Metabolic diversity in commensal protists regulates intestinal immunity and trans-kingdom competition.

The microbiota influences intestinal health and physiology, yet the contributions of commensal protists to the gut environment have been largely overlooked. Here, we discover human- and rodent-associated parabasalid protists, revealing substantial diversity and prevalence in nonindustrialized human populations. Genomic and metabolomic analyses of murine parabasalids from the genus Tritrichomonas revealed species-level differences in excretion of the metabolite succinate, which results in distinct small intestinal immune responses. Metabolic differences between Tritrichomonas species also determine their ecological niche within the microbiota. By manipulating dietary fibers and developing in vitro protist culture, we show that different Tritrichomonas species prefer dietary polysaccharides or mucus glycans. These polysaccharide preferences drive trans-kingdom competition with specific commensal bacteria, which affects intestinal immunity in a diet-dependent manner. Our findings reveal unappreciated diversity in commensal parabasalids, elucidate differences in commensal protist metabolism, and suggest how dietary interventions could regulate their impact on gut health.

Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak.

An outbreak of over 1,000 COVID-19 cases in Provincetown, Massachusetts (MA), in July 2021-the first large outbreak mostly in vaccinated individuals in the US-prompted a comprehensive public health response, motivating changes to national masking recommendations and raising questions about infection and transmission among vaccinated individuals. To address these questions, we combined viral genomic and epidemiological data from 467 individuals, including 40% of outbreak-associated cases. The Delta variant accounted for 99% of cases in this dataset; it was introduced from at least 40 sources, but 83% of cases derived from a single source, likely through transmission across multiple settings over a short time rather than a single event. Genomic and epidemiological data supported multiple transmissions of Delta from and between fully vaccinated individuals. However, despite its magnitude, the outbreak had limited onward impact in MA and the US overall, likely due to high vaccination rates and a robust public health response.

Genome-wide functional screen of 3'UTR variants uncovers causal variants for human disease and evolution.

3' untranslated region (3'UTR) variants are strongly associated with human traits and diseases, yet few have been causally identified. We developed the massively parallel reporter assay for 3'UTRs (MPRAu) to sensitively assay 12,173 3'UTR variants. We applied MPRAu to six human cell lines, focusing on genetic variants associated with genome-wide association studies (GWAS) and human evolutionary adaptation. MPRAu expands our understanding of 3'UTR function, suggesting that simple sequences predominately explain 3'UTR regulatory activity. We adapt MPRAu to uncover diverse molecular mechanisms at base pair resolution, including an adenylate-uridylate (AU)-rich element of LEPR linked to potential metabolic evolutionary adaptations in East Asians. We nominate hundreds of 3'UTR causal variants with genetically fine-mapped phenotype associations. Using endogenous allelic replacements, we characterize one variant that disrupts a miRNA site regulating the viral defense gene TRIM14 and one that alters PILRB abundance, nominating a causal variant underlying transcriptional changes in age-related macular degeneration.

A Metabolic Pathway for Activation of Dietary Glucosinolates by a Human Gut Symbiont.

Consumption of glucosinolates, pro-drug-like metabolites abundant in Brassica vegetables, has been associated with decreased risk of certain cancers. Gut microbiota have the ability to metabolize glucosinolates, generating chemopreventive isothiocyanates. Here, we identify a genetic and biochemical basis for activation of glucosinolates to isothiocyanates by Bacteroides thetaiotaomicron, a prominent gut commensal species. Using a genome-wide transposon insertion screen, we identified an operon required for glucosinolate metabolism in B. thetaiotaomicron. Expression of BT2159-BT2156 in a non-metabolizing relative, Bacteroides fragilis, resulted in gain of glucosinolate metabolism. We show that isothiocyanate formation requires the action of BT2158 and either BT2156 or BT2157 in vitro. Monocolonization of mice with mutant BtΔ2157 showed reduced isothiocyanate production in the gastrointestinal tract. These data provide insight into the mechanisms by which a common gut bacterium processes an important dietary nutrient.

Direct Identification of Hundreds of Expression-Modulating Variants using a Multiplexed Reporter Assay.

