A gut-to-brain signal of fluid osmolarity controls thirst satiation.

Satiation is the process by which eating and drinking reduce appetite. For thirst, oropharyngeal cues have a critical role in driving satiation by reporting to the brain the volume of fluid that has been ingested1-12. By contrast, the mechanisms that relay the osmolarity of ingested fluids remain poorly understood. Here we show that the water and salt content of the gastrointestinal tract are precisely measured and then rapidly communicated to the brain to control drinking behaviour in mice. We demonstrate that this osmosensory signal is necessary and sufficient for satiation during normal drinking, involves the vagus nerve and is transmitted to key forebrain neurons that control thirst and vasopressin secretion. Using microendoscopic imaging, we show that individual neurons compute homeostatic need by integrating this gastrointestinal osmosensory information with oropharyngeal and blood-borne signals. These findings reveal how the fluid homeostasis system monitors the osmolarity of ingested fluids to dynamically control drinking behaviour

Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions

Unlocking the bottleneck in forward genetics using whole-genome sequencing and identity by descent to isolate causative mutations

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.The High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics is funded by Wellcome Trust grant reference 090532/Z/09/Z and MRC Hub grant G0900747 91070. This study was supported by Wellcome Trust Strategic Award 082030 (CCG), Wellcome Trust Studentship 094446/Z/10/Z (KRB), the Oxford NIHR Biomedical Research Centre, and the MRC Human Immunology Unit (RJC). AJR and GL were supported by Wellcome Trust grant 090532/Z/ 09/Z, CCG and AE by a Major initiative Award from the Clive and Vera Ramaciotti Foundation, and AE by an NHMRC Career Development Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Severely Impaired Learning and Altered Neuronal Morphology in Mice Lacking NMDA Receptors in Medium Spiny Neurons

The striatum is composed predominantly of medium spiny neurons (MSNs) that integrate excitatory, glutamatergic inputs from the cortex and thalamus, and modulatory dopaminergic inputs from the ventral midbrain to influence behavior. Glutamatergic activation of AMPA, NMDA, and metabotropic receptors on MSNs is important for striatal development and function, but the roles of each of these receptor classes remain incompletely understood. Signaling through NMDA-type glutamate receptors (NMDARs) in the striatum has been implicated in various motor and appetitive learning paradigms. In addition, signaling through NMDARs influences neuronal morphology, which could underlie their role in mediating learned behaviors. To study the role of NMDARs on MSNs in learning and in morphological development, we generated mice lacking the essential NR1 subunit, encoded by the Grin1 gene, selectively in MSNs. Although these knockout mice appear normal and display normal 24-hour locomotion, they have severe deficits in motor learning, operant conditioning and active avoidance. In addition, the MSNs from these knockout mice have smaller cell bodies and decreased dendritic length compared to littermate controls. We conclude that NMDAR signaling in MSNs is critical for normal MSN morphology and many forms of learning

Alternative splicing of the Anopheles gambiae Dscam gene in diverse Plasmodium falciparum infections

Background: In insects, including Anopheles mosquitoes, Dscam (Down syndrome cell adhesion molecule) appears to be involved in phagocytosis of pathogens, and shows pathogen-specific splice-form expression between divergent pathogen (or parasite) types (e.g. between bacteria and Plasmodium or between Plasmodium berghei and Plasmodium falciparum). Here, data are presented from the first study of Dscam expression in response to genetic diversity within a parasite species. Methods: In independent field and laboratory studies, a measure of Dscam splice-form diversity was compared between mosquitoes fed on blood that was free of P. falciparum to mosquitoes exposed to either single or mixed genotype infections of P. falciparum. Results: Significant increases in Anopheles gambiae Dscam (AgDscam) receptor diversity were observed in parasite-exposed mosquitoes, but only weak evidence that AgDscam diversity rises further upon exposure to mixed genotype parasite infections was found. Finally, a cluster of AgDscam exon 4 variants that become especially common during Plasmodium invasion was identified. Conclusions: While the data clearly indicate that AgDscam diversity increases with P. falciparum exposure, they do not suggest that AgDscam diversity rises further in response to increased parasite diversit

Genome-Wide Association Study Identifies Genetic Loci Associated with Iron Deficiency

The existence of multiple inherited disorders of iron metabolism in man, rodents and other vertebrates suggests genetic contributions to iron deficiency. To identify new genomic locations associated with iron deficiency, a genome-wide association study (GWAS) was performed using DNA collected from white men aged ≥25 y and women ≥50 y in the Hemochromatosis and Iron Overload Screening (HEIRS) Study with serum ferritin (SF) ≤ 12 µg/L (cases) and iron replete controls (SF>100 µg/L in men, SF>50 µg/L in women). Regression analysis was used to examine the association between case-control status (336 cases, 343 controls) and quantitative serum iron measures and 331,060 single nucleotide polymorphism (SNP) genotypes, with replication analyses performed in a sample of 71 cases and 161 controls from a population of white male and female veterans screened at a US Veterans Affairs (VA) medical center. Five SNPs identified in the GWAS met genome-wide statistical significance for association with at least one iron measure, rs2698530 on chr. 2p14; rs3811647 on chr. 3q22, a known SNP in the transferrin (TF) gene region; rs1800562 on chr. 6p22, the C282Y mutation in the HFE gene; rs7787204 on chr. 7p21; and rs987710 on chr. 22q11 (GWAS observed P<1.51×10−7 for all). An association between total iron binding capacity and SNP rs3811647 in the TF gene (GWAS observed P = 7.0×10−9, corrected P = 0.012) was replicated within the VA samples (observed P = 0.012). Associations with the C282Y mutation in the HFE gene also were replicated. The joint analysis of the HEIRS and VA samples revealed strong associations between rs2698530 on chr. 2p14 and iron status outcomes. These results confirm a previously-described TF polymorphism and implicate one potential new locus as a target for gene identification

Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS-like viruses

The emergence of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed. Here, we utilized a targeted donor selection strategy to isolate a large panel of human broadly neutralizing antibodies (bnAbs) to sarbecoviruses. Many of these bnAbs are remarkably effective in neutralizing a diversity of sarbecoviruses and against most SARS-CoV-2 VOCs, including the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor-binding domain (RBD). Consistent with targeting of conserved sites, select RBD bnAbs exhibited protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model in vivo. These bnAbs provide new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and provide a molecular basis for effective design of pan-sarbecovirus vaccines

Human malarial disease: a consequence of inflammatory cytokine release

Malaria causes an acute systemic human disease that bears many similarities, both clinically and mechanistically, to those caused by bacteria, rickettsia, and viruses. Over the past few decades, a literature has emerged that argues for most of the pathology seen in all of these infectious diseases being explained by activation of the inflammatory system, with the balance between the pro and anti-inflammatory cytokines being tipped towards the onset of systemic inflammation. Although not often expressed in energy terms, there is, when reduced to biochemical essentials, wide agreement that infection with falciparum malaria is often fatal because mitochondria are unable to generate enough ATP to maintain normal cellular function. Most, however, would contend that this largely occurs because sequestered parasitized red cells prevent sufficient oxygen getting to where it is needed. This review considers the evidence that an equally or more important way ATP deficency arises in malaria, as well as these other infectious diseases, is an inability of mitochondria, through the effects of inflammatory cytokines on their function, to utilise available oxygen. This activity of these cytokines, plus their capacity to control the pathways through which oxygen supply to mitochondria are restricted (particularly through directing sequestration and driving anaemia), combine to make falciparum malaria primarily an inflammatory cytokine-driven disease

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes