Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy.

Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 µm and report voltage correlations in pairs of neurons.

Clustering predicted structures at the scale of the known protein universe.

Proteins are key to all cellular processes and their structure is important in understanding their function and evolution. Sequence-based predictions of protein structures have increased in accuracy1, and over 214 million predicted structures are available in the AlphaFold database2. However, studying protein structures at this scale requires highly efficient methods. Here, we developed a structural-alignment-based clustering algorithm-Foldseek cluster-that can cluster hundreds of millions of structures. Using this method, we have clustered all of the structures in the AlphaFold database, identifying 2.30 million non-singleton structural clusters, of which 31% lack annotations representing probable previously undescribed structures. Clusters without annotation tend to have few representatives covering only 4% of all proteins in the AlphaFold database. Evolutionary analysis suggests that most clusters are ancient in origin but 4% seem to be species specific, representing lower-quality predictions or examples of de novo gene birth. We also show how structural comparisons can be used to predict domain families and their relationships, identifying examples of remote structural similarity. On the basis of these analyses, we identify several examples of human immune-related proteins with putative remote homology in prokaryotic species, illustrating the value of this resource for studying protein function and evolution across the tree of life.

Microglial Function Is Distinct in Different Anatomical Locations during Retinal Homeostasis and Degeneration.

Microglia from different nervous system regions are molecularly and anatomically distinct, but whether they also have different functions is unknown. We combined lineage tracing, single-cell transcriptomics, and electrophysiology of the mouse retina and showed that adult retinal microglia shared a common developmental lineage and were long-lived but resided in two distinct niches. Microglia in these niches differed in their interleukin-34 dependency and functional contribution to visual-information processing. During certain retinal-degeneration models, microglia from both pools relocated to the subretinal space, an inducible disease-associated niche that was poorly accessible to monocyte-derived cells. This microglial transition involved transcriptional reprogramming of microglia, characterized by reduced expression of homeostatic checkpoint genes and upregulation of injury-responsive genes. This transition was associated with protection of the retinal pigmented epithelium from damage caused by disease. Together, our data demonstrate that microglial function varies by retinal niche, thereby shedding light on the significance of microglia heterogeneity.

Structural architecture of the human NALCN channelosome.

Depolarizing sodium (Na+) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity1-8. NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood3,7,9-12. NALCN, UNC79 and UNC80 are essential in rodents2,9,13, and mutations in human NALCN and UNC80 cause severe developmental and neurological disease14,15. Here we determined the structure of the NALCN channelosome, an approximately 1-MDa complex, as fundamental aspects about the composition, assembly and gating of this channelosome remain obscure. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly, which docks intracellularly onto the NALCN-FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single-channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6 gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79-UNC80 subcomplex and the NALCN DI-DII and DII-DIII linkers were identified, leading to a model of channelosome gating. Our results provide a structural blueprint to understand the physiology of the NALCN channelosome and a template for drug discovery to modulate the resting membrane potential.

Correlative operando microscopy of oxygen evolution electrocatalysts.

Transition metal (oxy)hydroxides are promising electrocatalysts for the oxygen evolution reaction1-3. The properties of these materials evolve dynamically and heterogeneously4 with applied voltage through ion insertion redox reactions, converting materials that are inactive under open circuit conditions into active electrocatalysts during operation5. The catalytic state is thus inherently far from equilibrium, which complicates its direct observation. Here, using a suite of correlative operando scanning probe and X-ray microscopy techniques, we establish a link between the oxygen evolution activity and the local operational chemical, physical and electronic nanoscale structure of single-crystalline ß-Co(OH)2 platelet particles. At pre-catalytic voltages, the particles swell to form an α-CoO2H1.5·0.5H2O-like structure-produced through hydroxide intercalation-in which the oxidation state of cobalt is +2.5. Upon increasing the voltage to drive oxygen evolution, interlayer water and protons de-intercalate to form contracted ß-CoOOH particles that contain Co3+ species. Although these transformations manifest heterogeneously through the bulk of the particles, the electrochemical current is primarily restricted to their edge facets. The observed Tafel behaviour is correlated with the local concentration of Co3+ at these reactive edge sites, demonstrating the link between bulk ion-insertion and surface catalytic activity.

