Aldehyde dehydrogenase superfamily in sorghum: genome-wide identification, evolution, and transcript profiling during development stages and stress conditions.

BACKGROUND: Aldehyde dehydrogenases (ALDHs) are a family of NAD(P)+ dependent enzymes that detoxify aldehydes by promoting their oxidation to respective carboxylic acids. The role of ALDH enzymes in various plant species has been extensively studied, revealing their critical role in salinity, drought, heat, and heavy metal stress tolerance. Despite their physiological significance, ALDH genes in Sorghum bicolor have yet to be studied thoroughly. RESULTS: In this study, a total of 19 ALDH genes have been identified that have been grouped into ten families based on the criteria of the ALDH gene nomenclature committee. Segmental duplication assisted more in the enhancement of SbALDH gene family members than tandem duplication. All the identified SbALDH members made a cluster with monocot rice and maize in the phylogenetic tree rather than dicot species, suggesting the pre-eudicot-monocot separation of the ALDH superfamily members. The gene structure and protein domain were found to be mostly conserved in separate phylogenetic classes, indicating that each family played an important role in evolution. Expression analysis revealed that several SbALDHs were expressed in various tissues, developmental stages, and in response to abiotic stresses, indicating that they can play roles in plant growth, development, or stress adaptation. Interestingly, the majority of the SbALDH genes were found to be highly responsive to drought stress, and the SbALDH18B1 transcript showed maximum enhancement in all the stress conditions. The presence of cis-acting elements (mainly ABRE and MBS) in the promoter region of these genes might have a significant role in drought tolerance. CONCLUSIONS: Our findings add to the current understanding, evolutionary history, and contribution of SbALDHs in stress tolerance, and smooth the path of further functional validation of these genes.

Glyoxalase III enhances salinity tolerance through reactive oxygen species scavenging and reduced glycation.

Methylglyoxal (MG) is a metabolically generated highly cytotoxic compound that accumulates in all living organisms, from Escherichia coli to humans, under stress conditions. To detoxify MG, nature has evolved reduced glutathione (GSH)-dependent glyoxalase and NADPH-dependent aldo-keto reductase systems. But both GSH and NADPH have been reported to be limiting in plants under stress conditions, and thus detoxification might not be performed efficiently. Recently, glyoxalase III (GLY III)-like enzyme activity has been reported from various species, which can detoxify MG without any cofactor. In the present study, we have tested whether an E. coli gene, hchA, encoding a functional GLY III, could provide abiotic stress tolerance to living systems. Overexpression of this gene showed improved tolerance in E. coli and Saccharomyces cerevisiae cells against salinity, dicarbonyl, and oxidative stresses. Ectopic expression of the E. coli GLY III gene (EcGLY-III) in transgenic tobacco plants confers tolerance against salinity at both seedling and reproductive stages as indicated by their height, weight, membrane stability index, and total yield potential. Transgenic plants showed significantly increased glyoxalase and antioxidant enzyme activity that resisted the accumulation of excess MG and reactive oxygen species (ROS) during stress. Moreover, transgenic plants showed more anti-glycation activity to inhibit the formation of advanced glycation end product (AGE) that might prevent transgenic plants from stress-induced senescence. Taken together, all these observations indicate that overexpression of EcGLYIII confers salinity stress tolerance in plants and should be explored further for the generation of stress-tolerant plants.

Transcript profiling of glutathione metabolizing genes reveals abiotic stress and glutathione-specific alteration in Arabidopsis and rice.

Homoeostasis of glutathione (GSH) is crucial for plant survival and adaptability against stress. Despite the presence of complete Arabidopsis and rice genome sequence, the comprehensive analysis of the GSH metabolizing genes is still missing. This research concentrated on the comprehensive understanding of GSH metabolizing genes in two model plants-Arabidopsis and rice in terms of their subcellular localization, exon-intron distribution, protein domain structure, and transcript abundance. Expression profiling using the microarray data provided significant evidence of their participation in response to various abiotic stress conditions. Besides, some of these GSH metabolizing genes revealed their expression alteration in several developmental changes and tissue diversification. The presence of various stress-specific cis-regulatory elements in the promoter region of GSH metabolizing genes could be directly correlated with their stress-specific transcript alteration. Moreover, the application of exogenous GSH significantly downregulated GSH synthesizing genes and upregulated GSH metabolizing genes in Arabidopsis with few exceptions indicating a product-dependent regulation of GSH metabolizing genes. Interestingly, validation of rice GSH metabolizing genes in response to drought and salinity showed an almost similar pattern of expression in quantitative real-time as observed by microarray data. Altogether, GSH metabolizing members are a promising and underutilized genetic source for plant improvement that could be used to enhance stress tolerance in plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01220-5.

