Physicochemical modulation of huntingtin aggregation on lipid membranes: Implications for Huntington\u27s disease

Huntington disease is a genetic, neurodegenerative disease caused by an expanded polyglutamine (polyQ) in the first exon of the huntingtin (htt) protein, facilitating its aggregation, leading to the formation of a diverse population of potentially toxic aggregate species, such as oligomers, fibrils, and annular aggregates. Htt interacts with a variety of membranous structures within the cell, and the first seventeen amino acids (Nt17) of htt directly flanking the polyQ domain is an amphipathic alpha-helix (AH) lipid-binding domain. AHs are also known to detect membrane curvature. Nt17 also promotes diverse aggregate species of htt and undergoes a number of posttranslational modifications that can modulate htt\u27s toxicity, subcellular localization, and trafficking of vesicles. To get in-depth mechanistic insights of huntingtin aggregation, both chemical and physical modulators of the aggregation process were explored. Specifically, the importance of htt acetylation and the role of membrane curvature on htt aggregation were investigated using atomic force microscopy (AFM), which has become a robust technique to obtain physical insights into the formation of toxic protein aggregates associated with amyloid diseases on lipid membranes. Acetylation of htt exon 1, and synthetic truncated htt exon1 mimicking peptide (Nt 17Q35P10KK) was achieved using a selective covalent label sulfo-N-hydroxysuccinimide (NHSA) in molar ratios of 1x, 2x, and 3x NHSA per peptide. Htt acetylation was found to decrease fibril formation in solution and promoted the formation of larger globular aggregates. Acetylation strongly altered htt\u27s ability to bind lipid membranes. However, one of the several limitations associated with using these current flat, supported bilayers as model surfaces is the absence of membrane curvature, which can heavily influence the interaction of proteins at lipid interfaces. Using an AFM force reconstruction technique, silicon substrate, and silica nanobeads, model lipid bilayer system was developed and validated in which the underlying solid support is comprised of flat and curved regions to induce regions of curvature in the bilayer. This model bilayer system was exposed to Nt17Q35P10KK peptide, and this peptide preferentially bound and accumulated to curved membranes, consistent with the ability of AHs to sense membrane curvature

Assessment From Plasma Amino Acid Imbalance

A CAJM article on the nutrition status of black Zimbabwean children. (formerly Rhodesians)Children living on a poor diet and showing no clinical signs of protein deficiency may nevertheless have an imbalance of certain amino acids in their plasma (Whitehead, 1964; Whitehead and Dean, 1964). A small pilot survey has been undertaken in which the relative concentrations of these amino acids have been investigated in serum samples taken from children at schools either near Salisbury or in a rather remote country area, in order to obtain some idea of their nutritional status

The comparison of in vitro release methods for the evaluation of oxytocin release from Pluronic® F127 parenteral formulations

The objective of these studies was to develop a discriminatory in vitro release test for assessing formulation factors that may affect oxytocin (OT) release during formulation development studies of a Pluronic® F127 OT in situ gel-forming parenteral dosage form. An appropriate release assessment method should be able to discriminate between the performance of different formulation compositions (1, 2), and this was the primary criterion used for selection of an appropriate test procedure during the test method development process. ANOVA and the difference (f1) and similarity (f2)factors were used to evaluate the discriminatory behavior of different test methods that were investigated in these studies. The in vitro release tests that were investigated included the use of USP Apparatus 1, 2, and 3; a dialysis bag in USP Apparatus 2; and a membrane-less diffusion method. It was concluded that the use of USP Apparatus 3 was best able to discriminate between OT release for the different formulations tested. USP Apparatus 3 was thus considered the most suitable in vitro release test apparatus for studying formulation factors affecting OT release during the development of a parenteral dosage form prepared using Pluronic® F127

Development and validation of a stability-indicating analytical method for the quantitation of oxytocin in pharmaceutical dosage forms

A single stability-indicating assay for oxytocin (OT) in pharmaceutical dosage forms using gradient elution over 21 min has been reported in the literature. Furthermore, published and compendial methods for the analysis of OT containing dosage forms also involve using HPLC with gradient elution and complicated mobile phases that include hydrophobic ion pairing agents. A simple isocratic and stability-indicating assay was developed and validated. The conditions are as follows, column: Phenomenex® C18 Hypersil, 5 μm packing, 4.6 mm × 150 mm with acetonitrile–phosphate buffer (pH 5; 0.08 M) (20:80) as the mobile phase with UV detection at 220 nm The method was found to be specific for OT in the presence of degradation products and chlorbutol (preservative) with an overall analytical run time of 16 min. Accuracy was determined to be 0.77–1.18% bias for all samples tested. Intra-assay precision (repeatability) was found to be 0.22–1.04%R.S.D. while the inter-day precision (intermediate precision) was found to be 1.27–1.68%R.S.D. for the samples studied. The calibration curve was found to be linear with the equation y = 1.81x + 0.02 and a linear regression coefficient of 0.9991 over the range 0.4–12.0 IU/ml. The LOD and the LOQ were determined to be 0.1 and 0.4 IU/ml, respectively. Syntocinon®, a commercially available dosage form of OT was assayed resulting in 100.5–106.6% recovery of the label claim and an average of 10.04 IU/ml

Analytical methods for the quantitative determination of oxytocin

Oxytocin is a clinically important nonapeptide that is used for the induction and/or augmentation of labor and is normally administered as a slow intravenous infusion diluted with normal saline or Ringer’s lactate solution. Oxytocin is also indicated for use in the prevention and treatment of post partum hemorrhage and may be administered via either the intramuscular or intravenous routes in order to increase uterine tone and/or reduce bleeding. The analysis of oxytocin in different media has evolved over the past 30 years with the result that more sophisticated, selective and sensitive techniques are used for the determination of the compound. A variety of techniques have been applied to the determination of oxytocin in different matrices ranging from simple paper chromatography to hyphenated liquid chromatographic such as liquid chromatography coupled with mass-spectrometry. Additionally enzyme linked immuno-sorbent assays (ELISA) and radio immuno-assays (RIA) are used for the determination of low concentrations of oxytocin in biological matrices. This manuscript provides a systematic survey of the analytical methods that have been reported for isolation and quantitation of oxytocin in different matrices

Sphingomyelin and GM1 Influence Huntingtin Binding to, Disruption of, and Aggregation on Lipid Membranes

Huntington disease (HD) is an inherited neurodegenerative disease caused by the expansion beyond a critical threshold of a polyglutamine (polyQ) tract near the N-terminus of the huntingtin (htt) protein. Expanded polyQ promotes the formation of a variety of oligomeric and fibrillar aggregates of htt that accumulate into the hallmark proteinaceous inclusion bodies associated with HD. htt is also highly associated with numerous cellular and subcellular membranes that contain a variety of lipids. As lipid homeostasis and metabolism abnormalities are observed in HD patients, we investigated how varying both the sphingomyelin (SM) and ganglioside (GM1) contents modifies the interactions between htt and lipid membranes. SM composition is altered in HD, and GM1 has been shown to have protective effects in animal models of HD. A combination of Langmuir trough monolayer techniques, vesicle permeability and binding assays, and in situ atomic force microscopy (AFM) were used to directly monitor the interaction of a model, synthetic htt peptide and a full-length htt-exon1 recombinant protein with model membranes comprised of total brain lipid extract (TBLE) and varying amounts of exogenously added SM or GM1. The addition of either SM or GM1 decreased htt insertion into the lipid monolayers. However, TBLE vesicles with an increased SM content were more susceptible to htt-induced permeabilization, whereas GM1 had no effect on permeablization. Pure TBLE bilayers and TBLE bilayers enriched with GM1 developed regions of roughened, granular morphologies upon exposure to htt-exon1, but plateau-like domains with a smoother appearance formed in bilayers enriched with SM. Oligomeric aggregates were observed on all bilayer systems regardless of induced morphology. Collectively, these observations suggest that the lipid composition and its subsequent effects on membrane material properties strongly influence htt binding and aggregation on lipid membranes

Reliability of an icterometer in black neonates with hyperbilirubinaemia

A perspex icterometer previously graded for White neonates was evaluated in an unselected Black newborn population. Grades of icterus showed a close correlation with levels of total serum bilirubin (TSB) as determined colorimetrically in a bilirubinometer. Previously reported data on White babies follow the same pattern, but are slightly higher for each grade.S. Afr. Med. J., 48, 1533 (1974)

Insights on the potential of RNA-Seq on improving pomological traits of African indigenous fruit trees: a mini review

Fruit tree improvement has taken great strides by roping in improved and efficient biotechnological tools to increase fruit yield and quality to meet local and export demands. For the past decade, the RNA-Seq tool has successfully been used in fruit tree improvement programs to identify genes, dissect complex traits, and understand different molecular pathways and differential expression of genes. However, despite their growing importance in food and nutrition security, medicinal uses, and climate change mitigation strategies, very little has been done to improve the pomological traits of African indigenous fruits, especially at the molecular level. African indigenous fruit trees exhibit unexplained variation in flowering, fruit load, fruit size, fruit ripening, fruit taste, fruit nutritional composition and shelf-life. The booming local commercial companies and export markets are demanding consistent quality indigenous fruits. This has necessitated the need for fast and effective tools that will hasten the understanding and improvement of fruiting qualities. The improvement of fruiting and fruit qualities will go a long way in accelerating the domestication and commercialization of African indigenous fruit trees. This review paper gives molecular biology insights on how RNA-Seq has been successfully used in fruit improvement of exotic fruits through gene identification, comparative transcriptome analysis under different conditions, and understanding molecular pathways that influence important pomological traits. The review article also unearths opportunities where RNA-Seq can improve our knowledge and improvement of undesirable traits common in African indigenous fruit

Optimization of salbutamol sulfate dissolution from sustained release matrix formulations using an artificial neural network

An artificial neural network was used to optimize the release of salbutamol sulfate from hydrophilic matrix formulations. Model formulations to be used for training, testing and validating the neural network were manufactured with the aid of a central composite design with varying the levels of Methocel® K100M, xanthan gum, Carbopol® 974P and Surelease® as the input factors. In vitro dissolution time profiles at six different sampling times were used as target data in training the neural network for formulation optimization. A multi layer perceptron with one hidden layer was constructed using Matlab®, and the number of nodes in the hidden layer was optimized by trial and error to develop a model with the best predictive ability. The results revealed that a neural network with nine nodes was optimal for developing and optimizing formulations. Simulations undertaken with the training data revealed that the constructed model was useable. The optimized neural network was used for optimization of formulation with desirable release characteristics and the results indicated that there was agreement between the predicted formulation and the manufactured formulation. This work illustrates the possible utility of artificial neural networks for the optimization of pharmaceutical formulations with desirable performance characteristics