3D Protein Models Research Articles

Phylogenetic analysis of selected species of Asteraceae on the basis of RPS 11 Gene

The Asteraceae family is a prominent group of flowering plants found across the globe, with the exception of Antarctica. The Asteraceae family is a largest flowering family pivotal group in plant evolution and diversification. Despite its importance, the genetic diversity within this family remains understudied. We focused on the rps-11 gene, a chloroplast marker previously utilized in phylogenetic studies, to investigate its potential in resolving Asteraceae relationships. The focus was on examining genetic diversity within sixteen specifically chosen species from the Asteraceae family. This assessment was based on an analysis of a chloroplast gene responsible for encoding the ribosomal protein of the smaller subunit 11 (rps 11). Nearly 417 bp of rps 11 gene was amplified, sequenced, computationally translated into amino acid sequence and the data was used for phylogenetic analysis as well as for rps 11 protein structure predictions. Based on nucleotide and amino acid sequences phylograms were drawn with the help of Molecular Evolutionary Genetic Analysis (MEGA 6), which exhibited clear genetic relationship among species under investigation. The observed genetic distance was 0.02 for Maximum likelihood tree based on nucleotide sequences whereas it was 0.05 for phylogram based on amino acid sequences. These values revealed that amino acid-based tree has demonstrated greater diversity among selected species in comparison to nucleotides-based tree. On the basis of pair wise distance calculations, genetic divergence values were found within the range of 0.015–0.309. Moreover, 3D protein modeling for rps 11 protein of sixteen selected species was also carried out by iterative threading assembly refinement (I-Tasser) software. The models exhibiting the highest C-score were picked with satisfactory plot statistics (> 90%) and structurally validated by PROCHECK. Furthermore, Ramachandran plots displayed that the rps 11 protein structures of Tagetes minuta, Xanthium strumarium, Lactuca sativa and Chrysanthemum indicum have best feature models with > 90% of residues in the allowed region and ≤ 2% in the disallowed region. The research is not enough to stand alone to validate the viability of the rps11 gene as a prospective contender for phylogenetic analysis. İn future we will focus on the maximum genetic diversity theory for phylogenetic analysis of this family.

Genomic Analysis of Aeromonas salmonicida ssp. salmonicida Isolates Collected During Multiple Clinical Outbreaks Supports Association with a Single Epidemiological Unit.

Outbreaks of furunculosis cause significant losses in salmonid aquaculture worldwide. With a recent rise in antimicrobial resistance, regulatory measures to minimize the use of antibiotics in animal husbandry, including aquaculture, have increased scrutiny and availability of veterinary medical products to control this disease in production facilities. In such a regulatory environment, the utility of autogenous vaccines to assist with disease prevention and control as a veterinary-guided prophylactic measure is of high interest to the producers and veterinary services alike. However, evolving concepts of epidemiological units and epidemiological links need to be considered during approval and acceptance procedures for the application of autogenous vaccines in multiple aquaculture facilities. Here, we present the results of solid-state nanopore sequencing (Oxford Nanopore Technologies, ONT) performed on 54 isolates of Aeromonas salmonicida ssp. salmonicida sampled during clinical outbreaks of furunculosis in different aquaculture facilities from Bavaria, Germany, from 2017 to 2020. All of the performed analyses (phylogeny, single nucleotide polymorphism and 3D protein modeling for major immunogenic proteins) support a high probability that all studied isolates belong to the same epidemiological unit. Simultaneously, we describe a cost/effective method of whole genome analysis with the usage of ONT as a viable strategy to study outbreaks of other pathogens in the field of aquatic veterinary medicine for the purpose of developing the best autogenous vaccine candidates applicable to multiple aquaculture establishments.

Analysis of Phylogenetics and Structural Relationships of the Cytochrome b Gene Protein in Mahseer Fish (Tor sp.)