Although studies have identified hundreds of loci associated with human traits and diseases, pinpointing causal alleles remains difficult, particularly for non-coding variants. To address this challenge, we adapted the massively parallel reporter assay (MPRA) to identify variants that directly modulate gene expression. We applied it to 32,373 variants from 3,642 cis-expression quantitative trait loci and control regions. Detection by MPRA was strongly correlated with measures of regulatory function. We demonstrate MPRA's capabilities for pinpointing causal alleles, using it to identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. We investigated one in detail, a risk allele for ankylosing spondylitis, and provide direct evidence of a non-coding variant that alters expression of the prostaglandin EP4 receptor. These results create a resource of concrete leads and illustrate the promise of this approach for comprehensively interrogating how non-coding polymorphism shapes human biology.

Translating energy balance research from the bench to the clinic to the community: Parallel animal-human studies in cancer.

Advances in energy balance and cancer research to date have largely occurred in siloed work in rodents or patients. However, substantial benefit can be derived from parallel studies in which animal models inform the design of clinical and population studies or in which clinical observations become the basis for animal studies. The conference Translating Energy Balance from Bench to Communities: Application of Parallel Animal-Human Studies in Cancer, held in July 2021, convened investigators from basic, translational/clinical, and population science research to share knowledge, examples of successful parallel studies, and strong research to move the field of energy balance and cancer toward practice changes. This review summarizes key topics discussed to advance research on the role of energy balance, including physical activity, body composition, and dietary intake, on cancer development, cancer outcomes, and healthy survivorship.

Machine-guided design of cell-type-targeting cis-regulatory elements.

Cis-regulatory elements (CREs) control gene expression, orchestrating tissue identity, developmental timing and stimulus responses, which collectively define the thousands of unique cell types in the body1-3. While there is great potential for strategically incorporating CREs in therapeutic or biotechnology applications that require tissue specificity, there is no guarantee that an optimal CRE for these intended purposes has arisen naturally. Here we present a platform to engineer and validate synthetic CREs capable of driving gene expression with programmed cell-type specificity. We take advantage of innovations in deep neural network modelling of CRE activity across three cell types, efficient in silico optimization and massively parallel reporter assays to design and empirically test thousands of CREs4-8. Through large-scale in vitro validation, we show that synthetic sequences are more effective at driving cell-type-specific expression in three cell lines compared with natural sequences from the human genome and achieve specificity in analogous tissues when tested in vivo. Synthetic sequences exhibit distinct motif vocabulary associated with activity in the on-target cell type and a simultaneous reduction in the activity of off-target cells. Together, we provide a generalizable framework to prospectively engineer CREs from massively parallel reporter assay models and demonstrate the required literacy to write fit-for-purpose regulatory code.

The evolution of lineage-specific regulatory activities in the human embryonic limb.

The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find that 13% of promoters and 11% of enhancers have gained activity on the human lineage since the human-rhesus divergence. These gains largely arose by modification of ancestral regulatory activities in the limb or potential co-option from other tissues and are likely to have heterogeneous genetic causes. Most enhancers that exhibit gain of activity in humans originated in mammals. Gains at promoters and enhancers in the human limb are associated with increased gene expression, suggesting they include molecular drivers of human morphological evolution.

Coexpression networks implicate human midfetal deep cortical projection neurons in the pathogenesis of autism.

Autism spectrum disorder (ASD) is a complex developmental syndrome of unknown etiology. Recent studies employing exome- and genome-wide sequencing have identified nine high-confidence ASD (hcASD) genes. Working from the hypothesis that ASD-associated mutations in these biologically pleiotropic genes will disrupt intersecting developmental processes to contribute to a common phenotype, we have attempted to identify time periods, brain regions, and cell types in which these genes converge. We have constructed coexpression networks based on the hcASD "seed" genes, leveraging a rich expression data set encompassing multiple human brain regions across human development and into adulthood. By assessing enrichment of an independent set of probable ASD (pASD) genes, derived from the same sequencing studies, we demonstrate a key point of convergence in midfetal layer 5/6 cortical projection neurons. This approach informs when, where, and in what cell types mutations in these specific genes may be productively studied to clarify ASD pathophysiology.

Multicenter integrated analysis of noncoding CRISPRi screens.

