Molecular Methodologies Research Articles

An Integrated Approach to Develop a Potent Vaccine Candidate Construct Against Prostate Cancer by Utilizing Machine Learning and Bioinformatics.

Prostate cancer is the most common malignancy among males. Prostaglandin G/H synthase (PGHS) is an essential enzyme in the synthesis of prostaglandins, and its activation has been linked to many malignancies, including colorectal cancer. Due to the limited effectiveness and specificity of existing prostate cancer therapies, this study was designed to formulate improved treatment techniques. Several immunoinformatic, reverse vaccinology, and molecular modeling methodologies were used to discover B- and T-cell epitopes for the glioblastoma multiforme tumor PGH2_HUMAN. This research evaluated Prostaglandin G/H synthase 2 protein as a potential vaccine candidate against the malignancy. The multi-epitope vaccine architecture is engineered to activate the immune system, with each epitope docked to its respective HLAs. Further, MD simulations analysis was performed to validate the findings. A multi-epitope subunit vaccine candidate was developed by concatenating the chosen B- and T-cell epitopes. Results yield a codon adaptive index (CAI) of 0.93 and a GC content of 56.77%. Thus, it conforms to a biological requirement for effective protein expression, suggesting competent vaccine efficacy inside the Escherichia coli system. Significant interleukin and cytokine responses were seen, characterized by elevated levels of IL-2 and IFN-γ in the immune system's response to the immunization. Molecular docking demonstrated an efficient binding affinity of -278 kcal/mol, with hydrogen bonding to several residues. Furthermore, the system total root mean square deviation (RMSD) reached 3.23 Å, with a maximum of up to 5.0 Å at the 100 ns time point but remains stable till 400 ns time intervals followed by stable root mean square fluctuation (RMSF) and radius of gyration values. The hydrogen bond cloud residues are the critical sites that significantly influence the binding energies of MMPBSA and MMGBSA via substantial van der Waals interactions. It has been determined that these in silico analyses will further augment the comprehension necessary for advancing the creation of targeted therapies for chemotherapeutic cancer treatments.

Exploring the potential anti-diabetic peripheral neuropathy mechanisms of Huangqi Guizhi Wuwu Decoction by network pharmacology and molecular docking.

Diabetic peripheral neuropathy (DPN) is the most prevalent microvascular complication of diabetes and Huangqi Guizhi Wuwu Decoction (HGWD) is frequently employed in classical Chinese medicine for treating DPN. This study aims to investigate the potential therapeutic targets and mechanisms of HGWD for treating DPN using network pharmacology and molecular docking methodologies. The intersection targets of DPN and HGWD were retrieved from the databases, with the resulting intersection targets being imported into the STRING database to construct the protein-protein interaction (PPI) network. Cytoscape 3.9.1 was used to screen the core targets and plot the herb-active ingredient-target (H-A-T) network. To identify the pivotal signaling pathways, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on intersection targets. Molecular docking was subsequently conducted with AutoDock Vina to validate the binding energy between the core active ingredients and the core targets. 91 potential targets of HGWD were identified for the treatment of DPN. Topological analysis revealed core targets, including AKT1, TNF, PPARG, NFKB1, TP53, STAT3, PTGS2, HIF1A, ESR1, and GSK3B, alongside core active ingredients such as protoporphyrin, jaranol, kaempferol, quercetin, and isorhamnetin. GO and KEGG analyses indicated that PI3K/AKT, HIF-1, and AGE/RAGE signaling pathways could be crucial in treating DPN using HGWD. Furthermore, molecular docking results demonstrated robust binding activities between the active ingredients in HGWD and the identified core targets. The above results indicated that HGWD may exerting an anti-DPN effect by modulating the PI3K/AKT, HIF-1, and AGE/RAGE signaling pathways.

The Genomic SSR Millets Database (GSMDB): enhancing genetic resources for sustainable agriculture.