Identification and expression profiling of proline metabolizing genes in Arabidopsis thaliana and Oryza sativa to reveal their stress-specific transcript alteration.

The amino acid, proline, is utilized by different organisms to offset cellular imbalances caused by environmental stresses. The wide use of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. A comprehensive analysis of the transcript abundance of proline metabolizing genes is fundamental for the assessment of function and regulation of each gene. Using available microarray data and quantitative real-time RT-PCR, the expression profiles of gene encoding key proline biosynthesis and degradation enzymes i.e., OAT, P5CS, P5CR and PDH were examined. Interestingly, validation of candidate genes in rice using in-silico data provided strong evidence for their involvement in stress response. Note that, OsOAT, OsP5CS1, OsP5CS2, OsP5CR showed similar expression pattern in quantitative real-time RT-PCR results as compared to microarray data. However, OsPDH showed a different expression pattern which may be due to the genotypic variation. Furthermore, a biochemical assay measuring proline content gave us a proper indication of the accumulation of proline under stressed conditions. Identification of key proline metabolizing genes from rice and Arabidopsis provides insights on the molecular regulation of proline homeostasis, to initiate metabolic engineering to develop stress-resilient plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01023-0.

Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes.

To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLIN-DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18 -StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies.

A nuclear-localized rice glyoxalase I enzyme, OsGLYI-8, functions in the detoxification of methylglyoxal in the nucleus.

The cellular levels of methylglyoxal (MG), a toxic byproduct of glycolysis, rise under various abiotic stresses in plants. Detoxification of MG is primarily through the glyoxalase pathway. The first enzyme of the pathway, glyoxalase I (GLYI), is a cytosolic metalloenzyme requiring either Ni2+ or Zn2+ for its activity. Plants possess multiple GLYI genes, of which only some have been partially characterized; hence, the precise molecular mechanism, subcellular localization and physiological relevance of these diverse isoforms remain enigmatic. Here, we report the biochemical properties and physiological role of a putative chloroplast-localized GLYI enzyme, OsGLYI-8, from rice, which is strikingly different from all hitherto studied GLYI enzymes in terms of its intracellular localization, metal dependency and kinetics. In contrast to its predicted localization, OsGLYI-8 was found to localize in the nucleus along with its substrate, MG. Further, OsGLYI-8 does not show a strict requirement for metal ions for its activity, is functional as a dimer and exhibits unusual biphasic steady-state kinetics with a low-affinity and a high-affinity substrate-binding component. Loss of AtGLYI-2, the closest Arabidopsis ortholog of OsGLYI-8, results in severe germination defects in the presence of MG and growth retardation under salinity stress conditions. These defects were rescued upon complementation with AtGLYI-2 or OsGLYI-8. Our findings thus provide evidence for the presence of a GLYI enzyme and MG detoxification in the nucleus.

Manipulation of glyoxalase pathway confers tolerance to multiple stresses in rice.

Crop plants face a multitude of diverse abiotic and biotic stresses in the farmers' fields. Although there now exists a considerable knowledge of the underlying mechanisms of response to individual stresses, the crosstalk between response pathways to various abiotic and biotic stresses remains enigmatic. Here, we investigated if the cytotoxic metabolite methylglyoxal (MG), excess of which is generated as a common consequence of many abiotic and biotic stresses, may serve as a key molecule linking responses to diverse stresses. For this, we generated transgenic rice plants overexpressing the entire two-step glyoxalase pathway for MG detoxification. Through assessment of various morphological, physiological and agronomic parameters, we found that glyoxalase-overexpression imparts tolerance towards abiotic stresses like salinity, drought and heat and also provides resistance towards damage caused by the sheath blight fungus (Rhizoctonia solani) toxin phenylacetic acid. We show that the mechanism of observed tolerance of the glyoxalase-overexpressing plants towards these diverse abiotic and biotic stresses involves improved MG detoxification and reduced oxidative damage leading to better protection of chloroplast and mitochondrial ultrastructure and maintained photosynthetic efficiency under stress conditions. Together, our findings indicate that MG may serve as a key link between abiotic and biotic stress response in plants.