Abstract The Cytochrome b gene from mitochondrial DNA encodes a protein that can be used as a genetic marker to identify species. This study aims to determine the bioinformatic characteristics of the gene encoding Cyt b isolated from various Mahseer species, Tor tambroides, Tor douronensis, Tor khudree, and Tor putitora. Mahseer Cyt b sequences can be used for detailed computational investigation of the properties of 3D protein models and phylogenetics. The data analysis technique uses nucleotides with a sequence length of 330 bp obtained from NCBI. The MEGA X program was used for phylogenetic analysis. 3D protein structure predictions were obtained through the Swiss model and protein homology program using PYMOL software. The results of phylogenetics show close kinship relationships with the closest genetic distance of 0.000, while the farthest distance is 0.068. The nucleotide composition contains Thymine (30.23%), Cytosine (25.81%), Arginine (28.05%), and Guanine (15.92%). 3D modeling of the Cyt b protein Mahseer referring to the species Neolissochilus hexagonolepis (McClelland, 1839). The 3D protein structure prediction was assessed via a Ramachandran plot of 99.07%. This study can be used as a reference to support sustainable conservation efforts by observing the populations and habitat conditions.

Bioinformatics Study on Site-Specific Variations of Eotaxin-3, a Key Chemokine in Eosinophilic Esophagitis (EoE).

Eotaxin-3 is a key chemokine with a relevant role in eosinophilic esophagitis, a rare chronic immune/antigen-mediated inflammatory disorder. Eotaxin-3 is a potent activator of eosinophil emergence and migration, which may lead to allergic airway inflammation. We investigated, using bioinformatics tools, the protein structure and the possible effects of the known variations reported in public databases. Following a procedure already established, we created a 3D model of the whole protein and modeled the structure of 105 protein variants due to known point mutations. The effects of the amino acid substitution at the level of impact on protein structure, stability, and possibly function were detected by the bioinformatics procedure and described in detail. A web application was implemented to browse the results of the analysis and visualize the 3D models, with the opportunity of downloading the models and analyzing them using their own software. Among 105 amino acid substitutions investigated, the study evidenced in 44 cases at least one change in any of the investigated structural parameters. Other six variations are also relevant, although a structural effect was not detected by our analysis, because they affected amino acids highly conserved, which suggests a possible function role. All these variations should be the object of particular attention, as they may induce a loss of functionality in the protein.

Mutated neuron navigator 3 as a candidate gene for a rare neurodevelopmental disorder.

Neuron navigator 3 (NAV3) is characterized as one of the neuron navigator family (NAV1, NAV2, NAV3) proteins predominantly expressed in the nervous system. The NAV3-encoded protein comprises a conserved AAA and coiled-coil domains characteristic of ATPases, which are associated with different cellular activities. We describe a Saudi proband presenting a complex recessive neurodevelopmental disorder (NDD). Whole exome sequencing (WES) followed by Sanger sequencing, 3D protein modeling and RT-qPCR was performed. WES revealed a bi-allelic frameshift variant (c.2604_2605delAG; p.Val870SerfsTer12) in exon 12 of the NAV3 gene. Furthermore, RT-qPCR revealed a significant decrease in the NAV3 mRNA expression in the patient sample, and 3D protein modeling revealed disruption of the overall secondary structure. For the time, we associate a bi-allelic variant in the NAV3 gene causing NDD in humans.

A novel homozygous FAM92A gene (CIBAR1) variant further confirms its association with non-syndromic postaxial polydactyly type A9 (PAPA9).

Polydactyly is a very common digit anomaly, having extra digits in hands and/or toes. Non-syndromic polydactyly in both autosomal dominant and autosomal recessive forms are caused by disease-causing variants in several genes, including GLI1, GLI3, ZNF141, FAM92A, IQCE, KIAA0825, MIPOL1, STKLD1, PITX1, and DACH1. Whole exome sequencing (WES) followed by bi-directional Sanger sequencing was performed for the single affected individual (II-1) of the family to reveal the disease causative variant/gene. 3D protein modeling and structural molecular docking was performed to determine the effect of the identified mutation on the overall protein structure. WES revealed a novel biallelic missense variant (c.472G>C; p.Ala158Pro) in exon 6 of the FAM92A gene. The identified variant segregated perfectly with the disease phenotype using Sanger sequencing. Furthermore, Insilco analysis revealed that the variant significantly changes the protein secondary structure, and substantially impact the stability of FAM92A. We report the second FAM92A disease-causing mutation associated with recessive non-syndromic postaxial polydactyly. The data further confirms the contribution of FAM92A in limb development and patterning.

