downstream signalling and proliferation3, 4, 5-6. Long-standing efforts to restore the hydrolase activity of RAS mutants have been unsuccessful, extinguishing any consideration towards a viable therapeutic strategy7. Here we show that tri-complex inhibitors-that is, molecular glues with the ability to recruit cyclophilin A (CYPA) to the active state of RAS-have a dual mechanism of action: not only do they prevent activated RAS from binding to its effectors, but they also stimulate GTP hydrolysis. Drug-bound CYPA complexes modulate residues in the switch II motif of RAS to coordinate the nucleophilic attack on the gamma-phosphate of GTP in a mutation-specific manner. RAS mutants that were most sensitive to stimulation of GTPase activity were more susceptible to treatment than mutants in which the hydrolysis could not be enhanced, suggesting that pharmacological stimulation of hydrolysis potentiates the therapeutic effects of tri-complex inhibitors for specific RAS mutants. This study lays the foundation for developing a class of therapeutics that inhibit cancer growth by stimulating mutant GTPase activity.

Advanced genomic technologies such as whole exome or whole genome sequencing have improved diagnoses and disease outcomes for individuals with genetic diseases. Yet, variants of unknown significance (VUS) require rigorous validation to establish disease causality or modification, or to exclude them from further analysis. Here, we describe a young individual of Polynesian ancestry who in the first 13 mo of life presented with SARS-CoV-2 pneumonia, severe enterovirus meningitis and adenovirus gastroenteritis, and severe adverse reaction to MMR vaccination. Genomic analysis identified a previously reported pathogenic homozygous variant in IFNAR1 (c.1156G > T, p.Glu386* LOF), which is common in Western Polynesia. Moreover, a new and putatively deleterious canonical splice site variant in DOCK8 was also found in homozygosity (c.3234 + 2T > C). This DOCK8 variant is common in Polynesians and other under-represented ancestries in large genomic databases. Despite in silico bioinformatic predictions, extensive in vitro and ex vivo analysis revealed the DOCK8 variant likely be neutral. Thus, our study reports a novel case of IFNAR1 deficiency, but also highlights the importance of functional validation of VUS, including those predicted to be deleterious, and the pressing need to expand our knowledge of the genomic architecture and landscape of under-represented populations and ancestries.

Although the etiological relevance of the detection of microsporidia in human stool samples remains uncertain, the immunological status of patients has been posited as an important determinant of potential clinical impact of these parasites. To further assess the interplay between the epidemiology of microsporidia and immunological markers, we conducted a study utilizing real-time PCR targeting Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis, combined in a single fluorescence channel. The study involved a cohort of 595 clinically and immunologically well-characterized Ghanaian HIV patients, alongside 82 HIV-negative control individuals from Ghana. While microsporidial DNA was absent in HIV-negative controls, among people living with HIV, its prevalence was inversely correlated with CD4+ lymphocyte counts: 6.0% in those with >500 cells/mu L, 9.5% in those with 200-499 cells/mu L, 13.8% in those with 50-199 cells/mu L, and 27.5% in those with <50 cells/L, respectively. Correspondingly, microsporidia were more frequently detected in HIV patients who were not receiving antiretroviral therapy. There were no associations with clinical symptoms including gastroenteritis with the exception of a non-significant trend towards weight loss. HLA-DR+CD38+ on CD4+ T lymphocytes, a marker of immune activation, as well as Ki67, a marker of cell proliferation, were increased on CD4+ T lymphocytes in HIV patients with microsporidia, suggesting an immune response may be triggered. In conclusion, our assessment indicates a higher prevalence of microsporidia in the stool of Ghanaian HIV patients, which varies with their immunological status. However, given the lack of clear associations with clinical symptoms, the detection of microsporidia in the stool of HIV patients needs to be cautiously interpreted in clinical settings.

A measurement is performed of Higgs bosons produced with high transverse momentum (p(T)) via vector boson or gluon fusion in proton-proton collisions. The result is based on a data set with a center-of-mass energy of 13 TeV collected in 2016-2018 with the CMS detector at the LHC and corresponds to an integrated luminosity of 138 fb(-1). The decay of a high-p(T) Higgs boson to a boosted bottom quark-antiquark pair is selected using large-radius jets and employing jet substructure and heavy-flavor taggers based on machine learning techniques. Independent regions targeting the vector boson and gluon fusion mechanisms are defined based on the topology of two quark-initiated jets with large pseudorapidity separation. The signal strengths for both processes are extracted simultaneously by performing a maximum likelihood fit to data in the large-radius jet mass distribution. The observed signal strengths relative to the standard model expectation are 4.9(-1.6)(+1.9) and 1.6(-1.5)(+1.7) for the vector boson and gluon fusion mechanisms, respectively. A differential cross section measurement is also reported in the simplified template cross section framework.