Genome-wide analysis and expression profiling of glyoxalase gene families in soybean (Glycine max) indicate their development and abiotic stress specific response.

BACKGROUND: Glyoxalase pathway consists of two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII) which detoxifies a highly cytotoxic metabolite methylglyoxal (MG) to its non-toxic form. MG may form advanced glycation end products with various cellular macro-molecules such as proteins, DNA and RNA; that ultimately lead to their inactivation. Role of glyoxalase enzymes has been extensively investigated in various plant species which showed their crucial role in salinity, drought and heavy metal stress tolerance. Previously genome-wide analysis of glyoxalase genes has been conducted in model plants Arabidopsis and rice, but no such study was performed in any legume species. RESULTS: In the present study, a comprehensive genome database analysis of soybean was performed and identified a total of putative 41 GLYI and 23 GLYII proteins encoded by 24 and 12 genes, respectively. Detailed analysis of these identified members was conducted including their nomenclature and classification, chromosomal distribution and duplication, exon-intron organization, and protein domain(s) and motifs identification. Expression profiling of these genes has been performed in different tissues and developmental stages as well as under salinity and drought stresses using publicly available RNAseq and microarray data. The study revealed that GmGLYI-7 and GmGLYII-8 have been expressed intensively in all the developmental stages and tissues; while GmGLYI-6, GmGLYI-9, GmGLYI-20, GmGLYII-5 and GmGLYII-10 were highly abiotic stress responsive members. CONCLUSIONS: The present study identifies the largest family of glyoxalase proteins to date with 41 GmGLYI and 23 GmGLYII members in soybean. Detailed analysis of GmGLYI and GmGLYII genes strongly indicates the genome-wide segmental and tandem duplication of the glyoxalase members. Moreover, this study provides a strong basis about the biological role and function of GmGLYI and GmGLYII members in soybean growth, development and stress physiology.

A glutathione responsive rice glyoxalase II, OsGLYII-2, functions in salinity adaptation by maintaining better photosynthesis efficiency and anti-oxidant pool.

Glyoxalase II (GLY II), the second enzyme of glyoxalase pathway that detoxifies cytotoxic metabolite methylglyoxal (MG), belongs to the superfamily of metallo-ß-lactamases. Here, detailed analysis of one of the uncharacterized rice glyoxalase II family members, OsGLYII-2 was conducted in terms of its metal content, enzyme kinetics and stress tolerance potential. Functional complementation of yeast GLY II mutant (∆GLO2) and enzyme kinetics data suggested that OsGLYII-2 possesses characteristic GLY II activity using S-lactoylglutathione (SLG) as the substrate. Further, Inductively Coupled Plasma Atomic Emission spectroscopy and modelled structure revealed that OsGLYII-2 contains a binuclear Zn/Fe centre in its active site and chelation studies indicated that these are essential for its activity. Interestingly, reconstitution of chelated enzyme with Zn(2+), and/or Fe(2+) could not reactivate the enzyme, while addition of Co(2+) was able to do so. End product inhibition study provides insight into the kinetics of GLY II enzyme and assigns hitherto unknown function to reduced glutathione (GSH). Ectopic expression of OsGLYII-2 in Escherichia coli and tobacco provides improved tolerance against salinity and dicarbonyl stress indicating towards its role in abiotic stress tolerance. Maintained levels of MG and GSH as well as better photosynthesis rate and reduced oxidative damage in transgenic plants under stress conditions seems to be the possible mechanism facilitating enhanced stress tolerance.

A unique Ni2+ -dependent and methylglyoxal-inducible rice glyoxalase I possesses a single active site and functions in abiotic stress response.