AlphaKnot 2.0: a web server for the visualization of proteins' knotting and a database of knotted AlphaFold-predicted models.

The availability of 3D protein models is rapidly increasing with the development of structure prediction algorithms. With the expanding availability of data, new ways of analysis, especially topological analysis, of those predictions are becoming necessary. Here, we present the updated version of the AlphaKnot service that provides a straightforward way of analyzing structure topology. It was designed specifically to determine knot types of the predicted structure models, however, it can be used for all structures, including the ones solved experimentally. AlphaKnot 2.0 provides the user's ability to obtain the knowledge necessary to assess the topological correctness of the model. Both probabilistic and deterministic knot detection methods are available, together with various visualizations (including a trajectory of simplification steps to highlight the topological complexities). Moreover, the web server provides a list of proteins similar to the queried model within AlphaKnot's database and returns their knot types for direct comparison. We pre-calculated the topology of high-quality models from the AlphaFold Database (4th version) and there are now more than 680.000 knotted models available in the AlphaKnot database. AlphaKnot 2.0 is available at https://alphaknot.cent.uw.edu.pl/.

Decoding LY6G6D in colorectal cancer: Unraveling biomarker potential and therapeutic insights.

Colorectal cancer (CRC) poses a significant global health challenge with high morbidity and mortality rates. This study investigates the role of LY6G6D, a member of the LY6/uPAR superfamily, in CRC. Employing a bioinformatic approach, we analyzed LY6G6D expression across different cancer types, compared it with known oncogenes in CRC, explored the involved genomic alterations, and assessed associated clinicopathological characteristics. LY6G6D exhibited aberrant expression, particularly elevated in CRC adenocarcinoma and highly specific to tumor tissues when compared with other oncogenes, despite its comparatively low frequency of genomic alteration. Subsequently, tumor immune infiltration analysis revealed distinct associations, primarily indicating a negative correlation, suggesting immune down-regulation. Survival analysis in context of LY6G6D was conducted with Kaplan-Meier (KM) curves, indicating a 10% risk of disease recurrence in the case of elevated expression. Additionally, we constructed a 3D protein model of LY6G6D through ab-inito approach. The protein model was validated, followed by conservation analysis and active site identification. Active site identification of LY6G6D's final predicted model revealed some similar sites that were estimated to be conserved. Target-guided drug molecules were collected and molecular docking was executed, proposing Cardigin (Digitoxin) and Manzamine A as potential therapeutic candidates. In conclusion, LY6G6D emerges as a significant biomarker for diagnostic and therapeutic applications in CRC, highlighting its multifaceted role in tumorigenesis. The proposed drugs present avenues for further investigations.

Predicting protein retention in ion-exchange chromatography using an open source QSPR workflow.

Protein-based biopharmaceuticals require high purity before final formulation to ensure product safety, making process development time consuming. Implementation of computational approaches at the initial stages of process development offers a significant reduction in development efforts. By preselecting process conditions, experimental screening can be limited to only a subset. One such computational selection approach is the application of Quantitative Structure Property Relationship (QSPR) models that describe the properties exploited during purification. This work presents a novel open-source Python tool capable of extracting a range of features from protein 3D models on a local computer allowing total transparency of the calculations. As open-source tool, it also impacts initial investments in constructing a QSPR workflow for protein property prediction for third parties, making it widely applicable within the field of bioprocess development. The focus of current calculated molecular features is projection onto the protein surface by constructing surface grid representations. Linear regression models were trained with the calculated features to predict chromatographic retention times/volumes. Model validation shows a high accuracy for anion and cation exchange chromatography data (cross-validated R2 of 0.87 and 0.95). Hence, these models demonstrate the potential of the use of QSPR to accelerate process design.

