Characterization, Antioxidant, and Antimicrobial Properties of Mulberry Lattices.

In order to find the most advantageous bioactive compounds from mulberry latex for drug development in the near future, this study was conducted to characterize and evaluate antioxidant and antimicrobial properties from four different mulberry lattices (BR-2, S-1, AR-14, and S-146). The characterization of the lattices was performed by scanning electron microscopy with energy-dispersive X-ray spectroscopy, gas chromatography coupled to mass spectroscopy, and Fourier transform infrared spectroscopy. Further, screenings of the antioxidant and antimicrobial potential of selected lattices were performed in vitro using 2,2-diphenyl-1-picrylhydrazyl assay and agar well diffusion methods, respectively. Interestingly, the outcome of the current study revealed that tested mulberry lattices contain a considerable amount of bioactive phytoconstituents, particularly antimicrobial and antioxidant compounds, as revealed by chromatographic analysis. BR-2 latex was found to have significant antioxidant activity (75%) followed by S-146 (64.6%) and AR-14 (52.9%). The maximum antimicrobial activity was found in BR-2 latex compared to other tested latex varieties. The results of this investigation showed that mulberry latex from the BR-2 type may successfully control both bacterial and fungal infections, with the added benefit of having enhanced antioxidant capabilities.

Tabu search problem application based economic impact of coronavirus pandemic on sugar industry inventory system for deteriorating objects with two-distribution center and waste-material treatment.

In this paper, the determined economic impact of the Sugar industry of the Coronavirus pandemic for aggravating items with a ramp-type demand with inflation effects in two-Distribution center storage devices and waste-material treatment cost using Taboo Search is developed. The owned Distribution center has a permanent ability of W units; rented Distribution center has unlimited capacity. Here, we hypothesized that the Block chain Economic Impact of the Coronavirus Pandemic Sugar Industry in Inventory Cost of Inventory in RW is greater than that in owned Distribution center using Taboo Search. The shortcomings of the economic impact of the Coronavirus pandemic Sugar industry are allowed and partially lagged behind, and it is assumed that Block chain's economic impact of the Coronavirus Sugar pandemic industry decreases over time with a variable deterioration rate and waste-material treatment cost using Taboo Search. The effect of inflation was also considered due to the different costs associated with Taboo Search applying the Economic Impact of the Coronavirus Sugar Industry Inventory System and waste-material treatment cost using Taboo Search. The mathematical sample is as well used to study the performance of the model using particle size optimization. The cost minimization technique is second-hand to get hold of expressions for total costs and erstwhile parameters.

Draft Genome of the Liver Fluke Fasciola gigantica.

Fascioliasis, a neglected foodborne disease caused by liver flukes (genus Fasciola), affects more than 200 million people worldwide. Despite technological advances, little is known about the molecular biology and biochemistry of these flukes. We present the draft genome of Fasciola gigantica for the first time. The assembled draft genome has a size of ∼1.04 Gb with an N50 and N90 of 129 and 149 kb, respectively. A total of 20 858 genes were predicted. The de novo repeats identified in the draft genome were 46.85%. The pathway included all of the genes of glycolysis, Krebs cycle, and fatty acid metabolism but lacked the key genes of the fatty acid biosynthesis pathway. This indicates that the fatty acid required for survival of the fluke may be acquired from the host bile. It may be hypothesized that the relatively larger F. gigantica genome did not evolve through genome duplications but rather is interspersed with many repetitive elements. The genomic information will provide a comprehensive resource to facilitate the development of novel interventions for fascioliasis control.

Identification of potential inhibitors of Fasciola gigantica thioredoxin1: computational screening, molecular dynamics simulation, and binding free energy studies.

Fasciola gigantica is the causative organism of fascioliasis and is responsible for major economic losses in livestock production globally. F. gigantica thioredoxin1 (FgTrx1) is an important redox-active enzyme involved in maintaining the redox homeostasis in the cell. To identify a potential anti-fasciolid compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,740) against the FgTrx1 structure. The ligands were docked against FgTrx1 and 309 ligands were found to have better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 30 compounds were found to fit well for re-docking studies. After refinement by molecular docking and drug-likeness analysis, three potential inhibitors (ZINC15970091, ZINC9312362, and ZINC9312661) were identified. These three ligands were further subjected to molecular dynamics simulation (MDS) to compare the dynamics and stability of the protein structure after binding of the ligands. The binding free energy analyses were calculated to determine the intermolecular interactions. The results suggested that the two compounds had a binding free energy of -82.237, and -109.52 kJ.mol-1 for compounds with IDs ZINC9312362 and ZINC9312661, respectively. These predicted compounds displayed considerable pharmacological and structural properties to be drug candidates. We concluded that these two compounds could be potential drug candidates to fight against F. gigantica parasites.

