The roles of serotonin, human urotensin II and calcium sensitivity in the regulation of pulmonary arterial tone

Pulmonary arterial hypertension (PAH) is a rare, progressive fatal disease characterised by persistent vasoconstriction and vascular remodelling of the small pulmonary arteries. 1) The endogenous peptide human urotensin II (hU-II) has recently been found to be a potent vasoconstrictor of the rat main pulmonary artery and the response was reported to be augmented in the chronically hypoxic rat model of PAH. 2) Increased basal Ca2+ sensitivity and enhanced contractile responses to 5-HT, have been reported to be augmented in the chronically hypoxic rat model of PAH. Additionally expression of the 5-HT transporter, 5-HTT, has been reported to be enhanced following exposure to chronic hypoxia and its over expression linked to PAH. These reports are controversial. Aims 1) To profile the vasoconstrictor and vasodilator responses to hU-II along the C57BL/6xCBA mouse and Wistar rat pulmonary arterial trees (extra/intra-lobar) and determine if the contractile activity of hU-II is augmented in rodent models of PAH. 2) To determine if basal Ca2+ sensitivity in the Wistar rat small pulmonary artery is augmented following exposure to chronic hypoxia. 3) To determine the influence of 5-HT and 5-HTT over-expression on Ca2+ sensitivity in Wistar rat, and C57BL/6xCBA mouse, small pulmonary arteries respectively. Results hU-II caused a potent but low efficacy contractile response in Wistar rat extra-lobar (Main pulmonary artery: pEC50; 8.64 +/- 0.17, Emax: 19 +/- 4% of the response to 50mM KC1 11=9, 1st branch; pEC50; 8.57 +/- 0.16, Emax: 9 +/- 2%, n=13) and 1st generation intra-lobar (pECso 8.54 +/- 0.19, Emax: 13 + 3%, n==14) vessels but had essentially no action on the smaller pulmonary arteries. hU-II failed to dilate any pre-constricted rat vessel tested and the contractile responses to hU-II were not augmented in rat models of PAH. Furthermore, hU-II had no effect on any mouse pulmonary vessel tested. 2) Basal Ca2+ sensitivity in beta-escin permeabilised, Wistar rat small pulmonary arteries was found to be slightly attenuated (pECso: Control; 6.33 +/- 0.02 vs CH; 6.24 +/- 0.02, n=10-11) following exposure to 2weeks chi'onic hypoxia, consistent with a decrease in Rho Kinase activity/Rho A expression. Neither 5-HT, nor 5-HTT over-expression augmented Ca2+ sensitivity. Conclusion hU-II can increase pulmonary vascular tone in the normal Wistar rat but the data was not consistent with a role in the pathogenesis of PAH in rodents. Altered Ca2+ sensitivity was not found to be responsible for the persistent elevated basal tone of the chronically hypoxic rat model of PAH. Furthermore 5-HT and 5-HTT overexpression did not exert their effects on vasomotor tone through altered Ca2+ sensitivity

Comparing sediment DNA extraction methods for assessing organic enrichment associated with marine aquaculture

Marine sediments contain a high diversity of micro- and macro-organisms which are important in the functioning of biogeochemical cycles. Traditionally, anthropogenic perturbation has been investigated by identifying macro-organism responses along gradients. Environmental DNA (eDNA) analyses have recently been advocated as a rapid and cost-effective approach to measuring ecological impacts and efforts are underway to incorporate eDNA tools into monitoring. Before these methods can replace or complement existing methods, robustness and repeatability of each analytical step has to be demonstrated. One area that requires further investigation is the selection of sediment DNA extraction method. Environmental DNA sediment samples were obtained along a disturbance gradient adjacent to a Chinook (Oncorhynchus tshawytscha) salmon farm in Otanerau Bay, New Zealand. DNA was extracted using four extraction kits (Qiagen DNeasy PowerSoil, Qiagen DNeasy PowerSoil Pro, Qiagen RNeasy PowerSoil Total RNA/DNA extraction/elution and Favorgen FavorPrep Soil DNA Isolation Midi Kit) and three sediment volumes (0.25, 2, and 5 g). Prokaryotic and eukaryotic communities were amplified using primers targeting the 16S and 18S ribosomal RNA genes, respectively, and were sequenced on an Illumina MiSeq. Diversity and community composition estimates were obtained from each extraction kit, as well as their relative performance in established metabarcoding biotic indices. Differences were observed in the quality and quantity of the extracted DNA amongst kits with the two Qiagen DNeasy PowerSoil kits performing best. Significant differences were observed in both prokaryotes and eukaryotes (p < 0.001) richness among kits. A small proportion of amplicon sequence variants (ASVs) were shared amongst the kits (~3%) although these shared ASVs accounted for the majority of sequence reads (prokaryotes: 59.9%, eukaryotes: 67.2%). Differences were observed in the richness and relative abundance of taxonomic classes revealed with each kit. Multivariate analysis showed that there was a significant interaction between “distance” from the farm and “kit” in explaining the composition of the communities, with the distance from the farm being a stronger determinant of community composition. Comparison of the kits against the bacterial and eukaryotic metabarcoding biotic index suggested that all kits showed similar patterns along the environmental gradient. Overall, we advocate for the use of Qiagen DNeasy PowerSoil kits for use when characterizing prokaryotic and eukaryotic eDNA from marine farm sediments. We base this conclusion on the higher DNA quality values and richness achieved with these kits compared to the other kits/amounts investigated in this study. The additional advantage of the PowerSoil Kits is that DNA extractions can be performed using an extractor robot, offering additional standardization and reproducibility of results.publishedVersio

Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms

We have studied a cohort of nemaline myopathy (NM) patients with mutations in the muscle α-skeletal actin gene (ACTA1). Immunoblot analysis of patient muscle demonstrates increased γ-filamin, myotilin, desmin and α-actinin in many NM patients, consistent with accumulation of Z line-derived nemaline bodies. We demonstrate that nebulin can appear abnormal secondary to a primary defect in actin, and show by isoelectric focusing that mutant actin isoforms are present within insoluble actin filaments isolated from muscle from two ACTA1 NM patients. Transfection of C2C12 myoblasts with mutant actinEGFP constructs resulted in abnormal cytoplasmic and intranuclear actin aggregates. Intranuclear aggregates were observed with V163L-, V163M- and R183G-actinEGFP constructs, and modeling shows these residues to be adjacent to the nuclear export signal of actin. V163L and V163M actin mutants are known to cause intranuclear rod myopathy, however, intranuclear bodies were not reported in patient R183G. Transfection studies in C2C12 myoblasts showed significant alterations in the ability of V136L and R183G actin mutants to polymerize and contribute to insoluble actin filaments. Thus, we provide direct evidence for a dominant-negative effect of mutant actin in NM. In vitro studies suggest that abnormal folding, altered polymerization and aggregation of mutant actin isoforms are common properties of NM ACTA1 mutants. Some of these effects are mutation-specific, and likely result in variations in the severity of muscle weakness seen in individual patients. A combination of these effects contributes to the common pathological hallmarks of NM, namely intranuclear and cytoplasmic rod formation, accumulation of thin filaments and myofibrillar disorganizatio

Nemaline myopathy type 6: clinical and myopathological features

Nemaline myopathy (NEM) is one of the most common congenital myopathies. A unique subtype, NEM6, maps to chromosome 15q21-q23 in two pedigrees, but the causative gene has not been determined. We conducted clinical examination and myopathological studies in a new NEM family. Genotyping and gene screening were accomplished by searching known and 18 new candidate genes. The disease started in childhood by affecting proximal and distal muscles and causing slowness of movements. Muscle biopsies showed numerous nemaline rods and core-like formations. Suggestive linkage to chromosome 15q22-q23 was established. Genes known to be mutated in NEM or core-rod myopathy were screened and excluded. No pathogenic mutations were identified in other candidate genes. The disease in this Spanish family was classified as NEM6. It is phenotypically similar and probably allelic to the two previously reported NEM6 pedigrees. Further studies of these families will lead to the identification of the NEM6 gene

Motor neuron diseases caused by a novel VRK1 variant – A genotype/phenotype study

Background: Motor neuron disorders involving upper and lower neurons are a genetically and clinically heterogenous group of rare neuromuscular disorders with overlap among spinal muscular atrophies (SMAs) and amyotrophic lateral sclerosis (ALS). Classical SMA caused by recessive mutations in SMN1 is one of the most common genetic causes of mortality in infants. It is characterized by degeneration of anterior horn cells in the spinal cord, leading to progressive muscle weakness and atrophy. Non-SMN1-related spinal muscular atrophies are caused by variants in a number of genes, including VRK1, encoding the vaccinia- related kinase 1 (VRK1). VRK1 variants have been segregated with motor neuron diseases including SMA phenotypes or hereditary complex motor and sensory axonal neuropathy (HMSN), with or without pontocerebellar hypoplasia or microcephaly. Results: Here, we report an association of a novel homozygous splice variant in VRK1 (c.1159 + 1G>A) with childhood-onset SMA or juvenile lower motor disease with brisk tendon reflexes without pontocerebellar hypoplasia and normal intellectual ability in a family with five affected individuals. We show that the VRK1 splice variant in patients causes decreased splicing efficiency and a mRNA frameshift that escapes the nonsensemediated decay machinery and results in a premature termination codon. Conclusions: Our findings unveil the impact of the variant on the VRK1 transcript and further support the implication of VRK1 in the pathogenesis of lower motor neuron diseases

