Introduction
Mutational analysis is commonly used to support the diagnosis and management of haemophilia. This has allowed for the generation of large mutation databases which provide unparalleled ...insight into genotype–phenotype relationships. Haemophilia is associated with inversions, deletions, insertions, nonsense and missense mutations. Both synonymous and non‐synonymous mutations influence the base pairing of messenger RNA (mRNA), which can alter mRNA structure, cellular half‐life and ribosome processivity/elongation. However, the role of mRNA structure in determining the pathogenicity of point mutations in haemophilia has not been evaluated.
Aim
To evaluate mRNA thermodynamic stability and associated RNA prediction software as a means to distinguish between neutral and disease‐associated mutations in haemophilia.
Methods
Five mRNA structure prediction software programs were used to assess the thermodynamic stability of mRNA fragments carrying neutral vs. disease‐associated and synonymous vs. non‐synonymous point mutations in F8, F9 and a third X‐linked gene, DMD (dystrophin).
Results
In F8 and DMD, disease‐associated mutations tend to occur in more structurally stable mRNA regions, represented by lower MFE (minimum free energy) levels. In comparing multiple software packages for mRNA structure prediction, a 101–151 nucleotide fragment length appears to be a feasible range for structuring future studies.
Conclusion
mRNA thermodynamic stability is one predictive characteristic, which when combined with other RNA and protein features, may offer significant insight when screening sequencing data for novel disease‐associated mutations. Our results also suggest potential utility in evaluating the mRNA thermodynamic stability profile of a gene when determining the viability of interchanging codons for biological and therapeutic applications.
Recently, pretreatment with immune checkpoint inhibitors (ICIs) has been shown to enhance the therapeutic effects of the combination therapy of ramucirumab (RAM) and docetaxel (DTX); however, its ...influence on the drug's side effects remains unclear. This study investigated the influence
of pretreatment with ICIs on the incidence of neutropenia caused by RAM + DTX therapy in patients with non-small cell lung cancer (NSCLC). Patients with NSCLC who received RAM + DTX therapy at Gifu Prefectural General Medical Center between April 2016 and December 2020 were enrolled. Retrospective
data regarding age, sex, performance status and detailed treatment history, among others, at treatment initiation were collected from the patients' electronic medical records. Additionally, data on the course number of RAM + DTX therapy, supportive therapy and blood biochemical parameters,
including leukocyte and neutrocyte counts, during the treatment period were collected. We identified 41 patients receiving RAM + DTX therapy. Among the more than grade 3 adverse events caused by this therapy, neutropenia was the most common (78.1%). Despite the fact that all previous risk
factors influencing this incidence rate had corresponded, the only factor influencing the incidence rate of neutropenia more than grade 3 was ICI treatment history. A difference in the incidence of neutropenia more than grade 3 in the Kaplan-Meier curve was observed between patients with and
without ICI pretreatment history (p = 0.037). The pretreatment history of ICI therapy affects the incidence of neutropenia caused by RAM + DTX therapy in patients with NSCLC.
Summary
Inhibitors are an impediment to the effective management of haemophilia B (HB), but there is limited understanding of the underlying genetic risk factors. In this study we aim to understand ...the role of F9 gene mutations on inhibitor development in patients with HB. Mutations in the F9 gene were identified and HLA typing performed for five boys with severe HB. Data from the CDC Haemophilia B Mutation Project (CHBMP) database were used to assess association between F9 gene mutation type and inhibitor development. Analysis of the CHBMP database showed that larger disruptions in the F9 gene are associated with a higher life‐time prevalence of inhibitors. We detected the following mutations in the five subjects, including four novel mutations: Nonsense in three patients (c.223 C>T; p.Arg75* in two siblings, c.553 C>T; p.Glu185*); Splice site in two patients (c.723 + 1 G>A, c.278‐27 A>G); Missense in one patient (c.580 A>G, p.Thr194Ala; c.723 G>T; p.Gln241His). Of the two siblings only one responded to immune tolerance induction (ITI). These siblings have identical F9 gene mutations but differ with respect to the HLA alleles. Interestingly, an analysis of peptide‐MHC binding affinities shows a significantly higher (one‐sided unpaired t‐test, P = 0.0018) median affinity for FIX‐derived peptides in the sibling that responded to ITI. We conclude that the nature of the F9 gene mutation may be an important risk factor for the development of inhibitors. In addition, the HLA alleles of the individual patients, in conjunction with the mutation type, could be a predictor for the development of inhibitors as well as the response to ITI.
The dynamic behaviors of unstirred dead-end microfiltration were explored under constant pressure conditions using a binary dispersion of oil-in-water (O/W) emulsion and polymethyl-methacrylate ...(PMMA) slurry, which is referred to as emulsion–slurry. The Ruth filtration coefficient determined from the flux decline data increased steadily during the course of filtration, and then a bilayer filter cake formed composed of the upper emulsion cake and the lower PMMA cake. Each average specific filtration resistance of the emulsion cake and the PMMA cake was well evaluated in terms of the resistance-in-series model in filtration. The PMMA cake was nearly incompressible, while the emulsion cake formed on the PMMA cake was highly compressible. Only the emulsion cake was consolidated without the cake cracking or the coalescence of oil droplets. The compressed cake of gel–emulsion had a porosity that was much lower than that of hexagonal close packing of undistorted spheres because of the remarkable deformation of oil droplets under the action of the compressive pressure. It was clearly demonstrated that the gel–emulsion cake formed in filtration of emulsion–slurry was exactly similar in nature to that formed in filtration of mono-dispersed O/W emulsion.
