A Monoallelic Variant in REST Is Associated with Non-Syndromic Autosomal Dominant Hearing Impairment in a South African Family

Hearing impairment (HI) is a sensory disorder with a prevalence of 0.0055 live births in South Africa. DNA samples from a South African family presenting with progressive, autosomal dominant non-syndromic HI were subjected to whole-exome sequencing, and a novel monoallelic variant in REST [c.1244GC; p.(C415S)], was identified as the putative causative variant. The co-segregation of the variant was confirmed with Sanger Sequencing. The variant is absent from databases, 103 healthy South African controls, and 52 South African probands with isolated HI. In silico analysis indicates that the p.C415S variant in REST substitutes a conserved cysteine and results in changes to the surrounding secondary structure and the disulphide bonds, culminating in alteration of the tertiary structure of REST. Localization studies using ectopically expressed GFP-tagged Wild type (WT) and mutant REST in HEK-293 cells show that WT REST localizes exclusively to the nucleus; however, the mutant protein localizes throughout the cell. Additionally, mutant REST has an impaired ability to repress its known target AF1q. The data demonstrates that the identified mutation compromises the function of REST and support its implication in HI. This study is the second report, worldwide, to implicate REST in HI and suggests that it should be included in diagnostic HI panels.

Poultry feather disulphide bond breakdown to enable bio-based polymer production

With oil supplies, needed for plastic production, decreasing dramatically, there is a clear driver for alterative polymers from sustainable resources. Poultry feathers, containing ∼90% keratin, are one source of natural polymer with huge potential for biopolymer production. However, the presence of crosslinks, known as disulphide bonds, hinders processability. This paper reviews techniques to enable breakage of disulphide bonds through use of reduction agents (sodium sulphite and sodium sulphate) and hydrolysis. Samples were analysed using FTIR and DSC to quantify achievable bond breakage, effect on thermal properties and changes in protein concentration. A review on the effect of particle size on disulphide bond breakage was also conducted, along with quantifying the reformation of bonds post-processing. Finally, a bicinchoninic acid (BCA) protein assay was used to quantify changes to soluble protein content, key to predicting if biopolymer formation can occur. The results showed a final disulphide bond breakage of between 48% and 67% was achievable using these techniques. It was also shown that disulphide bond content exhibited up to 60% bond reformation post treatment. These reductions in disulphide bonds increased the thermoplastic nature and apparent protein content. Despite achieving the highest bond breakage percentage, hydrolysis caused degradation of useful proteins, rendering the material unsuitable for biopolymer production. Results suggested that treatment with sodium sulphite (4.3% wt. of feathers) and use of a small particle size (0–100 µm), sufficiently altered the properties of raw feathers to enable feather biopolymer production.

Influence of the Presence of Disulphide Bonds in Aromatic or Aliphatic Dicarboxylic Acid Hardeners Used to Produce Reprocessable Epoxidized Thermosets

The design of polymers from renewable resources with recycling potential comes from economic and environmental problems. This work focused on the impact of disulphide bonds in the dicarboxylic acids reactions with three epoxidized vegetable oils (EVOs). For the first time, the comparison between aromatic vs. aliphatic dicarboxylic acids, containing or not S–S bonds with EVOs was discussed and evaluated by dynamic scanning calorimetry. The obtained thermosets showed reprocessability, by the dual dynamic exchange mechanism. The virgin and reprocessed materials were characterized and the thermomechanical properties were compared. The thermosets derived from EVOs with high epoxy content combined with aromatic diacids containing disulphide bridges showed high glass transition values (~111 °C), high crosslink densities and good solvent stability.

Impact of microfluidization on colloidal properties of insoluble pea protein fractions

Microfluidization is a technique commonly used to disrupt and homogenize dispersions such as oil-in-water emulsions or cellular suspensions. In this study, we investigated its ability to alter the physicochemical properties of plant-derived insoluble protein aggregates such as those found in pea protein extracts. Insoluble pea protein dispersions (5% w/w, pH 7) were homogenized at 25–150 MPa for 1–5 cycles. Increasing the homogenization pressure and cycles decreased the particle size (d43) of the unhomogenized insoluble pea proteins from 180 ± 40 μm to 0.2 ± 0.0 μm (at ≥ 125 MPa), leading to more transparent dispersions. Furthermore, the solubility of the insoluble pea proteins increased from 23 ± 1% to 86 ± 4%. Treatments with chaotropic agents, dithiothreitol and urea, revealed that insoluble pea protein aggregates were stabilized not only by disulphide bonds but also by hydrogen bonds and hydrophobic interactions. These molecular interactions were disrupted by microfluidization. The study provides insights into the disruption mechanism of insoluble pea proteins by applying microfluidization and offers a mean to improve their technofunctional properties to facilitate further use in food manufacture.