The glyoxalase system constitutes the major pathway for the detoxification of metabolically produced cytotoxin methylglyoxal (MG) into a non-toxic metabolite D-lactate. Glyoxalase I (GLY I) is an evolutionarily conserved metalloenzyme requiring divalent metal ions for its activity: Zn(2+) in the case of eukaryotes or Ni(2+) for enzymes of prokaryotic origin. Plant GLY I proteins are part of a multimember family; however, not much is known about their physiological function, structure and metal dependency. In this study, we report a unique GLY I (OsGLYI-11.2) from Oryza sativa (rice) that requires Ni(2+) for its activity. Its biochemical, structural and functional characterization revealed it to be a monomeric enzyme, possessing a single Ni(2+) coordination site despite containing two GLY I domains. The requirement of Ni(2+) as a cofactor by an enzyme involved in cellular detoxification suggests an essential role for this otherwise toxic heavy metal in the stress response. Intriguingly, the expression of OsGLYI-11.2 was found to be highly substrate inducible, suggesting an important mode of regulation for its cellular levels. Heterologous expression of OsGLYI-11.2 in Escherichia coli and model plant Nicotiana tabacum (tobacco) resulted in improved adaptation to various abiotic stresses caused by increased scavenging of MG, lower Na(+) /K(+) ratio and maintenance of reduced glutathione levels. Together, our results suggest interesting links between MG cellular levels, its detoxification by GLY I, and Ni(2+) - the heavy metal cofactor of OsGLYI-11.2, in relation to stress response and adaptation in plants.

Glyoxalases and stress tolerance in plants.

The glyoxalase pathway is required for detoxification of cytotoxic metabolite MG (methylglyoxal) that would otherwise increase to lethal concentrations under adverse environmental conditions. Since its discovery 100 years ago, several roles have been assigned to glyoxalases, but, in plants, their involvement in stress response and tolerance is the most widely accepted role. The plant glyoxalases have emerged as multigene family and this expansion is considered to be important from the perspective of maintaining a robust defence machinery in these sessile species. Glyoxalases are known to be differentially regulated under stress conditions and their overexpression in plants confers tolerance to multiple abiotic stresses. In the present article, we review the importance of glyoxalases in plants, discussing possible roles with emphasis on involvement of the glyoxalase pathway in plant stress tolerance.

Exploring the evolutionary landscape and structural resonances of ferritin with insights into functional significance in plant.

The mineral iron plays a crucial role in facilitating the optimal functioning of numerous biological processes within the cellular environment. These processes involve the transportation of oxygen, energy production, immune system functioning, cognitive abilities, and muscle function. However, it is crucial to note that excessive levels of iron can result in oxidative damage within cells, primarily through Fenton reactions. Iron availability and toxicity present significant challenges that have been addressed through evolution. Ferritin is an essential protein that stores iron and is divided into different subfamilies, including DNA-binding proteins under starvation (Dps), bacterioferritin, and classical ferritin. Ferritin plays a critical role in maintaining cellular balance and protecting against oxidative damage. This study delves into ferritin's evolutionary dynamics across diverse taxa, emphasizing structural features and regulatory mechanisms. Insights into ferritin's evolution and functional diversity are gained through phylogenetic and structural analysis in bacterial Dps, bacterioferritin, and classical ferritin proteins. Additionally, the involvement of ferritin in plant stress responses and development is explored. Analysis of ferritin gene expression across various developmental stages and stress conditions provides insights into its regulatory roles. This comprehensive exploration enhances our understanding of ferritin's significance in plant biology, offering insights into its evolutionary history, structural diversity, and protective mechanisms against oxidative stress.

Evolutionary expansion, functional diversification, and transcript profiling of plant Glutathione Peroxidases.