Identification of novel pathogenic variants of CUBN in patients with isolated proteinuria.

Although proteinuria is long recognized as an independent risk factor for progressive chronic kidney diseases, not all forms of proteinuria are detrimental to kidney function, one of which is isolated proteinuria caused by cubilin (CUBN)-specific mutations. CUBN encodes an endocytic receptor, initially found to be responsible for the Imerslund-Gräsbeck syndrome (IGS; OMIM #261100) characterized by a combined phenotype of megaloblastic anemia and proteinuria. After analyzing their clinical and pathological characterizations, next-generation sequencing for renal disease genes or whole-exome sequencing (WES) was performed on four patients with non-progressive isolated proteinuria. CUBN biallelic pathogenic variants were identified and further analyzed by cDNA-PCR sequencing, immunohistochemistry, minigene assay, and multiple in silico prediction tools, including 3D protein modeling. Here, we present four patients with isolated proteinuria caused by CUBN C-terminal biallelic pathogenic variants, all of which showed no typical IGS symptoms, such as anemia and vitamin B12 deficiency. Their urine protein levels fluctuated between +~++ and estimated glomerular filtration rate (eGFR) were normal or slightly higher. Mild mesangial hypercellularity was found in three children's renal biopsies. A homozygous splice-site variant of CUBN (c.6821+3 (IVS44) A>G) was proven to result in the exon 44 skipping and premature translation termination by cDNA sequencing and immunohistochemistry. Compound heterozygous mutations were identified among the other three children, including another novel splice-site variant (c.10764+1 (IVS66) G>A) causing the retention of first 4 nucleotides in intron 66 by minigene assay, two unreported missense mutations (c.4907G>A (p.R1636Q); c. 9095 A>G (p.Y3032C)), and two reported missense mutations in China (c.8938G>A (p.D2980N); c. 9287T>C (p.L3096P)), locating behind the vitamin B12-binding domain, affecting CUB11, CUB16, CUB22, CUB23, and CUB27 domains, respectively. These results demonstrate that above CUBN mutations may cause non-progressive and isolated proteinuria, expanding the variant spectrum of CUBN and benefiting our understanding of proteinuria and renal function.

Clinical features and molecular genetic investigation of infantile-onset ascending hereditary spastic paralysis (IAHSP) in two Chinese siblings caused by a novel splice site ALS2 variation

ObjectiveALS2-related disorder involves retrograde degeneration of the upper motor neurons of the pyramidal tracts, among which autosomal recessive Infantile-onset ascending hereditary spastic paralysis (IAHSP) is a rare phenotype. In this study, we gathered clinical data from two Chinese siblings who were affected by IAHSP. Our aim was to assess the potential pathogenicity of the identified variants and analyze their clinical and genetic characteristics.MethodHere, Whole-exome sequencing (WES) was performed on proband to identify the candidate variants. Subsequently, Sanger sequencing was used to verify identified candidate variants and to assess co-segregation among available family members. Utilizing both silico prediction and 3D protein modeling, an analysis was conducted to evaluate the potential functional implications of the variants on the encoded protein, and minigene assays were performed to unravel the effect of the variants on the cleavage of pre-mRNA.ResultsBoth patients were characterized by slurred speech, astasia, inability to walk, scoliosis, lower limb hypertonia, ankle clonus, contracture of joint, foot pronation and no psychomotor retardation was found. Genetic analysis revealed a novel homozygous variant of ALS2, c.1815G > T(p.Lys605Asn) in two Chinese siblings. To our knowledge, it is the first confirmed case of a likely pathogenic variant leading to IAHSP in a Chinese patient.ConclusionThis study broadens the range of ALS2 variants and has practical implications for prenatal and postnatal screening of IAHSR. Symptom-based diagnosis of IAHSP is frequently difficult for medical practitioners. WES can be a beneficial resource to identify a particular disorder when the diagnosis cannot be determined from the symptoms alone.