Structure of the human sodium leak channel NALCN.

Persistently depolarizing sodium (Na+) leak currents enhance electrical excitability1,2. The ion channel responsible for the major background Na+ conductance in neurons is the Na+ leak channel, non-selective (NALCN)3,4. NALCN-mediated currents regulate neuronal excitability linked to respiration, locomotion and circadian rhythm4-10. NALCN activity is under tight regulation11-14 and mutations in NALCN cause severe neurological disorders and early death15,16. NALCN is an orphan channel in humans, and fundamental aspects of channel assembly, gating, ion selectivity and pharmacology remain obscure. Here we investigate this essential leak channel and determined the structure of NALCN in complex with a distinct auxiliary subunit, family with sequence similarity 155 member A (FAM155A). FAM155A forms an extracellular dome that shields the ion-selectivity filter from neurotoxin attack. The pharmacology of NALCN is further delineated by a walled-off central cavity with occluded lateral pore fenestrations. Unusual voltage-sensor domains with asymmetric linkages to the pore suggest mechanisms by which NALCN activity is modulated. We found a tightly closed pore gate in NALCN where the majority of missense patient mutations cause gain-of-function phenotypes that cluster around the S6 gate and distinctive π-bulges. Our findings provide a framework to further study the physiology of NALCN and a foundation for discovery of treatments for NALCN channelopathies and other electrical disorders.

Structure of the essential inner membrane lipopolysaccharide-PbgA complex.

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.

UFCG: database of universal fungal core genes and pipeline for genome-wide phylogenetic analysis of fungi.

In phylogenomics the evolutionary relationship of organisms is studied by their genomic information. A common approach to phylogenomics is to extract related genes from each organism, build a multiple sequence alignment and then reconstruct evolution relations through a phylogenetic tree. Often a set of highly conserved genes occurring in single-copy, called core genes, are used for this analysis, as they allow efficient automation within a taxonomic clade. Here we introduce the Universal Fungal Core Genes (UFCG) database and pipeline for genome-wide phylogenetic analysis of fungi. The UFCG database consists of 61 curated fungal marker genes, including a novel set of 41 computationally derived core genes and 20 canonical genes derived from literature, as well as marker gene sequences extracted from publicly available fungal genomes. Furthermore, we provide an easy-to-use, fully automated and open-source pipeline for marker gene extraction, training and phylogenetic tree reconstruction. The UFCG pipeline can identify marker genes from genomic, proteomic and transcriptomic data, while producing phylogenies consistent with those previously reported, and is publicly available together with the UFCG database at https://ufcg.steineggerlab.com.

Practical guidelines for the use of scanning electrochemical cell microscopy (SECCM).

Scanning electrochemical cell microscopy (SECCM) has emerged as a transformative technology for electrochemical materials characterisation and the study of single entities, garnering global adoption by numerous research groups. While details on the instrumentation and operational principles of SECCM are readily available, the growing need for practical guidelines, troubleshooting strategies, and a systematic overview of applications and trends has become increasingly evident. This tutorial review addresses this gap by offering a comprehensive guide to the practical application of SECCM. The review begins with a discussion of recent developments and trends in the application of SECCM, before providing systematic approaches to (and the associated troubleshooting associated with) instrumental set up, probe fabrication, substrate preparation and the deployment of environmental (e.g., atmosphere and humidity) control. Serving as an invaluable resource, this tutorial review aims to equip researchers and practitioners entering the field with a comprehensive guide to essential considerations for conducting successful SECCM experiments.

The potential influence of genome-wide adaptive divergence on conservation translocation outcome in an isolated greater sage-grouse population.