Loss of Cysteine Rich domain was critical for evolution of heterodimerization in Toll proteins

Toll proteins play roles in immunity/development which have largely remained conserved. However, there are differences in Toll biology as mammalian TLRs recognise pathogen associated molecular patterns (PAMPs) but not their invertebrate homologues. The reason for the same is not known. One critical molecular difference is absence of Cysteine Rich Domain (CRD) in vertebrate Tolls and their presence in invertebrates. Interestingly, in Drosophila, all Toll proteins have CRD except Toll9. This provided us the appropriate model to investigate significance of loss of CRD in Toll evolution. CRDs nudge protein dimerization by forming disulphide bonds hence we asked if they did same in Drosophila Toll-proteins. We tested if, Toll-1(which forms homodimer) can heterodimerize with Toll-9. We found that wildtype Toll-1 didn’t interact with Toll9 however; when CRD of Toll1 was deleted/mutated it formed heterodimer with Toll9. This indicates that presence of CRD limits Toll proteins to form homodimer and thus its loss was a critical event which pushed Toll proteins towards heterodimerization. We further show that Drosophila Toll9 can directly bind dsRNA, a PAMP. Interestingly, dsRNA affinity for toll-9 monomer was twice as that for the dimer, which can be attributed to CRD loss. Thus, we show that loss of CRD was a major step in Toll evolution as it resulted in functional diversity and was a first step towards heterodimer formation. Therefore, we propose that CRD loss was under positive selection and also that heterodimerization of Toll-proteins is an evolved property.One line summaryLoss of Cysteine Rich Domain in Drosophila Toll9 and recognition of dsRNA.

Effect of disulphide bonds and sulphhydryl concentrations on properties of wheat flour

Disulphide bonds and sulphhydryl concentrations were evaluated to determine the effects on rheological, thermodynamic, pasting, and dynamic rheological characteristics of mixed flours. Gluten samples, first treated with sodium sulphite of different concentrations, were added into flour at a 4% level, which had a significant impact on free sulphhydryl, disulphide bonds, and the ratio of the two indices. There was no relevance between the ratio and other parameters except for free sulphhydryl. The mixed flour doughs had reduced water absorption, dough development time, dough stability time as well as degree of weakening (P < 0.05). Disulphide bonds were associated negatively with the rate of starch gelatinisation (C3–C2), peak, and setback and these characteristics were correlated strongly with dough development time, dough stability time, and progressive protein weakening (C2–C1). The stability of starch gelatinisation and cooking stability of mixed flours did not remain significantly different. The larger the concentration of sodium sulphite, the higher the peak, breakdown, final viscosity, and setback values, but there were no significant differences between samples. For all samples, storage modulus and loss modulus increased with increasing scanning frequency. For mixed doughs, the trend lines of moduli decreased with increasing levels of reduction in added gluten. There was no substantial effect on thermal properties of flours.

Isolation, Production, Purification, Assay and Characterization of Insulin From Goat (Capra Hircus) Pancreas By RDT Using Saccharomyces Cerevisae Vector

Insulin is a polypeptide hormone secreted by the β-cell of Islet’s of langerhan’s of the pancreas and cosists of two polypeptide chain-A and chain- B. It is linked by two inter chain disulphide bonds (A7- B7 and A20-B19) and also has an intra-chain disulphide bond in the A-Chain (A6-A11). For production of Insulin from goat pancreas saccharomyces cerevisae using as a vector. In this study Recombinant DNA technology, chromatography, Elecrophoresis, Spectrophotometer, SDS-PAGE, zymography etc techaniques were used. Humulin was tanken as a marker. It was found, goat insulin showed good result. 5.8 – 6.5 KDa recombinant insulin was calculated. It did not show antigenic proterties.