Yeast cells were immobilized on calcium alginate beads prepared using different calcium concentrations. The compression properties of the immobilized beads (e.g., softness index and retardation time ...for compression) were strongly affected by the calcium concentration. The effects of the bead properties on filtration characteristics, such as cake porosity, specific cake filtration resistance, cake compression creeping effect and cake compressibility, were analysed using a dead-end filtration system. The filtration curve of yeast-immobilized beads had an "S" shape, similar to that of soft gel particles. The cake compression behaviour and variation in cake properties were directly reflected on the curve trend. The Voigt in the series model was employed to describe variation in cake porosity with time during a compression. The yeast immobilization increased the bead softness; therefore, the porosity of a cake formed by yeast-immobilized beads was lower than that formed by pure calcium alginate beads. The cakes formed by yeast-immobilized beads possessed a high compressibility of approximately 1.0 and a high softness index of approximately 1.5. The beads prepared using lower calcium concentrations had higher softness, shorter retardation times for compression, higher cake compressibility, lower cake porosity and higher specific cake filtration resistance. The results demonstrated that immobilizing yeast cells on calcium alginate beads is beneficial for retaining higher yeast activity than that of freely suspended yeast. However, the activity levels of yeast immobilized using different calcium concentrations were nearly the same after 3 h. Therefore, using high concentrations of calcium for yeast immobilization is beneficial for improving yeast activity and filtration characteristics.
Summary
Haemophilia B is an X‐linked recessive disorder caused by deficiency of functional coagulation factor IX, which results almost exclusively from mutations in the F9 gene. We sought to ...determine features, which could distinguish between mutations that cause severe disease symptoms from those that cause non‐severe disease symptoms. Towards this objective, we have performed a statistical analysis of reported point mutations in F9. These include: potential local changes in mRNA free energy, codon usage, charge and type of mutated amino acid, location of the mutation with regard to protein secondary structure and functional domain and amino acids' evolutionary conservation scores. Wilcoxon signed‐rank tests showed highly significant differences between severe and non‐severe disease causing mutations in their effect on free energy of small mRNA fragments and evolutionarily conserved amino acids. Our results suggest that information at the mRNA level as well as conservation of the amino acid correlate well with disease severity. This study demonstrates that computational tools may be used to characterize the severity of haemophilia B associated with point mutations and suggests their utility in predicting the outcome of sequence changes in recombinant proteins.
The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G ...protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p). Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 μm and Hill coefficient of 1.9. Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch. The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein. Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes. The ncs1 deletion mutant (ncs1Δ) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular Ca2+ (0.1 m CaCl2). Analysis of Ncs1p mutants lacking myristoylation (Ncs1pG2A) or deficient in Ca2+ binding (Ncs1pE84Q/E120Q/E168Q) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1Δ, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p. Starvation-independent sexual development of ncs1Δ was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin. In contrast, the Ca2+ intolerance of ncs1Δ was not affected by cAMP or recoverin, suggesting that the two ncs1Δ phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization of Git3p.
Abstract We performed a combined neurochemical and behavioral study to determine the effects of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BnTIQ) on the extracellular dopamine concentrations in the ...striatum. Single dose administration of 1-BnTIQ (20, 40, and 80 mg/kg i.p.) increased striatal dopamine extracellular levels in a dose-dependent manner when an in vivo microdialysis technique was used to assess dopamine levels in the striatum of rats. Enhancement of striatal dopamine levels by systemic administration of a single dose of 1-BnTIQ was suppressed by perfusion of tetrodotoxin and a calcium ion-free solution into the striatum. This 1-BnTIQ-induced increase in extracellular dopamine concentration was also inhibited by pre-treatment with a dopamine uptake inhibitor, GBR12909 (1-(2-bis(4-Fluorophenyl)-4-(3-phenylpropyl)piperazine dihydrochloride). Local application of 1-BnTIQ into the striatum via a dialysis probe failed to enhance the extracellular concentration of dopamine. However, microinjection of 1-BnTIQ into the substantia nigra pars compacta increased the extracellular dopamine levels in the striatum. Locomotor activity was increased by systemic administration of a single dose of 1-BnTIQ in a dose-dependent manner. This 1-BnTIQ-induced locomotor activity was attenuated by pre-treatment with SCH23390 (R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochlodride) and raclopride, D1 and D2 dopaminergic receptor antagonists, respectively. Moreover, 1-BnTIQ induced ipsilateral rotational behavior in 6-hydroxydopamine–lesioned rats. These results suggest that systemic administration of a single dose of 1-BnTIQ increases striatal extracellular dopamine concentration through activation of dopaminergic nigra striatal neurons via the dopamine transporter.
Abstract
Background
We are developing a novel autologous tissue-engineered heart valve with a unique in-body tissue engineering. This is expected to be a viable bioprosthesis keeping better ...biocompatibility.
Purpose
We developed a conduit-type valve without any foreign materials and tested the feasibility and long-term availability in large animal experiments.
Methods
We created plastic molds for Biovalves with 3D printer easily and quickly considering the recipient character. We embedded them in the subcutaneous spaces of adult goats about 2 months. After extracting the molds with the tissue en-block and removing the plastic molds only, Biovalve with tri-leaflets similar to those of the native valves were constituted from completely autologous connective tissues and fibroblasts. Total 21 conduit-type Biovalves were implanted in the apico-aortic bypass or the pulmonary artery of goats, (8 and 13, respectively). No anticoagurants were used after implantation.
Results
The valves were successfully implanted and showed smooth movement of the leaflets with a little regurgitation in angiogram, and the maximum duration reached to 3 years 7 months. Histological examination of the Biovalves showed the autologous cells covering the laminar surface of the valve leaflets as the endothelium and also getting inside to construct characteristic tissues like native leaflets.
Conclusion
The valves have a potential to be used for viable bioprosthetic valves and to keep better function and biocompatibility longer than current ones.
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