Glutathione peroxidases (GPXs) play a crucial role in combating activated oxygen species and have been widely studied for their involvement in stress responses. In addition to their stress-related functions, GPXs exhibit diverse roles such as immunological response, and involvement in growth and development. These enzymes are found in both animals and plants, with multiple families identified in the evolutionarily diverse species. These families consist of conserved genes as well as unique members, highlighting the evolutionary diversification of GPX members. While animals have eight GPX families, plants possess five families. Notably, plant genomes undergo duplication and expansion events, leading to an increase in the number of GPX genes and the overall size of the GPX superfamily. This expansion suggests a wide range of functional roles for GPX. In this study, the evolutionary diversification, family expansion, and diverse functional roles of GPX enzymes have been investigated. Additionally, the expression profile of Arabidopsis and Oryza sativa GPX genes were analyzed in different developmental stages, tissues, and abiotic stress conditions. Further extensive research has been required to unravel the intricate interplay between GPX and other proteins, to gain the comprehensive mechanism governing the physiological and developmental roles of GPX.

Methylglyoxal detoxifying gene families in tomato: Genome-wide identification, evolution, functional prediction, and transcript profiling.

Methylglyoxal (MG) is a highly cytotoxic molecule produced in all biological systems, which could be converted into non-toxic D-lactate by an evolutionarily conserved glyoxalase pathway. Glutathione-dependent glyoxalase I (GLYI) and glyoxalase II (GLYII) are responsible for the detoxification of MG into D-lactate in sequential reactions, while DJ-1 domain containing glyoxalase III (GLYIII) catalyzes the same reaction in a single step without glutathione dependency. Afterwards, D-lactate dehydrogenase (D-LDH) converts D-lactate into pyruvate, a metabolically usable intermediate. In the study, a comprehensive genome-wide investigation has been performed in one of the important vegetable plants, tomato to identify 13 putative GLYI, 4 GLYII, 3 GLYIII (DJ-1), and 4 D-LDH genes. Expression pattern analysis using microarray data confirmed their ubiquitous presence in different tissues and developmental stages. Moreover, stress treatment of tomato seedlings and subsequent qRT-PCR demonstrated upregulation of SlGLYI-2, SlGLYI-3, SlGLYI-6A, SlGLYII-1A, SlGLYII-3B, SlDJ-1A, SlDLDH-1 and SlDLDH-4 in response to different abiotic stresses, whereas SlGLYI-6B, SlGLYII-1B, SlGLYII-3A, SlDJ-1D and SlDLDH-2 were downregulated. Expression data also revealed SlGLYII-1B, SlGLYI-1A, SlGLYI-2, SlDJ-1D, and SlDLDH-4 were upregulated in response to various pathogenic infections, indicating the role of MG detoxifying enzymes in both plant defence and stress modulation. The functional characterization of each of these members could lay the foundation for the development of stress and disease-resistant plants promoting sustainable agriculture and production.

Identification of m6A RNA methylation genes in Oryza sativa and expression profiling in response to different developmental and environmental stimuli.

Eukaryotic messenger RNAs (mRNAs) transcend their predominant function of protein encoding by incorporating auxiliary components that ultimately contribute to their processing, transportation, translation, and decay. In doing so, additional layers of modifications are incorporated in mRNAs at post-transcriptional stage. Among them, N6-methyladenosine (m6A) is the most frequently found mRNA modification that plays crucial roles in plant development and stress response. In the overall mechanism of m6A methylation, key proteins classified based on their functions such as writers, readers, and erasers dynamically add, read, and subtract methyl groups respectively to deliver relevant functions in response to external stimuli. In this study, we identified 30 m6A regulatory genes (9 writers, 5 erasers, and 16 readers) in rice that encode 53 proteins (13 writers, 7 erasers, and 33 readers) where segmental duplication was found in one writer and four reader gene pairs. Reproductive cells such as sperm, anther and panicle showed high levels of expression for most of the m6A regulatory genes. Notably, writers like OsMTA, OsMTD, and OsMTC showed varied responses in different stress and infection contexts, with initial upregulation in response to early exposure followed by downregulation later. OsALKBH9A, a noteworthy eraser, displayed varied expression in response to different stresses at different time intervals, but upregulation in certain infections. Reader genes like OsECT5, OsCPSF30-L3, and OsECT8 showed continuous upregulation in exertion of all kinds of stress relevant here. Conversely, other reader genes along with OsECT11 and OsCPSF30-L2 were observed to be consistently downregulated. The apparent correlation between the expression patterns of m6A regulatory genes and stress modulation pathways in this study underscores the need for additional research to unravel their intricate regulatory mechanisms that could ultimately contribute to the substantial development of enhanced stress tolerance in rice through mRNA modification.

