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.National Institutes of Health (U.S.) (grant DP2OD006514)National Institutes of Health (U.S.) (grant K99HG0081)National Institutes of Health (U.S.) (grant R01HG006785

Reducing neuroinflammation by delivery of IL‐10 encoding lentivirus from multiple‐channel bridges

The spinal cord is unable to regenerate after injury largely due to growth‐inhibition by an inflammatory response to the injury that fails to resolve, resulting in secondary damage and cell death. An approach that prevents inhibition by attenuating the inflammatory response and promoting its resolution through the transition of macrophages to anti‐inflammatory phenotypes is essential for the creation of a growth permissive microenvironment. Viral gene delivery to induce the expression of anti‐inflammatory factors provides the potential to provide localized delivery to alter the host inflammatory response. Initially, we investigated the effect of the biomaterial and viral components of the delivery system to influence the extent of cell infiltration and the phenotype of these cells. Bridge implantation reduces antigen‐presenting cell infiltration at day 7, and lentivirus addition to the bridge induces a transient increase in neutrophils in the spinal cord at day 7 and macrophages at day 14. Delivery of a lentivirus encoding IL‐10, an anti‐inflammatory factor that inhibits immune cell activation and polarizes the macrophage population towards anti‐inflammatory phenotypes, reduced neutrophil infiltration at both day 7 and day 28. Though IL‐10 lentivirus did not affect macrophages number, it skewed the macrophage population toward an anti‐inflammatory M2 phenotype and altered macrophage morphology. Additionally, IL‐10 delivery resulted in improved motor function, suggesting reduced secondary damage and increased sparing. Taken together, these results indicate that localized expression of anti‐inflammatory factors, such as IL‐10, can modulate the inflammatory response following spinal cord injury, and may be a key component of a combinatorial approach that targets the multiple barriers to regeneration and functional recovery.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134909/1/btm210018.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134909/2/btm210018_am.pd

Identifying Risk Factors That Distinguish Symptomatic Severe Acute Respiratory Syndrome Coronavirus 2 Infection From Common Upper Respiratory Infections in Children

Background Demographic and clinical risk factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children presenting with respiratory viral symptoms are not well defined. An understanding of risk factors for SARS-CoV-2 infection can help prioritize testing. Methodology We evaluated potential demographic and clinical factors in children who had respiratory viral symptoms and were tested by polymerase chain reaction (PCR) for SARS-CoV-2 and other respiratory viral infections. Results Among the 263 symptomatic children tested for routine seasonal respiratory viruses by PCR, 18 (6.8%) tested positive for SARS-CoV-2. Overall, 22.2% of SARS-CoV-2-infected children and 37.1% of SARS-CoV-2-uninfected children had infection with one or more non-SARS-CoV-2 pathogens (p = 0.31). Higher proportions of children with compared to without SARS-CoV-2 infection were male (77.8 vs. 51.8%, p = 0.05), Hispanic (44.4% vs. 9.8%, p < 0.001), or had the symptoms of fatigue (22.2% vs. 2.5%, p = 0.003) or anosmia/ageusia (11.1% vs. 0%, p = 0.004). History of hypoxic-ischemic encephalopathy (HIE) and obesity were more common in children with versus without SARS-CoV-2 infection (11.1% vs. 1.2%, p = 0.04, and 11.1% vs. 0%, p = 0.004, respectively). In a multivariate analysis, Hispanic ethnicity, symptoms of fatigue or anosmia/ageusia, and presence of obesity (as noted on physical examination) or HIE were independently associated with SARS-CoV-2 infection. Numbers in each category were small, and these preliminary associations require confirmation in future studies. Conclusions In this area of the United States, infection with other viruses did not rule out infection with SARS-CoV-2. Additionally, children with respiratory viral symptoms who were of Hispanic ethnicity, had symptoms of weakness/fatigue, or had obesity or HIE were at an increased risk for SARS-CoV-2 infection. Future studies should assess if these factors are associated with risk in populations in other areas of the United States

A Defective Pentose Phosphate Pathway Reduces Inflammatory Macrophage Responses during Hypercholesterolemia

Metabolic reprogramming has emerged as a crucial regulator of immune cell activation, but how systemic metabolism influences immune cell metabolism and function remains to be investigated. To investigate the effect of dyslipidemia on immune cell metabolism, we performed in-depth transcriptional, metabolic, and functional characterization of macrophages isolated from hypercholesterolemic mice. Systemic metabolic changes in such mice alter cellular macrophage metabolism and attenuate inflammatory macrophage responses. In addition to diminished maximal mitochondrial respiration, hypercholesterolemia reduces the LPS-mediated induction of the pentose phosphate pathway (PPP) and the Nrf2-mediated oxidative stress response. Our observation that suppression of the PPP diminishes LPS-induced cytokine secretion supports the notion that this pathway contributes to inflammatory macrophage responses. Overall, this study reveals that systemic and cellular metabolism are strongly interconnected, together dictating macrophage phenotype and function

Macrophage fumarate hydratase restrains mtRNA-mediated interferon production

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-β production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses

Nrf2 activation reprograms macrophage intermediary metabolism and suppresses the I interferon

To overcome oxidative, inflammatory, and metabolic stress, cells have evolved cytoprotective protein networks controlled by nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) and its negative regulator, Kelch-like ECH associated protein 1 (Keap1). Here, using high-resolution mass spectrometry we characterize the proteomes of macrophages with altered Nrf2 status revealing significant differences among the genotypes in metabolism and redox homeostasis, which were validated with respirometry and metabolomics. Nrf2 affected the proteome following lipopolysaccharide (LPS) stimulation, with alterations in redox, carbohydrate and lipid metabolism, and innate immunity. Notably, Nrf2 activation promoted mitochondrial fusion. The Keap1 inhibitor, 4-octyl itaconate remodeled the inflammatory macrophage proteome, increasing redox and suppressing type I interferon (IFN) response. Similarly, pharmacologic or genetic Nrf2 activation inhibited the transcription of IFN-β and its downstream effector IFIT2 during LPS stimulation. These data suggest that Nrf2 activation facilitates metabolic reprogramming and mitochondrial adaptation, and finetunes the innate immune response in macrophages

Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons

Phytoplankton composition from sPACE: Requirements, opportunities, and challenges

Ocean color satellites have provided a synoptic view of global phytoplankton for over 25 years through near surface measurements of the concentration of chlorophyll a. While remote sensing of ocean color has revolutionized our understanding of phytoplankton and their role in the oceanic and freshwater ecosystems, it is important to consider both total phytoplankton biomass and changes in phytoplankton community composition in order to fully understand the dynamics of the aquatic ecosystems. With the upcoming launch of NASA\u27s Plankton, Aerosol, Clouds, ocean Ecosystem (PACE) mission, we will be entering into a new era of global hyperspectral data, and with it, increased capabilities to monitor phytoplankton diversity from space. In this paper, we analyze the needs of the user community, review existing approaches for detecting phytoplankton community composition in situ and from space, and highlight the benefits that the PACE mission will bring. Using this three-pronged approach, we highlight the challenges and gaps to be addressed by the community going forward, while offering a vision of what global phytoplankton community composition will look like through the “eyes” of PACE

Multidimensional individualised Physical ACTivity (Mi-PACT) - a technology-enabled intervention to promote physical activity in primary care: Study protocol for a randomised controlled trial

© 2015 Peacock et al. Background: Low physical activity is a major public health problem. New cost-effective approaches that stimulate meaningful long-term changes in physical activity are required, especially within primary care settings. It is becoming clear that there are various dimensions to physical activity with independent health benefits. Advances in technology mean that it is now possible to generate multidimensional physical activity 'profiles' that provide a more complete representation of physical activity and offer a variety of options that can be tailored to the individual. Mi-PACT is a randomised controlled trial designed to examine whether personalised multidimensional physical activity feedback and self-monitoring alongside trainer-supportive sessions increases physical activity and improves health outcomes in at-risk men and women. Methods/Design: We aim to recruit 216 patients from within primary care aged 40 to 70years and at medium or high risk of cardiovascular disease and/or type II diabetes mellitus. Adopting an unequal allocation ratio (intervention: control) of 2:1, participants will be randomised to one of two groups, usual care or the intervention. The control group will receive usual care from their general practitioner (GP) and standardised messages about physical activity for health. The intervention group will receive physical activity monitors and access to a web-based platform for a 3-month period to enable self-monitoring and the provision of personalised feedback regarding the multidimensional nature of physical activity. In addition, this technology-enabled feedback will be discussed with participants on 5 occasions during supportive one-to-one coaching sessions across the 3-month intervention. The primary outcome measure is physical activity, which will be directly assessed using activity monitors for a 7-day period at baseline, post intervention and at 12months. Secondary measures (at these time-points) include weight loss, fat mass, and markers of metabolic control, motivation and well-being. Discussion: Results from this study will provide insight into the effects of integrated physical activity profiling and self-monitoring combined with in-person support on physical activity and health outcomes in patients at risk of future chronic disease. Trial registration:ISRCTN18008011Trial registration date: 31 July 201