Immunoinformatics based designing a multi-epitope vaccine against pathogenic Chandipura vesiculovirus.

Chandipura vesiculovirus (CHPV) is a rapidly emerging pathogen responsible for causing acute encephalitis. Due to its widespread occurrence in Asian and African countries, this has become a global threat, and there is an urgent need to design an effective and nonallergenic vaccine against this pathogen. The present study aimed to develop a multi-epitope vaccine using an immunoinformatics approach. The conventional method of vaccine design involves large proteins or whole organism which leads to unnecessary antigenic load with increased chances of allergenic reactions. In addition, the process is also very time-consuming and labor-intensive. These limitations can be overcome by peptide-based vaccines comprising short immunogenic peptide fragments that can elicit highly targeted immune responses, avoiding the chances of allergenic reactions, in a relatively shorter time span. The multi-epitope vaccine constructed using CTL, HTL, and IFN-γ epitopes was able to elicit specific immune responses when exposed to the pathogen, in silico. Not only that, molecular docking and molecular dynamics simulation studies confirmed a stable interaction of the vaccine with the immune receptors. Several physicochemical analyses of the designed vaccine candidate confirmed it to be highly immunogenic and nonallergic. The computer-aided analysis performed in this study suggests that the designed multi-epitope vaccine can elicit specific immune responses and can be a potential candidate against CHPV.

Thermodynamics of ferredoxin binding to cyanobacterial nitrate reductase.

The role of the seven negatively charged amino acids of Synechocystis sp. PCC 6803 ferredoxin (Fd), i.e., Glu29, Glu30, Asp60, Asp65, Asp66, Glu92, and Glu93, predicted to form complex with nitrate reductase (NR), was investigated using site-directed mutagenesis and isothermal titration calorimetry (ITC). These experiments identified four Fd amino acids, i.e., Glu29, Asp60, Glu92, and Glu93, that are essential for the Fd binding and efficient electron transfer to the NR. ITC measurements showed that the most likely stoichiometry for the wild-type NR/wild-type Fd complex is 1:1, a Kd value 4.7 µM for the complex at low ionic strength residues and both the enthalpic and entropic components are associated with complex formation. ITC titrations of wild-type NR with four Fd variants, E29N, D60N, E92Q, and E93N demonstrated that the complex formation, although favorable, was less energetically favorable when compared to complex formation between the two wild-type proteins, suggesting that these negatively charged Fd residues at these positions are important for the effective and productive interaction with wild-type enzyme.

Identification of the Ferredoxin-Binding Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase.

An in silico model for the 1:1 ferredoxin (Fd)/nitrate reductase (NR) complex, using the known structure of Synechocystis sp. PCC 6803 Fd and the in silico model of Synechococcus sp. PCC 7942 NR, is used to map the interaction sites that define the interface between Fd and NR. To test the electrostatic interactions predicted by the model complex, five positively charged NR amino acids (Arg43, Arg46, Arg197, Lys201, and Lys614) and a negatively charged amino acid (Glu219) were altered using site-directed mutagenesis and characterized by activity measurements, metal analysis, and electron paramagnetic resonance (EPR) studies. All of the charge replacement variants retained wild-type levels of activity with reduced methyl viologen (MV), but a significant decrease in activity was observed for the R43Q, R46Q, K201Q, and K614Q variants when reduced Fd served as the electron donor. EPR analysis as well as the Fe and Mo analyses showed that loss of activity observed with these variants was not the consequence of perturbation of the Mo center or [4Fe-4S] cluster. Therefore, the loss of the Fd-linked specific activity observed with these variants can be explained only by invoking a role for Arg43, Arg46, Lys201, and Lys614 in Fd binding. The R43Q, R46Q, K201Q, and K614Q NR variants also showed a decreased binding affinity for Fd, compared to that of wild-type NR, supporting a key role of these four positively charged residues in the productive binding of Fd.

Identification of the ferredoxin interaction sites on ferredoxin-dependent glutamate synthase from Synechocystis sp. PCC 6803.