The global population surge demands increased food production and nutrient-rich options to combat rising food insecurity. Climate-resilient crops are vital, with millets emerging as superfoods due to nutritional richness and stress tolerance. Given limited genomic information, a comprehensive genetic resource is crucial to advance millet research. Whole-genome sequencing provides an unprecedented opportunity, and molecular genetic methodologies, particularly simple sequence repeats (SSRs), play a pivotal role in DNA fingerprinting, constructing linkage maps, and conducting population genetic studies. SSRs are composed of repetitive DNA sequences where one to six nucleotides are repeated in tandem and distributed throughout the genome. Different millet species exhibit genomic variations attributed to the presence of SSRs. While SSRs have been identified in a few millet species, the existing information only covers some of the sequenced genomes. Moreover, there is an absence of complete gene annotation and visualization features for SSRs. Addressing this disparity and leveraging the de-novo millet genome assembly available from the NCBI, we have developed the Genomic SSR Millets Database (GSMDB; https://bioinfo.icgeb.res.in/gsmdb/). This open-access repository provides a web-based tool offering search functionalities and comprehensive details on 6.747645 million SSRs mined from the genomic sequences of seven millet species. The database, featuring unrestricted public access and JBrowse visualization, is a pioneering resource for the research community dedicated to advancing millet cultivars and related species. GSMDB holds immense potential to support myriad studies, including genetic diversity assessments, genetic mapping, marker-assisted selection, and comparative population investigations aiming to facilitate the millet breeding programs geared toward ensuring global food security. Database URL: https://bioinfo.icgeb.res.in/gsmdb/.

Abstract A062: Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification

Abstract Retinoblastoma (RB) the most common intraocular malignancy in children poses a significant challenge as traditional tissue biopsies are not feasible. Due to its critical location within the eye and the high risk of tumor seeding, clinicians are limited in performing molecular analysis-based risk stratification prior to enucleation. In this study, we explored the potential of aqueous humor (AH) as a source for minimally invasive liquid biopsies, aiming to establish a molecular profiling methodology for circulating tumor DNA (ctDNA) for risk stratification. We analyzed AH samples and corresponding tumor tissues from 19 enucleated eyes of patients diagnosed with RB. The extracted ctDNA from AH underwent low-coverage whole-genome sequencing (lcWGS) and methylation profiling. These data were then compared with the genomic and methylation landscapes of the matched tumor DNA. Additionally, we assessed the ability of AH- derived ctDNA to predict retinoblastoma methylation cluster. Our findings demonstrate a high concordance between ctDNA from AH and the primary tumor DNA in both methylation patterns and genomic alterations detected by lcWGS allowing the correct prediction of the retinoblastoma molecular group. The shared aberrations underscore the potential of AH as a reliable surrogate for tumor tissue in retinoblastoma. Additionally, the methylation profiles revealed consistent epigenetic modifications, further supporting the accuracy of AH-derived ctDNA in reflecting the tumor’s molecular characteristics. This study highlights the feasibility and effectiveness of using AH for molecular ctDNA genetic analysis in retinoblastoma. The overall high concordance between ctDNA profiles with primary tumor DNA and the ability to accurately predict RB methylation classes, highlights the superiority of the liquid biopsy approach overcoming the limitations of traditional biopsies and transforming the potential for early molecular analysis and risk stratification. This revolutionary technique could redefine pre-enucleation diagnostics, offering unprecedented insights and paving the way for tailored, precision medicine in retinoblastoma care. Citation Format: Sophia H Montigel, Tatsiana Ryl, Stefanie Volz, Elena Afanasyeva, Tatjana Wedig, Nathalie Schwarz, Stefan M Pfister, Kristian W Pajtler, Petra Ketteler, Kendra K Maaß. Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A062.

Phenotypic and molecular characterization of Prototheca wickerhamii from a Brazilian case of human systemic protothecosis.

