Ever-Changing Landscapes: Transcriptional Enhancers in Development and Evolution.

A class of cis-regulatory elements, called enhancers, play a central role in orchestrating spatiotemporally precise gene-expression programs during development. Consequently, divergence in enhancer sequence and activity is thought to be an important mediator of inter- and intra-species phenotypic variation. Here, we give an overview of emerging principles of enhancer function, current models of enhancer architecture, genomic substrates from which enhancers emerge during evolution, and the influence of three-dimensional genome organization on long-range gene regulation. We discuss intricate relationships between distinct elements within complex regulatory landscapes and consider their potential impact on specificity and robustness of transcriptional regulation.

Multiple Origins of Virus Persistence during Natural Control of HIV Infection.

Targeted HIV cure strategies require definition of the mechanisms that maintain the virus. Here, we tracked HIV replication and the persistence of infected CD4 T cells in individuals with natural virologic control by sequencing viruses, T cell receptor genes, HIV integration sites, and cellular transcriptomes. Our results revealed three mechanisms of HIV persistence operating within distinct anatomic and functional compartments. In lymph node, we detected viruses with genetic and transcriptional attributes of active replication in both T follicular helper (TFH) cells and non-TFH memory cells. In blood, we detected inducible proviruses of archival origin among highly differentiated, clonally expanded cells. Linking the lymph node and blood was a small population of circulating cells harboring inducible proviruses of recent origin. Thus, HIV replication in lymphoid tissue, clonal expansion of infected cells, and recirculation of recently infected cells act together to maintain the virus in HIV controllers despite effective antiviral immunity.

A Functional Genomics Approach to Understand Variation in Cytokine Production in Humans.

As part of the Human Functional Genomics Project, which aims to understand the factors that determine the variability of immune responses, we investigated genetic variants affecting cytokine production in response to ex vivo stimulation in two independent cohorts of 500 and 200 healthy individuals. We demonstrate a strong impact of genetic heritability on cytokine production capacity after challenge with bacterial, fungal, viral, and non-microbial stimuli. In addition to 17 novel genome-wide significant cytokine QTLs (cQTLs), our study provides a comprehensive picture of the genetic variants that influence six different cytokines in whole blood, blood mononuclear cells, and macrophages. Important biological pathways that contain cytokine QTLs map to pattern recognition receptors (TLR1-6-10 cluster), cytokine and complement inhibitors, and the kallikrein system. The cytokine QTLs show enrichment for monocyte-specific enhancers, are more often located in regions under positive selection, and are significantly enriched among SNPs associated with infections and immune-mediated diseases. PAPERCLIP.

Host and Environmental Factors Influencing Individual Human Cytokine Responses.

Differences in susceptibility to immune-mediated diseases are determined by variability in immune responses. In three studies within the Human Functional Genomics Project, we assessed the effect of environmental and non-genetic host factors of the genetic make-up of the host and of the intestinal microbiome on the cytokine responses in humans. We analyzed the association of these factors with circulating mediators and with six cytokines after stimulation with 19 bacterial, fungal, viral, and non-microbial metabolic stimuli in 534 healthy subjects. In this first study, we show a strong impact of non-genetic host factors (e.g., age and gender) on cytokine production and circulating mediators. Additionally, annual seasonality is found to be an important environmental factor influencing cytokine production. Alpha-1-antitrypsin concentrations partially mediate the seasonality of cytokine responses, whereas the effect of vitamin D levels is limited. The complete dataset has been made publicly available as a comprehensive resource for future studies. PAPERCLIP.

Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity.

Gut microbial dysbioses are linked to aberrant immune responses, which are often accompanied by abnormal production of inflammatory cytokines. As part of the Human Functional Genomics Project (HFGP), we investigate how differences in composition and function of gut microbial communities may contribute to inter-individual variation in cytokine responses to microbial stimulations in healthy humans. We observe microbiome-cytokine interaction patterns that are stimulus specific, cytokine specific, and cytokine and stimulus specific. Validation of two predicted host-microbial interactions reveal that TNFα and IFNγ production are associated with specific microbial metabolic pathways: palmitoleic acid metabolism and tryptophan degradation to tryptophol. Besides providing a resource of predicted microbially derived mediators that influence immune phenotypes in response to common microorganisms, these data can help to define principles for understanding disease susceptibility. The three HFGP studies presented in this issue lay the groundwork for further studies aimed at understanding the interplay between microbial, genetic, and environmental factors in the regulation of the immune response in humans. PAPERCLIP.

Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises. PAPERCLIP.

Temporal dynamics of the multi-omic response to endurance exercise training.