A computational approach to identify phytochemicals as potential inhibitor of acetylcholinesterase: Molecular docking, ADME profiling and molecular dynamics simulations.

Inhibition of acetylcholinesterase (AChE) is a crucial target in the treatment of Alzheimer's disease (AD). Common anti-acetylcholinesterase drugs such as Galantamine, Rivastigmine, Donepezil, and Tacrine have significant inhibition potential. Due to side effects and safety concerns, we aimed to investigate a wide range of phytochemicals and structural analogues of these compounds. Compounds similar to the established drugs, and phytochemicals were investigated as potential inhibitors for AChE in treating AD. A total of 2,270 compound libraries were generated for further analysis. Initial virtual screening was performed using Pyrx software, resulting in 638 molecules showing higher binding affinities compared to positive controls Tacrine (-9.0 kcal/mol), Donepezil (-7.3 kcal/mol), Galantamine (-8.3 kcal/mol), and Rivastigmine (-6.4 kcal/mol). Subsequently, ADME properties were assessed, including blood-brain barrier permeability and Lipinski's rule of five violations, leading to 88 compounds passing the ADME analysis. Among the rivastigmine analogous, [3-(1-methylpiperidin-2-yl)phenyl] N,N-diethylcarbamate showed interaction with Tyr123, Tyr336, Tyr340, Phe337, Trp285 residues of AChE. Tacrine similar compounds, such as 4-amino-2-styrylquinoline, exhibited bindings with Tyr123, Phe337, Tyr336, Trp285, Trp85, Gly119, and Gly120 residues. A phytocompound (bisdemethoxycurcumin) showed interaction with Trp285, Tyr340, Trp85, Tyr71, and His446 residues of AChE with favourable binding. These findings underscore the potential of these compounds as novel inhibitors of AChE, offering insights into alternative therapeutic avenues for AD. A 100ns simulation analysis confirmed the stability of protein-ligand complex based on the RMSD, RMSF, ligand properties, PCA, DCCM and MMGBS parameters. The investigation suggested 3 ligands as a potent inhibitor of AChE which are [3-(1-methylpiperidin-2-yl)phenyl] N,N-diethylcarbamate, 4-Amino-2-styrylquinoline and bisdemethoxycurcumin. Furthermore, investigation, including in-vitro and in-vivo studies, is needed to validate the efficacy, safety profiles, and therapeutic potential of these compounds for AD treatment.

Retention of methicillin susceptibility in Staphylococcus aureus using natural adjuvant as an allosteric modifier of penicillin-binding protein 2a.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant global public health challenge due to its resistance to conventional antibiotics, primarily mediated by the mutated penicillin-binding protein, PBP2a. This study aims to investigate the potential of phytochemicals derived from medicinal plants in the Indian subcontinent to serve as adjuvants, enhancing the efficacy of methicillin against MRSA through allosteric modification of PBP2a using molecular docking and molecular dynamics (MD) simulation. After comprehensive Absorption, Distribution, Metabolism, and Excretion (ADME) profiling, along with AMES and hepatotoxicity tests, 9 compounds were shortlisted as suitable adjuvant candidates. Among them, nimbolide, quercetin, emodin, daidzein, eriodictyol, luteolin, and apigenin exhibited strong binding affinity to the allosteric site of PBP2a, with docking scores ranging from -8.7 to -7.3 kcal/mol. These phytochemicals facilitated enhanced methicillin binding, as evidenced by improved docking scores ranging from -6.1 to -6.8 kcal/mol, compared to -5.6 kcal/mol for methicillin alone. Molecular dynamics simulations confirmed the stability and favorable conformations of phytochemical-PBP2a complexes. Quercetin and daidzein were identified as the most promising adjuvant candidates, forming stable and energetically favorable complexes with PBP2a. Experimental validation showed that quercetin, at 30 mg/mL, effectively retained methicillin's antibacterial efficacy against MRSA. This study underscores the potential of natural compounds in overcoming antibiotic resistance and suggests that phytochemical-antibiotic synergism could be a viable strategy to combat multidrug-resistant bacterial infections.