The genus Prototheca (alga) comprises a unique group of achlorophyllic saprotrophic and mammalian pathogen species. Despite its rare occurrence in humans and animals, protothecosis is considered an emerging clinical entity with relevance in immunocompromised patients. In this study, the characterization of spherical structures with endospores recovered from a blood culture in an HIV patient was investigated using phenotypic and molecular methodologies. On 2% Sabouraud dextrose agar, the isolate displayed morphological and biochemical characteristics found on isolates identified as Prototheca wickerhamii. To validate these analyses, molecular phylogeny of the internal transcript space (ITS) partial gene confirmed the identity of the isolate as P. wickerhamii. This is the first case of systemic human protothecosis in Brazil. The present case of human Prototheca and those reported in the medical literature highlight the need for novel methodologies to identify pathogenic algae in the clinical laboratory, improving in this way the diagnosis and treatment of this group of neglected pathogens.

Yi-Qi-Jian-Pi-Xiao-Yu formula inhibits cisplatin-induced acute kidney injury through suppressing ferroptosis via STING-NCOA4-mediated ferritinophagy

BackgroundThe kidneys are the primary excretory organs for platinum drugs, making them susceptible to damage from these drugs. Cisplatin-induced acute kidney injury (CIAKI) is the most common side effect observed in patients undergoing clinical cisplatin treatment. A traditional Chinese medicinal preparation, the Yi-Qi-Jian-Pi-Xiao-Yu formula (YQJPXY), which is a modified formulation of the classical Chinese medicine formula Buyang Huanwu Decoction, has long been used in the treatment of clinical kidney diseases. It is expected to be used to ameliorate cisplatin-induced acute kidney injury. However, the mechanism of this YQJPXY for the treatment of cisplatin-induced acute kidney injury remains unclear. PurposeThe objective of this study is to examine the impact of the YQJPXY on the inhibition of ferroptosis in cisplatin-induced acute kidney injury and to elucidate the underlying mechanisms. MethodsThe active components of YQJPXY were analysed using UPLC-MS/MS. A comprehensive investigation was conducted to elucidate the effects and regulatory mechanisms of YQJPXY on CIAKI and ferroptosis in mice subjected to acute cisplatin treatment and in mice receiving cisplatin treatment after STING expression was inhibited using the STING inhibitor C176. The renoprotective effect of YQJPXY on cisplatin-treated mice was evaluated by measuring tissue damage, inflammation and pro-fibrosis. In addition, we employed network pharmacology and molecular docking methodologies to analyse the principal regulatory targets of YQJPXY. Furthermore, the expression of key proteins and markers of ferroptosis and iron metabolism, as well as the levels of key indicators related to STING-associated ferritinophagy, were examined by immunoblotting, immunohistochemistry, immunoprecipitation, quantitative real-time PCR (qPCR) and specific probes. ResultsThe results demonstrated that YQJPXY reduced the levels of indicators of injury, inflammation and pro-fibrosis in CIAKI mice, with renoprotective effects. Network pharmacological analyses revealed that ferroptosis might be the main biological process regulated by YQJPXY. Furthermore, molecular docking results indicated that STING might be a potential regulatory target of YQJPXY. Furthermore, YQJPXY treatment resulted in a significant reduction in MDA and 4-HNE levels, as well as the inhibition of ferroptosis and improvement in iron metabolic processes. Concomitantly, YQJPXY exhibited a robust protective effect on ferroptosis and iron metabolism homeostasis, as evidenced by its inhibitory action on ferritinophagy. Validation experiments utilising the cisplatin inhibitor C176 demonstrated that YQJPXY inhibits cisplatin-induced ferroptosis in kidney via STING-mediated ferritinophagy. ConclusionThese suggest that YQJPXY alleviates cisplatin-induced acute kidney injury through suppressing ferroptosis via STING-NCOA4-mediated Ferritinophagy.

Latin America: A hub for agrobiotechnological innovations.