Regular exercise promotes whole-body health and prevents disease, but the underlying molecular mechanisms are incompletely understood1-3. Here, the Molecular Transducers of Physical Activity Consortium4 profiled the temporal transcriptome, proteome, metabolome, lipidome, phosphoproteome, acetylproteome, ubiquitylproteome, epigenome and immunome in whole blood, plasma and 18 solid tissues in male and female Rattus norvegicus over eight weeks of endurance exercise training. The resulting data compendium encompasses 9,466 assays across 19 tissues, 25 molecular platforms and 4 training time points. Thousands of shared and tissue-specific molecular alterations were identified, with sex differences found in multiple tissues. Temporal multi-omic and multi-tissue analyses revealed expansive biological insights into the adaptive responses to endurance training, including widespread regulation of immune, metabolic, stress response and mitochondrial pathways. Many changes were relevant to human health, including non-alcoholic fatty liver disease, inflammatory bowel disease, cardiovascular health and tissue injury and recovery. The data and analyses presented in this study will serve as valuable resources for understanding and exploring the multi-tissue molecular effects of endurance training and are provided in a public repository ( https://motrpac-data.org/ ).

Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans.

Multiple sensory cues emanating from humans are thought to guide blood-feeding female mosquitoes to a host. To determine the relative contribution of carbon dioxide (CO2) detection to mosquito host-seeking behavior, we mutated the AaegGr3 gene, a subunit of the heteromeric CO2 receptor in Aedes aegypti mosquitoes. Gr3 mutants lack electrophysiological and behavioral responses to CO2. These mutants also fail to show CO2-evoked responses to heat and lactic acid, a human-derived attractant, suggesting that CO2 can gate responses to other sensory stimuli. Whereas attraction of Gr3 mutants to live humans in a large semi-field environment was only slightly impaired, responses to an animal host were greatly reduced in a spatial-scale-dependent manner. Synergistic integration of heat and odor cues likely drive host-seeking behavior in the absence of CO2 detection. We reveal a networked series of interactions by which multimodal integration of CO2, human odor, and heat orchestrates mosquito attraction to humans.

Temporal response of the human virome to immunosuppression and antiviral therapy.

There are few substantive methods to measure the health of the immune system, and the connection between immune strength and the viral component of the microbiome is poorly understood. Organ transplant recipients are treated with posttransplant therapies that combine immunosuppressive and antiviral drugs, offering a window into the effects of immune modulation on the virome. We used sequencing of cell-free DNA in plasma to investigate drug-virome interactions in a cohort of organ transplant recipients (656 samples, 96 patients) and find that antivirals and immunosuppressants strongly affect the structure of the virome in plasma. We observe marked virome compositional dynamics at the onset of the therapy and find that the total viral load increases with immunosuppression, whereas the bacterial component of the microbiome remains largely unaffected. The data provide insight into the relationship between the human virome, the state of the immune system, and the effects of pharmacological treatment and offer a potential application of the virome state to predict immunocompetence.

Virological assessment of hospitalized patients with COVID-2019.

Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.

Whole blood stimulation as a tool for studying the human immune system.

The human immune system is best accessible via tissues and organs not requiring major surgical intervention, such as blood. In many circumstances, circulating immune cells correlate with an individual's health state and give insight into physiological and pathophysiological processes. Stimulating whole blood ex vivo is a powerful tool to investigate immune responses. In the context of clinical research, the applications of whole blood stimulation include host immunity, disease characterization, diagnosis, treatment, and drug development. Here, we summarize different setups and readouts of whole blood assays and discuss applications for preclinical research and clinical practice. Finally, we propose combining whole blood stimulation with high-throughput technologies, such as single-cell RNA-sequencing, to comprehensively analyze the human immune system for the identification of biomarkers, therapeutic interventions as well as companion diagnostics.

A High-Dimensional Atlas of Human T Cell Diversity Reveals Tissue-Specific Trafficking and Cytokine Signatures.

Depending on the tissue microenvironment, T cells can differentiate into highly diverse subsets expressing unique trafficking receptors and cytokines. Studies of human lymphocytes have primarily focused on a limited number of parameters in blood, representing an incomplete view of the human immune system. Here, we have utilized mass cytometry to simultaneously analyze T cell trafficking and functional markers across eight different human tissues, including blood, lymphoid, and non-lymphoid tissues. These data have revealed that combinatorial expression of trafficking receptors and cytokines better defines tissue specificity. Notably, we identified numerous T helper cell subsets with overlapping cytokine expression, but only specific cytokine combinations are secreted regardless of tissue type. This indicates that T cell lineages defined in mouse models cannot be clearly distinguished in humans. Overall, our data uncover a plethora of tissue immune signatures and provide a systemic map of how T cell phenotypes are altered throughout the human body.

Single-cell analysis reveals T cell infiltration in old neurogenic niches.

