Elevated amyloid beta disrupts the nanoscale organization and function of synaptic vesicle pools in hippocampal neurons.

Alzheimer's disease is linked to increased levels of amyloid beta (Aß) in the brain, but the mechanisms underlying neuronal dysfunction and neurodegeneration remain enigmatic. Here, we investigate whether organizational characteristics of functional presynaptic vesicle pools, key determinants of information transmission in the central nervous system, are targets for elevated Aß. Using an optical readout method in cultured hippocampal neurons, we show that acute Aß42 treatment significantly enlarges the fraction of functional vesicles at individual terminals. We observe the same effect in a chronically elevated Aß transgenic model (APPSw,Ind) using an ultrastructure-function approach that provides detailed information on nanoscale vesicle pool positioning. Strikingly, elevated Aß is correlated with excessive accumulation of recycled vesicles near putative endocytic sites, which is consistent with deficits in vesicle retrieval pathways. Using the glutamate reporter, iGluSnFR, we show that there are parallel functional consequences, where ongoing information signaling capacity is constrained. Treatment with levetiracetam, an antiepileptic that dampens synaptic hyperactivity, partially rescues these transmission defects. Our findings implicate organizational and dynamic features of functional vesicle pools as targets in Aß-driven synaptic impairment, suggesting that interventions to relieve the overloading of vesicle retrieval pathways might have promising therapeutic value.

A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli.

Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni  N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli.

Long COVID: mechanisms, risk factors and recovery.

NEW FINDINGS: What is the topic of this review? The emerging condition of long COVID, its epidemiology, pathophysiological impacts on patients of different backgrounds, physiological mechanisms emerging as explanations of the condition, and treatment strategies being trialled. The review leads from a Physiological Society online conference on this topic. What advances does it highlight? Progress in understanding the pathophysiology and cellular mechanisms underlying Long COVID and potential therapeutic and management strategies. ABSTRACT: Long COVID, the prolonged illness and fatigue suffered by a small proportion of those infected with SARS-CoV-2, is placing an increasing burden on individuals and society. A Physiological Society virtual meeting in February 2022 brought clinicians and researchers together to discuss the current understanding of long COVID mechanisms, risk factors and recovery. This review highlights the themes arising from that meeting. It considers the nature of long COVID, exploring its links with other post-viral illnesses such as myalgic encephalomyelitis/chronic fatigue syndrome, and highlights how long COVID research can help us better support those suffering from all post-viral syndromes. Long COVID research started particularly swiftly in populations routinely monitoring their physical performance - namely the military and elite athletes. The review highlights how the high degree of diagnosis, intervention and monitoring of success in these active populations can suggest management strategies for the wider population. We then consider how a key component of performance monitoring in active populations, cardiopulmonary exercise training, has revealed long COVID-related changes in physiology - including alterations in peripheral muscle function, ventilatory inefficiency and autonomic dysfunction. The nature and impact of dysautonomia are further discussed in relation to postural orthostatic tachycardia syndrome, fatigue and treatment strategies that aim to combat sympathetic overactivation by stimulating the vagus nerve. We then interrogate the mechanisms that underlie long COVID symptoms, with a focus on impaired oxygen delivery due to micro-clotting and disruption of cellular energy metabolism, before considering treatment strategies that indirectly or directly tackle these mechanisms. These include remote inspiratory muscle training and integrated care pathways that combine rehabilitation and drug interventions with research into long COVID healthcare access across different populations. Overall, this review showcases how physiological research reveals the changes that occur in long COVID and how different therapeutic strategies are being developed and tested to combat this condition.

A multi-hit hypothesis for an APOE4-dependent pathophysiological state.