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.

A metabolomics pipeline for the mechanistic interrogation of the gut microbiome.

Gut microorganisms modulate host phenotypes and are associated with numerous health effects in humans, ranging from host responses to cancer immunotherapy to metabolic disease and obesity. However, difficulty in accurate and high-throughput functional analysis of human gut microorganisms has hindered efforts to define mechanistic connections between individual microbial strains and host phenotypes. One key way in which the gut microbiome influences host physiology is through the production of small molecules1-3, yet progress in elucidating this chemical interplay has been hindered by limited tools calibrated to detect the products of anaerobic biochemistry in the gut. Here we construct a microbiome-focused, integrated mass-spectrometry pipeline to accelerate the identification of microbiota-dependent metabolites in diverse sample types. We report the metabolic profiles of 178 gut microorganism strains using our library of 833 metabolites. Using this metabolomics resource, we establish deviations in the relationships between phylogeny and metabolism, use machine learning to discover a previously undescribed type of metabolism in Bacteroides, and reveal candidate biochemical pathways using comparative genomics. Microbiota-dependent metabolites can be detected in diverse biological fluids from gnotobiotic and conventionally colonized mice and traced back to the corresponding metabolomic profiles of cultured bacteria. Collectively, our microbiome-focused metabolomics pipeline and interactive metabolomics profile explorer are a powerful tool for characterizing microorganisms and interactions between microorganisms and their host.

Neuroprosthetic baroreflex controls haemodynamics after spinal cord injury.

Spinal cord injury (SCI) induces haemodynamic instability that threatens survival1-3, impairs neurological recovery4,5, increases the risk of cardiovascular disease6,7, and reduces quality of life8,9. Haemodynamic instability in this context is due to the interruption of supraspinal efferent commands to sympathetic circuits located in the spinal cord10, which prevents the natural baroreflex from controlling these circuits to adjust peripheral vascular resistance. Epidural electrical stimulation (EES) of the spinal cord has been shown to compensate for interrupted supraspinal commands to motor circuits below the injury11, and restored walking after paralysis12. Here, we leveraged these concepts to develop EES protocols that restored haemodynamic stability after SCI. We established a preclinical model that enabled us to dissect the topology and dynamics of the sympathetic circuits, and to understand how EES can engage these circuits. We incorporated these spatial and temporal features into stimulation protocols to conceive a clinical-grade biomimetic haemodynamic regulator that operates in a closed loop. This 'neuroprosthetic baroreflex' controlled haemodynamics for extended periods of time in rodents, non-human primates and humans, after both acute and chronic SCI. We will now conduct clinical trials to turn the neuroprosthetic baroreflex into a commonly available therapy for people with SCI.

Dynamic Processing of Displacement Loops during Recombinational DNA Repair.

Displacement loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the activities controlling their metabolism. Nascent D-loops are detected within 2 h of DSB formation and extended in a delayed fashion in a genetic system designed to preclude downstream repair steps. The majority of nascent D-loops are disrupted by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a layer of quality control of HR relying on nascent D-loop dynamics.

Principal role of fungi in soil carbon stabilization during early pedogenesis in the high Arctic.

Climate warming is causing widespread deglaciation and pioneer soil formation over glacial deposits. Melting glaciers expose rocky terrain and glacial till sediment that is relatively low in biomass, oligotrophic, and depleted in nutrients. Following initial colonization by microorganisms, glacial till sediments accumulate organic carbon and nutrients over time. However, the mechanisms driving soil nutrient stabilization during early pedogenesis after glacial retreat remain unclear. Here, we traced amino acid uptake by microorganisms in recently deglaciated high-Arctic soils and show that fungi play a critical role in the initial stabilization of the assimilated carbon. Pioneer basidiomycete yeasts were among the predominant taxa responsible for carbon assimilation, which were associated with overall high amino acid use efficiency and reduced respiration. In intermediate- and late-stage soils, lichenized ascomycete fungi were prevalent, but bacteria increasingly dominated amino acid assimilation, with substantially decreased fungal:bacterial amino acid assimilation ratios and increased respiration. Together, these findings demonstrate that fungi are important drivers of pedogenesis in high-Arctic ecosystems that are currently subject to widespread deglaciation from global warming.