Conservation translocations are an important conservation tool commonly employed to augment declining or reestablish extirpated populations. One goal of augmentation is to increase genetic diversity and reduce the risk of inbreeding depression (i.e., genetic rescue). However, introducing individuals from significantly diverged populations risks disrupting coadapted traits and reducing local fitness (i.e., outbreeding depression). Genetic data are increasingly more accessible for wildlife species and can provide unique insight regarding the presence and retention of introduced genetic variation from augmentation as an indicator of effectiveness and adaptive similarity as an indicator of source and recipient population suitability. We used 2 genetic data sets to evaluate augmentation of isolated populations of greater sage-grouse (Centrocercus urophasianus) in the northwestern region of the species range (Washington, USA) and to retrospectively evaluate adaptive divergence among source and recipient populations. We developed 2 statistical models for microsatellite data to evaluate augmentation outcomes. We used one model to predict genetic diversity after augmentation and compared these predictions with observations of genetic change. We used the second model to quantify the amount of observed reproduction attributed to transplants (proof of population integration). We also characterized genome-wide adaptive divergence among source and recipient populations. Observed genetic diversity (HO = 0.65) was higher in the recipient population than predicted had no augmentation occurred (HO = 0.58) but less than what was predicted by our model (HO = 0.75). The amount of shared genetic variation between the 2 geographically isolated resident populations increased, which is evidence of periodic gene flow previously assumed to be rare. Among candidate adaptive genes associated with elevated fixation index (FST) (143 genes) or local environmental variables (97 and 157 genes for each genotype-environment association method, respectively), we found clusters of genes with related functions that may influence the ability of transplants to use local resources and navigate unfamiliar environments and their reproductive potential, all possible reasons for low genetic retention from augmentation.

Influencia potencial de la divergencia adaptativa a nivel genoma sobre el resultado de la reubicación para conservación en una población aislada de urogallo mayor Resumen Las reubicaciones para conservación son una herramienta importante que se usa con frecuencia para aumentar las poblaciones en declinación o reestablecer las poblaciones erradicadas. Una de las metas de este aumento es incrementar la diversidad genética y reducir el riesgo de depresión endogámica (es decir, rescate genético). Sin embargo, la introducción de individuos de una población con divergencia significativa puede perturbar los rasgos coadaptados y reducir la aptitud local (es decir, depresión exogámica). La información genética es cada vez más accesible para las especies silvestres y puede proporcionar conocimiento único con respecto a la presencia y retención de la variación genética introducida a partir del aumento como un indicador de eficiencia y las similitudes adaptativas como un indicador de la idoneidad de la población de origen y la receptora. Usamos dos conjuntos de datos genéticos para evaluar el aumento de las poblaciones aisladas del urogallo mayor (Centrocercus urophasianus) en la región noroeste de la distribución de la especie (Washington, EUA) y para evaluar de forma retrospectiva la divergencia adaptativa entre la población de origen y la receptora. Desarrollamos dos modelos estadísticos para los datos microsatelitales para así evaluar los resultados del aumento. Usamos un modelo para predecir la diversidad genética después del aumento y comparamos estas predicciones con observaciones del cambio genético. Usamos el segundo modelo para cuantificar el aumento de la reproducción observada atribuida a las reubicaciones (evidencia de la integración poblacional). También caracterizamos la divergencia adaptativa a nivel genoma entre la población de origen y la población receptora. La diversidad genética observada (HO = 0.65) fue mayor de lo que se predijo en la población receptora de no haber ocurrido el aumento (HO = 0.58) pero menor de lo que se predijo en nuestro modelo (HO = 0.75). El aumento de la variación genética compartida entre las dos poblaciones residentes geográficamente aisladas incrementó, lo cual es evidencia de un flujo génico periódico que antes se supuso casi no ocurría. Entre los genes adaptativos candidatos asociados a una FST elevada (143 genes) o a variables ambientales locales (97 y 157 genes para cada método de asociación entre el ambiente y el genotipo, respectivamente) encontramos grupos de genes con funciones relacionadas que pueden influir sobre la habilidad de cada reubicación para usar recursos locales y navegar ambientes desconocidos y su potencial reproductivo, todas posibles razones para la baja retención genética en el aumento.

Advanced Spatiotemporal Voltammetric Techniques for Kinetic Analysis and Active Site Determination in the Electrochemical Reduction of CO2.

