An N‐terminal fusion allele to study melanin concentrating hormone receptor 1

Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (mCherryMCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs

Large-scale phenotyping of patients with long COVID post-hospitalization reveals mechanistic subtypes of disease

One in ten severe acute respiratory syndrome coronavirus 2 infections result in prolonged symptoms termed long coronavirus disease (COVID), yet disease phenotypes and mechanisms are poorly understood1. Here we profiled 368 plasma proteins in 657 participants ≥3 months following hospitalization. Of these, 426 had at least one long COVID symptom and 233 had fully recovered. Elevated markers of myeloid inflammation and complement activation were associated with long COVID. IL-1R2, MATN2 and COLEC12 were associated with cardiorespiratory symptoms, fatigue and anxiety/depression; MATN2, CSF3 and C1QA were elevated in gastrointestinal symptoms and C1QA was elevated in cognitive impairment. Additional markers of alterations in nerve tissue repair (SPON-1 and NFASC) were elevated in those with cognitive impairment and SCG3, suggestive of brain–gut axis disturbance, was elevated in gastrointestinal symptoms. Severe acute respiratory syndrome coronavirus 2-specific immunoglobulin G (IgG) was persistently elevated in some individuals with long COVID, but virus was not detected in sputum. Analysis of inflammatory markers in nasal fluids showed no association with symptoms. Our study aimed to understand inflammatory processes that underlie long COVID and was not designed for biomarker discovery. Our findings suggest that specific inflammatory pathways related to tissue damage are implicated in subtypes of long COVID, which might be targeted in future therapeutic trials

SARS-CoV-2-specific nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination

BACKGROUND: Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. METHODS: In this follow up study, plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. FINDINGS: Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months (p < 0.0001). Nasal and plasma anti-S IgG remained elevated for at least 12 months (p < 0.0001) with plasma neutralising titres that were raised against all variants compared to controls (p < 0.0001). Of 323 with complete data, 307 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal (1.46-fold change after 10 months, p = 0.011) and the median remained below the positive threshold determined by pre-pandemic controls. Samples 12 months after admission showed no association between nasal IgA and plasma IgG anti-S responses (R = 0.05, p = 0.18), indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. INTERPRETATION: The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. FUNDING: This study has been supported by ISARIC4C and PHOSP-COVID consortia. ISARIC4C is supported by grants from the National Institute for Health and Care Research and the Medical Research Council. Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research. The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research. The funders were not involved in the study design, interpretation of data or the writing of this manuscript

Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

<div><p>Perturbation of endoplasmic reticulum (ER) homeostasis triggers the ER stress response (also known as Unfolded Protein Response), a hallmark of many pathological disorders. However the connection between ER stress and inflammation remains largely unexplored. Recent data suggest that ER stress controls the activity of inflammasomes, key signaling platforms that mediate innate immune responses. Here we report that expression of NLRP1, a core inflammasome component, is specifically up-regulated during severe ER stress conditions in human cell lines. Both IRE1α and PERK, but not the ATF6 pathway, modulate <i>NLRP1</i> gene expression. Furthermore, using mutagenesis, chromatin immunoprecipitation and CRISPR-Cas9-mediated genome editing technology, we demonstrate that ATF4 transcription factor directly binds to <i>NLRP1</i> promoter during ER stress. Although involved in different types of inflammatory responses, XBP-1 splicing was not required for <i>NLRP1</i> induction. This study provides further evidence that links ER stress with innate</p></div

NLRP1 mRNA and protein are up-regulated upon ER stress.

<p>(A) Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 levels were measured by quantitative real-time PCR (qPCR) using cyclophillin A as an endogenous control. Semi-quantitative RT-PCR using a different NLRP1 primer set and GAPDH as a control is also shown. (B) HeLa cells were treated either with BFA or TG for the indicated times. NLRP1 mRNA levels were measured by qPCR and RT-PCR. Spliced and un-spliced XBP-1 forms were also evaluated by RT-PCR. (C) HCT116 cells were treated with the indicated stimuli for 24 hours. NLRP1 and NOD1 mRNA levels were measured by qPCR. (D) Cell lysates from wild-type or <i>NLRP1</i>^{<i>−/−</i>} HeLa, THP-1 and K562 cells, untreated or treated with BFA for 20 hours, were normalized for total protein content. Cell extracts were then subjected to SDS-PAGE/immunoblot analysis before and after immunoprecipitation with NLRP1 antibody. Vinculin was detected as loading control. NLRP1 mRNA levels were also measured by RT-PCR. Each panel is representative of at least three independent experiments. (DMSO: dimethyl sulfoxide, TM: tunicamycin, TG: thapsigargin, MSU: monosodium urate crystals, BFA: brefeldin A, PolyI:C: polyinosinic-polycytidylic acid, FLA: flagellin, MDP: muramyl dipeptide, R837: Imiquimod)</p

Atf4 but not Xbp-1s stimulates <i>NLRP1</i> gene expression during ER stress.

<p>(A) HeLa cells were infected with increasing concentrations of murine Xbp-1s and Atf4 adenovirus for 24 hours and NLRP1 mRNA was measured by qPCR. (B) IRE1α, PERK, ATF6 and XBP-1s were down-regulated using siRNA in HeLa cells. Cells were treated with BFA for 20 hours and mRNA levels were measured by qPCR. IRE1α, PERK, ATF6 and XBP-1s knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.</p

NLRP1 mRNA up-regulation is dependent on both IRE1α and PERK pathways.

<p>(A) IRE1α, PERK and ATF6 levels were reduced using siRNA. Upon treatment with ER stress, mRNA levels were measured by qPCR and RT-PCR. IRE1α, PERK and ATF6 knock-down was verified by SDS-PAGE/immunoblotting. (B) Stably transduced HeLa cells were cultured in presence or absence of doxycycline (Dox) for 24 hours and then treated overnight with 2μM BFA. mRNA levels were measured by qPCR and RT-PCR. IRE1α, PERK and ATF6 knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.</p

The Superiority of an Ideal Consumption Tax over an Ideal Income Tax

This paper considers the arguments regarding the choice between an ideal income tax and an ideal consumption tax, focusing on an argument made by Atkinson and Stiglitz regarding neutral taxation of commodities. The argument shows that a properly designed consumption tax is Pareto superior to an income tax: it is more efficient and at least as good at redistribution. The major exception to the Atkinson and Stiglitz result is if individuals with equal wages have different propensities to save. In that event, a consumption tax may no longer be Pareto superior to an income tax. A consumption tax will continue, however, to be more desirable than an income tax. It will be strictly more efficient than an income tax, and under reasonable assumptions, better at redistributing from those who are better off to those who are worse off. This result holds true even if one heavily weights the welfare of the poor