Bioinformatics analysis and validation of the interaction between PML protein and TAB1 protein

To analyze the interaction between PML protein and TAB1 protein using bioinformatic approaches and experimentally verify the results. Using Rosetta software, a 3D model of TAB1 protein was constructed through a comparative modeling approach; the secondary structure of PML protein was retrieved in the PDB database and its crystal structure and 3D structure were resolved. Zdock 3.0.2 software was used to perform protein-protein docking of PML and TAB1, and the best conformation was extracted for molecular structure analysis of the docking model. The interaction between the two proteins was detected using immunoprecipitation in α-MMC-treated M1 inflammatory macrophages. When 6IMQ of PML was used as the docking site, PML protein formed 3 salt bridges, 6 hydrogen bonds and 6 hydrophobic interactions with TAB1 proteins; when 5YUF of PML was used as the docking site, PML protein formed 1 hydrogen bond, 3 electrostatic interactions and 9 hydrophobic interactions with TAB1 proteins, and both of the docking modes formed good molecular docking and interactions. In the M1 inflammatory macrophages treated with α-MMC for 4 h, positive protein bands of PML and TAB1 were detected in the cell lysates in PML-IP group. PML protein can interact strongly with TAB1 protein.

Molecular insights into STAT1a protein in rohu (Labeo rohita): unveiling expression profiles, SRC homology domain recognition, and protein-protein interactions triggered by poly I: C.

STAT1a is an essential signal transduction protein involved in the interferon pathway, playing a vital role in IFN-alpha/beta and gamma signaling. Limited information is available about the STAT protein in fish, particularly in Indian major carps (IMC). This study aimed to identify and characterize the STAT1a protein in Labeo rohita (LrSTAT1a). The full-length CDS of LrSTAT1a transcript was identified and sequenced. Phylogenetic analyses were performed based on the nucleotide sequences. The in-vivo immune stimulant poly I: C was used to treat various tissues, and the expression of LrSTAT1a was determined using quantitative real-time polymerase chain reaction (qRT-PCR). A 3D model of the STAT1a protein was generated using close structure homologs available in the database and checked using molecular dynamics (MD) simulations. The full-length CDS of Labeo rohita STAT1a (LrSTAT1a) transcript consisted of 3238 bp that encoded a polypeptide of 721 amino acids sequence was identified. Phylogenetic analyses were performed based on the nucleotide sequences. Based on our findings, other vertebrates share a high degree of conservation with STAT1a. Additionally, we report that the in vivo immune stimulant poly I: C treatment of various tissues resulted in the expression of LrSTAT1a as determined by quantitative real-time polymerase chain reaction (qRT-PCR). In the current investigation, treatment with poly I: C dramatically increased the expression of LrSTAT1a in nearly every organ and tissue, with the brain, muscle, kidney, and intestine showing the highest levels of expression compared to the control. We made a 3D model of the STAT1a protein by using close structure homologs that were already available in the database. The model was then checked using molecular dynamics (MD) simulations. Consistent with previous research, the MD study highlighted the significance of the STAT1a protein, which is responsible for Src homology 2 (SH2) recognition. An important H-bonding that successfully retains SH2 inside the STAT1a binding cavity was determined to be formed by the conserved residues SER107, GLN530, SER583, LYS584, MET103, and ALA106. This study provides molecular insights into the STAT1a protein in Rohu (Labeo rohita) and highlights the potential role of STAT1a in the innate immune response in fish. The high degree of conservation of STAT1a among other vertebrates suggests its crucial role in the immune response. The in-vivo immune stimulation results indicate that STAT1a is involved in the immune response in various tissues, with the brain, muscle, kidney, and intestine being the most responsive. The 3D model and MD study provide further evidence of the significance of STAT1a in the immune response, specifically in SH2 recognition. Further research is necessary to understand the specific mechanisms involved in the IFN pathway and the role of STAT1a in the immune response of IMC.

