Understanding the complex formation of falstatin; an endogenous macromolecular inhibitor of falcipains.

Proteolytic activity constitutes a fundamental process essential for the survival of the malaria parasite and is thus highly regulated. Falstatin, a protease inhibitor of Plasmodium falciparum, tightly regulates the activity of cysteine hemoglobinases, falcipain-2 and 3 (FP2, FP3), by inhibiting FP2 through a single surface exposed loop. However, the multimeric nature of falstatin and its interaction with FP2 remained unexplored. Here we report that the N-terminal falstatin region is highly disordered, and needs chaperone activity (heat-shock protein 70, HSP70) for its folding. Protein-protein interaction assays showed a significant interaction between falstatin and HSP70. Further, characterization of the falstatin multimer through a series of biophysical techniques identified the formation of a falstatin decamer, which was extremely thermostable. Computational analysis of the falstatin decamer showed the presence of five falstatin dimers, with each dimer aligned in a head-to-tail orientation. Further, the falstatin C-terminal region was revealed to be primarily involved in the oligomerization process. Stoichiometric analysis of the FP2-falstatin multimer showed the formation of a heterooligomeric complex in a 1:1 ratio, with the participation of ten subunits of each protein. Taken together, our results report a novel protease-inhibitor complex and strengthens our understanding of the regulatory mechanisms of major plasmodium hemoglobinases.

A Quality by Design Approach for Developing SNEDDS Loaded with Vemurafenib for Enhanced Oral Bioavailability.

Vemurafenib (VMF) is a practically insoluble (< 0.1 µg/mL) and least bioavailable (1%) drug. To enhance its oral bioavailability and solubility, we formulated a reliable self-nano emulsifying drug delivery system (SNEDDS). A Quality by Design (QbD) approach was used to optimize the ratio of Capryol 90, Tween 80, and Transcutol HP. VMF-loaded SNEDDS was characterized for its size, polydispersity index (PDI), zeta potential, drug content, and transmittance. The in vitro release profile of the drug loaded in SNEDDS was compared to the free drug in two media, pH 6.8 and 1.2, and the data obtained were analyzed with different mathematical models. A reverse-phase ultra-pressure liquid chromatography (UPLC) technique with high sensitivity and selectivity was developed and validated for the quantification of VMF in analytical and bioanalytical samples. Dissolution efficiency for SNEDDS was estimated using different models, which proved that the developed novel SNEDDS formulation had a better in vitro dissolution profile than the free drug. A 2.13-fold enhanced oral bioavailability of VMF-loaded SNEDDS compared to the free drug demonstrates the superiority of the developed formulation. This work thus presents an overview of VMF-loaded SNEDDS as a promising alternative to improve the oral bioavailability of the drug.

Transcriptional regulation of suppressors of cytokine signaling during infection with Mycobacterium tuberculosis in human THP-1-derived macrophages and in mice.

Mycobacterium tuberculosis (Mtb) infection leads to upregulation of Suppressors of Cytokine signaling (SOCS) expression in host macrophages (Mϕ). SOCS proteins inhibit cytokine signaling by negatively regulating JAK/STAT. We investigated this host-pathogen dialectic at the level of transcription. We used phorbol-differentiated THP-1 Mϕ infected with Mtb to investigate preferential upregulation of some SOCS isoforms that are known to inhibit signaling by IFN-γ, IL-12, and IL-6. We examined time kinetics of likely transcription factors and signaling molecules upstream of SOCS transcription, and survival of intracellular Mtb following SOCS upregulation. Our results suggest a plausible mechanism that involves PGE2 secretion during infection to induce the PKA/CREB axis, culminating in nuclear translocation of C/EBPß to induce expression of SOCS1. Mtb-infected Mϕ secreted IL-10, suggesting a mechanism of induction of STAT3, which may subsequently induce SOCS3. We provide evidence of temporal variation in SOCS isoform exspression and decay. Small-interfering RNA-mediated knockdown of SOCS1 and SOCS3 restored the pro-inflammatory milieu and reduced Mtb viability. In mice infected with Mtb, SOCS isoforms persisted across Days 28-85 post infection. Our results suggest that differential temporal regulation of SOCS isoforms by Mtb drives the host immune response towards a phenotype that facilitates the pathogen's survival.

Inhaled Adjunct Therapy with Second-Line Drug Candidates for Dose Reduction in Chemotherapeutic Regimens for Multi-drug-Resistant Tuberculosis.