Structure-based screening and molecular dynamics simulations offer novel natural compounds as potential inhibitors of Mycobacterium tuberculosis isocitrate lyase.

Mycobacterium tuberculosis is the etiological agent of tuberculosis in humans and is responsible for more than two million deaths annually. M. tuberculosis isocitrate lyase (MtbICL) catalyzes the first step in the glyoxylate cycle, plays a pivotal role in the persistence of M. tuberculosis, which acts as a potential target for an anti-tubercular drug. To identify the potential anti-tuberculosis compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,748) against the MtbICL structure. The ligands were docked against MtbICL in three sequential docking modes that resulted in 340 ligands having better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 27 compounds were found to fit well with re-docking studies. After refinement by molecular docking and drug-likeness analyses, three potential inhibitors (ZINC1306071, ZINC2111081, and ZINC2134917) were identified. These three ligands and the reference compounds were further subjected to molecular dynamics simulation and binding energy analyses to compare the dynamic structure of protein after ligand binding and the stability of the MtbICL and bound complexes. The binding free energy analyses were calculated to validate and capture the intermolecular interactions. The results suggested that the three compounds had a negative binding energy with -96.462, -143.549, and -122.526 kJ mol-1 for compounds with IDs ZINC1306071, ZINC2111081, and ZINC2134917, respectively. These lead compounds displayed substantial pharmacological and structural properties to be drug candidates. We concluded that ZINC2111081 has a great potential to inhibit MtbICL and would add to the drug discovery process against tuberculosis.

Distant Phe345 mutation compromises the stability and activity of Mycobacterium tuberculosis isocitrate lyase by modulating its structural flexibility.

Isocitrate lyase (ICL), a potential anti-tubercular drug target, catalyzes the first step of the glyoxylate shunt. In the present investigation, we studied the conformational flexibility of MtbICL to better understand its stability and catalytic activity. Our biochemical results showed that a point mutation at Phe345, which is topologically distant (>10 Å) to the active site signature sequence (189KKCGH193), completely abolishes the activity of the enzyme. In depth computational analyses were carried out for understanding the structural alterations using molecular dynamics, time-dependent secondary structure and principal component analysis. The results showed that the mutated residue increased the structural flexibility and induced conformational changes near the active site (residues 170-210) and in the C-terminal lid region (residues 411-428). Both these regions are involved in the catalytic activity of MtbICL. Upon mutation, the residual mobility of the enzyme increased, resulting in a decrease in the stability, which was confirmed by the lower free energy of stabilization in the mutant enzyme suggesting the destabilization in the structure. Our results have both biological importance and chemical novelty. It reveals internal dynamics of the enzyme structure and also suggests that regions other than the active site should be exploited for targeting MtbICL inhibition and development of novel anti-tuberculosis compounds.

UDP-N-Acetylglucosamine enolpyruvyl transferase (MurA) of Acinetobacter baumannii (AbMurA): Structural and functional properties.

Peptidoglycan (PG) is the key component of the bacterial cell wall. The enzyme UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridinediphospho-N-acetylglucosamine (UNAG), which is the first committed step of PG biosynthesis. Here, we present the biochemical and structural features of the MurA enzyme of the opportunistic pathogen Acinetobacter baumannii (AbMurA). The recombinant AbMurA exists as a monomer in solution and shows optimal activity at pH 7.5 and 37°C. The Km for UDP-N-acetylglucosamine was 1.062±0.09mM and for PEP was 1.806±0.23mM. The relative enzymatic activity was inhibited ∼3 fold in the presence of 50mM fosfomycin (FFQ). Superimposition of the AbMurA model with E. coli demonstrated key structural similarity in the FFQ-binding site. AbMurA also has a surface loop that contains the active site Cys116 that interact with FFQ. Sequence analysis indicates the presence of the five conserved amino acids, i.e., K22, C116, D306, D370 and L371, required for the functional activity like other MurA enzymes from different bacteria. MurA enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be a promising drug target.

A combined biochemical and computational studies of the rho-class glutathione s-transferase sll1545 of Synechocystis PCC 6803.