The mammalian brain contains neurogenic niches that comprise neural stem cells and other cell types. Neurogenic niches become less functional with age, but how they change during ageing remains unclear. Here we perform single-cell RNA sequencing of young and old neurogenic niches in mice. The analysis of 14,685 single-cell transcriptomes reveals a decrease in activated neural stem cells, changes in endothelial cells and microglia, and an infiltration of T cells in old neurogenic niches. T cells in old brains are clonally expanded and are generally distinct from those in old blood, which suggests that they may experience specific antigens. T cells in old brains also express interferon-γ, and the subset of neural stem cells that has a high interferon response shows decreased proliferation in vivo. We find that T cells can inhibit the proliferation of neural stem cells in co-cultures and in vivo, in part by secreting interferon-γ. Our study reveals an interaction between T cells and neural stem cells in old brains, opening potential avenues through which to counteract age-related decline in brain function.

The microbiota regulate neuronal function and fear extinction learning.

Multicellular organisms have co-evolved with complex consortia of viruses, bacteria, fungi and parasites, collectively referred to as the microbiota1. In mammals, changes in the composition of the microbiota can influence many physiologic processes (including development, metabolism and immune cell function) and are associated with susceptibility to multiple diseases2. Alterations in the microbiota can also modulate host behaviours-such as social activity, stress, and anxiety-related responses-that are linked to diverse neuropsychiatric disorders3. However, the mechanisms by which the microbiota influence neuronal activity and host behaviour remain poorly defined. Here we show that manipulation of the microbiota in antibiotic-treated or germ-free adult mice results in significant deficits in fear extinction learning. Single-nucleus RNA sequencing of the medial prefrontal cortex of the brain revealed significant alterations in gene expression in excitatory neurons, glia and other cell types. Transcranial two-photon imaging showed that deficits in extinction learning after manipulation of the microbiota in adult mice were associated with defective learning-related remodelling of postsynaptic dendritic spines and reduced activity in cue-encoding neurons in the medial prefrontal cortex. In addition, selective re-establishment of the microbiota revealed a limited neonatal developmental window in which microbiota-derived signals can restore normal extinction learning in adulthood. Finally, unbiased metabolomic analysis identified four metabolites that were significantly downregulated in germ-free mice and have been reported to be related to neuropsychiatric disorders in humans and mouse models, suggesting that microbiota-derived compounds may directly affect brain function and behaviour. Together, these data indicate that fear extinction learning requires microbiota-derived signals both during early postnatal neurodevelopment and in adult mice, with implications for our understanding of how diet, infection, and lifestyle influence brain health and subsequent susceptibility to neuropsychiatric disorders.

Passage of exogeneous fine particles from the lung into the brain in humans and animals.

There are still significant knowledge gaps in understanding the intrusion and retention of exogeneous particles into the central nervous system (CNS). Here, we uncovered various exogeneous fine particles in human cerebrospinal fluids (CSFs) and identified the ambient environmental or occupational exposure sources of these particles, including commonly found particles (e.g., Fe- and Ca-containing ones) and other compositions that have not been reported previously (such as malayaite and anatase TiO2), by mapping their chemical and structural fingerprints. Furthermore, using mouse and in vitro models, we unveiled a possible translocation pathway of various inhaled fine particles from the lung to the brain through blood circulation (via dedicated biodistribution and mechanistic studies). Importantly, with the aid of isotope labeling, we obtained the retention kinetics of inhaled fine particles in mice, indicating a much slower clearance rate of localized exogenous particles from the brain than from other main metabolic organs. Collectively, our results provide a piece of evidence on the intrusion of exogeneous particles into the CNS and support the association between the inhalation of exogenous particles and their transport into the brain tissues. This work thus provides additional insights for the continued investigation of the adverse effects of air pollution on the brain.

Discrete roles of Ir76b ionotropic coreceptor impact olfaction, blood feeding, and mating in the malaria vector mosquito Anopheles coluzzii.

Anopheline mosquitoes rely on their highly sensitive chemosensory apparatus to detect diverse chemical stimuli that drive the host-seeking and blood-feeding behaviors required to vector pathogens for malaria and other diseases. This process incorporates a variety of chemosensory receptors and transduction pathways. We used advanced in vivo gene-editing and -labeling approaches to localize and functionally characterize the ionotropic coreceptor AcIr76b in the malaria mosquito Anopheles coluzzii, where it impacts both olfactory and gustatory systems. AcIr76b has a broad expression pattern in female adult antennal grooved pegs, coeloconic sensilla, and T1 and T2 sensilla on the labellum, stylets, and tarsi, as well as the larval sensory peg. AcIr76b is colocalized with the Orco odorant receptor (OR) coreceptor in a subset of cells across the female antennae and labella. In contrast to Orco and Ir8a, chemosensory coreceptors that appear essential for the activity of their respective sets of chemosensory neurons in mosquitoes, AcIr76b−/− mutants maintain wild-type peripheral responses to volatile amines on the adult palps, labellum, and larval sensory cone. Interestingly, AcIr76b−/− mutants display significantly increased responses to amines in antennal grooved peg sensilla, while coeloconic sensilla reveal significant deficits in responses to several acids and amines. Behaviorally, AcIr76b mutants manifest significantly female-specific insemination deficits, and although AcIr76b−/− mutant females can locate, alight on, and probe artificial blood hosts, they are incapable of blood feeding successfully. Taken together, our findings reveal a multidimensional functionality of Ir76b in anopheline olfactory and gustatory pathways that directly impacts the vectorial capacity of these mosquitoes.