The APOE gene encoding the Apolipoprotein E protein is the single most significant genetic risk factor for late-onset Alzheimer's disease. The APOE4 genotype confers a significantly increased risk relative to the other two common genotypes APOE3 and APOE2. Intriguingly, APOE4 has been associated with neuropathological and cognitive deficits in the absence of Alzheimer's disease-related amyloid or tau pathology. Here, we review the extensive literature surrounding the impact of APOE genotype on central nervous system dysfunction, focussing on preclinical model systems and comparison of APOE3 and APOE4, given the low global prevalence of APOE2. A multi-hit hypothesis is proposed to explain how APOE4 shifts cerebral physiology towards pathophysiology through interconnected hits. These hits include the following: neurodegeneration, neurovascular dysfunction, neuroinflammation, oxidative stress, endosomal trafficking impairments, lipid and cellular metabolism disruption, impaired calcium homeostasis and altered transcriptional regulation. The hits, individually and in combination, leave the APOE4 brain in a vulnerable state where further cumulative insults will exacerbate degeneration and lead to cognitive deficits in the absence of Alzheimer's disease pathology and also a state in which such pathology may more easily take hold. We conclude that current evidence supports an APOE4 multi-hit hypothesis, which contributes to an APOE4 pathophysiological state. We highlight key areas where further study is required to elucidate the complex interplay between these individual mechanisms and downstream consequences, helping to frame the current landscape of existing APOE-centric literature.

SARS-CoV-2 variants of concern alpha, beta, gamma and delta have extended ACE2 receptor host ranges.

Following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in PR China in late 2019 a number of variants have emerged, with two of these - alpha and delta - subsequently growing to global prevalence. One characteristic of these variants are changes within the spike protein, in particular the receptor-binding domain (RBD). From a public health perspective, these changes have important implications for increased transmissibility and immune escape; however, their presence could also modify the intrinsic host range of the virus. Using viral pseudotyping, we examined whether the variants of concern (VOCs) alpha, beta, gamma and delta have differing host angiotensin-converting enzyme 2 (ACE2) receptor usage patterns, focusing on a range of relevant mammalian ACE2 proteins. All four VOCs were able to overcome a previous restriction for mouse ACE2, with demonstrable differences also seen for individual VOCs with rat, ferret or civet ACE2 receptors, changes that we subsequently attributed to N501Y and E484K substitutions within the spike RBD.

The impact of the COVID-19 pandemic on presentations to health services following self-harm: systematic review.

BACKGROUND: Evidence on the impact of the pandemic on healthcare presentations for self-harm has accumulated rapidly. However, existing reviews do not include studies published beyond 2020. AIMS: To systematically review evidence on presentations to health services following self-harm during the COVID-19 pandemic. METHOD: A comprehensive search of databases (WHO COVID-19 database; Medline; medRxiv; Scopus; PsyRxiv; SocArXiv; bioRxiv; COVID-19 Open Research Dataset, PubMed) was conducted. Studies published from 1 January 2020 to 7 September 2021 were included. Study quality was assessed with a critical appraisal tool. RESULTS: Fifty-one studies were included: 57% (29/51) were rated as 'low' quality, 31% (16/51) as 'moderate' and 12% (6/51) as 'high-moderate'. Most evidence (84%, 43/51) was from high-income countries. A total of 47% (24/51) of studies reported reductions in presentation frequency, including all six rated as high-moderate quality, which reported reductions of 17-56%. Settings treating higher lethality self-harm were overrepresented among studies reporting increased demand. Two of the three higher-quality studies including study observation months from 2021 reported reductions in self-harm presentations. Evidence from 2021 suggests increased numbers of presentations among adolescents, particularly girls. CONCLUSIONS: Sustained reductions in numbers of self-harm presentations were seen into the first half of 2021, although this evidence is based on a relatively small number of higher-quality studies. Evidence from low- and middle-income countries is lacking. Increased numbers of presentations among adolescents, particularly girls, into 2021 is concerning. Findings may reflect changes in thresholds for help-seeking, use of alternative sources of support and variable effects of the pandemic across groups.

Choice of method of place cell classification determines the population of cells identified.