Reduced levels of MRE11 cause disease phenotypes distinct from ataxia telangiectasia-like disorder.

The MRE11/RAD50/NBS1 (MRN) complex plays critical roles in cellular responses to DNA double-strand breaks. MRN is involved in end binding and processing, and it also induces cell cycle checkpoints by activating the ataxia-telangiectasia mutated (ATM) protein kinase. Hypomorphic pathogenic variants in the MRE11, RAD50, or NBS1 genes cause autosomal recessive genome instability syndromes featuring variable degrees of dwarfism, neurological defects, anemia, and cancer predisposition. Disease-associated MRN alleles include missense and nonsense variants, and many cause reduced protein levels of the entire MRN complex. However, the dramatic variability in the disease manifestation of MRN pathogenic variants is not understood. We sought to determine if low protein levels are a significant contributor to disease sequelae and therefore generated a transgenic murine model expressing MRE11 at low levels. These mice display dramatic phenotypes including small body size, severe anemia, and impaired DNA repair. We demonstrate that, distinct from ataxia telangiectasia-like disorder caused by MRE11 pathogenic missense or nonsense variants, mice and cultured cells expressing low MRE11 levels do not display the anticipated defects in ATM activation. Our findings indicate that ATM signaling can be supported by very low levels of the MRN complex and imply that defective ATM activation results from perturbation of MRN function caused by specific hypomorphic disease mutations. These distinct phenotypic outcomes underline the importance of understanding the impact of specific pathogenic MRE11 variants, which may help direct appropriate early surveillance for patients with these complicated disorders in a clinical setting.

Pitavastatin to Prevent Cardiovascular Disease in HIV Infection.

BACKGROUND: The risk of cardiovascular disease is increased among persons with human immunodeficiency virus (HIV) infection, so data regarding primary prevention strategies in this population are needed. METHODS: In this phase 3 trial, we randomly assigned 7769 participants with HIV infection with a low-to-moderate risk of cardiovascular disease who were receiving antiretroviral therapy to receive daily pitavastatin calcium (at a dose of 4 mg) or placebo. The primary outcome was the occurrence of a major adverse cardiovascular event, which was defined as a composite of cardiovascular death, myocardial infarction, hospitalization for unstable angina, stroke, transient ischemic attack, peripheral arterial ischemia, revascularization, or death from an undetermined cause. RESULTS: The median age of the participants was 50 years (interquartile range, 45 to 55); the median CD4 count was 621 cells per cubic millimeter (interquartile range, 448 to 827), and the HIV RNA value was below quantification in 5250 of 5997 participants (87.5%) with available data. The trial was stopped early for efficacy after a median follow-up of 5.1 years (interquartile range, 4.3 to 5.9). The incidence of a major adverse cardiovascular event was 4.81 per 1000 person-years in the pitavastatin group and 7.32 per 1000 person-years in the placebo group (hazard ratio, 0.65; 95% confidence interval [CI], 0.48 to 0.90; P = 0.002). Muscle-related symptoms occurred in 91 participants (2.3%) in the pitavastatin group and in 53 (1.4%) in the placebo group; diabetes mellitus occurred in 206 participants (5.3%) and in 155 (4.0%), respectively. CONCLUSIONS: Participants with HIV infection who received pitavastatin had a lower risk of a major adverse cardiovascular event than those who received placebo over a median follow-up of 5.1 years. (Funded by the National Institutes of Health and others; REPRIEVE ClinicalTrials.gov number, NCT02344290.).

Adaptive Evolution of Nearctic Deepwater Fish Vision: Implications for Assessing Functional Variation for Conservation.

Intraspecific functional variation is critical for adaptation to rapidly changing environments. For visual opsins, functional variation can be characterized in vitro and often reflects a species' ecological niche but is rarely considered in the context of intraspecific variation or the impact of recent environmental changes on species of cultural or commercial significance. Investigation of adaptation in postglacial lakes can provide key insight into how rapid environmental changes impact functional evolution. Here, we report evidence for molecular adaptation in vision in 2 lineages of Nearctic fishes that are deep lake specialists: ciscoes and deepwater sculpin. We found depth-related variation in the dim-light visual pigment rhodopsin that evolved convergently in these 2 lineages. In vitro characterization of spectral sensitivity of the convergent deepwater rhodopsin alleles revealed blue-shifts compared with other more widely distributed alleles. These blue-shifted rhodopsin alleles were only observed in deep clear postglacial lakes with underwater visual environments enriched in blue light. This provides evidence of remarkably rapid and convergent visual adaptation and intraspecific functional variation in rhodopsin. Intraspecific functional variation has important implications for conservation, and these fishes are of conservation concern and great cultural, commercial, and nutritional importance to Indigenous communities. We collaborated with the Saugeen Ojibway Nation to develop and test a metabarcoding approach that we show is efficient and accurate in recovering the ecological distribution of functionally relevant variation in rhodopsin. Our approach bridges experimental analyses of protein function and genetics-based tools used in large-scale surveys to better understand the ecological extent of adaptive functional variation.

The endosomal system of primary human vascular endothelial cells and albumin-FcRn trafficking.

Human serum albumin (HSA) has a long circulatory half-life owing, in part, to interaction with the neonatal Fc receptor (FcRn or FCGRT) in acidic endosomes and recycling of internalised albumin. Vascular endothelial and innate immune cells are considered the most relevant cells for FcRn-mediated albumin homeostasis in vivo. However, little is known about endocytic trafficking of FcRn-albumin complexes in primary human endothelial cells. To investigate FcRn-albumin trafficking in physiologically relevant endothelial cells, we generated primary human vascular endothelial cell lines from blood endothelial precursors, known as blood outgrowth endothelial cells (BOECs). We mapped the endosomal system in BOECs and showed that BOECs efficiently internalise fluorescently labelled HSA predominantly by fluid-phase macropinocytosis. Pulse-chase studies revealed that intracellular HSA molecules co-localised with FcRn in acidic endosomal structures and that the wildtype HSA, but not the non-FcRn-binding HSAH464Q mutant, was excluded from late endosomes and/or lysosomes. Live imaging revealed that HSA is partitioned into FcRn-positive tubules derived from maturing macropinosomes, which are then transported towards the plasma membrane. These findings identify the FcRn-albumin trafficking pathway in primary vascular endothelial cells, relevant to albumin homeostasis.

A mechanistic model of in vitro plasma activation to evaluate therapeutic kallikrein-kinin system inhibitors.

BACKGROUND: The kallikrein-kinin system (KKS) is a complex biochemical pathway that plays a crucial role in regulating several physiological processes, including inflammation, coagulation, and blood pressure. Dysregulation of the KKS has been associated with several pathological conditions such as hereditary angioedema (HAE), hypertension, and stroke. Developing an accurate quantitative model of the KKS may provide a better understanding of its role in health and disease and facilitate the rapid and targeted development of effective therapies for KKS-related disorders. OBJECTIVES: Here, we present a novel, detailed mechanistic model of the plasma KKS, elucidating the processes of Factor XII (FXII) activation, the kallikrein feedback loop, cleavage of high molecular weight kininogen leading to bradykinin (BK) production, and the impact of inhibitors. METHODS: The model incorporates both surface and solution-phase reactions of all proteins in the KKS, describing how binding site concentration affects the rate of surface reactions. The model was calibrated and validated using a variety of published and in-house experimental datasets, which encompass a range of dextran sulphate (DXS) concentrations to initiate contact activation and various KKS inhibitors to block bradykinin production. RESULTS: Our mathematical model showed that a trace amount of activated FXII is required for subsequent FXII activation. The model also reveals a bell-shaped curve relationship between the activation of the KKS and the number of DXS surface binding sites. Simulations of BK generation in healthy and HAE plasma demonstrated the impact of C1 esterase inhibitor (C1inh) deficiency via increased peak BK levels and accelerated formation in HAE plasma. The efficacy of KKS inhibitors, such as CSL312, ecallantide, and C1inh, was also evaluated, with CSL312 showing the most potent inhibition of BK generation. CONCLUSIONS: The present model represents a valuable framework for studying the intricate interactions within the plasma KKS and provides a better understanding of the mechanism of action of various KKS-targeted therapies.