Studies of the synthesis, environmental occurrence and toxicity of unresolved complex mixtures (UCMs) of hydrocarbons

Merged with duplicate record (10026.1/606) on 03.01.2017 by CS (TIS)This is a digitised version of a thesis that was deposited in the University Library. If you are the author please contact PEARL Admin ([email protected]) to discuss options.The occurrence of unresolved complex mixtures of hydrocarbons (UCMs) in the aliphatic fraction of marine sediments and organisms from areas impacted by petroleum hydrocarbons is well documented and widely accepted as an indication of fossil fuel contamination. In contrast, the presence of an aromatic UCM is often ignored and environmental concentrations of aromatic UCM hydrocarbons in marine biota and sediments are rarely reported. The aims of this study were to establish the quantitative significance of aromatic UCMs in environmental samples and to assess the toxicological significance of both aliphatic and aromatic UCMs. A reproducible method was developed and validated for the analysis and quantification of petroleum hydrocarbons in mussel (Mytilus edulis) tissue. Emphasis was placed upon development of a method which minimized losses of more volatile, lower molecular weight, toxicologically significant hydrocarbons, without compromising recovery of higher molecular weight compounds which are useful for source identification in environmental monitoring schemes. Analysis of mussels from a small number of U. K. coastal locations indicated that aromatic hydrocarbon UCMs may form a significant proportion (ca 20 %) of the total hydrocarbon body burden of mussels from areas contaminated with petroleum hydrocarbons. Aromatic UCM hydrocarbons were not observed in mussels from relatively uncontaminated areas but concentrations of 430 μg g'' (dry wt tissue) were measured in mussels from heavily impacted areas. Aliphatic UCM concentrations ranged from 7- 3445 μg gg' (dry wt tissue). For the purposes of toxicological studies, a low molecular weight model aliphatic UCM hydrocarbon, 4-propyloctane (4-PO) was synthesised. Two low molecular weight model aromatic hydrocarbons 7- cyclohexyltetralin and 7-cyclohexyl-l-propyltetralin were also synthesised using a modification of the Haworth synthesis. All three target compounds and synthetic intermediates were characterised by NMR, MSandIR. Exposure of M. edulis to 4-PO caused a significant reduction in mussel ciliary feeding activity indicating that 4-PO was indeed toxic as measured by this bioassay. The demonstrable narcotic activity of 4-PO is presumably related to the greater aqueous solubility of branched hydrocarbons compared with similar straight chain hydrocarbons. Further experiments investigating the effect of 4- PO over exposure periods up to 120h provided a unique and detailed insight into the relationship between concentration of toxicant in the gills of M. edulis and observed feeding rate. The established method of mussel feeding rate determination was modified in light of this relationship to produce an " improved rapid and reproducible screening technique. Both of the model aromatic UCM hydrocarbons were also found to be toxic to mussel ciliary feeding activity. This appears to be the first report of investigations into the toxicity of the aromatic UCM and suggests that previous studies have ignored an environmental burden of toxicological significance. Estimates of the tissue effective concentration (TEC50) for the model UCM hydrocarbons gave comparable values with those reported for the effect of other narcotic hydrocarbons upon mussel feeding rate, providing support for the theory that non-specific narcosis occurs at a relatively constant tissue concentration of toxicant. The demonstrated narcotic activity of each of the three model UCM hydrocarbons has extended the molecular weight range of narcotic hydrocarbons studied to date. The results presented herein suggest that a small proportion of low molecular weight aliphatic UCMs and perhaps a greater proportion of aromatic UCMs are of toxicological significance.the Natural Environment Research Council in collaboration with Plymouth Marine Laborator

Surgery for scoliosis in Duchenne muscular dystrophy

published_or_final_versio

Surgery for scoliosis in Duchenne muscular dystrophy

Reviewpublished_or_final_versio

Congenital Insensitivity to Pain: Novel SCN9A Missense and In-Frame Deletion Mutations

SCN9A encodes the voltage-gated sodium channel Nav1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Nav1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Nav1.7-ΔR1370-L1374). Both of these mutations map to the pore region of the Nav1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Nav1.7. © 2010 Wiley-Liss, Inc