Place cells, spatially responsive hippocampal cells, provide the neural substrate supporting navigation and spatial memory. Historically most studies of these neurons have used electrophysiological recordings from implanted electrodes but optical methods, measuring intracellular calcium, are becoming increasingly common. Several methods have been proposed as a means to identify place cells based on their calcium activity but there is no common standard and it is unclear how reliable different approaches are. Here we tested four methods that have previously been applied to two-photon hippocampal imaging or electrophysiological data, using both model datasets and real imaging data. These methods use different parameters to identify place cells, including the peak activity in the place field, compared to other locations (the Peak method); the stability of cells' activity over repeated traversals of an environment (Stability method); a combination of these parameters with the size of the place field (Combination method); and the spatial information held by the cells (Information method). The methods performed differently from each other on both model and real data. In real datasets, vastly different numbers of place cells were identified using the four methods, with little overlap between the populations identified as place cells. Therefore, choice of place cell detection method dramatically affects the number and properties of identified cells. Ultimately, we recommend the Peak method be used in future studies to identify place cell populations, as this method is robust to moderate variations in place field within a session, and makes no inherent assumptions about the spatial information in place fields, unless there is an explicit theoretical reason for detecting cells with more narrowly defined properties.

SARS-CoV-2 Infection and the Risk of Suicidal and Self-Harm Thoughts and Behaviour: A Systematic Review.

OBJECTIVE: The COVID-19 pandemic has had a complex impact on risks of suicide and non-fatal self-harm worldwide with some evidence of increased risk in specific populations including women, young people, and people from ethnic minority backgrounds. This review aims to systematically address whether SARS-CoV-2 infection and/or COVID-19 disease confer elevated risk directly. METHOD: As part of a larger Living Systematic Review examining self-harm and suicide during the pandemic, automated daily searches using a broad list of keywords were performed on a comprehensive set of databases with data from relevant articles published between January 1, 2020 and July 18, 2021. Eligibility criteria for our present review included studies investigating suicide and/or self-harm in people infected with SARS-CoV-2 with or without manifestations of COVID-19 disease with a comparator group who did not have infection or disease. Suicidal and self-harm thoughts and behaviour (STBs) were outcomes of interest. Studies were excluded if they reported data for people who only had potential infection/disease without a confirmed exposure, clinical/molecular diagnosis or self-report of a positive SARS-CoV-2 test result. Studies of news reports, treatment studies, and ecological studies examining rates of both SARS-CoV-2 infections and suicide/self-harm rates across a region were also excluded. RESULTS: We identified 12 studies examining STBs in nine distinct samples of people with SARS-CoV-2. These studies, which investigated STBs in the general population and in subpopulations, including healthcare workers, generally found positive associations between SARS-CoV-2 infection and/or COVID-19 disease and subsequent suicidal/self-harm thoughts and suicidal/self-harm behaviour. CONCLUSIONS: This review identified some evidence that infection with SARS-CoV-2 and/or COVID-19 disease may be associated with increased risks for suicidal and self-harm thoughts and behaviours but a causal link cannot be inferred. Further research with longer follow-up periods is required to confirm these findings and to establish whether these associations are causal.

The Emergence of a Stable Neuronal Ensemble from a Wider Pool of Activated Neurons in the Dorsal Medial Prefrontal Cortex during Appetitive Learning in Mice.

Animals selectively respond to environmental cues associated with food reward to optimize nutrient intake. Such appetitive conditioned stimulus-unconditioned stimulus (CS-US) associations are thought to be encoded in select, stable neuronal populations or neuronal ensembles, which undergo physiological modifications during appetitive conditioning. These ensembles in the medial prefrontal cortex (mPFC) control well-established, cue-evoked food seeking, but the mechanisms involved in the genesis of these ensembles are unclear. Here, we used male Fos-GFP mice that express green fluorescent protein (GFP) in recently behaviorally activated neurons, to reveal how dorsal mPFC neurons are recruited and modified to encode CS-US memory representations using an appetitive conditioning task. In the initial conditioning session, animals did not exhibit discriminated, cue-selective food seeking, but did so in later sessions indicating that a CS-US association was established. Using microprism-based in vivo 2-Photon imaging, we revealed that only a minority of neurons activated during the initial session was consistently activated throughout subsequent conditioning sessions and during cue-evoked memory recall. Notably, using ex vivo electrophysiology, we found that neurons activated following the initial session exhibited transient hyperexcitability. Chemogenetically enhancing the excitability of these neurons throughout subsequent conditioning sessions interfered with the development of reliable cue-selective food seeking, indicated by persistent, nondiscriminated performance. We demonstrate how appetitive learning consistently activates a subset of neurons to form a stable neuronal ensemble during the formation of a CS-US association. This ensemble may arise from a pool of hyperexcitable neurons activated during the initial conditioning session.SIGNIFICANCE STATEMENT Appetitive conditioning endows cues associated with food with the ability to guide food-seeking, through the formation of a food-cue association. Neuronal ensembles in the mPFC control established cue-evoked food-seeking. However, how neurons undergo physiological modifications and become part of an ensemble during conditioning remain unclear. We found that only a minority of dorsal mPFC neurons activated on the initial conditioning session became consistently activated during conditioning and memory recall. These initially activated neurons were also transiently hyperexcitable. We demonstrate the following: (1) how stable neuronal ensemble formation in the dorsal mPFC underlies appetitive conditioning; and (2) how this ensemble may arise from hyperexcitable neurons activated before the establishment of cue-evoked food seeking.

Extinction of cue-evoked food-seeking recruits a GABAergic interneuron ensemble in the dorsal medial prefrontal cortex of mice.

Animals must quickly adapt food-seeking strategies to locate nutrient sources in dynamically changing environments. Learned associations between food and environmental cues that predict its availability promote food-seeking behaviors. However, when such cues cease to predict food availability, animals undergo "extinction" learning, resulting in the inhibition of food-seeking responses. Repeatedly activated sets of neurons, or "neuronal ensembles," in the dorsal medial prefrontal cortex (dmPFC) are recruited following appetitive conditioning and undergo physiological adaptations thought to encode cue-reward associations. However, little is known about how the recruitment and intrinsic excitability of such dmPFC ensembles are modulated by extinction learning. Here, we used in vivo 2-Photon imaging in male Fos-GFP mice that express green fluorescent protein (GFP) in recently behaviorally activated neurons to determine the recruitment of activated pyramidal and GABAergic interneuron dmPFC ensembles during extinction. During extinction, we revealed a persistent activation of a subset of interneurons which emerged from a wider population of interneurons activated during the initial extinction session. This activation pattern was not observed in pyramidal cells, and extinction learning did not modulate the excitability properties of activated pyramidal cells. Moreover, extinction learning reduced the likelihood of reactivation of pyramidal cells activated during the initial extinction session. Our findings illuminate novel neuronal activation patterns in the dmPFC underlying extinction of food-seeking, and in particular, highlight an important role for interneuron ensembles in this inhibitory form of learning.

Capillary pericytes regulate cerebral blood flow in health and disease.

Increases in brain blood flow, evoked by neuronal activity, power neural computation and form the basis of BOLD (blood-oxygen-level-dependent) functional imaging. Whether blood flow is controlled solely by arteriole smooth muscle, or also by capillary pericytes, is controversial. We demonstrate that neuronal activity and the neurotransmitter glutamate evoke the release of messengers that dilate capillaries by actively relaxing pericytes. Dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress vasoconstricting 20-HETE synthesis. In vivo, when sensory input increases blood flow, capillaries dilate before arterioles and are estimated to produce 84% of the blood flow increase. In pathology, ischaemia evokes capillary constriction by pericytes. We show that this is followed by pericyte death in rigor, which may irreversibly constrict capillaries and damage the blood-brain barrier. Thus, pericytes are major regulators of cerebral blood flow and initiators of functional imaging signals. Prevention of pericyte constriction and death may reduce the long-lasting blood flow decrease that damages neurons after stroke.

Genotypic determinants of fluoroquinolone and macrolide resistance in Neisseria gonorrhoeae.

Background High rates of antimicrobial resistance (AMR) in Neisseria gonorrhoeae hinder effective treatment, but molecular AMR diagnostics may help address the challenge. This study aimed to appraise the literature for resistance-associated genotypic markers linked to fluoroquinolones and macrolides, to identify and review their use in diagnostics. METHODS: Medline and EMBASE databases were searched and data pooled to evaluate associations between genotype and phenotypic resistance. The minimum inhibitory concentration (MIC) cut-offs were ≤ 0.06 mg L-1 for non-resistance to ciprofloxacin and ≤ 0.5 mg L-1 for non-resistance to azithromycin. RESULTS: Diagnostic accuracy estimates were limited by data availability and reporting. It was found that: 1) S91 and D95 mutations in the GyrA protein independently predicted ciprofloxacin resistance and, used together, gave 98.6% (95% confidence interval (CI) 98.0-99.0%) sensitivity and 91.4% (95%CI 88.6-93.7%) specificity; 2) the number of 23S rRNA gene alleles with C2611T or A2059G mutations was highly correlated with azithromycin resistance, with mutation in any allele giving a sensitivity and specificity of 66.1% (95%CI 62.1-70.0%) and 98.9% (95%CI 97.5-99.5%) respectively. Estimated negative (NPV) and positive predictive values (PPV) for a 23S rRNA diagnostic were 98.6% (95%CI 96.8-99.4%) and 71.5% (95%CI 68.0-74.8%) respectively; 3) mutation at amino acid position G45 in the MtrR protein independently predicted azithromycin resistance; however, when combined with 23S rRNA, did not improve the PPV or NPV. CONCLUSIONS: Viable candidates for markers of resistance detection for incorporation into diagnostics were demonstrated. Such tests may enhance antibiotic stewardship and treatment options.

Small vessels, dementia and chronic diseases - molecular mechanisms and pathophysiology.

Cerebral small vessel disease (SVD) is a major contributor to stroke, cognitive impairment and dementia with limited therapeutic interventions. There is a critical need to provide mechanistic insight and improve translation between pre-clinical research and the clinic. A 2-day workshop was held which brought together experts from several disciplines in cerebrovascular disease, dementia and cardiovascular biology, to highlight current advances in these fields, explore synergies and scope for development. These proceedings provide a summary of key talks at the workshop with a particular focus on animal models of cerebral vascular disease and dementia, mechanisms and approaches to improve translation. The outcomes of discussion groups on related themes to identify the gaps in knowledge and requirements to advance knowledge are summarized.

Pharmacogenomics and Patient Treatment Parameters to Opioid Treatment in Chronic Pain: A Focus on Morphine, Oxycodone, Tramadol, and Fentanyl.

OBJECTIVE: Opioids are one of the most commonly prescribed medicines for chronic pain. However, their use for chronic pain has been controversial. The objective of this literature review was to identify the role of genetic polymorphisms on patient treatment parameters (opioid dose requirements, response, and adverse effects) for opioids used in malignant and nonmalignant chronic pain. The opioids that this review focuses on are codeine, morphine, oxycodone, tramadol, and fentanyl. METHOD: A literature search of databases Medline and Embase was carried out, and studies up to April 2016 were included in this review. Studies were included based on a combination of key words: chronic pain and related terms, pharmacogenetics and related terms, and opioids and related terms. RESULTS: Among the 1,408 individual papers retrieved from the search in Medline and Embase, 32 original articles were included in this review, with none related to codeine. The 32 papers reported various study designs, opioids, and polymorphisms being studied for associations with treatment outcomes. This literature review reveals that variants in ABCB1, OPRM1, and COMT have been replicated for opioid dosing and variants in ABCB1 have been replicated for both treatment response and adverse effects. CONCLUSIONS: Currently, there are few validated studies to form a strong evidence base to support pharmacogenomics testing when initiating opioid therapy. However, the field of pharmacogenomics in chronic pain is likely to expand over the coming years, with the increasing number of treatment options available and larger cohorts being assembled in order to identify true associations.

Accurate detection of Neisseria gonorrhoeae ciprofloxacin susceptibility directly from genital and extragenital clinical samples: towards genotype-guided antimicrobial therapy.

INTRODUCTION: Increasing use of nucleic acid amplification tests (NAATs) as the primary means of diagnosing gonococcal infection has resulted in diminished availability of Neisseria gonorrhoeae antimicrobial susceptibility data. We conducted a prospective diagnostic assessment of a real-time PCR assay (NGSNP) enabling direct detection of gonococcal ciprofloxacin susceptibility from a range of clinical sample types. METHODS: NGSNP, designed to discriminate an SNP associated with ciprofloxacin resistance within the N. gonorrhoeae genome, was validated using a characterized panel of geographically diverse isolates (n = 90) and evaluated to predict ciprofloxacin susceptibility directly on N. gonorrhoeae-positive NAAT lysates derived from genital (n = 174) and non-genital (n = 116) samples (n = 290), from 222 culture-confirmed clinical episodes of gonococcal infection. RESULTS: NGSNP correctly genotyped all phenotypically susceptible (n = 49) and resistant (n = 41) panel isolates. Ciprofloxacin-resistant N. gonorrhoeae was responsible for infection in 29.7% (n = 66) of clinical episodes evaluated. Compared with phenotypic susceptibility testing, NGSNP demonstrated sensitivity and specificity of 95.8% (95% CI 91.5%-98.3%) and 100% (95% CI 94.7%-100%), respectively, for detecting ciprofloxacin-susceptible N. gonorrhoeae, with a positive predictive value of 100% (95% CI 97.7%-100%). Applied to urogenital (n = 164), rectal (n = 40) and pharyngeal samples alone (n = 30), positive predictive values were 100% (95% CI 96.8%-100%), 100% (95% CI 87.2%-100%) and 100% (95% CI 82.4%-100%), respectively. CONCLUSIONS: Genotypic prediction of N. gonorrhoeae ciprofloxacin susceptibility directly from clinical samples was highly accurate and, in the absence of culture, will facilitate use of tailored therapy for gonococcal infection, sparing use of current empirical treatment regimens and enhancing acquisition of susceptibility data for surveillance.

Conformational heterogeneity in antibody-protein antigen recognition: implications for high affinity protein complex formation.

Specific, high affinity protein-protein interactions lie at the heart of many essential biological processes, including the recognition of an apparently limitless range of foreign proteins by natural antibodies, which has been exploited to develop therapeutic antibodies. To mediate biological processes, high affinity protein complexes need to form on appropriate, relatively rapid timescales, which presents a challenge for the productive engagement of complexes with large and complex contact surfaces (∼600-1800 Å(2)). We have obtained comprehensive backbone NMR assignments for two distinct, high affinity antibody fragments (single chain variable and antigen-binding (Fab) fragments), which recognize the structurally diverse cytokines interleukin-1ß (IL-1ß, ß-sheet) and interleukin-6 (IL-6, α-helical). NMR studies have revealed that the hearts of the antigen binding sites in both free anti-IL-1ß Fab and anti-IL-6 single chain variable exist in multiple conformations, which interconvert on a timescale comparable with the rates of antibody-antigen complex formation. In addition, we have identified a conserved antigen binding-induced change in the orientation of the two variable domains. The observed conformational heterogeneity and slow dynamics at protein antigen binding sites appears to be a conserved feature of many high affinity protein-protein interfaces structurally characterized by NMR, suggesting an essential role in protein complex formation. We propose that this behavior may reflect a soft capture, protein-protein docking mechanism, facilitating formation of high affinity protein complexes on a timescale consistent with biological processes.

Risk factors for severe COVID-19 disease increase SARS-CoV-2 infectivity of endothelial cells and pericytes.

Coronavirus disease 2019 (COVID-19) was initially considered a primarily respiratory disease but is now known to affect other organs including the heart and brain. A major route by which COVID-19 impacts different organs is via the vascular system. We studied the impact of apolipoprotein E (APOE) genotype and inflammation on vascular infectivity by pseudo-typed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses in mouse and human cultured endothelial cells and pericytes. Possessing the APOE4 allele or having existing systemic inflammation is known to enhance the severity of COVID-19. Using targeted replacement human APOE3 and APOE4 mice and inflammation induced by bacterial lipopolysaccharide (LPS), we investigated infection by SARS-CoV-2. Here, we show that infectivity was higher in murine cerebrovascular pericytes compared to endothelial cells and higher in cultures expressing APOE4. Furthermore, increasing the inflammatory state of the cells by prior incubation with LPS increased infectivity into human and mouse pericytes and human endothelial cells. Our findings provide insights into the mechanisms underlying severe COVID-19 infection, highlighting how risk factors such as APOE4 genotype and prior inflammation may exacerbate disease severity by augmenting the virus's ability to infect vascular cells.