Chemotherapy of multi-drug-resistant tuberculosis (TB) requires prolonged administration of multiple drugs. We investigated whether pulmonary delivery of minute doses of drugs, along with reduced oral doses of the same agents, would affect preclinical efficacy. We prepared dry powder inhalation (DPI) formulations comprising sutezolid (SUT), the second-generation pretomanid analog TBA-354 (TBA), or a fluorinated derivative of TBA-354 (32,625) in a matrix of the biodegradable polymer poly(L-lactide). We established formulation characteristics, doses inhaled by healthy mice, and preclinical efficacy in a mouse model of TB. Oral doses of 100 mg/kg/day or DPI doses of 0.25-0.5 mg/kg/day of drugs SUT, TBA-354, or 32,625 administered over 28 days were sub-optimally effective in reducing lung and spleen burden of Mycobacterium tuberculosis (Mtb) in infected mice. The addition of 0.25-0.5 mg/kg/day of SUT, TBA-354, or 32,625 as DPI to oral doses of 50 mg/kg/day was non-inferior in clearing Mtb from the lungs of infected mice. We concluded that adjunct therapy with inhaled second-line agents has the potential to reduce the efficacious oral dose.

Exploring the structural basis to develop efficient multi-epitope vaccines displaying interaction with HLA and TAP and TLR3 molecules to prevent NIPAH infection, a global threat to human health.

Nipah virus (NiV) is an emerging zoonotic virus that caused several serious outbreaks in the south asian region with high mortality rates ranging from 40 to 90% since 2001. NiV infection causes lethal encephalitis and respiratory disease with the symptom of endothelial cell-cell fusion. No specific and effective vaccine has yet been reported against NiV. To address the urgent need for a specific and effective vaccine against NiV infection, in the present study, we have designed two Multi-Epitope Vaccines (MEVs) composed of 33 Cytotoxic T lymphocyte (CTL) epitopes and 38 Helper T lymphocyte (HTL) epitopes. Out of those CTL and HTL combined 71 epitopes, 61 novel epitopes targeting nine different NiV proteins were not used before for vaccine design. Codon optimization for the cDNA of both the designed MEVs might ensure high expression potential in the human cell line as stable proteins. Both MEVs carry potential B cell linear epitope overlapping regions, B cell discontinuous epitopes as well as IFN-γ inducing epitopes. Additional criteria such as sequence consensus amongst CTL, HTL and B Cell epitopes was implemented for the design of final constructs constituting MEVs. Hence, the designed MEVs carry the potential to elicit cell-mediated as well as humoral immune response. Selected overlapping CTL and HTL epitopes were validated for their stable molecular interactions with HLA class I and II alleles and in case of CTL epitopes with human Transporter Associated with antigen Processing (TAP) cavity. The structure based epitope cross validation for interaction with TAP cavity was used as another criteria choosing final epitopes for NiV MEVs. Finally, human Beta-defensin 2 and Beta-defensin 3 were used as adjuvants to enhance the immune response of both the MEVs. Molecular dynamics simulation studies of MEVs-TLR3 ectodomain (Human Toll-Like Receptor 3) complex indicated the stable molecular interaction. We conclude that the MEVs designed and in silico validated here could be highly potential vaccine candidates to combat NiV infections, with great effectiveness, high specificity and large human population coverage worldwide.

Recognition of fold- and function-specific sites in the ligand-binding domain of the thyroid hormone receptor-like family.