Mutation update and genotype-phenotype correlations of novel and previously described mutations in TPM2 and TPM3 causing congenital myopathies

Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy, cap myopathy, core-rod myopathy, congenital fiber-type disproportion, distal arthrogryposes, and Escobar syndrome. We correlate the clinical picture of these diseases with novel (19) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Thirty distinct pathogenic variants of TPM2 and 20 of TPM3 have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal-dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca2+ sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotype more often had contractures of the limb joints (18/19) and jaw (6/19) than those with nonhypercontractile ones (2/22 and 1/22), whereas patients with the non-hypercontractile molecular phenotype more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin–actin association or tropomyosin head-to-tail binding

Surgical management of non-metastatic pancreatic cancer in the United Kingdom: results of a nationwide survey on current practice

Background: It is presently unclear what clinical pathways are followed for patients with non-metastatic PDAC in specialised centres for pancreatic surgery across the United Kingdom (UK). Methods: Between August 2019 and August 2020 an electronic survey was conducted aiming at a national cohort of pancreatic surgeons in the UK. Participants replied to a list of standardised questions and clinical vignettes, and data were collected and analysed focusing on management preferences, resectability criteria, and contraindications to surgery. Results: Within the study period, 65 pancreatic surgeons from 27 specialist centres in the UK (96%) completed the survey. Multidisciplinary team meetings are utilised universally for the management of patients with PDAC, however, different staging systems for resectability classification are being applied. In borderline resectable PDAC, most surgeons were keen to proceed with surgical exploration post NAT, but differences were noted in preferred chemotherapy regimens. Surgeons from standard volume institutions performed fewer vein resections annually and were more likely to deem patients with locally advanced PDAC as unresectable. Intra-institutional variability in patient management was also present and ranging between 20-80%. Conclusions: Significant variability in the surgical management of non-metastatic PDAC was identified both on inter- and intra-institutional level

Nemaline Myopathy in Brazilian Patients: Molecular and Clinical Characterization

Nemaline myopathy (NM), a structural congenital myopathy, presents a significant clinical and genetic heterogeneity. Here, we compiled molecular and clinical data of 30 Brazilian patients from 25 unrelated families. Next-generation sequencing was able to genetically classify all patients: sixteen families (64%) with mutation in NEB, five (20%) in ACTA1, two (8%) in KLHL40, and one in TPM2 (4%) and TPM3 (4%). In the NEB-related families, 25 different variants, 11 of them novel, were identified; splice site (10/25) and frame shift (9/25) mutations were the most common. Mutation c.24579 G>C was recurrent in three unrelated patients from the same region, suggesting a common ancestor. Clinically, the “typical” form was the more frequent and caused by mutations in the different NM genes. Phenotypic heterogeneity was observed among patients with mutations in the same gene. Respiratory involvement was very common and often out of proportion with limb weakness. Muscle MRI patterns showed variability within the forms and genes, which was related to the severity of the weakness. Considering the high frequency of NEB mutations and the complexity of this gene, NGS tools should be combined with CNV identification, especially in patients with a likely non-identified second mutation

Nemaline Myopathy in Brazilian Patients: Molecular and Clinical Characterization

Nemaline myopathy (NM), a structural congenital myopathy, presents a significant clinical and genetic heterogeneity. Here, we compiled molecular and clinical data of 30 Brazilian patients from 25 unrelated families. Next-generation sequencing was able to genetically classify all patients: sixteen families (64%) with mutation in NEB, five (20%) in ACTA1, two (8%) in KLHL40, and one in TPM2 (4%) and TPM3 (4%). In the NEB-related families, 25 different variants, 11 of them novel, were identified; splice site (10/25) and frame shift (9/25) mutations were the most common. Mutation c.24579 G>C was recurrent in three unrelated patients from the same region, suggesting a common ancestor. Clinically, the “typical” form was the more frequent and caused by mutations in the different NM genes. Phenotypic heterogeneity was observed among patients with mutations in the same gene. Respiratory involvement was very common and often out of proportion with limb weakness. Muscle MRI patterns showed variability within the forms and genes, which was related to the severity of the weakness. Considering the high frequency of NEB mutations and the complexity of this gene, NGS tools should be combined with CNV identification, especially in patients with a likely non-identified second mutation

Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing

More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. We describe the use of programmable targeted long-read sequencing with Oxford Nanopore's ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort (n = 37) including patients with various neurogenetic diseases (n = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care