ConspectusElectrochemical reduction of the greenhouse gas CO2 offers prospects for the sustainable generation of fuels and industrially useful chemicals when powered by renewable electricity. However, this electrochemical process requires the use of highly stable, selective, and active catalysts. The development of such catalysts should be based on a detailed kinetic and mechanistic understanding of the electrochemical CO2 reduction reaction (eCO2RR), ideally through the resolution of active catalytic sites in both time (i.e., temporally) and space (i.e., spatially). In this Account, we highlight two advanced spatiotemporal voltammetric techniques for electrocatalytic studies and describe the considerable insights they provide on the eCO2RR. First, Fourier transformed large-amplitude alternating current voltammetry (FT ac voltammetry), as applied by the Monash Electrochemistry Group, enables the resolution of rapid underlying electron-transfer processes in complex reactions, free from competing processes, such as the background double-layer charging current, slow catalytic reactions, and solvent/electrolyte electrolysis, which often mask conventional voltammetric measurements of the eCO2RR. Crucially, FT ac voltammetry allows details of the catalytically active sites or the rate-determining step to be revealed under catalytic turnover conditions. This is well illustrated in investigations of the eCO2RR catalyzed by Bi where formate is the main product. Second, developments in scanning electrochemical cell microscopy (SECCM) by the Warwick Electrochemistry and Interfaces Group provide powerful methods for obtaining high-resolution activity maps and potentiodynamic movies of the heterogeneous surface of a catalyst. For example, by coupling SECCM data with colocated microscopy from electron backscatter diffraction (EBSD) or atomic force microscopy, it is possible to develop compelling correlations of (precatalyst) structure-activity at the nanoscale level. This correlative electrochemical multimicroscopy strategy allows the catalytically more active region of a catalyst, such as the edge plane of two-dimensional materials and the grain boundaries between facets in a polycrystalline metal, to be highlighted. The attributes of SECCM-EBSD are well-illustrated by detailed studies of the eCO2RR on polycrystalline gold, where carbon monoxide is the main product. Comparing SECCM maps and movies with EBSD images of the same region reveals unambiguously that the eCO2RR is enhanced at surface-terminating dislocations, which accumulate at grain boundaries and slip bands. Both FT ac voltammetry and SECCM techniques greatly enhance our understanding of the eCO2RR, significantly boosting the electrochemical toolbox and the information available for the development and testing of theoretical models and rational catalyst design. In the future, it may be possible to further enhance insights provided by both techniques through their integration with in situ and in operando spectroscopy and microscopy methods.

A genetic warning system for a hierarchically structured wildlife monitoring framework.

Genetic variation is a well-known indicator of population fitness yet is not typically included in monitoring programs for sensitive species. Additionally, most programs monitor populations at one scale, which can lead to potential mismatches with ecological processes critical to species' conservation. Recently developed methods generating hierarchically nested population units (i.e., clusters of varying scales) for greater sage-grouse (Centrocercus urophasianus) have identified population trend declines across spatiotemporal scales to help managers target areas for conservation. The same clusters used as a proxy for spatial scale can alert managers to local units (i.e., neighborhood-scale) with low genetic diversity, further facilitating identification of management targets. We developed a genetic warning system utilizing previously developed hierarchical population units to identify management-relevant areas with low genetic diversity within the greater sage-grouse range. Within this warning system we characterized conservation concern thresholds based on values of genetic diversity and developed a statistical model for microsatellite data to robustly estimate these values for hierarchically nested populations. We found that 41 of 224 neighborhood-scale clusters had low genetic diversity, 23 of which were coupled with documented local population trend decline. We also found evidence of cross-scale low genetic diversity in the small and isolated Washington population, unlikely to be reversed through typical local management actions alone. The combination of low genetic diversity and a declining population suggests relatively high conservation concern. Our findings could further facilitate conservation action prioritization in combination with population trend assessments and (or) local information, and act as a base-line of genetic diversity for future comparison. Importantly, the approach we used is broadly applicable across taxa.

Phosphine and Selenoether peri-Substituted Acenaphthenes and Their Transition-Metal Complexes: Structural and NMR Investigations.

A series of peri-substituted acenaphthene-based phosphine selenoether bidentate ligands Acenap(iPr2P)(SeAr) (L1-L4, Acenap = acenaphthene-5,6-diyl, Ar = Ph, mesityl, 2,4,6-trisopropylphenyl and supermesityl) were prepared. The rigid acenaphthene framework induces a forced overlap of the phosphine and selenoether lone pairs, resulting in a large magnitude of through-space 4JPSe coupling, ranging from 452 to 545 Hz. These rigid ligands L1-L4 were used to prepare a series of selected late d-block metals, mercury, and borane complexes, which were characterized, including by multinuclear NMR and single-crystal X-ray diffraction. The Lewis acidic motifs (BH3, Mo(CO)4, Ag+, PdCl2, PtCl2, and HgCl2) bridge the two donor atoms (P and Se) in all but one case in the solid-state structures. Where the bridging motif contained NMR-active nuclei (11B, 107Ag, 109Ag, 195Pt, and 199Hg), JPM and JSeM couplings are observed directly, in addition to the altered JPSe in the respective NMR spectra. The solution NMR data are correlated with single-crystal diffraction data, and in the case of mercury(II) complexes, they are also correlated with the solid-state NMR data and coupling deformation density calculations. The latter indicate that the through-space interaction dominates in free L1, while in the L1HgCl2 complex, the main coupling pathway is via the metal atom and not through the carbon framework of the acenaphthene ring system.

GPR40 deficiency worsens metabolic syndrome-associated periodontitis in mice.

BACKGROUND AND OBJECTIVE: G protein-coupled receptor 40 (GPR40) is a receptor for medium- and long-chain free fatty acids (FFAs). GPR40 activation improves type 2 diabetes mellitus (T2DM), metabolic syndrome (MetS), and the complications of T2DM and MetS. Periodontitis, a common oral inflammatory disease initiated by periodontal pathogens, is another complication of T2DM and MetS. Since FFAs play a key role in the pathogenesis of MetS which exacerbates periodontal inflammation and GPR40 is a FFA receptor with anti-inflammatory properties, it is important to define the role of GPR40 in MetS-associated periodontitis. MATERIALS AND METHODS: We induced MetS and periodontitis by high-fat diet and periodontal injection of lipopolysaccharide (LPS), respectively, in wild-type and GPR40-deficient mice and determined alveolar bone loss and periodontal inflammation using micro-computed tomography, histology, and osteoclast staining. We also performed in vitro study to determine the role of GPR40 in the expression of proinflammatory genes. RESULTS: The primary outcome of the study is that GPR40 deficiency increased alveolar bone loss and enhanced osteoclastogenesis in control mice and the mice with both MetS and periodontitis. GPR40 deficiency also augmented periodontal inflammation in control mice and the mice with both MetS and periodontitis. Furthermore, GPR40 deficiency led to increased plasma lipids and insulin resistance in control mice but had no effect on the metabolic parameters in mice with MetS alone. For mice with both MetS and periodontitis, GPR40 deficiency increased insulin resistance. Finally, in vitro studies with macrophages showed that deficiency or inhibition of GPR40 upregulated proinflammatory genes while activation of GPR40 downregulated proinflammatory gene expression stimulated synergistically by LPS and palmitic acid. CONCLUSION: GPR40 deficiency worsens alveolar bone loss and periodontal inflammation in mice with both periodontitis and MetS, suggesting that GPR40 plays a favorable role in MetS-associated periodontitis. Furthermore, GPR40 deficiency or inhibition in macrophages further upregulated proinflammatory and pro-osteoclastogenic genes induced by LPS and palmitic acid, suggesting that GPR40 has anti-inflammatory and anti-osteoclastogenic properties.

Adipocyte Na, K-ATPase Signaling Attenuates Experimental Uremic Cardiomyopathy.