Background: The thyroid hormone receptor-like (THR-like) family is the largest transcription factors family belonging to the nuclear receptor superfamily, which directly binds to DNA and regulates the gene expression and thereby controls various metabolic processes in a ligand-dependent manner. The THR-like family contains receptors THRs, RARs, VDR, PPARs, RORs, Rev-erbs, CAR, PXR, LXRs, and others. THR-like receptors are involved in many aspects of human health, including development, metabolism and homeostasis. Therefore, it is considered an important therapeutic target for various diseases such as osteoporosis, rickets, diabetes, etc. Methods: In this study, we have performed an extensive sequence and structure analysis of the ligand-binding domain (LBD) of the THR-like family spanning multiple taxa. We have use different computational tools (information-theoretic measures; relative entropy) to predict the key residues responsible for fold and functional specificity in the LBD of the THR-like family. The MSA of THR-like LBDs was further used as input in conservation studies and phylogenetic clustering studies. Results: Phylogenetic analysis of the LBD domain of THR-like proteins resulted in the clustering of eight subfamilies based on their sequence homology. The conservation analysis by relative entropy (RE) revealed that structurally important residues are conserved throughout the LBDs in the THR-like family. The multi-harmony conservation analysis further predicted specificity in determining residues in LBDs of THR-like subfamilies. Finally, fold and functional specificity determining residues (residues critical for ligand, DBD and coregulators binding) were mapped on the three-dimensional structure of thyroid hormone receptor protein. We then compiled a list of natural mutations in THR-like LBDs and mapped them along with fold and function-specific mutations. Some of the mutations were found to have a link with severe diseases like hypothyroidism, rickets, obesity, lipodystrophy, epilepsy, etc. Conclusion: Our study identifies fold and function-specific residues in THR-like LBDs. We believe that this study will be useful in exploring the role of these residues in the binding of different drugs, ligands, and protein-protein interaction among partner proteins. So this study might be helpful in the rational design of either ligands or receptors.

Preparation and Evaluation of Low-Dose Calcitriol Dry Powder Inhalation as Host-Directed Adjunct Therapy for Tuberculosis.

BACKGROUND: It is unclear whether Vitamin D is efficacious as a host-directed therapy (HDT) for patients of tuberculosis (TB). We investigated pulmonary delivery of the active metabolite of Vitamin D3, i.e., 1, 25-dihydroxy vitamin D3 (calcitriol) in a mouse model of infection with Mycobacterium tuberculosis (Mtb). METHODS: We optimized a spray drying process to prepare a dry powder inhalation (DPI) of calcitriol using a Quality by Design (QbD) approach. We then compared outcomes when Mtb-infected mice were treated with inhaled calcitriol at 5 ng/kg as a stand-alone intervention versus DPI as adjunct to standard oral anti-tuberculosis therapy (ATT). RESULTS: The DPI with or without concomitant ATT markedly improved the morphology of the lungs and mitigated histopathology in both the lungs and the spleens. The number of nodular lesions on the lung surface decreased from 43.7 ± 3.1 to 22.5 ± 3.9 with the DPI alone and to 9.8 ± 2.5 with DPI + ATT. However, no statistically significant induction of host antimicrobial peptide cathelicidin or reduction in bacterial burden was seen with the DPI alone. DPI + ATT did not significantly reduce the bacterial burden in the lungs compared to ATT alone. CONCLUSIONS: We concluded that HDT using the low dose calcitriol DPI contributed markedly to mitigation of pathology, but higher dose may be required to evoke significant induction of bactericidal host response and bactericidal activity in the lung.

Signature transcriptome analysis of stage specific atherosclerotic plaques of patients.

BACKGROUND: Inflammation plays an important role in all the stages of atherosclerotic plaque development. The current study aimed at assessing the altered expression of genes functioning in inflammation within the early stage (ES) and advanced stage (AS) atherosclerotic plaques obtained from patients undergoing coronary artery bypass grafting (CABG) surgery and identifying biomarker panel/s that may detect the status of plaque stages using peripheral blood samples. METHODS: A section of ES and AS plaques and normal left internal mammary arteries (LIMA) were obtained from 8 patients undergoing the CABG surgery. Total RNA isolated was analyzed for mRNA and miRNA expression profile by Affymetrix arrays. A significant number of mRNAs was found to be differentially expressed in ES and AS plaque tissues relative to LIMA. The pathway analysis of differentially expressed mRNAs in the two plaque stages was also performed using DAVID Bioinformatics Database. RESULTS: The mRNAs were found to be involved in critical inflammatory processes such as the toll-like receptor signaling pathway and cytokine-cytokine receptor interaction. Few miRNAs targeting these mRNAs were also altered in the two plaque conditions. QRT-PCR results showed a similar expression pattern of a few of the mRNAs and miRNAs in peripheral blood of the same patients relative to healthy controls. CONCLUSION: Changes in mRNA and miRNA expression associated with various inflammatory processes occur in different atherosclerotic stage plaques as well as peripheral blood. Detection of such variations in patients' blood can be used as a possible prognostic tool to detect and/or predict the risk and stage of atherosclerosis.

Transient, inhaled gene therapy with gamma interferon mitigates pathology induced by host response in a mouse model of tuberculosis.