Modern biotechnology is one of the last century's major advances in human science. Particularly in the agronomical field, the landscape of crop improvement technologies has witnessed a great expansion, driven by the integration of molecular and genetic engineering methodologies into the breeding toolbox. Latin America (LATAM) serves as a pioneering region in incorporating such techniques with several countries swiftly embracing these technologies. This review aims to give a comprehensive overview of the elements that influenced agrobiotech acceptance in LATAM countries and how such cases could provide support for upcoming technologies to be considered worldwide. Nearly 50 years of biotech breakthroughs have provided humankind with an impressive portfolio of tools already integrated into several life-sciences areas. The agronomical field has greatly progressed thanks to technologies derived from Genetically Modified Organisms (GMOs) and high promises are being made to also incorporate genome -editing products. LATAM's case is a prime example of how early introduction of novelties in the crop production chain can result in improved yields, paving the way for future developments to be easily integrated into the technological ecosystem of a region. The example set by LATAM can also be useful for the present gene-editing regulatory scenario. With several countries presently on the path to approving these methods in their current crop systems, basing their next steps on the southern continent's example, could represent a safe and practical pathway towards a new agronomical revolution.

A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus detection in oysters

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture.

Prevalence and Genetic Diversity of Entamoeba and Cryptosporidium in Pigs and Wild Boars in Central and Southern Vietnam: Implications for Zoonotic Risks and Surveillance.

Background: Parasites of Entamoeba and Cryptosporidium genera, prevalent among various vertebrates such as humans and pigs, pose a zoonotic threat as common protozoan pathogens. This study investigated the prevalence and genetic diversity of Entamoeba and Cryptosporidium species in pigs and wild boars across central and southern Vietnam, to ascertain parasite transmission dynamics. Methods: A total of 113 independent stool samples from 77 pigs and 36 wild boars were analyzed using PCR-based molecular methodologies to detect the presence of Entamoeba spp. and Cryptosporidium spp. The identified species were further characterized through Sanger sequencing, and phylogenetic relationships were analyzed. Results: The study revealed a high prevalence of Entamoeba spp. (62%, n = 70/113) and Cryptosporidium spp. (31%, n = 35/113). Entamoeba suis (57%, n = 40) was predominant, followed by Entamoeba polecki (40%, n = 40) and Entamoeba hartmanni (3%, n = 2). Among Cryptosporidium species, Cryptosporidium scrofarum (89%, n = 31) was the most common, followed by Cryptosporidium suis (11%, n = 4). Wild boars exhibited a higher prevalence of Entamoeba infection compared with domestic pigs (p = 0.019). Conclusions: The study highlights a high prevalence of Entamoeba and Cryptosporidium, suggesting a potential for zoonotic transmission in Vietnam. Further investigations are necessary to determine the extent to which these parasites in pigs and wild boars contribute to the burden in the human population.

Molecular Docking in Drug Discovery: Techniques, Applications, and Advancements.

The primary objective of this study is to conduct a comprehensive review of the significance of molecular docking in the field of drug discovery. This includes an examination of the various approaches and methods used in molecular docking, as well as an exploration of the techniques used for interpreting and validating docking results. To gather relevant data, a systematic search was conducted using Web of Science, PubMed, and Google Scholar. The search focused on articles related to molecular docking methodologies and their applications in drug discovery. Additionally, alternative techniques that can be used for more precise simulations of ligand-protein interactions were also considered. Molecular docking has proven to be an incredibly rich and valuable process in the field of drug discovery. Its flexibility allows for the incorporation of advanced computational techniques, thereby enhancing the reliability and efficiency of drug discovery processes. The results of the study highlights the significant strides made in the field of molecular docking, demonstrating its potential to revolutionize drug discovery. Molecular docking continues to evolve, with new advancements being made regularly. Despite the challenges faced, these advancements have significantly contributed to the enhancement of molecular docking, solidifying its position as a crucial tool in the field of drug discovery.

FMO-LC-TDDFTB method for excited states of large molecular assemblies in the strong light-matter coupling regime.