Specialized interferon action in COVID-19.

The impacts of interferon (IFN) signaling on COVID-19 pathology are multiple, with both protective and harmful effects being documented. We report here a multiomics investigation of systemic IFN signaling in hospitalized COVID-19 patients, defining the multiomics biosignatures associated with varying levels of 12 different type I, II, and III IFNs. The antiviral transcriptional response in circulating immune cells is strongly associated with a specific subset of IFNs, most prominently IFNA2 and IFNG. In contrast, proteomics signatures indicative of endothelial damage and platelet activation associate with high levels of IFNB1 and IFNA6. Seroconversion and time since hospitalization associate with a significant decrease in a specific subset of IFNs. Additionally, differential IFN subtype production is linked to distinct constellations of circulating myeloid and lymphoid immune cell types. Each IFN has a unique metabolic signature, with IFNG being the most associated with activation of the kynurenine pathway. IFNs also show differential relationships with clinical markers of poor prognosis and disease severity. For example, whereas IFNG has the strongest association with C-reactive protein and other immune markers of poor prognosis, IFNB1 associates with increased neutrophil to lymphocyte ratio, a marker of late severe disease. Altogether, these results reveal specialized IFN action in COVID-19, with potential diagnostic and therapeutic implications.

Quantitative detection of hydrogen peroxide in rain, air, exhaled breath, and biological fluids by NMR spectroscopy.

Hydrogen peroxide (H2O2) plays a key role in environmental chemistry, biology, and medicine. H2O2 concentrations typically are 6 to 10 orders of magnitude lower than that of water, making its quantitative detection challenging. We demonstrate that optimized NMR spectroscopy allows direct, interference-free, quantitative measurements of H2O2 down to submicromolar levels in a wide range of fluids, ranging from exhaled breath and air condensate to rain, blood, urine, and saliva. NMR measurements confirm the previously reported spontaneous generation of H2O2 in microdroplets that form when condensing water vapor on a hydrophobic surface, which can interfere with atmospheric H2O2 measurements. Its antimicrobial activity and strong seasonal variation speculatively could be linked to the seasonality of respiratory viral diseases.

Involvement of circulating factors in the transmission of paternal experiences through the germline.

Environmental factors can change phenotypes in exposed individuals and offspring and involve the germline, likely via biological signals in the periphery that communicate with germ cells. Here, using a mouse model of paternal exposure to traumatic stress, we identify circulating factors involving peroxisome proliferator-activated receptor (PPAR) pathways in the effects of exposure to the germline. We show that exposure alters metabolic functions and pathways, particularly lipid-derived metabolites, in exposed fathers and their offspring. We collected data in a human cohort exposed to childhood trauma and observed similar metabolic alterations in circulation, suggesting conserved effects. Chronic injection of serum from trauma-exposed males into controls recapitulates metabolic phenotypes in the offspring. We identify lipid-activated nuclear receptors PPARs as potential mediators of the effects from father to offspring. Pharmacological PPAR activation in vivo reproduces metabolic dysfunctions in the offspring and grand-offspring of injected males and affects the sperm transcriptome in fathers and sons. In germ-like cells in vitro, both serum and PPAR agonist induce PPAR activation. Together, these results highlight the role of circulating factors as potential communication vectors between the periphery and the germline.

DecoID improves identification rates in metabolomics through database-assisted MS/MS deconvolution.

Chimeric MS/MS spectra contain fragments from multiple precursor ions and therefore hinder compound identification in metabolomics. Historically, deconvolution of these chimeric spectra has been challenging and relied on specific experimental methods that introduce variation in the ratios of precursor ions between multiple tandem mass spectrometry (MS/MS) scans. DecoID provides a complementary, method-independent approach where database spectra are computationally mixed to match an experimentally acquired spectrum by using LASSO regression. We validated that DecoID increases the number of identified metabolites in MS/MS datasets from both data-independent and data-dependent acquisition without increasing the false discovery rate. We applied DecoID to publicly available data from the MetaboLights repository and to data from human plasma, where DecoID increased the number of identified metabolites from data-dependent acquisition data by over 30% compared to direct spectral matching. DecoID is compatible with any user-defined MS/MS database and provides automated searching for some of the largest MS/MS databases currently available.