Risdiplam in Spinal Muscular Atrophy: Safety Profile and Use Through The Early Access to Medicine Scheme for the Paediatric Cohort in Great Britain

BACKGROUND: Spinal muscular atrophy (SMA) is a progressive neuromuscular disease caused by mutations in Survival motor neuron 1 (SMN1) gene, leading to reduction in survival motor neuron protein (SMN), key for motor neuron survival and function in the brainstem and spinal cord. Risdiplam is an orally administered SMN2-splicing modifier which increases production of functional SMN protein. Risdiplam was offered in the UK under early access to medicines scheme (EAMS) to SMA type 1 and 2 patients aged 2 months and older, not suitable for authorised treatments from September 2020 to December 2021. OBJECTIVE: To describe the largest paediatric European real-world set of data on patients' characteristics and short-term safety for risdiplam in Great Britain through EAMS. METHODS: We collated data from SMA REACH UK a national clinical and research network for all patients enrolled onto EAMS and assessed all submitted adverse events. RESULTS: Of the 92 patients; 78% were Type 2 SMA, mean age 10.9 years, range 0-17 years. 56 were treatment naïve, 33 previously treated; of these 25 had received nusinersen, 3 previous treatment unknown. Sixty adverse events (AEs) were reported occurring in 34 patients. The commonest were respiratory tract infections and gastrointestinal disturbance. Four life-threatening events were reported with 2 deaths and permanent cessation of risdiplam in 3 patients.Overall, 38/60 AEs were considered unrelated to risdiplam, 10/60 related to risdiplam and for 12/60 causality not specified. CONCLUSIONS: This study found a safety profile similar to clinical trials with no new safety concerns identified. With the restricted eligibility of onasemnogene abeparvovec and complications of nusinersen administration, EAMS allowed access or continued treatment to naïve patients or patients no longer suitable for approved medications. Collection of longitudinal data for this complex population is needed, to provide greater insights into risdiplam's role in addressing patients' needs into the future

Efficacy and safety of onasemnogene abeparvovec in children with spinal muscular atrophy type 1: real-world evidence from 6 infusion centres in the United Kingdom

Background: Real-world data on the efficacy and safety of onasemnogene abeparvovec (OA) in spinal muscular atrophy (SMA) are needed, especially to overcome uncertainties around its use in older and heavier children. This study evaluated the efficacy and safety of OA in patients with SMA type 1 in the UK, including patients ≥2 years old and weighing ≥13.5 kg. / Methods: This observational cohort study used data from patients with genetically confirmed SMA type 1 treated with OA between May 2021 and January 2023, at 6 infusion centres in the United Kingdom. Functional outcomes were assessed using age-appropriate functional scales. Safety analyses included review of liver function, platelet count, cardiac assessments, and steroid requirements. / Findings: Ninety-nine patients (45 SMA therapy-naïve) were treated with OA (median age at infusion: 10 [range, 0.6–89] months; median weight: 7.86 [range, 3.2–20.2] kg; duration of follow-up: 3–22 months). After OA infusion, mean ± SD change in CHOP-INTEND score was 11.0 ± 10.3 with increased score in 66/78 patients (84.6%); patients aged 100 U/L (95% CI, 2.3–223.7; P = 0.008) and 21.2-fold increased odds of steroid doubling, as per treatment protocol (95% CI, 2.2–209.2; P = 0.009) in patients weighing ≥13.5 kg versus <8.5 kg. Weight at infusion was positively correlated with steroid treatment duration (r = 0.43; P < 0.001). Worsening transaminitis, despite doubling of oral prednisolone, led to treatment with intravenous methylprednisolone in 5 children. Steroid-sparing immunosuppressants were used in 5 children to enable steroid weaning. Two deaths apparently unrelated to OA were reported. / Interpretation: OA led to functional improvements and was well tolerated with no persistent clinical complications, including in older and heavier patients. / Funding: Novartis Innovative Therapies AG provided a grant for independent medical writing services

Identification of C 25 highly branched isoprenoid (HBI) alkenes in diatoms of the genus Rhizosolenia in polar and sub-polar marine phytoplankton.