We present a new methodology to calculate the strong light-matter coupling between photonic modes in microcavities and large molecular aggregates that consist of hundreds of molecular fragments. To this end, we combine our fragment molecular orbital long-range corrected time-dependent density functional tight-binding methodology with a generalized Tavis-Cummings Hamiltonian. We employ an excitonic Hamiltonian, which is built from a quasi-diabatic basis that is constructed from locally excited and charge-transfer states of all molecular fragments. To calculate polaritonic states, we extend our quasi-diabatic basis to include photonic states of a microcavity and derive and implement the couplings between the locally excited states and the cavity states and built a Tavis-Cummings Hamiltonian that incorporates the intermolecular excitonic couplings. Subsequently, we demonstrate the capability of our methodology by simulating the influence of the electric field polarization on the polaritonic spectra for a tetracene aggregate of 125 monomers. Furthermore, we investigate the dependence of the splitting of the upper and lower polaritonic branches on the system size by comparing the spectra of five different tetracene clusters. In addition, we investigate the polariton dispersion of a large tetracene aggregate for electric field polarizations in the x, y, and z directions. Our new methodology can facilitate the future study of exciton dynamics in complex molecular systems, which consist of up to hundreds of molecules that are influenced by strong light-matter coupling to microcavities.

Metal tolerance of Río Tinto fungi.

Southwest Spain's Río Tinto is a stressful acidic microbial habitat with a noticeably high concentration of toxic heavy metals. Nevertheless, it has an unexpected degree of eukaryotic diversity in its basin, with a high diversity of fungal saprotrophs. Although some studies on the eukaryotic diversity in Rio Tinto have been published, none of them used molecular methodologies to describe the fungal diversity and taxonomic affiliations that emerge along the river in different seasons. The aim of the present study was to isolate and describe the seasonal diversity of the fungal community in the Río Tinto basin and its correlation with the physicochemical parameters existing along the river's course. The taxonomic affiliation of 359 fungal isolates, based on the complete internal transcribed spacer DNA sequences, revealed a high degree of diversity, identifying species belonging primarily to the phylum Ascomycota, but representatives of the Basidiomycota and Mucoromycota phyla were also present. In total, 40 representative isolates along the river were evaluated for their tolerance to toxic heavy metals. Some of the isolates were able to grow in the presence of 1000 mM of Cu2+, 750 mM of As5+ and Cd2+, and 100 mM of Co2+, Ni2+, and Pb2+.

Molecular diagnostics in cerebrospinal fluid for the diagnosis of central nervous system infections.

SUMMARYCentral nervous system (CNS) infections can be caused by various pathogens, including bacteria, viruses, fungi, and parasites. Molecular diagnostic methods are pivotal for identifying the different causative pathogens of these infections in clinical settings. The efficacy and specificity of these methods can vary per pathogen involved, and in a substantial part of patients, no pathogen is identified in the cerebrospinal fluid (CSF). Over recent decades, various molecular methodologies have been developed and applied to patients with CNS infections. This review provides an overview of the accuracy of nucleic acid amplification methods in CSF for a diverse range of pathogens, examines the potential value of multiplex PCR panels, and explores the broad-range bacterial and fungal PCR/sequencing panels. In addition, it evaluates innovative molecular approaches to enhance the diagnosis of CNS infections.

Analysis of graphene as a potential filtration membrane for desalination at varying operating conditions using molecular dynamics simulation and response surface methodology

ABSTRACT Water scarcity is a critical global issue exacerbated by pollution and overuse, necessitating sustainable water management solutions. Desalination using membrane technology presents a promising approach for freshwater production. This study investigated the performance of nanoporous (NPG) membranes for desalination, focusing on the influence of pressure and temperature on water flux and ion rejection. Utilizing molecular dynamics (MD) simulations with the LAMMPS package, this study evaluates NPG membranes under various conditions of pressures and temperatures. The simulations demonstrate that increasing both the pressure and temperature enhances the water flux without compromising ion rejection. The results indicate that at 300 K and 50 MPa, the water flux exceeds 2000 L/m2 h bar, significantly outperforming traditional reverse osmosis membranes, which typically achieve a capacity of approximately 1 L/m2 h bar. These findings were validated experimentally, aligning with previous research and confirming the superior performance of NPG membranes. A statistical model derived from response surface methodology revealed a linear relationship between pressure, temperature, and water flux. The study concludes that NPG membranes offer a high efficiency and scalable solution for desalination, with significant potential for energy savings and cost reduction. This study underscores the viability of NPG membranes in addressing global freshwater shortages and provides a pathway for sustainable water production.