Based on in silico docking methods, five amino acids in glutamate synthase (Gln-467, His-1144, Asn-1147, Arg-1162, and Trp-676) likely constitute key binding residues in the interface of a glutamate synthase:ferredoxin complex. Although all interfacial mutants studied showed the ability to form a complex under low ionic strength, these docking mutations showed significantly less ferredoxin-dependent activities, while still retaining enzymatic activity. Furthermore, isothermal titration calorimetry showed a possible 1:2 molar ratio between the wild-type glutamate synthase and ferredoxin. However, each of our interfacial mutants showed only a 1:1 complex with ferredoxin, suggesting that the mutations directly affect the glutamate synthase:ferredoxin heterodimer interface.

Identification of Amino Acids at the Catalytic Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase.

An in silico model of the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942, and information about active sites in related enzymes, had identified Cys148, Met149, Met306, Asp163, and Arg351 as amino acids likely to be involved in either nitrate binding, prosthetic group binding, or catalysis. Site-directed mutagenesis was used to alter each of these residues, and differences in enzyme activity and substrate binding of the purified variants were analyzed. In addition, the effects of these replacements on the assembly and properties of the Mo cofactor and [4Fe-4S] centers were investigated using Mo and Fe determinations, coupled with electron paramagnetic resonance spectroscopy. The C148A, M149A, M306A, D163N, and R351Q variants were all inactive with either the physiological electron donor, reduced ferredoxin, or the nonphysiological electron donor, reduced methyl viologen, as the source of electrons, and all exhibited changes in the properties of the Mo cofactor. Charge-conserving D163E and R351K variants were also inactive, suggesting that specific amino acids are required at these two positions. The implications for the role of these five conserved active-site residues in light of these new results and previous structural, spectroscopic, and mutagenesis studies for related periplasmic nitrate reductases are discussed.

A loop unique to ferredoxin-dependent glutamate synthases is not absolutely essential for ferredoxin-dependent catalytic activity.

It had been proposed that a loop, typically containing 26 or 27 amino acids, which is only present in monomeric, ferredoxin-dependent, "plant-type" glutamate synthases and is absent from the catalytic α-subunits of both NADPH-dependent, heterodimeric glutamate synthases found in non-photosynthetic bacteria and NADH-dependent heterodimeric cyanobacterial glutamate synthases, plays a key role in productive binding of ferredoxin to the plant-type enzymes. Site-directed mutagenesis has been used to delete the entire 27 amino acid-long loop in the ferredoxin-dependent glutamate synthase from the cyanobacterium Synechocystis sp. PCC 6803. The specific activity of the resulting loopless variant of this glutamate synthase, when reduced ferredoxin serves as the electron donor, is actually higher than that of the wild-type enzyme, suggesting that this loop is not absolutely essential for efficient electron transfer from reduced ferredoxin to the enzyme. These results are consistent with the results of an in-silico study that suggests that the loop is unlikely to interact directly with ferredoxin in the energetically most favorable model of a 1:1 complex of ferredoxin with the wild-type enzyme.

Kinetic studies of a ferredoxin-dependent cyanobacterial nitrate reductase.

A flash photolysis study of electron transfer (ET) kinetics from reduced ferredoxin (photoreduced by Photosystem I) to the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been carried out. In the presence of nitrate, under conditions where only a single electron is transferred to nitrate reductase, the rate of enzyme reduction shows a biphasic concentration dependence: At low enzyme concentrations the dependence is approximately linear, with an estimated second-order rate constant of 7.4 ± 0.8 × 10(7) M(-1) s(-1); at concentrations above 2 µM, the rate increases nonlinearly to an asymptotic value of approximately 300 s(-1), indicating the presence of a rate-limiting step in the process. The spectrum of the one-electron reduced enzyme suggests that Mo centers are largely reduced with a minor contribution of iron-sulfur cluster reduction. Under conditions favoring two-electron reduction of the enzyme, the redox difference spectrum can be accounted for by the oxidation of two reduced ferredoxins, suggesting that the enzyme has completed one full catalytic cycle. The spectral changes observed in the absence of nitrate are significantly different from those seen in the presence of nitrate. Experiments in the absence of nitrate revealed that the singly reduced enzyme exhibits different absorption characteristics and reoxidation kinetics, compared to those observed with nitrate present, and exhibits a much faster binding by reduced ferredoxin than the oxidized enzyme. The implications of these observations for understanding the enzyme mechanism are discussed.

Roles of four conserved basic amino acids in a ferredoxin-dependent cyanobacterial nitrate reductase.