Structural studies on azadirachtin targeting the glucuronosyltransferase from lymphatic filariasis and its inhibition

Background and Aims: Brugia malayi, Brugia timori, and Wuchereria bancrofti are parasitic worms that cause lymphatic filariasis, also known as elephantiasis in some parts of the world. Because no effective vaccines and current treatments have adverse pharmacological properties, this condition has sparked much interest in the drug discovery community. This research aims to assess the efficacy of azadirachtin against a possible therapeutic target, intestine glucuronosyltransferase protein, in treating and preventing lymphatic filariasis. Methods: I-Tasser was used to create the protein, Uniprot was used to collect its FASTA sequences, and Galaxy Refine was used to refine the protein model. The protein 3D model was tested using PROCHECK and ERRAT. We used PubChem to get the three-dimensional structure of the azadirachtin ligand molecules. To study the toxicity profiles and pharmacokinetics, researchers employ SwissADME. Molecular docking studies were conducted using the AutoDock tool, and Pymol viewer was used to observe the interactions between the target protein and the ligand molecules. Results: The azadirachtin compound has a satisfactory range of pharmacokinetic parameters in terms of its absorption, distribution, metabolism, and excretion. When compared to the currently existing drug Diethylcarbamazine, molecular docking studies indicate that the azadirachtin molecule binds with the intestinal glucuronosyltransferase proteins of Brugia timori, Wuchereria bancrofti, and Brugia malayi strongly. Conclusions: In sum, our understanding of the mechanism by which azadirachtin, a traditional component derived from plants, inhibits intestinal glucuronosyltransferase protein is enhanced, and this could pave the way for the development of new targeted anti-filarial agents.

Design of Inhibitors That Target the Menin–Mixed-Lineage Leukemia Interaction

The prognosis of mixed-lineage leukemia (MLL) has remained a significant health concern, especially for infants. The minimal treatments available for this aggressive type of leukemia has been an ongoing problem. Chromosomal translocations of the KMT2A gene are known as MLL, which expresses MLL fusion proteins. A protein called menin is an important oncogenic cofactor for these MLL fusion proteins, thus providing a new avenue for treatments against this subset of acute leukemias. In this study, we report results using the structure-based drug design (SBDD) approach to discover potential novel MLL-mediated leukemia inhibitors from natural products against menin. The three-dimensional (3D) protein model was derived from Protein Databank (Protein ID: 4GQ4), and EasyModeller 4.0 and I-TASSER were used to fix missing residues during rebuilding. Out of the ten protein models generated (five from EasyModeller and I-TASSER each), one model was selected. The selected model demonstrated the most reasonable quality and had 75.5% of residues in the most favored regions, 18.3% of residues in additionally allowed regions, 3.3% of residues in generously allowed regions, and 2.9% of residues in disallowed regions. A ligand library containing 25,131 ligands from a Chinese database was virtually screened using AutoDock Vina, in addition to three known menin inhibitors. The top 10 compounds including ZINC000103526876, ZINC000095913861, ZINC000095912705, ZINC000085530497, ZINC000095912718, ZINC000070451048, ZINC000085530488, ZINC000095912706, ZINC000103580868, and ZINC000103584057 had binding energies of −11.0, −10.7, −10.6, −10.2, −10.2, −9.9, −9.9, −9.9, −9.9, and −9.9 kcal/mol, respectively. To confirm the stability of the menin–ligand complexes and the binding mechanisms, molecular dynamics simulations including molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) computations were performed. The amino acid residues that were found to be potentially crucial in ligand binding included Phe243, Met283, Cys246, Tyr281, Ala247, Ser160, Asn287, Asp185, Ser183, Tyr328, Asn249, His186, Leu182, Ile248, and Pro250. MI-2-2 and PubChem CIDs 71777742 and 36294 were shown to possess anti-menin properties; thus, this justifies a need to experimentally determine the activity of the identified compounds. The compounds identified herein were found to have good pharmacological profiles and had negligible toxicity. Additionally, these compounds were predicted as antileukemic, antineoplastic, chemopreventive, and apoptotic agents. The 10 natural compounds can be further explored as potential novel agents for the effective treatment of MLL-mediated leukemia.

