Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus system

We describe the expression, in insect cells using the baculovirus system, of two protein fragments derived from the C-terminus of merozoite surface protein 1(MSP-1) of the human malaria parasite Plasmodium falciparum, and their glycosylation and intracellular location. The transport and intracellular localisation of the intact C-terminal MSP-1 fragment, modified by addition of a signal sequence for secretion, was compared with that of a similar control protein in which translation of the GPI-cleavage/attachment site was abolished by insertion of a stop codon into the DNA sequence. Both proteins could only be detected intracellularly, most likely in the endoplasmic reticulum. This lack of transport to the cell surface or beyond, was confirmed for both proteins by immunofluorescence with a specific antibody and characterisation of their N-glycans. The N-glycans had not been processed by enzymes localised in post-endoplasmic reticulum compartments. In contrast to MSP-1, the surface antigen SAG-1 of Toxoplasma gondii was efficiently transported out of the endoplasmic reticulum of insect cells and was located, at least in part, on the cell surface. No GPI-anchor could be detected for either of the MSP-1 constructs or SAG-1, showing that the difference in transport is a property of the individual proteins and cannot be attributed to the lack of a GPI-anchor. The different intracellular location and post-translational modification of recombinant proteins expressed in insect cells, as compared to the native proteins expressed in parasites, and the possible implications for vaccine development are discussed.     

Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11

Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce 7-ethyl-10-hydroxycamptothecin (SN-38), a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and pH 6.8 and resolution of 2.0 A and 1.58 A, respectively. Est55 folds into three domains, a catalytic domain, an alpha/beta domain and a regulatory domain. The structure is in an inactive form; the side-chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side-chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy.     

Synthesis and molecular modeling of novel non-sulfonylureas as hypoglycemic agents and selective ALR2 inhibitors

Novel non-sulfonylureas derivatives bearing an acetamide linker between a spirohydantoin scaffold and a phenyl ring were prepared and their hypoglycemic activity was estimated in vivo. Their abilities to discriminate in vitro between aldehyde reductase (ALR1) and aldose reductase (ALR2) were determined. The molecular docking and the in silico prediction studies were performed to rationalize the obtained biological results and to predict the physicochemical properties and drug-likeness scores of the new compounds. N-(2,4-Dichlorophenyl)-2-(2′,4′-dioxospiro[fluorene-9,5′-imidazolidine]-3′-yl)acetamide (3e) displayed an 84% reduction in blood glucose level superior to that of repaglinide 66% and showed an IC₅₀ value of 0.37 μM against ALR2 that is superior to that of sorbinil 3.14 µM. Compound (3e) was selective 96 fold towards ALR2 which is closely related to serious diabetic complications. Based on the identification of this hit candidate, a new generation of safe and effective antidiabetic agents could be designed.     

Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8⁺ T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine.     

Covalent reaction intermediate revealed in crystal structure of the Geobacillus stearothermophilus carboxylesterase Est30

Est30 is a thermophilic carboxylesterase cloned from Geobacillus stearothermophilus that showed optimal hydrolysis of esters with short acyl chains at 70 degrees C. Est30 is a member of a new family of carboxylesterases with representatives in other Gram-positive bacteria. The crystal structure has been determined at 1.63A resolution using multiple anomalous dispersion data. The two-domain crystal structure showed a large domain with a modified alpha/beta hydrolase core including a seven, rather than an eight-stranded beta sheet, and a smaller cap domain comprising three alpha helices. The catalytic triad consists of residues Ser94, Asp193, and His223. A 100Da tetrahedral ligand was observed to be covalently bound to the side-chain of Ser94. The propyl acetate ligand represents the first tetrahedral intermediate in the reaction mechanism. Therefore, this Est30 crystal structure will help understand the mode of action of all enzymes in the serine hydrolase superfamily.     

Assessing the potential use of chitosan scaffolds for the sustained localized delivery of vitamin D

Vitamin D is a commonly used bone modulator in regenerative medicine. Several modalities have been explored for the delivery of vitamin D including nanoparticles and scaffold. The present study aimed to assess the potential use of a bio-degradable chitosan scaffold for the delivery of vitamin D. The objectives included fabrication of a bio-degradable chitosan scaffold, integration of vitamin D into the scaffold, characterization of the vitamin D integrated scaffold. Characterization was carried out using, X-ray diffraction, Fourier transform infrared spectroscopy, and differential scanning calorimetry. The structure of the scaffold was assessed by scanning electron microscopy. The scaffold was placed in phosphate buffer saline and the release duration of vitamin D was observed using UV spectrophotometry. Dental pulp mesenchymal stem cells were added to the scaffold to study the scaffold associated toxicity and the functionality of the scaffold released vitamin D. The vitamin D release period from the scaffold was estimated to be for 80 hrs. MTT assay of the stem cells was comparable to that of the control group (stem cells cultured in media) inferring that the scaffold is not toxic towards the stem cells. The positive alizarin red S staining, a higher expression of alkaline phosphatase, osteocalcin, and RunX2 confirmed the functional capability (osteogenic differentiation of the stem cells) of the released vitamin D. Based on the data from the present study, it can be inferred that chitosan scaffold can be used for the sustained delivery of functional vitamin D for 3–5 days.     