The roles of four conserved basic amino acids in the reaction catalyzed by the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 have been investigated using site-directed mutagenesis in combination with measurements of steady-state kinetics, substrate-binding affinities, and spectroscopic properties of the enzyme's two prosthetic groups. Replacement of either Lys58 or Arg70 by glutamine leads to a complete loss of activity, both with the physiological electron donor, reduced ferredoxin, and with a nonphysiological electron donor, reduced methyl viologen. More conservative, charge-maintaining K58R and R70K variants were also completely inactive. Replacement of Lys130 by glutamine produced a variant that retained 26% of the wild-type activity with methyl viologen as the electron donor and 22% of the wild-type activity with ferredoxin as the electron donor, while replacement by arginine produces a variant that retains a significantly higher percentage of the wild-type activity with both electron donors. In contrast, replacement of Arg146 by glutamine had minimal effect on the activity of the enzyme. These results, along with substrate-binding and spectroscopic measurements, are discussed in terms of an in silico structural model for the enzyme.

In-silico genome wide analysis of Mitogen activated protein kinase kinase kinase gene family in C. sinensis.

Mitogen activated protein kinase kinase kinase (MAPKKK) form the upstream component of MAPK cascade. It is well characterized in several plants such as Arabidopsis and rice however the knowledge about MAPKKKs in tea plant is largely unknown. In the present study, MAPKKK genes of tea were obtained through a genome wide search using Arabidopsis thaliana as the reference genome. Among 59 candidate MAPKKK genes in tea, 17 genes were MEKK-like, 31 genes were Raf-like and 11 genes were ZIK- like. Additionally, phylogenetic relationships were established along with structural analysis, which includes gene structure, its location as well as conserved motifs, cis-acting regulatory elements and functional domain signatures that were systematically examined. Also, on the basis of one orthologous gene found between tea and Arabidopsis, functional interaction was carried out in C. sinensis based on an Arabidopsis association model. The expressional profiles indicated major involvement of MAPKKK genes from tea in response to various abiotic stress factors. Taken together, this study provides the targets for additional inclusive identification, functional study, and provides comprehensive knowledge for a better understanding of the MAPKKK cascade regulatory network in C. sinensis.

From Infection to Immunity: Understanding the Response to SARS-CoV2 Through In-Silico Modeling.

Background: Immune system conditions of the patient is a key factor in COVID-19 infection survival. A growing number of studies have focused on immunological determinants to develop better biomarkers for therapies. Aim: Studies of the insurgence of immunity is at the core of both SARS-CoV-2 vaccine development and therapies. This paper attempts to describe the insurgence (and the span) of immunity in COVID-19 at the population level by developing an in-silico model. We simulate the immune response to SARS-CoV-2 and analyze the impact of infecting viral load, affinity to the ACE2 receptor, and age in an artificially infected population on the course of the disease. Methods: We use a stochastic agent-based immune simulation platform to construct a virtual cohort of infected individuals with age-dependent varying degrees of immune competence. We use a parameter set to reproduce known inter-patient variability and general epidemiological statistics. Results: By assuming the viremia at day 30 of the infection to be the proxy for lethality, we reproduce in-silico several clinical observations and identify critical factors in the statistical evolution of the infection. In particular, we evidence the importance of the humoral response over the cytotoxic response and find that the antibody titers measured after day 25 from the infection are a prognostic factor for determining the clinical outcome of the infection. Our modeling framework uses COVID-19 infection to demonstrate the actionable effectiveness of modeling the immune response at individual and population levels. The model developed can explain and interpret observed patterns of infection and makes verifiable temporal predictions. Within the limitations imposed by the simulated environment, this work proposes quantitatively that the great variability observed in the patient outcomes in real life can be the mere result of subtle variability in the infecting viral load and immune competence in the population. In this work, we exemplify how computational modeling of immune response provides an important view to discuss hypothesis and design new experiments, in particular paving the way to further investigations about the duration of vaccine-elicited immunity especially in the view of the blundering effect of immunosenescence.

In silico designing of vaccine candidate against Clostridium difficile.