Unravelling the impact of fat content on the microbial dynamics and spatial distribution of foodborne bacteria in tri-phasic viscoelastic 3D models

The aim of the current study is to develop and characterise novel complex multi-phase in vitro 3D models, for advanced microbiological studies. More specifically, we enriched our previously developed bi-phasic polysaccharide (Xanthan Gum)/protein (Whey Protein) 3D model with a fat phase (Sunflower Oil) at various concentrations, i.e., 10%, 20%, 40% and 60% (v/v), for better mimicry of the structural and biochemical composition of real food products. Rheological, textural, and physicochemical analysis as well as advanced microscopy imaging (including spatial mapping of the fat droplet distribution) of the new tri-phasic 3D models revealed their similarity to industrial food products (especially cheese products). Furthermore, microbial growth experiments of foodborne bacteria, i.e., Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Lactococcus lactis on the surface of the 3D models revealed very interesting results, regarding the growth dynamics and distribution of cells at colony level. More specifically, the size of the colonies formed on the surface of the 3D models, increased substantially for increasing fat concentrations, especially in mid- and late-exponential growth phases. Furthermore, colonies formed in proximity to fat were substantially larger as compared to the ones that were located far from the fat phase of the models. In terms of growth location, the majority of colonies were located on the protein/polysaccharide phase of the 3D models. All those differences at microscopic level, that can directly affect the bacterial response to decontamination treatments, were not captured by the macroscopic kinetics (growth dynamics), which were unaffected from changes in fat concentration. Our findings demonstrate the importance of developing structurally and biochemically complex 3D in vitro models (for closer proximity to industrial products), as well as the necessity of conducting multi-level microbial analyses, to better understand and predict the bacterial behaviour in relation to their biochemical and structural environment. Such studies in advanced 3D environments can assist a better/more accurate design of industrial antimicrobial processes, ultimately, improving food safety.

Structure-Based In Silico Screening of Marine Phlorotannins for Potential Walrus Calicivirus Inhibitor.

A new calicivirus isolated from a walrus was reported in 2004. Since unknown marine mammalian zoonotic viruses could pose great risks to human health, this study aimed to develop therapeutic countermeasures to quell any potential outbreak of a pandemic caused by this virus. We first generated a 3D model of the walrus calicivirus capsid protein and identified compounds from marine natural products, especially phlorotannins, as potential walrus calicivirus inhibitors. A 3D model of the target protein was generated using homology modeling based on two publicly available template sequences. The sequence of the capsid protein exhibited 31.3% identity and 42.7% similarity with the reference templates. The accuracy and reliability of the predicted residues were validated via Ramachandran plotting. Molecular docking simulations were performed between the capsid protein 3D model and 17 phlorotannins. Among them, five phlorotannins demonstrated markedly stable docking profiles; in particular, 2,7-phloroglucinol-6,6-bieckol showed favorable structural integrity and stability during molecular dynamics simulations. The results indicate that the phlorotannins are promising walrus calicivirus inhibitors. Overall, the study findings showcase the rapid turnaround of in silico-based drug discovery approaches, providing useful insights for developing potential therapies against novel pathogenic viruses, especially when the 3D structures of the viruses remain experimentally unknown.

