Unveiling the Potential of Covalent Organic Framework Electrocatalyst for Enhanced Oxygen Evolution.

The potential for sustainable energy and carbon neutrality has expanded with the development of a highly active electrocatalyst for the oxygen evolution reaction (OER). Covalent Organic Frameworks (COF) have recently garnered attention because of their enormous potential in a number of cutting-edge application sectors, such as gas storage, sensors, fuel cells, and active catalytic supports. A simple and effective COF constructed and integrated by post-alteration plasma modification facilitates high electrocatalytic OER activity under alkaline conditions. Variations in parameters such as voltage and treatment duration have been employed to enhance the factor that demonstrates high OER performance. The overpotential and Tafel slope are the lowest of all when using an optimized parameter, such as plasma treatment for 30 min utilizing 6 kV of voltage, PT-30 COF, measuring 390 mV at a current density of 10 mA.cm-2 and 69 mV.dec-1, respectively, as compared to 652 mV and 235 mV.dec-1 for the Pristine-COF. Our findings provide a method for broadening the scope by post-functionalizing the parent framework for effective water splitting.

Enabling Ultrahigh Surface Area of Covalently-linked Organic Framework for Boosted CO2 Capture: An Air Liquid Interfacial Plasma as Post-furnishing Protocol.

The cognitive intent of a highly ordered and robust adsorbent is extremely sensible and, in this context, Covalent Organic Framework (COF) materials have significantly burgeoned their scope in diverse applications. Herein, a simple time-competent hydrothermal procedure is presented to construct a covalent framework with an ultrahigh surface area of 1428 m2 /g that shows active adsorption of carbon dioxide (CO2 ) at variable temperature ranges. Moreover, a facile scalably controlled post-synthetic air liquid interfacial plasma (ALIP) induced protocol is substantiated that explicitly amplifies the surface area of the pristine framework even to a higher value of 2051 m2 /g. The post-synthetic plasma approach presented here led to the rapid enhancement of the surface area of the pristine COF by 43 %, which concurrently advances the CO2 uptake up to 67 %. Hence, the current study may open up a new frontier in the design as well as fine-tune the properties of the covalent framework that unfolds the advanced outlook in addressing the challenges of CO2 capture.

Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment.

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

Design of a peptide-based subunit vaccine against novel coronavirus SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that was first reported in Wuhan, China, and has subsequently spread worldwide. In the absence of any antiviral or immunomodulatory therapies, the disease is spreading at an alarming rate. A possibility of a resurgence of COVID-19 in places where lockdowns have already worked is also developing. Thus, for controlling COVID-19, vaccines may be a better option than drugs. An mRNA-based anti-COVID-19 candidate vaccine has entered a phase 1 clinical trial. However, its efficacy and potency have to be evaluated and validated. Since vaccines have high failure rates, as an alternative, we are presenting a new, designed multi-peptide subunit-based epitope vaccine against COVID-19. The recombinant vaccine construct comprises an adjuvant, cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes joined by linkers. The computational data suggest that the vaccine is non-toxic, non-allergenic, thermostable, with the capability to elicit a humoral and cell-mediated immune response. The stabilization of the vaccine construct is validated with molecular dynamics simulation studies. This unique vaccine is made up of 33 highly antigenic epitopes from three proteins that have a prominent role in host-receptor recognition, viral entry, and pathogenicity. We advocate this vaccine must be synthesized and tested urgently as a public health priority.

Conserved Arg451 residue is critical for maintaining the stability and activity of thioredoxin glutathione reductase.

Thioredoxin glutathione reductase (TGR), a potential anthelminthic drug target causes NADPH-dependent transfer of electrons to both thioredoxins and glutathione systems. In the present study, we showed that a single point mutation conserved at Arg451 position is critical for maintaining the structure-function of FgTGR. The current biochemical results showed that R451A mutation significantly decreases both oxidoreductase activities (glutathione reductase and thioredoxin reductase) of the enzyme. Computational analyses using molecular dynamics simulation provided an in-depth insight into the structural alterations caused as a result of the mutation. Furthermore, the different regions of the mutant FgTGR structure were found to be altered in flexibility/rigidity as a result of the mutation. This led to mutant-specific conformational alterations and dominant differential motions that contributed to the abrogated function of mutant FgTGR. These results were confirmed using GdnHCl-induced denaturation-based stability studies. Moreover, mutation reduced the free energy of stabilization of the protein, thereby destabilizing the mutant protein structure. Therefore, these findings displayed differential dynamics in the FgTGR structure and highlighted the relevance of residue-level interactions in the protein. Thus, the current study provided a basis for exploiting regions other than the active site of TGR for inhibitory effect and development of novel antihelminthics.

Structural insights into natural compounds as inhibitors of Fasciola gigantica thioredoxin glutathione reductase.

Fascioliasis is caused by the helminth parasites of genus Fasciola. Thioredoxin glutathione reductase (TGR) is an important enzyme in parasitic helminths and plays an indispensable role in its redox biology. In the present study, we conducted a structure-based virtual screening of natural compounds against the Fasciola gigantica TGR (FgTGR). The compounds were docked against FgTGR in four sequential docking modes. The screened ligands were further assessed for Lipinski and ADMET prediction so as to evaluate drug proficiency and likeness property. After refinement, three potential inhibitors were identified that were subjected to 50 ns molecular dynamics simulation and free energy binding analyses to evaluate the dynamics of protein-ligand interaction and the stability of the complexes. Key residues involved in the interaction of the selected ligands were also determined. The results suggested that three top hits had a negative binding energy greater than GSSG (-91.479 KJ · mol-1 ), having -152.657, -141.219, and -92.931 kJ · mol-1 for compounds with IDs ZINC85878789, ZINC85879991, and ZINC36369921, respectively. Further analysis showed that the compound ZINC85878789 and ZINC85879991 displayed substantial pharmacological and structural properties to be a drug candidate. Thus, the present study might prove useful for the future design of new derivatives with higher potency and specificity.

Role of the glutaredoxin domain and FAD in the stabilization of thioredoxin glutathione reductase.

Thioredoxin glutathione reductase (TGRsec) is a multi-domain flavoprotein that plays a principal role in redox homeostasis maintenance. We have previously demonstrated the role of selenocysteine in maintaining TGRsec structure-function, but the role of the glutaredoxin (Grx) domain and FAD is still unclear. In the present study, the urea-induced unfolding of recombinant Fasciola gigantica TGRsec (FgTGRsec) and its N-terminal truncated variant (ΔNTD-FgTGRsec) were examined to understand the role of the Grx domain and FAD in the stabilization of FgTGRsec and ΔNTD-FgTGRsec. Our results showed that both proteins underwent unfolding in a three state manner. First, the protein undergoes a conformational transition rendering a near-native state with no FAD bound, and then full unfolding of the apo-dimer occurs without dissociation. The Grx domain stabilized the global FgTGRsec structure and positively regulated FgTGRsec activity, and alteration in the FAD microenvironment was directly proportional to the loss of thioredoxin reductase (TrxR) and glutathione reductase activities. Based on these results, we concluded that the Grx domain stabilizes the full-length FgTGRsec protein for efficient catalysis. Thus, we suggest that in platyhelminth parasites, during evolution, the Grx domain merged with the TrxR domain to confer higher catalytic activity and provide additional structural stability to the full-length TGR.

Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica.

The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (∆NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ∆NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR.

Harnessing plasma-generated reactive species for the synthesis of different phases of molybdenum oxide to study adsorption and photocatalytic activity.

This study employs plasma-liquid interaction technique to synthesize different phases of molybdenum oxide using air and argon as plasma-forming gases. In situ plasma-generated nitrogen species primarily NO3-/NO2- and hydrogen species (H+) facilitate the reduction of the molybdenum precursor anion (Mo7O24-). The reduced Mo species subsequently reacts with reactive oxygen species, forming MoO6 octahedra, which is the building block of a molybdenum oxide crystal. Varied concentrations of NO3-/NO2- and H+ species in air and argon plasma treatment significantly influence the growth process. Air plasma synthesis yields hexagonal molybdenum oxide microrods, which upon calcination changes its phase to orthorhombic 2D layered structure. Moreover, the argon plasma synthesized sample exhibits a mixed phase of hexagonal and orthorhombic molybdenum oxide due to the heavy argon ion bombardment, inducing material porosity and surface oxygen vacancies. The mixed-phase material exhibits superior adsorption and photo-degradation towards cationic dye compared to the other two phases. The higher photocatalytic performance may be responsible for the extended lifetime of the photo-generated charge carriers possessed by the mixed-phase material. Radical scavenging tests have identified holes and hydroxyl radicals as the key reactive species that take part in the photo-degradation process.

Computational Protein Design for COVID-19 Research and Emerging Therapeutics.

As the world struggles with the ongoing COVID-19 pandemic, unprecedented obstacles have continuously been traversed as new SARS-CoV-2 variants continually emerge. Infectious disease outbreaks are unavoidable, but the knowledge gained from the successes and failures will help create a robust health management system to deal with such pandemics. Previously, scientists required years to develop diagnostics, therapeutics, or vaccines; however, we have seen that, with the rapid deployment of high-throughput technologies and unprecedented scientific collaboration worldwide, breakthrough discoveries can be accelerated and insights broadened. Computational protein design (CPD) is a game-changing new technology that has provided alternative therapeutic strategies for pandemic management. In addition to the development of peptide-based inhibitors, miniprotein binders, decoys, biosensors, nanobodies, and monoclonal antibodies, CPD has also been used to redesign native SARS-CoV-2 proteins and human ACE2 receptors. We discuss how novel CPD strategies have been exploited to develop rationally designed and robust COVID-19 treatment strategies.

Immunoinformatics Protocol to Design Multi-Epitope Subunit Vaccines.

With the development of scientific technologies, the accessibility of genomic data, computational tools, software, databases, and machine learning, the field of immunoinformatics has emerged as an effective technique for immunologists to design potential vaccines in a short time. A large number of tools and databases are available to screen the genome sequences of parasites/pathogens and identify the highly immunogenic peptides or epitopes that can be used to design effective vaccines. In this chapter, we provide an easy-to-use protocol for the design of multi-epitope-based subunit vaccines. Though the computational immunoinformatics-based approaches have demonstrated their competency in designing potentially effective vaccine candidates quickly, their immunogenicity and safety must be evaluated in laboratory settings before they are tested in clinical trials.

From De Novo Design to Redesign: Harnessing Computational Protein Design for Understanding SARS-CoV-2 Molecular Mechanisms and Developing Therapeutics.

The continuous emergence of novel SARS-CoV-2 variants and subvariants serves as compelling evidence that COVID-19 is an ongoing concern. The swift, well-coordinated response to the pandemic highlights how technological advancements can accelerate the detection, monitoring, and treatment of the disease. Robust surveillance systems have been established to understand the clinical characteristics of new variants, although the unpredictable nature of these variants presents significant challenges. Some variants have shown resistance to current treatments, but innovative technologies like computational protein design (CPD) offer promising solutions and versatile therapeutics against SARS-CoV-2. Advances in computing power, coupled with open-source platforms like AlphaFold and RFdiffusion (employing deep neural network and diffusion generative models), among many others, have accelerated the design of protein therapeutics with precise structures and intended functions. CPD has played a pivotal role in developing peptide inhibitors, mini proteins, protein mimics, decoy receptors, nanobodies, monoclonal antibodies, identifying drug-resistance mutations, and even redesigning native SARS-CoV-2 proteins. Pending regulatory approval, these designed therapies hold the potential for a lasting impact on human health and sustainability. As SARS-CoV-2 continues to evolve, use of such technologies enables the ongoing development of alternative strategies, thus equipping us for the "New Normal".

Functional expression, localization, and biochemical characterization of thioredoxin glutathione reductase from air-breathing magur catfish, Clarias magur.

The glutathione (GSH) and thioredoxin (Trx) systems regulate cellular redox homeostasis and maintain antioxidant defense in most eukaryotes. We earlier reported the absence of gene coding for the glutathione reductase (GR) enzyme of the GSH system in the facultative air-breathing catfish, Clarias magur. Here, we identified three thioredoxin reductase (TrxR) genes, one of which was later confirmed as a thioredoxin glutathione reductase (TGR). We then characterized the novel recombinant TGR enzyme of C. magur (CmTGR). The tissue-specific expression of the txnrd genes and the tissue-specific activity of the TrxR enzyme were analyzed. The recombinant CmTGR is a dimer of ~133 kDa. The protein showed TrxR activity with 5,5'-diothiobis (2-nitrobenzoic acid) reduction assay with a Km of 304.40 µM and GR activity with a Km of 58.91 µM. Phylogenetic analysis showed that the CmTGR was related to the TrxRs of fishes and distantly related to the TGRs of platyhelminth parasites. The structural analysis revealed the conserved glutaredoxin active site and FAD- and NADPH-binding sites. To our knowledge, this is the first report of the presence of a TGR in any fish. This unusual presence of TGR in C. magur is crucial as it helps maintain redox homeostasis under environmental stressors-induced oxidative stress.

Methodological advances in the design of peptide-based vaccines.

The tremendous advances in genomics, recombinant DNA technology, bioengineering and nanotechnology, in conjunction with the development of high-end computations, have been instrumental in the process of rational design of peptide-based vaccines. The use of peptide vaccines was limited owing to their inherent instability when systemically administered; however, advanced formulation techniques have been developed for their systemic delivery, thereby overcoming their degradation, clearance, cellular uptake and off-target effects. With the rise of sophisticated immunological predictors and experimental techniques, several methodological advances have occurred in this field. This review examines contemporary methods to identify and optimize epitopes, engineer their immunogenic properties and develop their safe and efficient delivery into the host.

Cold atmospheric plasma driven self-assembly in serum proteins: insights into the protein aggregation to biomaterials.

The self-assembly of proteins is crucial in many biomedical applications. This work deals with understanding the role of cold atmospheric plasma (CAP) on the self-assembly of two different proteins present in the serum - BSA and hemoglobin and to elucidate the process associated with the direct application of physical plasma on or in the human (or animal) body, which has implications in therapeutics. The work has been corroborated by several spectroscopic studies such as fluorescence spectroscopy, circular dichroism spectroscopy, and SEM analysis. Through steady-state fluorescence spectroscopy and by following the tryptophan fluorescence, we observed that the emission intensity was quenched for the protein when treated with plasma radiation. Circular dichroism spectroscopy revealed that the structure of the protein was altered both in the case of BSA and hemoglobin. N-Acetyl tryptophanamide (NATA), which resembles the tryptophan in the protein, was treated with CAP and we observed the similar quenching of fluorescence as in the proteins, indicating that the protein underwent self-assembly. Time-resolved fluorescence spectroscopy with a decrease in the lifetime revealed that the protein self-assembly was promoted with CAP treatment, which was also substantiated by SEM micrographs. The ROS/RNS produced in the CAP has been correlated with the protein self-assembly. This work will help to design protein self-assembled systems, and in the future, may bring possibilities of creating novel biomaterials with the help of plasma radiation.

Cold atmospheric pressure plasma for attenuation of SARS-CoV-2 spike protein binding to ACE2 protein and the RNA deactivation.

Cold atmospheric pressure (CAP) plasma has a profound effect on protein-protein interactions. In this work, we have highlighted the deactivation of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike protein by CAP plasma treatment. Complete deactivation of spike protein binding to the human ACE2 protein was observed within an exposure time of 5 minutes which is correlated to the higher concentration of hydrogen peroxide formation due to the interaction with the reactive oxygen species present in the plasma. On the other hand, we have established that CAP plasma is also capable of degrading RNA of SARS-CoV-2 virus which is also linked to hydrogen peroxide concentration. The reactive oxygen species is produced in the plasma by using noble gases such as helium, in the absence of any other chemicals. Therefore, it is a green process with no chemical waste generated and highly advantageous from the environmental safety prospects. Results of this work could be useful in designing plasma-based disinfection systems over those based on environmentally hazardous chemical-based disinfection and biomedical applications.

Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric SARS-CoV-2 Receptor Binding Domain Derivative.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80-100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Genome-wide identification and characterization of eukaryotic protein kinases.

The tropical liver fluke, Fasciola gigantica is a food-borne parasite responsible for the hepatobiliary disease fascioliasis. The recent completion of F. gigantica genome sequencing by our group has provided a platform for the systematic analysis of the parasite genome. Eukaryotic protein kinases (ePKs) are regulators of cellular phosphorylation. In the present study, we used various computational and bioinformatics tools to extensively analyse the ePKs in F. gigantica (FgePKs) genome. A total of 455 ePKs were identified that represent ~2% of the parasite genome. Out of these, 214 ePKs are typical kinases (Ser/Thr- and Tyr-specific ePKs), and 241 were other kinases. Several FgePKs were found to possess unusual domain architectures, which suggests the diverse nature of the proteins that can be exploited for designing novel inhibitors. 115 kinases showed <35% query coverage when compared to human ePKs highlighting significant divergences in their respective kinomes, further providing a platform for novel structure-based drug designing. This study provides a platform that may open new avenues into our understanding of helminth biochemistry and drug discovery.

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.