Transient transfection of the respiratory mucosa of mice infected with Mycobacterium tuberculosis (Mtb) with gamma interferon (IFN-γ) promises benefits in disease therapy. We investigated preclinical efficacy of a dry powder inhalation (DPI) as a stand-alone versus adjunct to oral anti-tuberculosis (TB) chemotherapy in mice. We observed that this host-directed therapy mitigates the gross organ pathology and histopathology of lung and spleen tissue of infected mice receiving the DPI, either alone or as adjunct therapy. However, no statistically significant reduction in Mtb colony forming units (CFU) occurred if mice were given only DPI; but not drugs. We compared one and three doses a week of the DPI over four weeks; with or without concomitant oral drugs. There was no significant difference in lung CFU after four or 12 doses of the DPI alone, but, surprisingly, four doses were qualitatively better than 12 doses in mitigating lung pathology. Nodular lesions on the lung surface and the area occupied by these was significantly reduced after four doses of the DPI, even without oral drugs. Transient transfection with IFN-γ did not induce pathological inflammation of the lungs and airways. We conclude that IFN-γ, as expected of host-directed therapy, 'heals the host; ' but does not 'kill the bug.'

Computationally validated SARS-CoV-2 CTL and HTL Multi-Patch vaccines, designed by reverse epitomics approach, show potential to cover large ethnically distributed human population worldwide.

The SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is responsible for the COVID-19 outbreak. The highly contagious COVID-19 disease has spread to 216 countries in less than six months. Though several vaccine candidates are being claimed, an effective vaccine is yet to come. A novel reverse epitomics approach, 'overlapping-epitope-clusters-to-patches' method is utilized to identify the antigenic regions from the SARS-CoV-2 proteome. These antigenic regions are named as 'Ag-Patch or Ag-Patches', for Antigenic Patch or Patches. The identification of Ag-Patches is based on the clusters of overlapping epitopes rising from SARS-CoV-2 proteins. Further, we have utilized the identified Ag-Patches to design Multi-Patch Vaccines (MPVs), proposing a novel method for the vaccine design. The designed MPVs were analyzed for immunologically crucial parameters, physiochemical properties and cDNA constructs. We identified 73 CTL (Cytotoxic T-Lymphocyte) and 49 HTL (Helper T-Lymphocyte) novel Ag-Patches from the proteome of SARS-CoV-2. The identified Ag-Patches utilized to design MPVs cover 768 overlapping epitopes targeting 55 different HLA alleles leading to 99.98% of world human population coverage. The MPVs and Toll-Like Receptor ectodomain complex shows stable complex formation tendency. Further, the cDNA analysis favors high expression of the MPVs constructs in a human cell line. We identified highly immunogenic novel Ag-Patches from the entire proteome of SARS CoV-2 by a novel reverse epitomics approach and utilized them to design MPVs. We conclude that the novel MPVs could be a highly potential novel approach to combat SARS-CoV-2, with greater effectiveness, high specificity and large human population coverage worldwide. Communicated by Ramaswamy H. Sarma.

Transcriptome Analysis of Insulin Signaling-Associated Transcription Factors in C. elegans Reveal Their Genome-Wide Target Genes Specificity and Complexity.

Insulin/IGF-1-like signaling (IIS) plays a crucial, conserved role in development, growth, reproduction, stress tolerance, and longevity. In Caenorhabditis elegans, the enhanced longevity under reduced insulin signaling (rIIS) is primarily regulated by the transcription factors (TFs) DAF-16/FOXO, SKN-1/Nrf-1, and HSF1/HSF-1. The specific and coordinated regulation of gene expression by these TFs under rIIS has not been comprehensively elucidated. Here, using RNA-sequencing analysis, we report a systematic study of the complexity of TF-dependent target gene interactions during rIIS under analogous genetic and experimental conditions. We found that DAF-16 regulates only a fraction of the C. elegans transcriptome but controls a large set of genes under rIIS; SKN-1 and HSF-1 show the opposite trend. Both of the latter TFs function as activators and repressors to a similar extent, while DAF-16 is predominantly an activator. For expression of the genes commonly regulated by TFs under rIIS conditions, DAF-16 is the principal determining factor, dominating over the other two TFs, irrespective of whether they activate or repress these genes. The functional annotations and regulatory networks presented in this study provide novel insights into the complexity of the gene regulatory networks downstream of the IIS pathway that controls diverse phenotypes, including longevity.