A-264 Validation of a Novel Real-Time PCR Assay for Identification of Mycobacterium species Isolates Recovered from Clinical Culture

Abstract Background Historically, clinical mycobacteriology laboratories used the nucleic acid hybridization-based AccuProbe assay manufactured by Hologic, Inc., to rapidly rule in or rule out Mycobacterium tuberculosis (MTB) and members of the M. avium-intracellulare complex (MAC). The clinical value of this test was in its ability to rapidly rule in/out MTB from positive broth cultures and allow the initiation of appropriate antimicrobial therapy and infection prevention practices. Recent discontinuation of this commercial assay left a gap in testing available for the rapid identification of these organisms, which allowed us to develop an in-house real-time PCR test. Methods A replacement assay was developed by Labcorp R & D based on real-time PCR amplification of extracted nucleic acids from positive culture growth. Aliquots from positive AFB VersaTREK broth cultures were heat inactivated, and nucleic acids were extracted using the MagNA Pure 24 (MP24) instrument (Roche). Real-time PCR using published primer probe sets (Rocchetti, et al 2016) was performed on the QuantStudio™ 7 (QS7) instrument (Applied Biosystems®). The assay was designed with three independent primer/probe sequence targets: one for MTB Complex (IS6110), one for MAC (ITS), and one pan-target for the Mycobacterium genus (16s rRNA). Results Procedural verification was performed using 100 residual clinical specimens previously identified using the Hologic AccuProbe® assay. Categorical concordance was 97% (97/100). The three discrepant results were identified as MAC positive by PCR; however, these were not identified as MAC on the predicate assay. Analytical sensitivity for the PCR assay was established to be 1,000 CFU/mL for each target. Analytical specificity was assessed in vitro for nine Mycobacterium species, with no cross-reactivity observed. In vitro analysis of target detection in the presence of yeast (Candida glabrata) was also performed, and we saw no inhibition. Analysis of the primer/probe sets for all targets was performed in silico using XploreDB database of microbial genomes, with confirmed 100% matches for both MAC and MTB genomes compared to hundreds of species. By comparison, the PAN-Myco primer set showed decreased performance, having three mismatches (two on forward primer and one on the probe). As this target sequence is used as an Internal Control (IC) this was not problematic for overall assay performance. The assay demonstrated excellent precision (%CV) for each target: MTB = 0.74%, MAC = 1.10%, PAN-Myco = 3.19%. Conclusions Utilizing the depth of molecular methodologies available in our laboratories, we developed a rapid method for identification of MAC and MTB from positive broth cultures, which had excellent performance compared to the predicate assay. The PCR ID LDT test is now available and allows testing to continue with no interruption in patient care.

Crotonylation of NAE1 Modulates Cardiac Hypertrophy via Gelsolin Neddylation.

