Early-life adversity increases morphine tolerance and persistent inflammatory hypersensitivity through upregulation of δ opioid receptors in mice

Supported by a NIAA (BJA/RCoA) grant WKR0-2017-0066 and WKR0-2019-0067 awarded to T. G. Hales. C. Sneddon and A. Bakina were supported by MRC DTP studentships. S. Singleton and T. G. Hales were supported by a UKRI and vs Arthritis Grant: MR/W002566/1.Exposure to severely stressful events during childhood is associated with poor health outcomes in later life, including chronic pain and substance use disorder. However, the mediators and mechanisms are unclear. We investigated the impact of a well-characterized mouse model of early-life adversity, fragmented maternal care (FC) between postnatal day 2 and 9, on nociception, inflammatory hypersensitivity, and responses to morphine. Male and female mice exposed to FC exhibited prolonged basal thermal withdrawal latencies and decreased mechanical sensitivity. In addition, morphine had reduced potency in mice exposed to FC and their development of tolerance to morphine was accelerated. Quantitative PCR analysis in several brain regions and the spinal cords of juvenile and adult mice revealed an impact of FC on the expression of genes encoding opioid peptide precursors and their receptors. These changes included enhanced abundance of δ opioid receptor transcript in the spinal cord. Acute inflammatory hypersensitivity (induced by hind paw administration of complete Freund's adjuvant) was unaffected by exposure to FC. However, after an initial recovery of mechanical hypersensitivity, there was a reappearance in mice exposed to FC by day 15, which was not seen in control mice. Changes in nociception, morphine responses, and hypersensitivity associated with FC were apparent in males and females but were absent from mice lacking δ receptors or β-arrestin2. These findings suggest that exposure to early-life adversity in mice enhances δ receptor expression leading to decreased basal sensitivity to noxious stimuli coupled with accelerated morphine tolerance and enhanced vulnerability to persistent inflammatory hypersensitivity.Publisher PDFPeer reviewe

Limits to the biofortification of leafy brassicas with zinc

Many humans lack sufficient zinc (Zn) in their diet for their wellbeing and increasing Zn concentrations in edible produce (biofortification) can mitigate this. Recent efforts have focused on biofortifying staple crops. However, greater Zn concentrations can be achieved in leafy vegetables than in fruits, seeds, or tubers. Brassicas, such as cabbage and broccoli, are widely consumed and might provide an additional means to increase dietary Zn intake. Zinc concentrations in brassicas are limited primarily by Zn phytotoxicity. To assess the limits of Zn biofortification of brassicas, the Zn concentration in a peat:sand (v/v 75:25) medium was manipulated to examine the relationship between shoot Zn concentration and shoot dry weight (DW) and thereby determine the critical shoot Zn concentrations, defined as the shoot Zn concentration at which yield is reduced below 90%. The critical shoot Zn concentration was regarded as the commercial limit to Zn biofortification. Experiments were undertaken over six successive years. A linear relationship between Zn fertiliser application and shoot Zn concentration was observed at low application rates. Critical shoot Zn concentrations ranged from 0.074 to 1.201 mg Zn g−1 DW among cabbage genotypes studied in 2014, and between 0.117 and 1.666 mg Zn g−1 DW among broccoli genotypes studied in 2015–2017. It is concluded that if 5% of the dietary Zn intake of a population is currently delivered through brassicas, then the biofortification of brassicas from 0.057 to > 0.100 mg Zn g−1 DW through the application of Zn fertilisers could increase dietary Zn intake substantially

Src Kinase Inhibition Attenuates Morphine Tolerance without Affecting Reinforcement or Psychomotor Stimulation

BACKGROUND: Prolonged opioid administration leads to tolerance characterized by reduced analgesic potency. Pain management is additionally compromised by the hedonic effects of opioids, the cause of their misuse. The multifunctional protein β-arrestin2 regulates the hedonic effects of morphine and participates in tolerance. These actions might reflect µ opioid receptor up-regulation through reduced endocytosis. β-Arrestin2 also recruits kinases to µ receptors. We explored the role of Src kinase in morphine analgesic tolerance, locomotor stimulation, and reinforcement in C57BL/6 mice.METHODS: Analgesic (tail withdrawal latency; percentage of maximum possible effect, n = 8 to 16), locomotor (distance traveled, n = 7 to 8), and reinforcing (conditioned place preference, n = 7 to 8) effects of morphine were compared in wild-type, µ, µ, and β-arrestin2 mice. The influence of c-Src inhibitors dasatinib (n = 8) and PP2 (n = 12) was examined.RESULTS: Analgesia in morphine-treated wild-type mice exhibited tolerance, declining by day 10 to a median of 62% maximum possible effect (interquartile range, 29 to 92%). Tolerance was absent from mice receiving dasatinib. Tolerance was enhanced in µ mice (34% maximum possible effect; interquartile range, 5 to 52% on day 5); dasatinib attenuated tolerance (100% maximum possible effect; interquartile range, 68 to 100%), as did PP2 (91% maximum possible effect; interquartile range, 78 to 100%). By contrast, c-Src inhibition affected neither morphine-evoked locomotor stimulation nor reinforcement. Remarkably, dasatinib not only attenuated tolerance but also reversed established tolerance in µ mice.CONCLUSIONS: The ability of c-Src inhibitors to inhibit tolerance, thereby restoring analgesia, without altering the hedonic effect of morphine, makes c-Src inhibitors promising candidates as adjuncts to opioid analgesics.</p

