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A food-sensitive olfactory circuit drives anticipatory satiety.
No full textFood sensory perception has emerged as a potent regulator of specialized feeding circuits; yet, the consequences on feeding behaviour and the underlying neuronal basis remain poorly understood. Here, we reveal a sensory pathway that co-ordinately integrates food odours to control forthcoming nutrient intake in male mice. Unbiased whole-brain mapping of food odour-induced brain activity revealed a potent activation of the medial septum (MS), where food odours selectively activate MS glutamatergic neurons (MSVGLUT2). Activity dynamics of MSVGLUT2 neurons uncovered a biphasic modulation of their neuronal activity with a transient activation after detection of food odours and a long-lasting inhibition following food ingestion, independent of the caloric value and identity of the food. MSVGLUT2 neurons receive direct projections from the olfactory bulb (OB) and acute optogenetic stimulation of OB→MS projections selectively before food ingestion decreased feeding in lean mice. However, acute OB→MS optogenetic stimulation in diet-induced obese mice failed to reduce feeding, suggesting the involvement of this pathway in calorie-rich diet-induced hyperphagia and obesity development. Altogether, our study uncovered a sensory circuit by which the organism integrates olfactory food cues to prime satiety at the outset of a meal
Accurate evaluation of live-virus microneutralisation for SARS-CoV-2 variant JN.1 in the assessment of vaccination and therapeutics.
No full textEmerging SARS-CoV-2 variants require rapid assessments of pathogenicity and evasion of existing immunity to inform policy. A crucial component of these assessments is accurate estimation of serum neutralising antibody titres using cultured live virus isolates. Here, we report a comparison of culture methods for Omicron sub-variant JN.1 and the subsequent evaluation of neutralising antibody titres (nAbTs) in recipients of BNT162b2-XBB.1.5 monovalent and the ancestral/BA.4/5 containing bivalent vaccines. We compared culture of JN.1 in either Vero V1 cells or Caco-2 cells, finding culture in Vero V1 either resulted in low-titre stocks or induced crucial mutations at the Spike furin cleavage site (FCS). Using sequence-clean culture stocks generated in Caco-2 cells, we assessed serum samples from 71 healthy adults eligible for a COVID-19 vaccination given as a 5th dose booster in the UK: all participants had detectable nAbs against JN.1 prior to vaccination, with baseline/pre-existing nAbTs between both vaccine groups comparable (p = 0.240). However, nAbTs against JN.1 post-vaccination were 2.6-fold higher for recipients of the monovalent XBB.1.5 vaccine than the BA.4/5 bivalent vaccine (p < 0.001). Further, at clinically relevant concentrations the therapeutic monoclonal antibody Sotrovimab marginally maintains neutralisation of JN.1. Regular re-appraisal of methods and policy outcomes as new variants arise is required to ensure robust data are used to underpin future severity assessments and vaccine strain selection decisions
TPM-with-cluster_updated 5825.csv
No full textThe carotid body (CB) chemoreceptors mediate rapid cardiorespiratory responses to hypoxia, which maintain systemic oxygen homeostasis, but CB dysfunction is also implicated in pathologies including hypertension, heart failure and sudden infant death.CB-mediated chemoreflexes mature during the perinatal period, with increasing sensitivity of the oxygen chemosensory response.We performed RNA-seq of CBs from sheep across 3 perinatal stages and adults, enabling us to identify gene expression changes that correlate with functional state. In parallel, we also analysed superior cervical ganglion (SCG) tissue at the same stages as an oxygen-insensitive control. n=3 biological replicates per stage, except for one d145 SCG that was omitted due to RNA quality.We then performed hierarchical clustering analysis on all genes showing significant differential expression between CB and SCG at any time point. This yielded 6 clusters of genes with different tissue-specific, temporal variation in expression across developmental time: three comprising genes that are CB-enriched (C1, C2, C3) and three with SCG-enriched genes (S1, S2, S3). Cluster C1 and S1 genes are predominantly enriched at early/fetal time points, while C3 and S3 genes increase with development and maturation. Genes in clusters C2 and S2 show more complex, intermediate temporal patterns of expression.The attached file shows expression levels (tpm) for a given gene in each sample and the overall cluster of that gene (if applicable).</p
Chemoenzymatic synthesis and purification of bioorthogonally tagged UDP-GlcNAc and UDP-GalNAc analogues.