Spectrofluorimetric study on fluorescence quenching of tyrosine and l‐tryptophan by the aniracetam cognition enhancer drug: quenching mechanism using Stern–Volmer and double‐log plots

A novel approach on fluorescence quenching of tyrosine and l ‐tryptophan is presented for spectrofluorimetric determination of aniracetam in drug substances and products. The quenching mechanism was investigated using Stern–Volmer plots and ultraviolet spectra figures of quencher–fluorophore mixtures. Binding constant and stoichiometry were calculated using double‐log plots. The spectrofluorimetric method was optimized for the experimental conditions affecting fluorescence quenching including fluorophore concentration, diluent, and reaction time. Moreover, the pH‐rate profile of aniracetam was studied using simple kinetics and found to be stable within the pH range 5–8. Fluorescence quenching of tyrosine and l ‐tryptophan were observed on addition of aniracetam in aqueous medium at pH 5.5–6.5. Aniracetam quenched the fluorescence of tyrosine and l ‐tryptophan in the concentration range 1–20 μg/ml and 0.3–20 μg/ml, respectively, with binomial relationships between quenching values (ΔF) and aniracetam concentration. Limits of detection were found to be 0.10 μg/ml for tyrosine–aniracetam and 0.14 μg/ml for l ‐tryptophan–aniracetam. Method validation was performed as per ICH guidelines and demonstrated that the developed spectrofluorimetric method was accurate, precise, specific, and suitable for analysis of aniracetam in routine quality control laboratories. All experimental materials and solvents used are eco‐friendly, indicating that the cited spectrofluorimetric procedure is an excellent green method.     

Tinospora cordifolia (Thunb.) Miers (Giloy) inhibits oral cancer cells in a dose-dependent manner by inducing apoptosis and attenuating epithelial-mesenchymal transition

BackgroundTinospora cordifolia (Thunb.) Miers (Giloy) has been applied successfully as an anti-inflammatory, anti-diabetic, and even as an anti-cancer agent. Yet, to date, the application of Giloy has not been explored concerning oral cancer.ObjectivesTo assess the effect of T cordifolia (Thunb.) Miers (Giloy) extract (TcE) on an oral cancer cell line.MethodsAW13516 (oral cancer cell line) cells were treated with the prepared aqueous extract of TcE for 24 h at various concentrations ranging between 5 μg/ml and 100 μg/ml and compared with control (cells without treatment). Thee effect of the extracts on apoptosis was assessed by through Annexin V flow cytometry assay and Luminometry based assessment of Caspase 8, 9 and caspase 3/7 activity. RNA was isolated from treated cells and gene expression of selected metastatic genes (MMP1, MMP10, and CXCL8); epithelial-mesenchymal stem cell genes (TWIST1, SNAIL, ZEB1, Oct4) and stemness related genses (Nanog, Sox2) were analyzed by using a quantitative real-time PCR system. The experiments were performed in triplicates.ResultsAqueous extract of TcE was found to induce apoptosis inducer in AW13516 cells in a concentration-dependent manner and was potent even at a low concentration of 5 μg/ml. The apoptosis induction was confirmed with the caspase activity assay. Treatment of the cells with the extract for 24 h exhibited a significant decrease in the expression of EMT genes in a dose-dependent manner without an effect on the metastatic genes.ConclusionAqueous extract of TcE induces apoptosis-mediated cell death in the oral cancer cell line AW13516 while attenuating its potential for epithelial mesenchymal transition.     

Application of homobrassinolide enhances growth,yield and quality of tomato

Brassinosteroids (BRs) have emerged as pleiotropic phytohormone owing to their wide function in crop growth and metabolism. Homobrassinolide (HBR) being an analogue of BRs is known to improve the growth, yield and quality parameters in many crop plants. Thus, an evaluation study was conducted for two years (2018 and 2019) to elucidate the performance of tomato plants (Solanum lycopersicum L.) to a novel group of phytohormone,HBR. The field experiment comprised of seven treatments with homobrassinolide 0.04% (Emulsifiable Concentrate) EC at four different concentrations (0.06, 0.08, 0.10 and 0.12 g active ingredient (a.i.) ha^?1) and two well-known growth promoters viz., Gibberellic acid (GA), Naphthalene Acetic Acid (NAA) along with the untreated control. Plant height and chlorophyll concentration were found significantly different in both years of experiment as well as among the different treatments. HBR at 0.12 g a.i. ha^?1 was found better with maximum number of fruits (77.36 plant^?1), fruit length (6.72 cm), fruit breadth (6.45 cm) and fruit weight (80.52 g) over other concentrations and treatments. Fruit yield was more pronounced in the plots treated with plant growth regulators compared to untreated control. However, significantly higher fruit yield of 91.07 t ha^?1 (62.58 t ha^?1 with untreated control) along with improved quality traits viz., fruit firmness (4.11 kg cm^?2), ascorbic acid content (24.09 mg 100 g^?1), total soluble solids (4.43°Brix) and keeping quality (12.50 days) was recorded in 0.12 g a.i. ha^?1 HBR treated plots. Thus, it can be inferred that HBRapplication would be a better option to enhance growth, yield as well as quality traits in tomato.     

An efficient method to express GPI-anchor proteins in insect cells

Glycosylphosphatidylinositols (GPIs) constitute a class of glycolipids that have various functions, the most basic being to attach proteins to the surface of eukaryotic cells. GPIs have to be taken into account, when expressing surface antigens from parasitic protozoa in heterologous systems. The synthesis of the GPI-anchors was previously reported to be drastically decreased to almost background level following baculovirus infection. Here we describe a new method to express GPI-anchor proteins in insect cells relying on using of a supplementary baculovirus construct that overexpresses the N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the enzyme catalyzing the second step in the GPI biosynthetic pathway.