Clostridium difficile is a spore-forming gram-positive bacterium, recognized as the primary cause of antibiotic-associated nosocomial diarrhoea. Clostridium difficile infection (CDI) has emerged as a major health-associated infection with increased incidence and hospitalization over the years with high mortality rates. Contamination and infection occur after ingestion of vegetative spores, which germinate in the gastro-intestinal tract. The surface layer protein and flagellar proteins are responsible for the bacterial colonization while the spore coat protein, is associated with spore colonization. Both these factors are the main concern of the recurrence of CDI in hospitalized patients. In this study, the CotE, SlpA and FliC proteins are chosen to form a multivalent, multi-epitopic, chimeric vaccine candidate using the immunoinformatics approach. The overall reliability of the candidate vaccine was validated in silico and the molecular dynamics simulation verified the stability of the vaccine designed. Docking studies showed stable vaccine interactions with Toll-Like Receptors of innate immune cells and MHC receptors. In silico codon optimization of the vaccine and its insertion in the cloning vector indicates a competent expression of the modelled vaccine in E. coli expression system. An in silico immune simulation system evaluated the effectiveness of the candidate vaccine to trigger a protective immune response.

A candidate multi-epitope vaccine against SARS-CoV-2.

In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2.

Bedaquiline inhibits the yeast and human mitochondrial ATP synthases.

Bedaquiline (BDQ, Sirturo) has been approved to treat multidrug resistant forms of Mycobacterium tuberculosis. Prior studies suggested that BDQ was a selective inhibitor of the ATP synthase from M. tuberculosis. However, Sirturo treatment leads to an increased risk of cardiac arrhythmias and death, raising the concern that this adverse effect results from inhibition at a secondary site. Here we show that BDQ is a potent inhibitor of the yeast and human mitochondrial ATP synthases. Single-particle cryo-EM reveals that the site of BDQ inhibition partially overlaps with that of the inhibitor oligomycin. Molecular dynamics simulations indicate that the binding mode of BDQ to this site is similar to that previously seen for a mycobacterial enzyme, explaining the observed lack of selectivity. We propose that derivatives of BDQ ought to be made to increase its specificity toward the mycobacterial enzyme and thereby reduce the side effects for patients that are treated with Sirturo.

Overexpression of the rice gene OsSIZ1 in Arabidopsis improves drought-, heat-, and salt-tolerance simultaneously.

Sumoylation is one of the post translational modifications, which affects cellular processes in plants through conjugation of small ubiquitin like modifier (SUMO) to target substrate proteins. Response to various abiotic environmental stresses is one of the major cellular functions regulated by SUMO conjugation. SIZ1 is a SUMO E3 ligase, facilitating a vital step in the sumoylation pathway. In this report, it is demonstrated that over-expression of the rice gene OsSIZ1 in Arabidopsis leads to increased tolerance to multiple abiotic stresses. For example, OsSIZ1-overexpressing plants exhibited enhanced tolerance to salt, drought, and heat stresses, and generated greater seed yields under a variety of stress conditions. Furthermore, OsSIZ1-overexpressing plants were able to exclude sodium ions more efficiently when grown in saline soils and accumulate higher potassium ions as compared to wild-type plants. Further analysis revealed that OsSIZ1-overexpressing plants expressed higher transcript levels of P5CS, a gene involved in the biosynthesis of proline, under both salt and drought stress conditions. Therefore, proline here is acting as an osmoprotectant to alleviate damages caused by drought and salt stresses. These results demonstrate that the rice gene OsSIZ1 has a great potential to be used for improving crop's tolerance to several abiotic stresses.

High-resolution cryo-EM analysis of the yeast ATP synthase in a lipid membrane.

Mitochondrial adenosine triphosphate (ATP) synthase comprises a membrane embedded Fo motor that rotates to drive ATP synthesis in the F1 subunit. We used single-particle cryo-electron microscopy (cryo-EM) to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin at 3.6- and 3.8-angstrom resolution, respectively. To limit conformational heterogeneity, we locked the rotor in a single conformation by fusing the F6 subunit of the stator with the δ subunit of the rotor. Assembly of the enzyme with the F6-δ fusion caused a twisting of the rotor and a 9° rotation of the Fo c10-ring in the direction of ATP synthesis, relative to the structure of isolated Fo Our cryo-EM structures show how F1 and Fo are coupled, give insight into the proton translocation pathway, and show how oligomycin blocks ATP synthesis.