In view of changing climatic conditions and disappearing natural resources such as fertile soil and water, exploring alternatives to today's industrial monocrop farming becomes essential. One promising farming practice is intercropping (IC), in which two or more crop species are grown together. Many experiments have shown that, under certain circumstances, IC can decrease soil erosion and fertilizer use, improve soil health and land management, while preserving crop production levels. However, there have been no quantitative approaches to predict, design, and control appropriate IC implementation for given particular environmental and farming conditions, and to assess its robustness. Here, we develop such an approach, based on methods and concepts developed in data science and systems biology. Our dataset groups the results of 2258 IC experiments, involving 274 pairs of 69 different plants. The data include 4 soil characteristics and 5 environmental and farming conditions, together with 8 traits for each of the two intercropped plants. We performed a dimensional reduction of the resulting 25-dimensional variable space and showed that, from a few quantities, one can predict IC yield relative to sole cultivation with good accuracy. For given environmental conditions, our computational approach can help to choose a companion plant and appropriate farming practices. It also indicates how to estimate the robustness of IC to external perturbations. This approach, together with its results, can be viewed as an initial step toward "systems agriculture," which would ultimately develop systems of multiple plant grown together in appropriately designed and controlled settings.

Hidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disease characterized by keratinized epithelial tunnels that grow deeply into the dermis. Here, we examined the immune microenvironment within human HS lesions. Multi-omics profiling and multiplexed imaging identified tertiary lymphoid structures (TLSs) near HS tunnels. These TLSs were enriched with proliferative T cells, including follicular helper (Tfh), regulatory (Treg), and pathogenic T cells ( IL17A + and IFNG +), alongside extensive clonal expansion of plasma cells producing antibodies reactive to keratinocytes. HS fibroblasts express CXCL13 or CCL19 in response to immune cytokines. Using a microfluidic system to mimic TLS on a chip, we found that HS fibroblasts critically orchestrated lymphocyte aggregation via tumor necrosis factor alpha (TNF-a)-CXCL13 and TNF-a-CCL19 feedback loops with B and T cells, respectively; early TNF-a blockade suppressed aggregate initiation. Our findings provide insights into TLS formation in the skin, suggest therapeutic avenues for HS, and reveal mechanisms that may apply to other autoimmune settings, including Crohn's disease.

Glioblastoma is incurable and in urgent need of improved therapeutics(1). Here we identify a small compound, gliocidin, that kills glioblastoma cells while sparing non-tumour replicative cells. Gliocidin activity targets a de novo purine synthesis vulnerability in glioblastoma through indirect inhibition of inosine monophosphate dehydrogenase 2 (IMPDH2). IMPDH2 blockade reduces intracellular guanine nucleotide levels, causing nucleotide imbalance, replication stress and tumour cell death(2). Gliocidin is a prodrug that is anabolized into its tumoricidal metabolite, gliocidin-adenine dinucleotide (GAD), by the enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) of the NAD(+) salvage pathway. The cryo-electron microscopy structure of GAD together with IMPDH2 demonstrates its entry, deformation and blockade of the NAD(+) pocket(3). In vivo, gliocidin penetrates the blood-brain barrier and extends the survival of mice with orthotopic glioblastoma. The DNA alkylating agent temozolomide induces Nmnat1 expression, causing synergistic tumour cell killing and additional survival benefit in orthotopic patient-derived xenograft models. This study brings gliocidin to light as a prodrug with the potential to improve the survival of patients with glioblastoma.

Early time-restricted eating (eTRE) is a dietary strategy that restricts caloric intake to the first 6-8 h of the day and can effect metabolic benefits independent of weight loss. However, the extent of these benefits is unknown. We conducted a randomized crossover feeding study to investigate the weight-independent effects of eTRE on glycemic variation, multiple time-in-range metrics, and levels of inflammatory markers. Ten adults with prediabetes were randomized to eTRE (8-h feeding window, 80% of calories consumed before 14:00 h) or usual feeding (50% of calories consumed after 16:00 h) for 1 week followed by crossover to the other schedule. Using continuous glucose monitoring, we showed that eTRE decreased glycemic variation (mean amplitude of glycemic excursion) and time in hyperglycemia greater than 140 mg/dL without affecting inflammatory markers (erythrocyte sedimentation rate and C-reactive protein). These data implicate eTRE as a candidate dietary intervention for the weight-independent management of dysglycemia in high-risk individuals.