A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient Cavalier King Charles Spaniels is amenable to exon 51 skipping

BACKGROUND Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion "hot spot" is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD "hot spot". METHODOLOGY/PRINCIPAL FINDINGS Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5' donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. CONCLUSIONS/SIGNIFICANCE Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD

Using dual-calibrated functional MRI to map brain oxygen supply and consumption in multiple sclerosis

Evidence suggests that cerebrovascular function and oxygen consumption are altered in multiple sclerosis (MS). Here, we quantified the vascular and oxygen metabolic MRI burden in patients with MS (PwMS) and assessed the relationship between these MRI measures of and metrics of damage and disability. In PwMS and in matched healthy volunteers, we applied a newly developed dual-calibrated fMRI method of acquisition and analysis to map grey matter (GM) cerebral blood flow (CBF), oxygen extraction fraction (OEF), cerebral metabolic rate of oxygen consumption (CMRO2) and effective oxygen diffusivity of the capillary network (DC). We also quantified physical and cognitive function in PwMS and controls. There was no significant difference in GM volume between 22 PwMS and 20 healthy controls (p=0.302). Significant differences in CBF (PwMS vs. controls: 44.91 ± 6.10 vs. 48.90 ± 5.87 ml/100g/min, p=0.010), CMRO2 (117.69 ± 17.31 vs. 136.49 ± 14.48 μmol/100g/min p<0.001) and DC (2.70 ± 0.51 vs. 3.18 ± 0.41 μmol/100g/mmHg/min, p=0.002) were observed in the PwMS. No significant between-group differences were observed for OEF (PwMS vs. controls: 0.38 ± 0.09 vs. 0.39 ± 0.02, p=0.358). Regional analysis showed widespread reductions in CMRO2 and DC for PwMS compared to healthy volunteers. There was a significant correlation between physiological measures and T2 lesion volume, but no association with current clinical disability. Our findings demonstrate concurrent reductions in oxygen supply and consumption in the absence of an alteration in oxygen extraction that may be indicative of a reduced demand for oxygen (O2), an impaired transfer of O2 from capillaries to mitochondria, and/or a reduced ability to utilise O2 that is available at the mitochondria. With no between-group differences in GM volume, our results suggest that changes in brain physiology may precede MRI-detectable GM loss and thus may be one of the pathological drivers of neurodegeneration and disease progression

Reduced brain oxygen metabolism in patients with multiple sclerosis: Evidence from dual-calibrated functional MRI

Cerebral energy deficiency is increasingly recognised as an important feature of multiple sclerosis (MS). Until now, we have lacked non-invasive imaging methods to quantify energy utilisation and mitochondrial function in the human brain. Here, we used novel dual-calibrated functional magnetic resonance imaging (dc-fMRI) to map grey-matter (GM) deoxy-haemoglobin sensitive cerebral blood volume (CBVdHb), cerebral blood flow (CBF), oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen consumption (CMRO2) in patients with MS (PwMS) and age/sex matched controls. By integrating a flow-diffusion model of oxygen transport, we evaluated the effective oxygen diffusivity of the capillary network (DC) and the partial pressure of oxygen at the mitochondria (PmO2). Significant between-group differences were observed as decreased CBF (p = 0.010), CMRO2 (p < 0.001) and DC (p = 0.002), and increased PmO2 (p = 0.043) in patients compared to controls. No significant differences were observed for CBVdHb (p = 0.389), OEF (p = 0.358), or GM volume (p = 0.302). Regional analysis showed widespread reductions in CMRO2 and DC for PwMS. Our findings may be indicative of reduced oxygen demand or utilisation in the MS brain and mitochondrial dysfunction. Our results suggest changes in brain physiology may precede MRI-detectable GM loss and may contribute to disease progression and neurodegeneration

Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterized samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries

AMI radio continuum observations of young stellar objects with known outflows

We present 16 GHz (1.9 cm) deep radio continuum observations made with the Arcminute Microkelvin Imager (AMI) of a sample of low-mass young stars driving jets. We combine these new data with archival information from an extensive literature search to examine spectral energy distributions (SEDs) for each source and calculate both the radio and sub-mm spectral indices in two different scenarios: (1) fixing the dust temperature (Td) according to evolutionary class; and (2) allowing Td to vary. We use the results of this analysis to place constraints on the physical mechanisms responsible for the radio emission. From AMI data alone, as well as from model fitting to the full SED in both scenarios, we find that 80 per cent of the objects in this sample have spectral indices consistent with freefree emission. We find an average spectral index in both Td scenarios, consistent with freefree emission. We examine correlations of the radio luminosity with bolometric luminosity, envelope mass and outflow force, and find that these data are consistent with the strong correlation with envelope mass seen in lower luminosity samples. We examine the errors associated with determining the radio luminosity and find that the dominant source of error is the uncertainty on the opacity index, beta. We examine the SEDs for variability in these young objects, and find evidence for possible radio flare events in the histories of L1551 IRS 5 and Serpens SMM 1

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Attitudinal & Knowledge Barriers towards Effective Pain Assessment & Management in Dementia: A Narrative Synthesis

Under-assessment and inadequate treatment of pain is a common problem for older adults, particularly those with dementia. This may be in part attributed to knowledge deficits and negative attitudes among healthcare staff and informal caregivers towards pain, its assessment and its management in dementia. Knowledge and attitudes have a significant predictive relationship with behavior, potentially impacting pain assessment and management practices. Despite this there remains a paucity of research in the area and a lack of clarity about existing knowledge levels and attitudes among dementia caregivers. Therefore, the aims of this review were to: identify what knowledge deficits and attitudinal barriers exist amongst dementia caregivers; and identify the scales available to measure these. A search was carried out in the following electronic databases: Academic Search Premier; CINAHL; Education Research Complete; Humanities International Journals; Psychology and Behavioral Sciences Collection; PsychINFO; PsychArticles; Teacher Reference Center; and MEDLINE. A total of 13 articles met the inclusion criteria. A number of knowledge deficits and negative attitudes were identified, particularly in the use self-reports and pain assessment tools in dementia, and the safety of opioids. Understanding and positive attitudes were demonstrated in some areas, such as non-narcotic pain medications and identifying behavioral pain indicators. Of the 4 scales identified, positive results were found for internal consistency and content validity, however further refinement and testing is necessary. It was concluded attitudinal and knowledge barriers exist which should be addressed given their influence over practice behavior, however, there is a willingness and knowledge base from which progress can build

Natural selection has driven the recurrent loss of an immunity gene that protects Drosophila against a major natural parasite

Polymorphisms in immunity genes can have large effects on susceptibility to infection. To understand the origins of this variation, we have investigated the genetic basis of resistance to the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster. We discovered that increased expression of the gene Lectin-24A after infection by parasitic wasps was associated with a faster cellular immune response and greatly increased rates of killing the parasite. Lectin-24A encodes a protein that is strongly upregulated in the fat body after infection and localizes to the surface of the parasite egg. In certain susceptible lines a deletion upstream of the Lectin-24A has largely abolished expression. Other mutations predicted to abolish the function of this gene have arisen recurrently in this gene, with multiple loss-of-expression alleles and premature stop codons segregating in natural populations. The frequency of these alleles varies greatly geographically, and in some southern African populations natural selection has driven them near to fixation. We conclude that natural selection has favored the repeated loss of an important component of the immune system, suggesting that in some populations a pleiotropic cost to Lectin-24A expression outweighs the benefits of resistance