Cardiac hypertrophy and its associated remodeling are among the leading causes of heart failure. Lysine crotonylation is a recently discovered posttranslational modification whose role in cardiac hypertrophy remains largely unknown. NAE1 (NEDD8 [neural precursor cell expressed developmentally downregulated protein 8]-activating enzyme E1 regulatory subunit) is mainly involved in the neddylation modification of protein targets. However, the function of crotonylated NAE1 has not been defined. This study aims to elucidate the effects and mechanisms of NAE1 crotonylation on cardiac hypertrophy. Crotonylation levels were detected in both human and mouse subjects with cardiac hypertrophy through immunoprecipitation and Western blot assays. Tandem mass tag (TMT)-labeled quantitative lysine crotonylome analysis was performed to identify the crotonylated proteins in a mouse cardiac hypertrophic model induced by transverse aortic constriction. We generated NAE1 knock-in mice carrying a crotonylation-defective K238R (lysine to arginine mutation at site 238) mutation (NAE1 K238R) and NAE1 knock-in mice expressing a crotonylation-mimicking K238Q (lysine to glutamine mutation at site 238) mutation (NAE1 K238Q) to assess the functional role of crotonylation of NAE1 at K238 in pathological cardiac hypertrophy. Furthermore, we combined coimmunoprecipitation, mass spectrometry, and dot blot analysis that was followed by multiple molecular biological methodologies to identify the target GSN (gelsolin) and corresponding molecular events contributing to the function of NAE1 K238 (lysine residue at site 238) crotonylation. The crotonylation level of NAE1 was increased in mice and patients with cardiac hypertrophy. Quantitative crotonylomics analysis revealed that K238 was the main crotonylation site of NAE1. Loss of K238 crotonylation in NAE1 K238R knock-in mice attenuated cardiac hypertrophy and restored the heart function, while hypercrotonylation mimic in NAE1 K238Q knock-in mice significantly enhanced transverse aortic constriction-induced pathological hypertrophic response, leading to impaired cardiac structure and function. The recombinant adenoviral vector carrying NAE1 K238R mutant attenuated, while the K238Q mutant aggravated Ang II (angiotensin II)-induced hypertrophy. Mechanistically, we identified GSN as a direct target of NAE1. K238 crotonylation of NAE1 promoted GSN neddylation and, thus, enhanced its protein stability and expression. NAE1 crotonylation-dependent increase of GSN promoted actin-severing activity, which resulted in adverse cytoskeletal remodeling and progression of pathological hypertrophy. Our findings provide new insights into the previously unrecognized role of crotonylation on nonhistone proteins during cardiac hypertrophy. We found that K238 crotonylation of NAE1 plays an essential role in mediating cardiac hypertrophy through GSN neddylation, which provides potential novel therapeutic targets for pathological hypertrophy and cardiac remodeling.

Exhaustive computational studies on pyrimidine derivatives as GPR119 agonist for the development of compounds against NIDDM

BackgroundType-2 Diabetes (T2DM) is a long-term medical disorder characterized by Insulin deficiency and high blood glucose levels. Among other medications to cure T2DM, the review of the literature found that various Pyrimidine derivatives act as an agonist for G-protein-coupled receptor 119 (GPR119) was proposed to control blood glucose levels by enhancing the function of pancreatic Beta-cells and its mechanism of action with fewer adverse effects. In the present research work, In-silico investigations were carried out to investigate the potential of the Pyrimidine analog as an agonist to the protein target GPR119 receptor. We performed exhaustive molecular modeling and protein modeling methodologies such as homology modeling, and molecular docking along with various drug designing tools such as 3D-QSAR and Pharmacophore Mapping to ascertain the design of better GPR119 agonists.ResultsBased on in-depth computational studies, we designed new pyrimidine moiety and analyzed them for GPR119 receptor agonist and further explored the ADMET properties. Designed compounds were found to exhibit better-predicted activities as compared to reference compound.ConclusionsThe current research on pyrimidine derivatives, using molecular docking, 3D-QSAR and Pharmacophore mapping demonstrated that the obtained computational model has significant properties and the designed molecules and Dataset from this model, produced antidiabetic compound against the target GPR119 i.e., compound 1S, 1Z and 1D with the docking score of − 11.696, − 9.314 and − 8.721, respectively. The pharmacokinetics and drug-likeness studies revealed that these compounds may be the future candidates for the treatment of diabetes acting via the GPR119 agonist mechanism.

Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in the flower of Paeonia lactiflora

Emergence of floral scents marked a significant milestone in biological development of plant, letting out olfactory signals that enable them to induce pollinators. In this research, molecular and metabolomics methodologies were used to explore terpene synthase (TPS) genes, which are responsible for synthesis of floral fragrance in Paeonia lactiflora ‘Wu Hua Long Yu’. Analyses on transcriptome data identified 11 PlTPS genes, among them 7 genes were isolated, and the functions of 5 genes were confirmed. PlTPS1 was identified as a single-product enzyme; PlTPS4, 8 and 9 were identified as bifunctional enzymes, capable of recognizing farnesyl pyrophosphate and geranyl pyrophosphate; PlTPS6 generated multiple products by recognizing farnesyl pyrophosphate. Enzymatic products of these PlTPS proteins closely matched the volatile terpenes emitted by flowers, revealing the relation between PlTPS genes and the release of such volatile compounds. Functions of five PlTPS genes in herbaceous peony were further elucidated through virus-induced gene silencing experiments, as well as overexpression in tobacco. PlTPS1 catalyzes geraniol production, while PlTPS4 catalyzes linalool. PlTPS6 is responsible for producing two sesquiterpenes, caryophyllene and α-humulene. PlTPS8, with the most varied products, mainly catalyzes the production of α-pinene, linalool, and germacrene D. Based on these findings, graphical models summarizing the biosynthesis of floral volatile terpenes in herbaceous peony have been proposed. The characterization of PlTPS genes has elucidated their specific roles in the synthesis of herbaceous peony fragrance, opening up possibilities for introducing or enhancing floral scents in herbaceous peony and other plants.

The study on characterization of interaction between casticin and model proteins using spectroscopic and computational methodologies

The present studies were conducted to investigate the interaction of casticin to model proteins (BSA, HSA, OVA and HEL) using fluorescence spectra, Fourier transform infrared spectra, molecular docking and molecular dynamics simulation methodologies. Our work displayed that the endogenous fluorescence intensities of BSA, HSA, OVA and HEL were quenched by various concentrations of casticin were the typical static quenching process. The binding constants (Ka) of casticin for these model proteins were in the range of 104–105 mol·L-1, with the highest affinity for HSA. On the basis of thermodynamic parameters and computational simulation studies, it was suggested that the binding force of casticin to BSA, HSA, OVA and HEL were mainly hydrogen bonds, van der Waals forces and hydrophobic interactions. The spectral results illustrated that the binding of casticin to BSA, HSA, OVA and HEL induced conformational and microenvironmental changes. Casticin could decrease the α-helix percentages of BSA, HSA and OVA, but increase that of HEL. Binding substitution studies confirmed that casticin bound to the Sudlow site I of BSA and HSA. Molecular docking predicted the binding models of casticin to BSA, HSA, OVA and HEL, followed closely by molecular dynamic simulations to assess the binding stability.

Exploring Chroman‐4‐One Derivatives as MAO‐B Inhibitors: A Comprehensive Computational Study

AbstractThe enzyme monoamine oxidase‐B (MAO‐B) significantly affects the neurotransmitter's metabolism, including norepinephrine, dopamine, and serotonin. Alzheimer's disease is the result of the dysregulation of MAO‐B activity, which is also attached to another neurological disorder, that is, Parkinson's disease. The MAO‐B antagonists are now seen as potential therapeutics for these conditions. A promising approach for improving the effectiveness of novel MAO inhibitors is using chromanone scaffolds. In this work, we used a multimodal computational approach to assess the inhibitory effects of chroman‐4‐one derivatives on MAO‐B. The study utilized molecular docking methodologies to investigate and evaluate the intricate binding interactions between MAO‐B and chroman‐4‐one derivatives. Moreover, the ADME study provided insightful information about the pharmacokinetic characteristics of chroman‐4‐one derivatives. The analysis of the temporal stability and dynamic behavior of ligand‐protein interactions has benefited from the application of MD simulations. To determine the binding free energies and gain a deeper comprehension of the binding affinity between chroman‐4‐one derivatives and MAO‐B, MM‐PBSA calculations were also performed. This study offers valuable information about logical design and the optimization of MAO‐B antagonists and highlights the potential of computational techniques in speeding up the drug discovery process.