Oxidative stress has been shown to cause an alteration of intracellular signaling in adipocytes that may lead to various comorbidities of obesity and cardiovascular complications. Evidence suggests that dysregulation of Na, K-ATPase signaling can contribute to systemic inflammation and redox signaling that leads to various metabolic disturbances. Hence the present study aims to explore the specific role of adipocyte Na, K-ATPase signaling in the amelioration of pathophysiological alterations of experimental uremic cardiomyopathy. Experimental uremic cardiomyopathy was induced by partial nephrectomy (PNx), and adipocyte-specific expression of NaKtide, a peptide that inhibits Na, K-ATPase signaling, was achieved using a lentivirus construct with NaKtide expression driven by an adiponectin promoter. Cardiomyopathy and anemia induced in partial nephrectomy mice were accompanied by an altered molecular phenotype of adipocytes, increased systemic inflammatory cytokines and oxidant stress within 4 weeks. These changes were significantly worsened by the addition of a Western diet (enriched in fat and fructose contents) but were prevented with specific expression of NaKtide in adipocytes. The skeletal muscle-specific expression of NaKtide did not ameliorate the disease phenotype. Adipocyte dysfunction and uremic cardiomyopathy developed in PNx mice, both were significantly ameliorated by the adipocyte-specific expression of NaKtide. These findings suggest that oxidative milieu in the adipocyte has a pivotal role in the development and progression of uremic cardiomyopathy in mice subjected to partial nephrectomy. If confirmed in humans, this may be a lead for future research to explore novel therapeutic targets in chronic renal failure.

Stereodivergent Hydroxylation of Berkeleylactones by Penicillium turbatum.

Penicillium turbatum has previously been reported to produce A26771B, a 16-membered macrocyclic polyketide with activity against Gram-positive bacteria, mycoplasma, and fungi, as well as the structurally related compounds berkeleylactone E and berkeleylactones I-O. In this work, large-scale cultivation of P. turbatum NRRL 5630 on rice yielded seven new berkeleylactone analogues, berkeleylactone E methyl ester, 14-epi-berkeleylactone F, berkeleylactones P-R, 12-epi-berkeleylactone Q, and 13-epi-berkeleylactone R, and six previously reported analogues, A26771B and berkeleylactones E-G and J-K. The structures of the berkeleylactones were elucidated by detailed analysis of spectroscopic data, molecular modeling, and comparison with literature values. Interestingly, six of the berkeleylactone analogues were isolated as pairs of hydroxy epimers, highlighting how Nature can exploit stereodivergence in biosynthetic pathways to increase chemical diversity. The genome of P. turbatum was sequenced, and a putative gene cluster (bekl) responsible for the biosynthesis of the berkeleylactones was identified. The new berkeleylactone analogues exhibited no significant biological activity against a panel of bacteria, fungi, the parasite Giardia duodenalis, or NS-1 murine myeloma cells, suggesting a hitherto undiscovered biological role.

Dicer-TRBP complex formation ensures accurate mammalian microRNA biogenesis.

RNA-mediated gene silencing in human cells requires the accurate generation of ∼22 nt microRNAs (miRNAs) from double-stranded RNA substrates by the endonuclease Dicer. Although the phylogenetically conserved RNA-binding proteins TRBP and PACT are known to contribute to this process, their mode of Dicer binding and their genome-wide effects on miRNA processing have not been determined. We solved the crystal structure of the human Dicer-TRBP interface, revealing the structural basis of the interaction. Interface residues conserved between TRBP and PACT show that the proteins bind to Dicer in a similar manner and by mutual exclusion. Based on the structure, a catalytically active Dicer that cannot bind TRBP or PACT was designed and introduced into Dicer-deficient mammalian cells, revealing selective defects in guide strand selection. These results demonstrate the role of Dicer-associated RNA binding proteins in maintenance of gene silencing fidelity.

Victorin, the host-selective cyclic peptide toxin from the oat pathogen Cochliobolus victoriae, is ribosomally encoded.