Integrin-linked kinase in muscle is necessary for the development of insulin resistance in diet-induced obese mice

Diet-induced muscle insulin resistance is associated with expansion of extracellular matrix (ECM) components, such as collagens, and the expression of collagen-binding integrin, α2β1. Integrins transduce signals from ECM via their cytoplasmic domains, which bind to intracellular integrin-binding proteins. The integrin-linked kinase (ILK)-PINCH-parvin (IPP) complex interacts with the cytoplasmic domain of β-integrin subunits and is critical for integrin signaling. In this study we defined the role of ILK, a key component of the IPP complex, in diet-induced muscle insulin resistance. Wild-type (ILK(lox/lox)) and muscle-specific ILK-deficient (ILK(lox/lox)HSAcre) mice were fed chow or a high-fat (HF) diet for 16 weeks. Body weight was not different between ILK(lox/lox) and ILK(lox/lox)HSAcre mice. However, HF-fed ILK(lox/lox)HSAcre mice had improved muscle insulin sensitivity relative to HF-fed ILK(lox/lox) mice, as shown by increased rates of glucose infusion, glucose disappearance, and muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. Improved muscle insulin action in the HF-fed ILK(lox/lox)HSAcre mice was associated with increased insulin-stimulated phosphorylation of Akt and increased muscle capillarization. These results suggest that ILK expression in muscle is a critical component of diet-induced insulin resistance, which possibly acts by impairing insulin signaling and insulin perfusion through capillaries

Mice Lacking beta2-Integrin Function Remain Glucose Tolerant in Spite of Insulin Resistance, Neutrophil Infiltration and Inflammation

Beta2-integrins are important in leukocyte trafficking and function, and are regulated through the binding of cytoplasmic proteins, such as kindlin-3, to their intracellular domain. Here, we investigate the involvement of beta2-integrins in the regulation of metabolic disease using mice where the kindlin-3 binding site in the beta2-integrin cytoplasmic tail has been mutated (TTT/AAA-beta2-integrin knock-in (KI) mice), leading to expressed but dysfunctional beta2-integrins and significant neutrophilia in vivo. Beta2-integrin KI mice fed on a high fat diet showed normal weight gain, and normal accumulation of macrophages and lymphocytes in white adipose tissue (WAT) and liver, but increased neutrophil numbers especially in WAT. In addition, beta2-integrin KI mice fed on a high fat diet showed significantly increased peripheral insulin resistance in response to high-fat feeding. However, this was associated with improved glucose disposal following glucose load. Interestingly, beta2-integrin KI neutrophils produced more elastase in vitro, in response to stimulation. Beta2-integrin KI mice displayed variability of tissue inflammatory status, with liver and WAT exhibiting little or no difference in inflammation compared to high fat fed controls, whereas skeletal muscle demonstrated a raised inflammatory profile in association with higher elastase levels and diminished signalling through the IRS1-PKB pathway. In conclusion, although expression of dysfunctional beta2-integrins increased neutrophil production and infiltration into tissue, skeletal muscle was the most affected tissue exhibiting evidence of higher neutrophil activity and insulin resistance. Thus, beta2-integrins modulate glucose homeostasis during high fat feeding predominantly through actions on skeletal muscle to affect metabolic phenotype in vivo.Peer reviewe

Antibiotic Prescribing Patterns in Ghana, Uganda, Zambia and Tanzania Hospitals: Results from the Global Point Prevalence Survey (G-PPS) on Antimicrobial Use and Stewardship Interventions Implemented

Antimicrobial resistance (AMR) remains an important global public health issue with antimicrobial misuse and overuse being one of the main drivers. The Global Point Prevalence Survey (G-PPS) of Antimicrobial Consumption and Resistance assesses the prevalence and the quality of antimicrobial prescriptions across hospitals globally. G-PPS was carried out at 17 hospitals across Ghana, Uganda, Zambia and Tanzania. The overall prevalence of antimicrobial use was 50% (30–57%), with most antibiotics prescribed belonging to the WHO ‘Access’ and ‘Watch’ categories. No ‘Reserve’ category of antibiotics was prescribed across the study sites while antimicrobials belonging to the ‘Not Recommended’ group were prescribed infrequently. Antimicrobials were most often prescribed for prophylaxis for obstetric or gynaecological surgery, making up between 12 and 18% of total prescriptions across all countries. The most prescribed therapeutic subgroup of antimicrobials was ‘Antibacterials for systemic use’. As a result of the programme, PPS data are now readily available for the first time in the hospitals, strengthening the global commitment to improved antimicrobial surveillance. Antimicrobial stewardship interventions developed included the formation of AMS committees, the provision of training and the preparation of new AMS guidelines. Other common interventions included the presentation of findings to clinicians for increased awareness, and the promotion of a multi-disciplinary approach to successful AMS programmes. Repeat PPS would be necessary to continually monitor the impact of interventions implemented. Broader participation is also encouraged to strengthen the evidence base

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)