Determination of Substrate Specificity of the Purified Novel Plant Cysteine Protease Solanain From the Latex of <em>Vallaris solanacea</em>

Peptide synthesis refers to the production of peptides. Proteases are the enzymes that degrade proteins. They hold first place in the world market of enzymes, estimated at ∼US$3 billion. Proteases are distributed widely in different parts of the biological sources. In occurrence of proteases, plant kingdom occupies the highest rank. The increasing demand for proteases and the need for economical production of commercially useful industrial proteases from novel sources are taken into consideration. A novel plant latex cysteine protease namely Vallaris solanacea was identified in Biodiversity Park, Visakhapatnam which showed maximum protease activity. It has been shown that the latex of Vallaris solanacea contains a high protease activity. The cysteine protease solanain was purified by fractionation with ammonium sulphate followed by DEAE-cellulose ion exchange and gel chromatography. Specificity studies towards synthetic peptide and ester substrates by the protease purified from the latex of Vallaris solanacea were performed. The purified solanain exhibited broad specificity similar to other cysteine proteases. However considerable differences were also noticed in the rate of hydrolysis and specificity towards simple peptide substrates

A Parallel Processing and Diversified-Hidden-Gene-based Genetic Algorithm Framework for Fuel-Optimal Trajectory Design for Interplanetary Spacecraft Missions

This thesis proposes a new parallel computing genetic algorithm framework for designing fuel-optimal trajectories for interplanetary spacecraft missions. The framework can capture the deep search space of the problem with the use of a fixed chromosome structure and hidden-genes concept, can explore the diverse set of candidate solutions with the use of the adaptive and twin-space crowding techniques and, can execute on any high-performance computing (HPC) platform with the adoption of the portable message passing interface (MPI) standard. The algorithm is implemented in C++ with the use of the MPICH implementation of the MPI standard. The algorithm uses a patched-conic approach with two-body dynamics assumptions. New procedures are developed for determining trajectories in the V-infinity-leveraging legs of the flight from the launch and non-launch planets and, deep-space maneuver legs of the flight from the launch and non-launch planets. The chromosome structure maintains the time of flight as a free parameter within certain boundaries. The fitness or the cost function of the algorithm uses only the mission $\Delta V$, and does not include time of flight. The optimization is conducted with two variations for the minimum mission gravity-assist sequence, the 4-gravity-assist, and the 3-gravity-assist, with a maximum of 5 gravity-assists allowed in both the cases. The optimal trajectories discovered using the framework in both of the cases demonstrate the success of this framework

Deferoxamine and Curcumin Loaded Nanocarriers Protect Against Rotenone-Induced Neurotoxicity

Background: Reduced glutathione and excess free iron within dopaminergic, substantia nigra neurons in Parkinson's disease (PD) can drive accumulation of toxic hydroxyl radicals resulting in sustained oxidative stress and cellular damage. Factors such as brain penetrance and bioavailability have limited the advancement of potential antioxidant and iron chelator therapies for PD. Objective: This study aimed to develop novel nanocarrier delivery systems for the antioxidant curcumin and/or iron chelator deferoxamine (DFO) to protect against rotenone-induced changes in cell viability and oxidative stress in SH-SY5Y cells. Methods: Nanocarriers of curcumin and/or DFO were prepared using Pluronic F68 (P68) with or without dequilinium (DQA) by modified thin-film hydration. Cell viability was assessed using an MTT assay and oxidative stress was measured using thiobarbituric acid reactive substances and cellular antioxidant activity assays. Results: All formulations demonstrated high encapsulation efficiency (65-96%) and nanocarrier size was <200 nm. 3-h pretreatment with P68 or P68+DQA nanocarriers containing various concentrations of curcumin and/or DFO significantly protected against rotenone-reduced cell viability. The addition of DFO to curcumin-loaded P68+DQA nanocarriers resulted in increased protection by at least 10%. All nanoformulations significantly protected against rotenone-induced lipid peroxidation (p < 0.0001). The addition of DQA, which targets mitochondria, resulted in up to 65% increase in cellular antioxidant activity. In nearly all preparations, the combination of 10 μM curcumin and 100 μM DFO had the most antioxidant activity. Conclusion: This study demonstrates for the first time the formulation and delivery using P68 and P68+DQA curcumin and/or DFO nanocarriers to protect against oxidative stress induced by a rotenone PD model. This strategy to combine antioxidants with iron chelators may provide a novel approach to fully utilise their therapeutic benefit for PD

Comparison study of iron preparations using a human intestinal model

Iron deficiency and related iron deficiency anaemia (IDA) are the most prevalent nutritional disorders worldwide. The standard treatment involves supple-mentation with solid or liquid iron supplement preparations, usually based on a ferrous salt such as ferrous sulphate, ferrous fumarate, or ferrous gluconate. In the present study, we compared iron uptake and absorption from various solid and liquid iron supplement preparations currently available in the United Kingdom using the well-characterised human epithelial adenocarcinoma cell line Caco-2. Intracellular ferritin protein formation by the Caco-2 cell was considered an indicator of cellular iron uptake and absorption. We investigated the effects of formulation ingredients at a defined pH on iron uptake and absorption, and designed a novel two-stage dissolution-absorption protocol that mimicked physiological conditions. Our experiments revealed wide variations in the rate of dissolution between the various solid iron preparations. Conventional-release ferrous iron tablets dissolved rapidly (48 ± 4 mins to 64 ± 4 mins), whereas modified-released tablets and capsules took significantly longer to undergo complete dissolution (274 ± 8 to 256 ± 8 mins). Among the solid iron preparations, ferrous sulphate conventional-release tablets demon-strated the highest iron absorption, whereas modified-release ferrous prepa-rations demonstrated uniformly low iron absorption, as compared to the control (P < 0.05). Taken together, our results demonstrate that there are wide-ranging variations in dissolution times and iron uptake from oral iron preparations, with the physical characteristics of the preparation as well as the form of iron playing a key role

Structural interrogation of phosphoproteome identified by mass spectrometry reveals allowed and disallowed regions of phosphoconformation

High-throughput mass spectrometric (HT-MS) study is the method of choice for monitoring global changes in proteome. Data derived from these studies are meant for further validation and experimentation to discover novel biological insights. Here we evaluate use of relative solvent accessible surface area (rSASA) and DEPTH as indices to assess experimentally determined phosphorylation events deposited in PhosphoSitePlus. Based on accessibility, we map these identifications on allowed (accessible) or disallowed (inaccessible) regions of phosphoconformation. Surprisingly a striking number of HT- MS/MS derived events (1461/5947 sites or 24.6%) are present in the disallowed region of conformation. By considering protein dynamics, autophosphorylation events and/or the sequence specificity of kinases, 13.8% of these phosphosites can be moved to the allowed region of conformation. We also demonstrate that rSASA values can be used to increase the confidence of identification of phosphorylation sites within an ambiguous MS dataset. While MS is a stand-alone technique for the identification of vast majority of phosphorylation events, identifications within disallowed region of conformation will benefit from techniques that independently probe for phosphorylation and protein dynamics. Our studies also imply that trapping alternate protein conformations may be a viable alternative to the design of inhibitors against mutation prone drug resistance kinases

Direct Correlation of Cell Toxicity to Conformational Ensembles of Genetic A<i>β </i>Variants

We report a systematic analysis of conformational ensembles generated from multiseed molecular dynamics simulations of all 15 known genetic variants of Aβ₄₂. We show that experimentally determined variant toxicities are largely explained by random coil content of the amyloid ensembles (correlation with smaller EC₅₀ values; <i>R</i>² = 0.54, <i>p</i> = 0.01), and to some extent the helix character (more helix-character is less toxic, <i>R</i>² = 0.32, <i>p</i> = 0.07) and hydrophobic surface (<i>R</i>² = 0.37, <i>p</i> = 0.04). Our findings suggest that qualitative structural features of the amyloids, rather than the quantitative levels, are fundamentally related to neurodegeneration. The data provide molecular explanations for the high toxicity of E22 variants and for the protective features of the recently characterized A2T variant. The identified conformational features, for example, the local helix–coil-strand transitions of the C-terminals of the peptides, are of likely interest in the direct targeting of amyloids by rational drug design

Micellar Nanocarriers of Hydroxytyrosol Are Protective against Parkinson’s Related Oxidative Stress in an In Vitro hCMEC/D3-SH-SY5Y Co-Culture System

Hydroxytyrosol (HT) is a natural phenolic antioxidant which has neuroprotective effects in models of Parkinson’s disease (PD). Due to issues such as rapid metabolism, HT is unlikely to reach the brain at therapeutic concentrations required for a clinical effect. We have previously developed micellar nanocarriers from Pluronic F68® (P68) and dequalinium (DQA) which have suitable characteristics for brain delivery of antioxidants and iron chelators. The aim of this study was to utilise the P68 + DQA nanocarriers for HT alone, or in combination with the iron chelator deferoxamine (DFO), and assess their physical characteristics and ability to pass the blood–brain barrier and protect against rotenone in a cellular hCMEC/D3-SH-SY5Y co-culture system. Both HT and HT + DFO formulations were less than 170 nm in size and demonstrated high encapsulation efficiencies (up to 97%). P68 + DQA nanoformulation enhanced the mean blood–brain barrier (BBB) passage of HT by 50% (p 0.0001, n = 6). This resulted in increased protection against rotenone induced cytotoxicity and oxidative stress by up to 12% and 9%, respectively, compared to the corresponding free drug treatments (p 0.01, n = 6). This study demonstrates for the first time the incorporation of HT and HT + DFO into P68 + DQA nanocarriers and successful delivery of these nanocarriers across a BBB model to protect against PD-related oxidative stress. These nanocarriers warrant further investigation to evaluate whether this enhanced neuroprotection is exhibited in in vivo PD models