The nucleotide specificity of succinyl-CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone.

Substrate specificity of an enzyme is an important characteristic of its mechanism of action. Investigation of the nucleotide specificity of Plasmodium falciparum succinyl-CoA synthetase (SCS; PfSCS) would provide crucial insights of its substrate recognition. Charged gatekeeper residues have been shown to alter the substrate specificity via electrostatic interactions with approaching substrates. The enzyme kinetics of recombinant PfSCS (wild-type), generated by refolding of the individual P. falciparum SCSß and Blastocystis SCSα subunits, demonstrated ADP-forming activity (KmATP  = 48 µm). Further, the introduction of charged gatekeeper residues, either positive (Lys and Lys) or negative (Glu and Asp), resulted in significant reductions in the ATP affinity of PfSCS. It is interesting to note that the recombinant PfSCSß subunit can be refolded to a functional enzyme conformation using Blastocystis SCSα, indicating the possibility of subunits swapping among different organisms. These results concluded that electrostatic interactions at the gatekeeper region alone are insufficient to alter the substrate specificity of PfSCS, and further structural analysis with a particular focus on binding site architecture is required.

Transient Transfection of the Respiratory Epithelium with Gamma Interferon for Host-Directed Therapy in Pulmonary Tuberculosis.

Nebulized gamma interferon (IFN-γ) protein has been studied for clinical safety and efficacy against pulmonary tuberculosis (TB). The protein is expensive, requires a cold chain, and is difficult to deploy in limited-resource, high-incidence settings. We generated a preclinical proof of concept (PoC) for a dry powder inhalation (DPI) containing DNA constructs to transiently transfect the lung and airway epithelium of mice with murine IFN-γ. Bacterial colony-forming units (CFU) in the lungs of mice infected with Mycobacterium tuberculosis (Mtb) reduced from about 106/g of tissue to ~104 after four doses given once a week. Nodular inflammatory lesions in the lungs reduced significantly in number. Immunohistochemistry of infected lung sections for LC3-1 and LAMP-1 indicated autophagy induction between 18 and 48 h after inhalation. ELISA on bronchoalveolar lavage (BAL) fluid showed differences in kinetics of IFN-γ concentrations in the epithelial lining fluid of healthy versus infected mice. Uninfected mice receiving DNA constructs expressing a fluorescent protein were live-imaged. The fluorescence signals from the intracellular protein peaked at about 36 h after inhalation and declined by 48 h. These results establish preclinical PoC of the efficacy of a DPI and dosing regimen as a host-directed and transient gene therapy of experimental pulmonary TB in mice, justifying preclinical development.

Structural Basis for Designing Multiepitope Vaccines Against COVID-19 Infection: In Silico Vaccine Design and Validation.

BACKGROUND: The novel coronavirus disease (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to the ongoing 2019-2020 pandemic. SARS-CoV-2 is a positive-sense single-stranded RNA coronavirus. Effective countermeasures against SARS-CoV-2 infection require the design and development of specific and effective vaccine candidates. OBJECTIVE: To address the urgent need for a SARS-CoV-2 vaccine, in the present study, we designed and validated one cytotoxic T lymphocyte (CTL) and one helper T lymphocyte (HTL) multi-epitope vaccine (MEV) against SARS-CoV-2 using various in silico methods. METHODS: Both designed MEVs are composed of CTL and HTL epitopes screened from 11 Open Reading Frame (ORF), structural and nonstructural proteins of the SARS-CoV-2 proteome. Both MEVs also carry potential B-cell linear and discontinuous epitopes as well as interferon gamma-inducing epitopes. To enhance the immune response of our vaccine design, truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 was used as an adjuvant at the N termini of both MEVs. The tertiary models for both the designed MEVs were generated, refined, and further analyzed for stable molecular interaction with toll-like receptor 3. Codon-biased complementary DNA (cDNA) was generated for both MEVs and analyzed in silico for high level expression in a mammalian (human) host cell line. RESULTS: In the present study, we screened and shortlisted 38 CTL, 33 HTL, and 12 B cell epitopes from the 11 ORF protein sequences of the SARS-CoV-2 proteome. Moreover, the molecular interactions of the screened epitopes with their respective human leukocyte antigen allele binders and the transporter associated with antigen processing (TAP) complex were positively validated. The shortlisted screened epitopes were utilized to design two novel MEVs against SARS-CoV-2. Further molecular models of both MEVs were prepared, and their stable molecular interactions with toll-like receptor 3 were positively validated. The codon-optimized cDNAs of both MEVs were also positively analyzed for high levels of overexpression in a human cell line. CONCLUSIONS: The present study is highly significant in terms of the molecular design of prospective CTL and HTL vaccines against SARS-CoV-2 infection with potential to elicit cellular and humoral immune responses. The epitopes of the designed MEVs are predicted to cover the large human population worldwide (96.10%). Hence, both designed MEVs could be tried in vivo as potential vaccine candidates against SARS-CoV-2.