No full textNucleotide-sugar donors containing bioorthogonal moieties are important tools to study cellular glycosylation. Typically, the acetamide moiety in N-acetylhexosamines such as GlcNAc and GalNAc is replaced by an acylamide with a clickable tag and converted to the corresponding uridine diphosphate analogue. These probes can then be tested for acceptance by glycosyltransferase enzymes in vitro. Lengthy procedures in synthetic chemistry currently limit the availability of bioorthogonal uridine diphosphate (UDP)-sugar analogues. Chemoenzymatic synthesis has proven to be a powerful and effective alternative, and multiple approaches have been published to date. In this protocol, we describe a streamlined method for the generation of bioorthogonal UDP-GlcNAc and UDP-GalNAc analogues. We describe the chemical modification of D-glucosamine and D-galactosamine to incorporate bioorthogonal acylamides, the subsequent one-pot multienzyme conversion to the corresponding UDP-sugar analogues, and reproducible purification. Our approach features the bacterial kinase NahK and human pyrophosphorylase AGX1 as well as a recombinantly expressed AGX1 variant with an expanded substrate profile. The approach further features an inorganic pyrophosphatase and an alkaline phosphatase to improve enzymatic turnover and aid the purification process, respectively. The use of biosynthetic enzymes with substrate promiscuity extends the scope of bioorthogonal nucleotide-sugar analogue structures to aid efforts in chemical glycobiology. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Chemical synthesis of bioorthogonally tagged acylamide analogues of D-GlcNAc and D-GalNAc Alternate Protocol 1: Chemical synthesis of bioorthogonally tagged acylamide analogues of D-GlcNAc and D-GalNAc from a protected GlcNH2 or GalNH2 precursor Basic Protocol 2: Conversion of D-GlcNAc or D-GalNAc analogues to UDP-sugars using analytical- (reaction scouting) and preparative-scale enzymatic synthesis and purification
DNGR-1 signalling limits dendritic cell activation for optimal antigen cross-presentation.
No full textInnate immune receptors often induce activation of conventional dendritic cells (cDCs) and enhance antigen (cross-)presentation, favouring immune responses. DNGR-1 (CLEC9A), a receptor expressed by type 1 cDCs (cDC1s) and implicated in immune responses to viruses and cancer, recognises F-actin exposed on dead cell remnants and promotes cross-presentation of associated antigens. Here, we show that recruitment of phosphatase SHIP1, a process governed by a single amino acid residue adjacent to the signalling motif of the receptor, partly explains how DNGR-1 fails to trigger cDC1 activation in vitro. Substituting this residue converts DNGR-1 into an activating receptor but decreases induction of cross-presentation of dead cell-associated antigens. Introducing the reverse mutation into the related receptor Dectin-1 impairs its activation capacity while enhancing its ability to promote cross-presentation. These findings reveal a functional trade-off in receptor signalling and suggest that DNGR-1 has evolved to prioritise antigen cross-presentation over cellular activation, possibly to minimise inflammatory responses to dead cells
HSC70 coordinates COP9 signalosome and SCF ubiquitin ligase activity to enable a prompt stress response.
No full textThe SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex plays a protective role against external stress, such as ultraviolet irradiation. The emergence of substrates activates SCF through neddylation, the covalent attachment of ubiquitin-like protein NEDD8 to CUL1. After substrate degradation, SCF is inactivated through deneddylation by COP9-signalosome (CSN), a solo enzyme that can deneddylate SCF. How the activity of CSN and SCF is coordinated within the cell is not fully understood. Here, we find that heat-shock cognate 70 (HSC70) chaperone coordinates SCF and CSN activation dependent on the neddylation status and substrate availability. Under basal conditions and low substrate availability, HCS70 directly enhances CSN deneddylation activity, thereby reducing SCF activity. Under SCF-activated conditions, HSC70 interacts with neddylated SCF and enhances its ubiquitination activity. The alternative interaction between HSC70 and CSN or neddylated SCF is regulated by the presence or absence of SCF substrates. The knockdown of HSC70 decreases SCF-mediated substrate ubiquitination, resulting in vulnerability against ultraviolet irradiation. Our work demonstrates the pivotal role of HSC70 in the alternative activation of CSN deneddylation and SCF substrate ubiquitination, which enables a prompt stress response
Feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in large animals.