Activation of the insulin receptor by insulin-like growth factor 2.

Insulin receptor (IR) controls growth and metabolism. Insulin-like growth factor 2 (IGF2) has different binding properties on two IR isoforms, mimicking insulin's function. However, the molecular mechanism underlying IGF2-induced IR activation remains unclear. Here, we present cryo-EM structures of full-length human long isoform IR (IR-B) in both the inactive and IGF2-bound active states, and short isoform IR (IR-A) in the IGF2-bound active state. Under saturated IGF2 concentrations, both the IR-A and IR-B adopt predominantly asymmetric conformations with two or three IGF2s bound at site-1 and site-2, which differs from that insulin saturated IR forms an exclusively T-shaped symmetric conformation. IGF2 exhibits a relatively weak binding to IR site-2 compared to insulin, making it less potent in promoting full IR activation. Cell-based experiments validated the functional importance of IGF2 binding to two distinct binding sites in optimal IR signaling and trafficking. In the inactive state, the C-terminus of α-CT of IR-B contacts FnIII-2 domain of the same protomer, hindering its threading into the C-loop of IGF2, thus reducing the association rate of IGF2 with IR-B. Collectively, our studies demonstrate the activation mechanism of IR by IGF2 and reveal the molecular basis underlying the different affinity of IGF2 to IR-A and IR-B.

Confirmation of COVID-19 infection status and reporting of Long COVID symptoms in a population-based birth cohort: No evidence of a nocebo effect.

Some patients with COVID-19 develop symptoms after the acute infection, known as 'Long COVID'. We examined whether or not confirmation of COVID-19 infection status could act as a nocebo, using data from questionnaires distributed to the Avon Longitudinal Study of Parents and Children cohort. We examined associations between confirmation of COVID-19 infection status (confirmed by a positive test vs unconfirmed) and reporting of Long COVID symptoms. We explored the roles of sex and anxiety as potential moderators. There was no clear evidence of a strong association between confirmation of COVID-19 infection status and the Long COVID composite score, physical or psychological symptoms or duration of symptoms. There was no clear evidence of moderation by sex or anxiety. We therefore found no evidence of a nocebo effect. Our data suggest that this psychological mechanism does not play a role in the medical symptomatology experienced by patients with Long COVID.

Distinct neurochemical influences on fMRI response polarity in the striatum.

The striatum, known as the input nucleus of the basal ganglia, is extensively studied for its diverse behavioral roles. However, the relationship between its neuronal and vascular activity, vital for interpreting functional magnetic resonance imaging (fMRI) signals, has not received comprehensive examination within the striatum. Here, we demonstrate that optogenetic stimulation of dorsal striatal neurons or their afferents from various cortical and subcortical regions induces negative striatal fMRI responses in rats, manifesting as vasoconstriction. These responses occur even with heightened striatal neuronal activity, confirmed by electrophysiology and fiber-photometry. In parallel, midbrain dopaminergic neuron optogenetic modulation, coupled with electrochemical measurements, establishes a link between striatal vasodilation and dopamine release. Intriguingly, in vivo intra-striatal pharmacological manipulations during optogenetic stimulation highlight a critical role of opioidergic signaling in generating striatal vasoconstriction. This observation is substantiated by detecting striatal vasoconstriction in brain slices after synthetic opioid application. In humans, manipulations aimed at increasing striatal neuronal activity likewise elicit negative striatal fMRI responses. Our results emphasize the necessity of considering vasoactive neurotransmission alongside neuronal activity when interpreting fMRI signal.