Peroxides are one of the most important radicals that cause oxidative stress. Certain Glutathione S-transferases (GSTs) have been reported to show peroxidase activity. We report a novel peroxidase activity of Synechocystis GST- sll1545. The recombinant protein was purified to homogeneity and characterized. Low Km (0.109µM) and high Vmax (0.663µmolmin-1) values suggest a high preference of sll1545 for cumenehydroperoxide. Disc inhibition assay confirmed the ability of the enzyme to protect cells against peroxide-induced damage. sll1545 has very low sequence and structural similarity with theta and alpha class GSTs that exhibit glutathione-dependent peroxidase activity. Recent data from our laboratory shows that sll1545 is also strongly active against dichloroacetate (DCA), which is a characteristic of zeta class GST. Interestingly, sll1545 shows less than 20% sequence identity with zeta class GST. Molecular dynamic simulation results show that sll1545 was much more structurally different from alpha/theta classes. Our results suggest that sll1545 shows structural variation from zeta, theta/alpha classes of GSTs but have related enzymatic activity. Phylogenetic analysis reveal that sll1545 is evolutionally very distinct from the known GSTs. Overall, the data suggest that Synechocystis sll1545 does not belong to any known GST class and represent a novel GST class, which we have named rho.

Preferential regeneration of thioredoxin from parasitic flatworm Fasciola gigantica using glutathione system.

The maintenance of cellular redox homeostasis is a crucial adaptive problem faced by parasites, and its disruption can shift the biochemical balance toward the host. The thioredoxin (Trx) system plays a key role in redox metabolism and defense against oxidative stress. In this study, biochemical experiments were performed on Fasciola gigantica Thioredoxin1 (FgTrx1). The recombinant FgTrx1 exists as a monomer and catalyzes the reduction of insulin. FgTrx1 is preferentially regenerated by the glutathione (GSH) system using glutathione reductase (GR). The regeneration of FgTrx1 by the conventional Trx system is much less as compared to the GSH system, suggesting that FgTrx1 could be acting as glutaredoxin (Grx). DNA nicking and hydroperoxide assay suggests that it protects the DNA from radical-induced oxidative damage. Thus, FgTrx1 might play a role in parasite survival as it can regenerate itself even in the absence of the canonical Trx system and also protect the cells from ROS induced damage. Further, we propose that the GR activity of FgTrx1 is not restricted to -CXXC- motif but is regulated by residues present in close proximity to the -CXXC- motif, through manipulation of the redox potential or the pKa of the active site Cys residues.

A novel male sterility-fertility restoration system in plants for hybrid seed production.

Hybrid seeds are used for stimulated crop production, as they harness heterosis. The achievement of complete male-sterility in the female-parent and the restored-fertility in F1-hybrids are the major bottlenecks in the commercial hybrid seed production. Here, we report a male sterility-fertility restoration system by engineering the in most nutritive anther wall layer tapetum of female and male parents. In the female parent, high-level, and stringent expression of Arabidopsis autophagy-related gene BECLIN1 was achieved in the tapetum, which altered the tapetal degeneration program, leading to male sterility. This works on our previously demonstrated expression cassette based on functional complementation of TATA-box mutant (TGTA) promoter and TATA-binding protein mutant3 (TBPm3), with modification by conjugating Long Hypocotyle in Far-Red1 fragment (HFR1(NT131)) with TBPm3 (HFR1(NT131)-TBPm3) to exercise regulatory control over it. In the male parent, tapetum-specific Constitutive photo-morphogenesis1 (COP1) was expressed. The F1 obtained by crossing these engineered parents showed decreased BECLIN1 expression, which was further completely abolished when COP1-mutant (COP1(L105A)) was used as a male parent, leading to normal tapetal development and restored fertility. The system works on COP1-HFR1 interaction and COP1-mediated degradation of TBPm3 pool (HFR1(NT131)-TBPm3). The system can be deployed for hybrid seed production in agricultural crops.

An improved method for high-level soluble expression and purification of recombinant amyloid-beta peptide for in vitro studies.

Amyloid-beta (Aß) peptide mediates several neurodegenerative diseases. The 42 amino acid (Aß1-42) is the predominant form of peptide found in the neuritic plaques and has been demonstrated to be neurotoxic in vivo and in vitro. The availability of large quantities of Aß peptide will help in several biochemical and biophysical studies that may help in exploring the aggregation mechanism and toxicity of Aß peptide. We report a convenient and economical method to obtain such a peptide biologically. Synthetic oligonucleotides encoding Aß1-42 were constructed and amplified through the polymerase cycling assembly (also known as assembly PCR), followed by the amplification PCR. Aß1-42 gene was cloned into pET41a(+) vector for expression. Interestingly, the addition of 3% (v/v) ethanol to the culture medium resulted in the production of large amounts of soluble Aß fusion protein. The Aß fusion protein was subjected to a Ni-NTA affinity chromatography followed by enterokinase digestion, and the Aß peptide was purified using glutathione Sepharose affinity chromatography. The peptide yield was ∼15mg/L culture, indicating the utility of this method for high-yield production of soluble Aß peptide. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and immunoblotting with anti-His antibody confirmed the identity of purified Aß fusion protein and Aß peptide. In addition, this method provides an advantage over the chemical synthesis and other conventional methods used for large-scale production of recombinant Aß peptide.