No full textPURPOSE: To assess the safety and feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in a large animal model. METHODS: The experimental Liběchov minipigs were used for subretinal delivery of a plasmid DNA vector (pS/MAR-CMV-copGFP) with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP) reporter (copGFP) and a scaffold/matrix attachment region (S/MAR) sequence. The eyes were randomized to subretinal injection of the vector following pars plana vitrectomy (control group) or a direct injection without prior vitrectomy surgery (experimental group). Intra- and post-operative observations up to 30 days after surgery were compared. RESULTS: Six eyes of three mini-pigs underwent surgery for delivery into the subretinal space. Two eyes in the control group were operated with a classical approach (lens-sparing vitrectomy and posterior hyaloid detachment). The other four eyes in the experimental group were injected directly with a subretinal cannula without vitrectomy surgery. No adverse events, such as endophthalmitis, retinal detachment and intraocular pressure elevation were observed post-operatively. The eyes in the experimental group had both shorter surgical time and recovery while achieving the same surgical goal. CONCLUSIONS: This pilot study demonstrates that successful subretinal delivery of gene therapy vectors is achievable using a direct injection without prior vitrectomy surgery
ETV4 and ETV5 orchestrate FGF-mediated lineage specification and epiblast maturation during early mouse development.
No full textCell fate decisions in early mammalian embryos are tightly regulated processes crucial for proper development. While FGF signaling plays key roles in early embryo patterning, its downstream effectors remain poorly understood. Our study demonstrates that the transcription factors Etv4 and Etv5 are critical mediators of FGF signaling in cell lineage specification and maturation in mouse embryos. We show that loss of Etv5 compromises primitive endoderm formation at pre-implantation stages. Furthermore, Etv4/5 deficiency delays naïve pluripotency exit and epiblast maturation, leading to elevated NANOG and reduced OTX2 expression within the blastocyst epiblast. As a consequence of delayed pluripotency progression, Etv4/5 deficient embryos exhibit anterior visceral endoderm migration defects post-implantation, a process essential for coordinated embryonic patterning and gastrulation initiation. Our results demonstrate the successive roles of these FGF signaling effectors in early lineage specification and embryonic body plan establishment, providing new insights into the molecular control of mammalian development
Baricitinib treatment in RNU7‐1‐associated Aicardi–Goutières syndrome in a South African child: A case report
No full textAicardi–Goutières syndrome (AGS) is a rare monogenic type I interferonopathy. Janus kinase (JAK) inhibition has emerged as a potential treatment for AGS. RNU7‐1 is one of the most recently discovered genes for AGS, and the clinical effects of JAK inhibition in these patients have not been reported. Here, we describe the diagnosis and treatment of a South African infant with RNU7‐1‐related AGS. The patient presented with developmental delay at age 5 months and was diagnosed with cerebral palsy due to a suspected congenital infection. By 18 months of age, he had a vasculitic rash, prominent generalized dystonia, persistent transaminitis, recurrent stomatitis, moderate‐range global developmental delay, and difficulty sleeping. AGS was considered after finding neuroimaging features of the disease; the diagnosis was confirmed when genetic investigations revealed two likely pathogenic RNU7‐1 compound heterozygous variants in the patient. Elevated interferon gene expression was noted in the patient and his mother who was a carrier of one RNU7‐1 variant. Baricitinib treatment was started, leading to modest, transient improvements in some clinical manifestations and a reduction in interferon‐stimulated gene expression. Liver function, dystonia, and neurological function did not improve even after increasing the baricitinib dose. Baricitinib was discontinued due to persistent and worsening adverse effects