Studies on the Neuromodulatory Effects of Ginkgo biloba on Alterations in Lipid Composition and Membrane Integrity of Rat Brain Following Aluminium Neurotoxicity.

Brain contains the highest lipid content involved in various structural and physiological activities such as structural development, neurogenesis, synaptogenesis, signal transduction and myelin sheath formation. Lipids bilayer is essential to maintain the structural integrity for the physiological functions of protein. Impairments in lipid metabolism and its composition can lead to the progression of various brain ailments such as neurodegenerative and neuropsychiatric disorders. Aluminium (Al), the potent neurotoxin has been linked to Alzheimer's disease (AD) like pathology. Al can bind to biomembrane and influence oligomerization and conformational changes of proteins by acting as cross-linkers. The present study evaluated the influence of Ginkgo biloba (GBE) on the lipid profile alterations induced by Al lactate in hippocampal and cortical regions using FTIR spectroscopy. Rats were exposed with 10 mg/kg b.w. (intraperitoneal) of Al lactate for 6 weeks. This was followed by a treatment protocol of GBE (100 mg/kg b.w.) both preexposure (2 weeks) and conjunctive (6 weeks) exposure. A self recovery group was also included, where Al withdrawal was done for 2 weeks post Al exposure. A significant decrease in peak areas of cholesterol, sphingolipids and phospholipids was observed in Al treated groups. Further, polyunsaturated fatty acids and membrane fluidity has also decreased, as revealed by olefinic and methyl asymmetric stretching bands. Al treatment significantly increased the fluorescence polarization, anisotropy and order parameter, which however were normalized following GBE supplementation. Results also showed that pretreatment with GBE provided more beneficial effects on the adverse changes following Al in membrane composition and behavioral outcome.

Effect of ethanolic extract of Solanum virginianum Linn. on neuropathic pain using chronic constriction injury rat model and molecular docking studies.

The present research work was designed to examine the neuroprotective effect of ethanolic extract of Solanum virginianum Linn. (SV) in chronic construction injury (CCI) of sciatic nerve-induced neuropathic pain in rats. The extract was initially standardized by high-performance thin-layer chromatography using solasodine as a biomarker and was then subjected to assess the degree of mechanical allodynia, thermal allodynia, mechanical hyperalgesia, thermal hyperalgesia and biochemical evaluations. Administration of SV (100 and 200 mg/kg; p.o.) and pregabalin (10 mg/kg; p.o.) as a reference standard significantly debilitated hyperalgesia and allodynia and notably restored the altered antioxidant level and pro-inflammatory cytokine (IL-1ß and TNF-α) expression in a dose-dependent manner. Further, to appraise the mechanistic approach of solasodine, docking simulation studies were done on the 3D structure of the voltage-gated N-type calcium channel (Cav 2.2), R-type calcium channel (Cav 2.3) and sodium channel (Nav 1.7), and the results revealed that solasodine properly positioned into Phe 19, Leu 32, Met 51 and Met 71 (FLMM pocket) of Cav 2.2 and Cav 2.3 and being a competitor of Ca2+/N-lobe it may inactivate these calcium channels but did not bind into the desired binding pocket of Nav 1.7. Thus, the study confirmed the role of solasodine as a major biomarker for the observed neuroprotective nature of Solanum virginianum.

Modulatory Effects of Ginkgo biloba Against Amyloid Aggregation Through Induction of Heat Shock Proteins in Aluminium Induced Neurotoxicity.