Characterization of a Highly pH Stable Chi-Class Glutathione S-Transferase from Synechocystis PCC 6803.

Glutathione S-transferases (GSTs) are multifunctional enzymes present in virtually all organisms. Besides having an essential role in cellular detoxification, they also perform various other functions, including responses in stress conditions and signaling. GSTs are highly studied in plants and animals; however, the knowledge regarding GSTs in cyanobacteria seems rudimentary. In this study, we report the characterization of a highly pH stable GST from the model cyanobacterium--Synechocystis PCC 6803. The gene sll0067 was expressed in Escherichia coli (E. coli), and the protein was purified to homogeneity. The expressed protein exists as a homo-dimer, which is composed of about 20 kDa subunit. The results of the steady-state enzyme kinetics displayed protein's glutathione conjugation activity towards its class specific substrate- isothiocyanate, having the maximal activity with phenethyl isothiocyanate. Contrary to the poor catalytic activity and low specificity towards standard GST substrates such as 1-chloro-2,4-dinitrobenzene by bacterial GSTs, PmGST B1-1 from Proteus mirabilis, and E. coli GST, sll0067 has broad substrate degradation capability like most of the mammalian GST. Moreover, we have shown that cyanobacterial GST sll0067 is catalytically efficient compared to the best mammalian enzymes. The structural stability of GST was studied as a function of pH. The fluorescence and CD spectroscopy in combination with size exclusion chromatography showed a highly stable nature of the protein over a broad pH range from 2.0 to 11.0. To the best of our knowledge, this is the first GST with such a wide range of pH related structural stability. Furthermore, the presence of conserved Proline-53, structural motifs such as N-capping box and hydrophobic staple further aid in the stability and proper folding of cyanobacterial GST-sll0067.

Functional classification and biochemical characterization of a novel rho class glutathione S-transferase in Synechocystis PCC 6803.

We report a novel class of glutathione S-transferase (GST) from the model cyanobacterium Synechocystis PCC 6803 (sll1545) which catalyzes the detoxification of the water pollutant dichloroacetate and also shows strong glutathione-dependent peroxidase activity representing the classical activities of zeta and theta/alpha class respectively. Interestingly, sll1545 has very low sequence and structural similarity with these classes. This is the first report of dichloroacetate degradation activity by any bacterial GST. Based on these results we classify sll1545 to a novel GST class, rho. The present data also indicate potential biotechnological and industrial applications of cyanobacterial GST in dichloroacetate-polluted areas.

Recombinant expression, purification and preliminary characterization of the mRNA export factor MEX67 of Saccharomyces cerevisiae.

The nuclear export of macromolecules is facilitated by the nuclear pore complexes (NPCs), embedded in the nuclear envelope and consists of multi-protein complexes. MEX67 is one of the nuclear export factor responsible for the transport of the majority of cellular mRNAs from the nucleus to the cytoplasm. The mechanism of mRNA transport through NPCs is unclear due to the unavailability of structures and the known interacting partners of MEX67. The mex67 gene was cloned in pQE30A and was expressed in Escherichia coli. A strategy has been developed to purify the insoluble MEX67 using a nickel affinity column with chelating Sepharose fast flow media, after solubilizing with sodium lauroyl sarcosinate (Sarkosyl). The IMAC purified recombinant MEX67 was further purified using SEC to apparent homogeneity (∼8 mg/L). Following SEC, MEX67 was stable and observed to be a 67 kDa monomeric protein as determined by PAGE and the size exclusion chromatography. The availability of large quantities of the protein will help in its biochemical and biophysical characterization, which may lead to the identification of new interaction partners of MEX67 or MEX67 complex.

BECLIN1 from Arabidopsis thaliana under the generic control of regulated expression systems, a strategy for developing male sterile plants.

Induction of male sterility followed by successful outcrossing is a prerequisite for hybrid seed production. In this article, we have identified a novel use of the BECLIN 1 gene of Arabidopsis, in inducing male sterility in plants, when expressed in the anther tapetum of tobacco. We also report a stringently regulated and high-level expression of the desired gene in tapetum by using a two-component transcription regulation system. The tapetum-specific, two-component transcription system utilizes the TGTA-TBPm³ complementation principle that has been demonstrated by us earlier. We also report a glucocorticoid-dependent expression of AtBECLIN 1 in tapetum, thereby developing glucocorticoid-inducible male sterility in plants.