Synthesis and biological evaluation of Ribo 7-N/O/S pyrimidine 9-deaza C-nucleoside analogs as new antiviral agents for inhibiting HCV RNA-dependent RNA polymerases.

Hepatitis C infection is caused by the bloodborne pathogen hepatitis C virus (HCV) and can lead to serious liver diseases and, ultimately, death if the treatment is ineffective. This work reports the synthesis and preclinical evaluation of 7 novel 9-O/N/S pyrimidine nucleosides, including compound 12, the triphosphate of known compound 7b. The nucleosides are 9-deaza modifications of adenosine and guanosine with ß-2'-C-methyl substituent on the ribose. Within this series of compounds, a 9-deaza furopyrimidine analog of adenosine, compound 7b, showed high anti-HCV activity in vitro, good stability, low toxicity, and low genotoxicity when administrated in low doses, and an adequate pharmacokinetics profile. An improved synthesis of compound 7b compared to a previous study is also reported. Compound 12 was synthesized as a control to verify phosphorylation of 7b occurred in vivo.

Synthesis of novel azasugar-containing 2'ß-C-Me 9-deaza nucleosides as potential anti-hepatitis C virus agents.

As a part of our ongoing discovery efforts exploring azasugar as agents for treating various unmet medical needs, we prepared analogs of azasugar as potential anti-hepatitis C virus (HCV) agents. Herein we describe the synthesis of novel 2'ß-C-Me 9-deazanucleoside azasugar analogs.

Variant-specific deleterious mutations in the SARS-CoV-2 genome reveal immune responses and potentials for prophylactic vaccine development.

Introduction: Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has had a disastrous effect worldwide during the previous three years due to widespread infections with SARS-CoV-2 and its emerging variations. More than 674 million confirmed cases and over 6.7 million deaths have been attributed to successive waves of SARS-CoV-2 infections as of 29th January 2023. Similar to other RNA viruses, SARS-CoV-2 is more susceptible to genetic evolution and spontaneous mutations over time, resulting in the continual emergence of variants with distinct characteristics. Spontaneous mutations of SARS-CoV-2 variants increase its transmissibility, virulence, and disease severity and diminish the efficacy of therapeutics and vaccines, resulting in vaccine-breakthrough infections and re-infection, leading to high mortality and morbidity rates. Materials and methods: In this study, we evaluated 10,531 whole genome sequences of all reported variants globally through a computational approach to assess the spread and emergence of the mutations in the SARS-CoV-2 genome. The available data sources of NextCladeCLI 2.3.0 (https://clades.nextstrain.org/) and NextStrain (https://nextstrain.org/) were searched for tracking SARS-CoV-2 mutations, analysed using the PROVEAN, Polyphen-2, and Predict SNP mutational analysis tools and validated by Machine Learning models. Result: Compared to the Wuhan-Hu-1 reference strain NC 045512.2, genome-wide annotations showed 16,954 mutations in the SARS-CoV-2 genome. We determined that the Omicron variant had 6,307 mutations (retrieved sequence:1947), including 67.8% unique mutations, more than any other variant evaluated in this study. The spike protein of the Omicron variant harboured 876 mutations, including 443 deleterious mutations. Among these deleterious mutations, 187 were common and 256 were unique non-synonymous mutations. In contrast, after analysing 1,884 sequences of the Delta variant, we discovered 4,468 mutations, of which 66% were unique, and not previously reported in other variants. Mutations affecting spike proteins are mostly found in RBD regions for Omicron, whereas most of the Delta variant mutations drawn to focus on amino acid regions ranging from 911 to 924 in the context of epitope prediction (B cell & T cell) and mutational stability impact analysis protruding that Omicron is more transmissible. Discussion: The pathogenesis of the Omicron variant could be prevented if the deleterious and persistent unique immunosuppressive mutations can be targeted for vaccination or small-molecule inhibitor designing. Thus, our findings will help researchers monitor and track the continuously evolving nature of SARS-CoV-2 strains, the associated genetic variants, and their implications for developing effective control and prophylaxis strategies.