Protein misfolding and aggregation of amyloid beta (Aß) peptide, as well as formation of neurofibrillary tangles (NFTs) are the signature hallmarks of Alzheimer's disease (AD) pathology. To prevent this, molecular chaperones come into play as they facilitate the refolding of the misfolded proteins and cell protection under stress. Here, we have evaluated the possible effects of Ginkgo biloba (GBE) against aggregation of the Aß through activation of heat shock proteins (HSPs) in the Aluminium (Al) induced AD based model. GBE (100 mg/kg body weight) was administered per oral to the female SD rats in conjunction with intraperitoneal (i.p.) injection of Al lactate (10 mg/kg body weight) for six weeks. Pretreated animals were administered GBE for additional two weeks prior to any exposure of Al. GBE administration resulted in decrease in Aß aggregation, ubiquitin deposition, accompanying a significant decline in APP & Tau protein hyperphosphorylation which can be attributed to activation of Heat shock factor (HSF-1) and upregulation in the protein expression of HSPs. Histopathological investigation studies have also shown the decrease in aggregation of Aß peptide by GBE administration. Additionally, the decrease in ROS levels and Aß aggregation by GBE administration prohibited the decline in the neurotransmitter levels and monoamine oxidase levels in hippocampus and cortex. This further caused improvement in learning and memory of the animals. In conclusion, our results indicate that GBE prevents the symptoms of Al induced AD like pathophysiology by upregulating the HSPs levels and decreasing the aggregation load.

Ginkgo biloba attenuates aluminum lactate-induced neurotoxicity in reproductive senescent female rats: behavioral, biochemical, and histopathological study.

Extensive use of aluminum (Al) in industry, cooking utensils, and wrapping or freezing the food items, due to its cheapness and abundance in the environment, has become a major concern. Growing evidence supports that environmental pollutant Al promotes the aggregation of amyloid beta (Aß) in the brain, which is the main pathological marker of Alzheimer's disease (AD). Further, AD- and Al-induced neurotoxic effects are more common among women following reproductive senescence due to decline in estrogen. Though clinically Ginkgo biloba extract (GBE) has been exploited as a memory enhancer, its role in Al-induced neurotoxicity in reproductive senescent female rats needs to be evaluated. Animals were exposed to intraperitoneal dose (10 mg/kg b.wt) of Al and oral dose (100 mg/kg b.wt.) of GBE daily for 6 weeks. A significant decline in the Al-induced Aß aggregates was observed in hippocampal and cortical regions of the brain with GBE supplementation, as confirmed by thioflavin (ThT) and Congo red staining. GBE administration significantly decreased the reactive oxygen species, lipid peroxidation, nitric oxide, and citrulline levels in comparison to Al-treated rats. On the contrary, a significant increase in the reduced glutathione, GSH/GSSG ratio as well as in the activities of antioxidant enzymes was observed with GBE administration. Based on the above results, GBE prevented the neuronal loss in the hippocampus and cortex, hence caused significant improvement in the learning and memory of the animals in terms of AChE activity, serotonin levels, Morris water maze, and active and passive avoidance tests. In conclusion, GBE has alleviated the behavioral, biochemical, and histopathological alterations due to Al toxicity in rats. However, molecular studies are going on to better understand the mechanism of GBE protection against the environmental toxicant Al exposure. Graphical abstract .

Metacaspase-3 of Plasmodium falciparum: An atypical trypsin-like serine protease.

Metacaspases are clan CD cysteine peptidases found in plants, fungi and protozoa that possess a conserved Peptidase_C14 domain, homologous to the human caspases and a catalytic His/Cys dyad. Earlier reports have indicated the role of metacaspases in cell death; however, metacaspases of human malaria parasite remains poorly understood. In this study, we aimed to functionally characterize a novel malarial protease, P. falciparum metacaspase-3 (PfMCA3). Unlike other clan CD peptidases, PfMCA3 has an atypical active site serine (Ser1865) residue in place of canonical cysteine and it phylogenetically forms a distinct branch across the species. To investigate whether this domain retains catalytic activity, we expressed, purified and refolded the Peptidase_C14 domain of PfMCA3 which was found to express in all asexual stages. PfMCA3 exhibited trypsin-like serine protease activity with ser1865 acting as catalytic residue to cleave trypsin oligopeptide substrate. PfMCA3 is inhibited by trypsin-like serine protease inhibitors. Our study found that PfMCA3 enzymatic activity was abrogated when catalytic serine1865 (S1865A) was mutated. Moreover, PfMCA3 was found to be inactive against caspase substrate. Overall, our study characterizes a novel metacaspase of P. falciparum, different from human caspases and not responsible for the caspase-like activity, therefore, could be considered as a potential chemotherapeutic target.