The necrotrophic fungal pathogen Cochliobolus victoriae produces victorin, a host-selective toxin (HST) essential for pathogenicity to certain oat cultivars with resistance against crown rust. Victorin is a mixture of highly modified heterodetic cyclic hexapeptides, previously assumed to be synthesized by a nonribosomal peptide synthetase. Herein, we demonstrate that victorin is a member of the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Analysis of a newly generated long-read assembly of the C. victoriae genome revealed three copies of precursor peptide genes (vicA1-3) with variable numbers of "GLKLAF" core peptide repeats corresponding to the victorin peptide backbone. vicA1-3 are located in repeat-rich gene-sparse regions of the genome and are loosely clustered with putative victorin biosynthetic genes, which are supported by the discovery of compact gene clusters harboring corresponding homologs in two distantly related plant-associated Sordariomycete fungi. Deletion of at least one copy of vicA resulted in strongly diminished victorin production. Deletion of a gene encoding a DUF3328 protein (VicYb) abolished the production altogether, supporting its predicted role in oxidative cyclization of the core peptide. In addition, we uncovered a copper amine oxidase (CAO) encoded by vicK, in which its deletion led to the accumulation of new glycine-containing victorin derivatives. The role of VicK in oxidative deamination of the N-terminal glycyl moiety of the hexapeptides to the active glyoxylate forms was confirmed in vitro. This study finally unraveled the genetic and molecular bases for biosynthesis of one of the first discovered HSTs and expanded our understanding of underexplored fungal RiPPs.

Impact of Home Monitoring Program and Early Gastrostomy Tube on Interstage Outcomes following Stage 1 Norwood Palliation.

Nutritional management and home monitoring programs (HMPs) may be beneficial for improving interstage morbidity and mortality following stage I Norwood palliation (S1P) for hypoplastic left heart syndrome (HLHS). We recognized an increasing trend towards early feeding gastrostomy tube (GT) placement prior to discharge in our institution, and we aimed to investigate the effect of HMPs and GTs on interstage mortality and growth parameters. Single-institutional review at a tertiary referral center between 2008 and 2018. Individual patient charts were reviewed in the electronic medical record. Those listed for transplant or hybrid procedures were excluded. Baseline demographics, operative details, and interstage outcomes were analyzed in GT and non-GT patients (nGT). Our HMP was instituted in 2009, and patients were analyzed by era: I (early, 2008-2012), II (intermediate, 2013-2016), and III (recent, 2017-2018). 79 patients were included in the study: 29 nGTs and 50 GTs. GTs had higher number of preoperative risk factors more S1P complications, longer ventilation times, longer lengths of stay, and shorter times to readmission. There were no differences in interstage mortality or overall mortality between groups. There was one readmission for a GT-related issue with no periprocedural complications in the group. Weight gain doubled after GT placement in the interstage period while waiting periods for placement decreased across Eras. HMPs and early GTs, especially for patients with high-risk features, provide a dependable mode of nutritional support to optimize somatic growth following S1P.

The Norms and Corporatization of Medicine Influence Physician Moral Distress in the United States.

PhenomenonMoral distress, which occurs when someone's moral integrity is seriously compromised because they feel unable to act in accordance with their core values and obligations, is an increasingly important concern for physicians. Due in part to limited understanding of the root causes of moral distress, little is known about which approaches are most beneficial for mitigating physicians' distress. Our objective was to describe system-level factors in United States (U.S.) healthcare that contribute to moral distress among pediatric hospitalist attendings and pediatric residents.ApproachIn this qualitative study, we conducted one-on-one semi-structured interviews with pediatric hospitalist attendings and pediatric residents from 4 university-affiliated, freestanding children's hospitals in the U.S. between August 2019 and February 2020. Data were coded with an iteratively developed codebook, categorized into themes, and then synthesized.FindingsWe interviewed 22 hospitalists and 18 residents. Participants described in detail how the culture of medicine created a context that cultivated moral distress. Norms of medical education and the practice of medicine created conflicts between residents' strong sense of professional responsibility to serve the best interests of their patients and the expectations of a hierarchical system of decision-making. The corporatization of the U.S. healthcare system created administrative and financial pressures that conflicted with the moral responsibility felt by both residents and hospitalists to provide the care that their patients and families needed.InsightsThese findings highlight the critical role of systemic sources of moral distress. These findings suggest that system-level interventions must supplement existing interventions that target individual health care providers. Preventing and managing moral distress will require a broad approach that addresses systemic drivers, such as the corporatization of medicine, which are entrenched in the culture of medicine.