We report the identification of a range of C25 highly branched isoprenoid (HBI) alkenes and certain sterols in filtered phytoplankton samples obtained from western Svalbard (Arctic) and near South Georgia (South Atlantic, sub-Antarctic) in 2016 and 2014, respectively. The C25 HBIs contained 3–5 double bonds and had structures identified previously from analysis of laboratory diatom cultures. The same HBIs were also identified in individual diatom taxa isolated from the mixed assemblages and with reasonably similar distributions. Thus, C25 HBIs were identified in Rhizosolenia setigera isolated from western Svalbard near-surface waters, while the same HBIs were also found in R. polydactyla f. polydactyla and R. hebetata f. semispina picked from seawater collected from a site in the South Atlantic. The main sterol composition was slightly different between the two locations, with cholesta-5,24-dien-3β-ol (desmosterol) identified as one of the major components in the sample from West Svalbard, consistent with the diatom assemblage being dominated by R. setigera. In contrast, the major sterol in the South Atlantic sample was cholesta-5,22-dien-3β-ol (22-dehydrocholesterol), likely reflecting the relatively high proportion of the genus Pseudo-nitzschia. For both locations, the suite of HBIs included a tri-unsaturated isomer (HBI III; 6Z-2,6,10,14-tetramethyl-9-(3'-methylpent-4-enylidene)-pentadec-6-ene), proposed in previous studies as a potential proxy measure of pelagic sea ice-edge conditions, and thus, a counterpart to the mono- and di-unsaturated HBIs IP25 and IPSO25, which have been used as seasonal sea ice proxies in the Arctic and Antarctic, respectively. HBI III has been reported previously in sediments from West Svalbard and we report here its occurrence in a small number of surface sediments from the South Atlantic. For both regions, HBI III was present as one of the major HBIs in sediments, which contrasts the HBI distributions in the filtered phytoplankton samples, where HBIs with four and five double bonds were the major components. Differences in HBI distributions between phytoplankton and sediment samples may potentially be due to the presence of other (unanalysed) diatoms in the filtered water samples, seasonal/annual variability in the production of HBIs by a range of diatoms, differential degradation of HBIs between sources and sediments, or a combination of these. Interestingly, we did not detect any C30 HBIs in the water samples, picked cells or sediments from either location, despite earlier reports of these lipids in laboratory cultures of R. setigera. This study represents the first source identification of certain C25 HBI lipids under in situ pelagic conditions

Mutations in GDP-mannose pyrophosphorylase b cause congenital and limb-girdle muscular dystrophies associated with hypoglycosylation of α-dystroglycan

Congenital muscular dystrophies with hypoglycosylation of α-dystroglycan (α-DG) are a heterogeneous group of disorders often associated with brain and eye defects in addition to muscular dystrophy. Causative variants in 14 genes thought to be involved in the glycosylation of α-DG have been identified thus far. Allelic mutations in these genes might also cause milder limb-girdle muscular dystrophy phenotypes. Using a combination of exome and Sanger sequencing in eight unrelated individuals, we present evidence that mutations in guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) can result in muscular dystrophy variants with hypoglycosylated α-DG. GMPPB catalyzes the formation of GDP-mannose from GTP and mannose-1-phosphate. GDP-mannose is required for O-mannosylation of proteins, including α-DG, and it is the substrate of cytosolic mannosyltransferases. We found reduced α-DG glycosylation in the muscle biopsies of affected individuals and in available fibroblasts. Overexpression of wild-type GMPPB in fibroblasts from an affected individual partially restored glycosylation of α-DG. Whereas wild-type GMPPB localized to the cytoplasm, five of the identified missense mutations caused formation of aggregates in the cytoplasm or near membrane protrusions. Additionally, knockdown of the GMPPB ortholog in zebrafish caused structural muscle defects with decreased motility, eye abnormalities, and reduced glycosylation of α-DG. Together, these data indicate that GMPPB mutations are responsible for congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-DG. © 2013 The American Society of Human Genetics.Funding for UK10K was provided by the Wellcome Trust under award WT091310

Digenic inheritance involving a muscle-specific protein kinase and the giant titin protein causes a skeletal muscle myopathy.

In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3-/-; ttn.1+/-) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases