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.

Role of Natural plant extracts for potential Antileishmanial targets-In-depth review of the molecular mechanism.

A group of protozoan parasites known as Leishmania species can cause a variety of chronic illnesses, ranging from self-healing lesions to fatal outcomes. Drug-resistant pathogens have become common due to the lack of safe and effective medications, which has sparked the development of new therapeutic interventions, particularly plant-based natural extracts. As a way to avoid chemotherapy's side effects, natural herbal remedies have drawn more attention. In addition to having anti-inflammatory, anticancer, and cosmetic properties, the secondary metabolites of plants, such as phenolic compounds, flavonoids, alkaloids, and terpenes, have a number of positive effects on our health. Natural metabolites such as naphthoquinone, alkaloids, benzophenones, etc. that have antileishmanial and antiprotozoal activity have been the subject of extensive research. In this review paper, it can be concluded that these natural extracts can be developed into excellent therapeutic agents against Leishmaniasis.

Higher Novel L-Cys Degradation Activity Results in Lower Organic-S and Biomass in Sarcocornia than the Related Saltwort, Salicornia.

Salicornia and Sarcocornia are almost identical halophytes whose edible succulent shoots hold promise for commercial production in saline water. Enhanced sulfur nutrition may be beneficial to crops naturally grown on high sulfate. However, little is known about sulfate nutrition in halophytes. Here we show that Salicornia europaea (ecotype RN) exhibits a significant increase in biomass and organic-S accumulation in response to supplemental sulfate, whereas Sarcocornia fruticosa (ecotype VM) does not, instead exhibiting increased sulfate accumulation. We investigated the role of two pathways on organic-S and biomass accumulation in Salicornia and Sarcoconia: the sulfate reductive pathway that generates Cys and l-Cys desulfhydrase that degrades Cys to H2S, NH3, and pyruvate. The major function of O-acetyl-Ser-(thiol) lyase (OAS-TL; EC 2.5.1.47) is the formation of l-Cys, but our study shows that the OAS-TL A and OAS-TL B of both halophytes are enzymes that also degrade l-Cys to H2S. This activity was significantly higher in Sarcocornia than in Salicornia, especially upon sulfate supplementation. The activity of the sulfate reductive pathway key enzyme, adenosine 5'-phosphosulfate reductase (APR, EC 1.8.99.2), was significantly higher in Salicornia than in Sarcocornia These results suggest that the low organic-S level in Sarcocornia is the result of high l-Cys degradation rate by OAS-TLs, whereas the greater organic-S and biomass accumulation in Salicornia is the result of higher APR activity and low l-Cys degradation rate, resulting in higher net Cys biosynthesis. These results present an initial road map for halophyte growers to attain better growth rates and nutritional value of Salicornia and Sarcocornia.

Characterization of leaf transcriptome, development and utilization of unigenes-derived microsatellite markers in sugarcane (Saccharum sp. hybrid).

Sugarcane (Saccharum species hybrid) is the major source of sugar (> 80% sugar) in the world and is cultivated in more than 115 countries. It has recently gained attention as a source of biofuel (ethanol). Due to genomic complexity, the development of new genomic resources is imperative in understanding the gene regulation and function, and to fine tune the genetic improvement of sugarcane. In this study, a cDNA library was constructed from mature leaves so as to develop ESTs resources which were further compared with nucleotide and protein databases to explore the functional identity of sugarcane genes. The non-redundant ESTs (unigenes) were categorized into 18 metabolic functions. The major categories were bioenergetics and photosynthesis (4%), cell metabolism (5%), development related protein (3%), membrane-related, mobile genetic elements (5%), signal transduction (2%), DNA (1%), RNA (1%) and protein (2%) metabolism, other metabolic processes (3%), transcription factors (1%), transport (4%) and proteins related to stress/defense (4%). From 540 unique ESTs, 212 simple sequence repeats were identified, of which 206 were from 463 singlets and six were mined from 77 contig sequences. A total of 540 unique EST sequences were used for SSR search of which 97 (17.9%) contained specified SSR motifs, generating 212 unique SSRs. The genes characterized in this study and the EST-derived microsatellite markers identified from the cDNA library will enrich genomic resources for association- and linkage-mapping studies in sugarcane.

Exploring the most promising anti - Depressant drug targeting Microtubule Affinity Receptor Kinase 4 involved in Alzheimer's Disease through molecular docking and molecular dynamics simulation.

Alzheimer's Disease (AD) is the prevailing type of neurodegenerative illness, characterised by the accumulation of amyloid beta plaques. The symptoms associated with AD are memory loss, emotional variability, and a decline in cognitive functioning. To date, the pharmaceuticals currently accessible in the marketplace are limited to symptom management. According to several research, antidepressants have demonstrated potential efficacy in the management of AD. In this particular investigation, a total of 24 anti-depressant medications were selected as ligands, while the Microtubule Affinity Receptor Kinase 4 (MARK4) protein was chosen as the focal point of our study. The selection of MARK4 was based on its known involvement in the advancement of AD and other types of malignancies, rendering it a highly prospective target for therapeutic interventions. The initial step involved doing ADMET analysis, which was subsequently followed by molecular docking of 24 drugs. This was succeeded by molecular dynamics simulation and molecular mechanics generalised Born surface area (MMGBSA) calculations. Upon conducting molecular docking experiments, it has been determined that the binding affinities observed fall within the range of -5.5 kcal/mol to -9.0 kcal/mol. In this study, we selected six anti-depressant compounds (CID ID - 4184, 2771, 4205, 5533, 4543, and 2160) based on their binding affinities, which were determined to be -9.0, -8.7, -8.4, -8.3, -8.2, and -8.2, respectively. Molecular dynamics simulations were conducted for all six drugs, with donepezil serving as the control drug. Various analyses were performed, including basic analysis and post-trajectory analysis such as free energy landscape (FEL), polarizable continuum model (PCM), and MMGBSA calculations. Based on the findings from molecular dynamics simulations and the MMGBSA analysis, it can be inferred that citalopram and mirtazapine exhibit considerable potential as anti-depressant agents. Consequently, these compounds warrant further investigation through in vitro and in vivo investigations in the context of treating AD.

Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors.

Selenium is a component of selenoproteins, which plays a crucial role in cellular redox homeostasis, thyroid metabolism, and DNA synthesis. Selenium has pleiotropic effects like antioxidant and anti-inflammatory activities; however, excess intake of selenium can imbalance such processes. The effects of selenium on human health are numerous and complex, demanding additional research to monitor the flux rate of selenium. Here, we have created a noninvasive and highly efficient genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor, SelFS (Selenium FRET-Sensor), for real-time monitoring of selenium at the cellular and subcellular levels. The construct of the nanosensor contains a selenium-binding protein (SeBP) as the selenium-detecting element inserted between the green fluorescent protein variants enhanced cyan fluorescent protein and Venus. In the presence of selenium, SelFS brings a conformational change, which is seen in the form of FRET. In vitro studies showed that SelFS is highly specific and selective for selenium and stable at an altered pH range from 5.0 to 8.0. SelFS is a flexible and dynamic tool for the detection of selenium in both prokaryotes and eukaryotes in a noninvasive way, with a binding constant (K d) of 0.198 × 10-6 M as compared to its mutants. The developed nanosensor can provide us a reporter tool for a wide range of industrial and environmental applications, which will help us to understand its functions in biological systems.

Rice Straw Waste-Based Biogas Production via Microbial Digestion: A Review.

The development of sustainable and renewable energy production is in high demand, and bioenergy production via microbial digestion of organic wastes is in prime focus. Biogas produced from the microbial digestion of organic waste is the most promising among existing biofuel options. In this context, biogas production from lignocellulosic biomass is one of the most viable and promising technologies for sustainable biofuel production. In the present review, an assessment and feasibility advancement have been presented towards the sustainable production of biogas from rice straw waste. Rice straw (RS) is abundantly available, contains a high composition of cellulose, and is found under the category of lignocellulosic waste, but it may cause severe environmental issues if not treated. Whereas, due to its high cellulose and inorganic content, lower cost, and huge availability, this waste can be effectively valorized into biogas production at a lower cost on a commercial scale. Therefore, the present review provides existing insight in this area by focusing on the operational parameter's improvement and advancement in the research for the expansion of mass-scale production at a lower cost. Thus, the presented review analyzed the processing parameters status, associated challenges, and positive endnote solutions for more sustainable viability for biogas production.

In-silico approaches for identification of compounds inhibiting SARS-CoV-2 3CL protease.

The world has witnessed of many pandemic waves of SARS-CoV-2. However, the incidence of SARS-CoV-2 infection has now declined but the novel variant and responsible cases has been observed globally. Most of the world population has received the vaccinations, but the immune response against COVID-19 is not long-lasting, which may cause new outbreaks. A highly efficient pharmaceutical molecule is desperately needed in these circumstances. In the present study, a potent natural compound that could inhibit the 3CL protease protein of SARS-CoV-2 was found with computationally intensive search. This research approach is based on physics-based principles and a machine-learning approach. Deep learning design was applied to the library of natural compounds to rank the potential candidates. This procedure screened 32,484 compounds, and the top five hits based on estimated pIC50 were selected for molecular docking and modeling. This work identified two hit compounds, CMP4 and CMP2, which exhibited strong interaction with the 3CL protease using molecular docking and simulation. These two compounds demonstrated potential interaction with the catalytic residues His41 and Cys154 of the 3CL protease. Their calculated binding free energies to MMGBSA were compared to those of the native 3CL protease inhibitor. Using steered molecular dynamics, the dissociation strength of these complexes was sequentially determined. In conclusion, CMP4 demonstrated strong comparative performance with native inhibitors and was identified as a promising hit candidate. This compound can be applied in-vitro experiment for the validation of its inhibitory activity. Additionally, these methods can be used to identify new binding sites on the enzyme and to design new compounds that target these sites.

Enhancing Immunogenicity of a Thermostable, Efficacious SARS-CoV-2 Vaccine Formulation through Oligomerization and Adjuvant Choice.

Currently deployed SARS-CoV-2 vaccines all require storage at refrigerated or sub-zero temperatures. We demonstrate that after month-long incubation at 37 °C, solubilization, and formulation with squalene-in-water emulsion adjuvant, a stabilized receptor binding domain retains immunogenicity and protective efficacy. We also examine the effects of trimerization of the stabilized RBD, as well as of additional adjuvants, on both B and T-cell responses. The additional emulsion or liposome-based adjuvants contained a synthetic TLR-4 ligand and/or the saponin QS-21. Trimerization enhanced immunogenicity, with significant antibody titers detectable after a single immunization. Saponin-containing adjuvants elicited enhanced immunogenicity relative to both emulsion and aluminum hydroxide adjuvanted formulations lacking these immunostimulants. Trimeric RBD formulated with liposomal based adjuvant containing both TLR-4 ligand and saponin elicited a strongly Th1 biased response, with ~10-fold higher neutralization titers than the corresponding aluminum hydroxide adjuvanted formulation. The SARS-CoV-2 virus is now endemic in humans, and it is likely that periodic updating of vaccine formulations in response to viral evolution will continue to be required to protect vulnerable individuals. In this context, it is desirable to have efficacious, thermostable vaccine formulations to facilitate widespread vaccine coverage, including in low- and middle-income countries, where global access rights to clinically de-risked adjuvants will be important moving forward.

QTL mapping and identification of candidate genes linked to red rot resistance in sugarcane.

Sugarcane (Saccharum species hybrid) is one of the most important commercial crops cultivated worldwide for products like white sugar, bagasse, ethanol, etc. Red rot is a major sugarcane disease caused by a hemi-biotrophic fungus, Colletotrichum falcatum Went., which can potentially cause a reduction in yield up to 100%. Breeding for red rot-resistant sugarcane varieties has become cumbersome due to its complex genome and frequent generation of new pathotypes of red rot fungus. In the present study, a genetic linkage map was developed using a selfed population of a popular sugarcane variety CoS 96268. A QTL linked to red rot resistance (qREDROT) was identified, which explained 26% of the total phenotypic variation for the trait. A genotype-phenotype network analysis performed to account for epistatic interactions, identified the key markers involved in red rot resistance. The differential expression of the genes located in the genomic region between the two flanking markers of the qREDROT as well as in the vicinity of the markers identified through the genotype-phenotype network analysis in a set of contrasting genotypes for red rot infection further confirmed the mapping results. Further, the expression analysis revealed that the plant defense-related gene coding 26S protease regulatory subunit is strongly associated with the red rot resistance. The findings can help in the screening of disease resistant genotypes for developing red rot-resistant varieties of sugarcane. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03481-7.

Ganoderma lucidum: Novel Insight into Hepatoprotective Potential with Mechanisms of Action.

Ganoderma lucidum (G. lucidum) has been widely used for its health benefits as an edible and traditional medicinal mushroom for thousands of years in Asian countries. It is currently used as a nutraceutical and functional food owing to its major bioactive compounds, polysaccharides and triterpenoids. G. lucidum exhibits a broad range of hepatoprotective impacts in various liver disorders, such as hepatic cancer, nonalcoholic fatty liver disease (NAFLD), alcohol-induced liver disease, hepatitis B, hepatic fibrosis, and liver injury induced by carbon tetrachloride (CCl4) and α-amanitin. G. lucidum protects the liver through a broad range of mechanisms that include the modulation of liver Phase I and II enzymes, the suppression of ß-glucuronidase, antifibrotic and antiviral actions, the regulation of the production of nitric oxide (NO), the maintenance of hepatocellular calcium homeostasis, immunomodulatory activity, and scavenging free radicals. G. lucidum could signify an encouraging approach for the management of various chronic hepatopathies, and its potential mechanisms make it a distinctive agent when used alone or with other drugs and applied as a functional food, nutraceutical supplement, or adjuvant to modern medicine. This review summarizes the hepatoprotective properties of G. lucidum with its various mechanisms of action on different liver ailments. Biologically active substances derived from G. lucidum are still being studied for their potential benefits in treating different liver ailments.

Knowledge, acceptance, motivators and barriers of booster dose of COVID-19 vaccination among dental patients: A cross-sectional study.

Given the lingering threat of COVID infection, questions are being raised if coronavirus disease 2019 (COVID-19) vaccine needs annual or regular boosters to maintain high levels of immunity against both the original virus and variants. This study was designed to evaluate the knowledge, acceptance, motivators and barriers of the booster dose of COVID-19 vaccine among the dental patients of District Lucknow, India. A total of 297 respondents were selected by a convenience sampling method in this cross-sectional study from various dental clinics. An anonymous, self-administered, closed-ended questionnaire was used. Overall 37.7% respondents reported to have taken all 3 doses and 57.9% had taken single/double doses. Correct information about booster doses shows a significant association with the number of doses taken. The majority had information about the availability of the Pfizer booster vaccine (69.0%). About 58% of participants had information about the technology used in booster doses. The hesitancy for booster doses and the development of natural immunity by infection show significant associations with the number of doses taken. Only 18.2% patients had hesitation about the booster dose and most of them 78.8% recommended others to take the booster vaccine as soon as possible. The majority assumed that previous COVID-19 vaccines can help them get immune (21.5%) followed by not much research has been done on the booster vaccines (15.5%) and their chronic diseases warn them against the booster dose administration (12.5%). Nearly 18.2% of respondents had hesitation about booster dose and less than one third of the respondents trusted a government source for information about booster dose of COVID vaccine. Nearly 36 % did not know that the booster dose of COVID vaccine is available at health centers. Dental health professionals and policymakers should implement and support strategies to ensure people are vaccinated for COVID-19 booster doses.

Advancement and New Trends in Analysis of Pesticide Residues in Food: A Comprehensive Review.

Food safety is a rising challenge worldwide due to the expanding population and the need to produce food to feed the growing population. At the same time, pesticide residues found in high concentrations in fresh agriculture pose a significant threat to food safety. Presently, crop output is being increased by applying herbicides, fungicides, insecticides, pesticides, fertilizers, nematicides, and soil amendments. A combination of factors, including bioaccumulation, widespread usage, selective toxicity, and stability, make pesticides among the most toxic compounds polluting the environment. They are especially harmful in vegetables and fruits because people are exposed to them. Thus, it is critical to monitor pesticide levels in fruits and vegetables using all analytical techniques available. Any evaluation of the condition of pesticide contamination in fruits and vegetables necessitates knowledge of maximum residue levels (MRLs). We set out the problems in determining various types of pesticides in vegetables and fruits, including the complexity and the diversity of matrices in biological materials. This review examines the different analytical techniques to determine the target analytes that must be isolated before final consumption. Many processes involved determining pesticide residues in fruits and vegetables and their advantages and disadvantages have been discussed with recommendations. Furthermore, MRLs of target pesticide residues in fruit and vegetable samples are discussed in the context of data from the literature. The review also examines MRLs' impact on the international trade of fruits and vegetables. Accurate, sensitive, and robust analytical procedures are critical to ensuring that pesticide levels in food products are effectively regulated. Despite advances in detection technology, effective sample preparation procedures for pesticide residue measurement in cereals and feedstuffs are still needed. In addition, these methods must be compatible with current analytical techniques. Multi-residue approaches that cover a wide range of pesticides are desired, even though pesticides' diverse natures, classes, and physio-chemical characteristics make such methods challenging to assemble. This review will be valuable to food analysts and regulatory authorities to monitor the quality and safety of fresh food products.

Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies against Delta and Omicron in Mice.

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We previously developed highly thermo-tolerant monomeric and trimeric receptor-binding domain derivatives that can withstand 100 °C for 90 min and 37 °C for four weeks and help eliminate cold-chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations and a 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for upcoming Phase I human clinical trials and that there is potential for increasing the efficacy with vaccine matching to improve the responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions, which show that, while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.

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.

A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern.

Saturation suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temperature of ~7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An additional derivative with three additional mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temperature exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 derivative showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qualitatively improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or additional components, and can be stored at room temperature. They are a useful modality to combat COVID-19, especially in remote and low-resource settings.

Subtractive hybridization-mediated analysis of genes and in silico prediction of associated microRNAs under waterlogged conditions in sugarcane (Saccharum spp.).

Sugarcane is an important tropical cash crop meeting 75% of world sugar demand and it is fast becoming an energy crop for the production of bio-fuel ethanol. A considerable area under sugarcane is prone to waterlogging which adversely affects both cane productivity and quality. In an effort to elucidate the genes underlying plant responses to waterlogging, a subtractive cDNA library was prepared from leaf tissue. cDNA clones were sequenced and annotated for their putative functions. Major groups of ESTs were related to stress (15%), catalytic activity (13%), cell growth (10%) and transport related proteins (6%). A few stress-related genes were identified, including senescence-associated protein, dehydration-responsive family protein, and heat shock cognate 70 kDa protein. A bioinformatics search was carried out to discover novel microRNAs (miRNAs) that can be regulated in sugarcane plants subjected to waterlogging stress. Taking advantage of the presence of miRNA precursors in the related sorghum genome, seven candidate mature miRNAs were identified in sugarcane. The application of subtraction technology allowed the identification of differentially expressed sequences and novel miRNAs in sugarcane under waterlogging stress. The comparative global transcript profiling in sugarcane plants undertaken in this study suggests that proteins associated with stress response, signal transduction, metabolic activity and ion transport play important role in conferring waterlogging tolerance in sugarcane.