Predicting the pathogenicity of missense variants based on protein instability to support diagnosis of patients with novel variants of ARSL

Rare diseases are estimated to affect 3.5%–5.9% of the population worldwide and are difficult to diagnose. Genome analysis is useful for diagnosis. However, since some variants, especially missense variants, are also difficult to interpret, tools to accurately predict the effect of missense variants are very important and needed. Here we developed a method, “VarMeter”, to predict whether a missense variant is damaging based on Gibbs free energy and solvent-accessible surface area calculated from the AlphaFold 3D protein model. We applied this method to the whole-exome sequencing data of 900 individuals with rare or undiagnosed disease in our in-house database, and identified four who were hemizygous for missense variants of arylsulfatase L (ARSL; known as the genetic cause of chondrodysplasia punctata 1, CPDX1). Two individuals had a novel Ser89 to Asn (Ser89Asn) or Arg469 to Trp (Arg469Trp) substitution, respectively predicted as “damaging” or “benign”; the other two had an Arg111 to His (Arg111His) or Gly117 to Arg (Gly117Arg) substitution, respectively predicted as “damaging” or “possibly damaging” and previously reported in patients showing clinical manifestations of CDPX1. Expression and analysis of the missense variant proteins showed that the predicted pathogenic variants (Ser89Asn, Arg111His, and Gly117Arg) had complete loss of sulfatase activity and reduced protease resistance due to destabilization of protein structure, while the predicted benign variant (Arg469Trp) had activity and protease resistance comparable to those of wild-type ARSL. The individual with the novel pathogenic Ser89Asn variant exhibited characteristics of CDPX1, while the individual with the benign Arg469Trp variant exhibited no such characteristics. These findings demonstrate that VarMeter may be used to predict the deleteriousness of variants found in genome sequencing data and thereby support disease diagnosis.

Molecular insight into endosulfan degradation by Ese protein from Arthrobacter: Evidence-based structural bioinformatics and quantum mechanical calculations.

Endosulfan is an organochlorine insecticide widely used for agricultural pest control. Many nations worldwide have restricted or completely banned it due to its extreme toxicity to fish and aquatic invertebrates. Arthrobacter sp. strain KW has the ability to degrade α, β endosulfan and its intermediate metabolite endosulfate; this degradation is associated with Ese protein, a two-component flavin-dependent monooxygenase (TC-FDM). Employing in silico tools, we obtained the 3D model of Ese protein, and our results suggest that it belongs to the Luciferase Like Monooxygenase family (LLM). Docking studies showed that the residues V59, V315, D316, and T335 interact with α-endosulfan. The residues: V59, T60, V315, D316, and T335 are implicated in the interacting site with β-endosulfan, and the residues: H17, V315, D316, T335, N364, and Q363 participate in the interaction with endosulfate. Topological analysis of the electron density by means of the Quantum Theory of Atoms in Molecules (QTAIM) and the Non-Covalent Interaction (NCI) index reveals that the Ese-ligands complexes are formed mainly by dispersive forces, where Cl atoms have a predominant role. As Ese is a monooxygenase member, we predict the homodimer formation. However, enzymatic studies must be developed to investigate the Ese protein's enzymatic and catalytic activity.

Truncated DNM1 variant underlines developmental delay and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) signify a group of heterogeneous neurodevelopmental disorder associated with early-onset seizures accompanied by developmental delay, hypotonia, mild to severe intellectual disability, and developmental regression. Variants in the DNM1 gene have been associated with autosomal dominant DEE type 31A and autosomal recessive DEE type 31B. In the current study, a consanguineous Pakistani family consisting of a proband (IV-2) was clinically evaluated and genetically analyzed manifesting in severe neurodevelopmental phenotypes. WES followed by Sanger sequencing was performed to identify the disease-causing variant. Furthermore, 3D protein modeling and dynamic simulation of wild-type and mutant proteins along with reverse transcriptase (RT)-based mRNA expression were checked using standard methods. Data analysis of WES revealed a novel homozygous non-sense variant (c.1402G>T; p. Glu468*) in exon 11 of the DNM1 gene that was predicted as pathogenic class I. Variants in the DNM1 gene have been associated with DEE types 31A and B. Different bioinformatics prediction tools and American College of Medical Genetics guidelines were used to verify the identified variant. Sanger sequencing was used to validate the disease-causing variant. Our approach validated the pathogenesis of the variant as a cause of heterogeneous neurodevelopmental disorders. In addition, 3D protein modeling showed that the mutant protein would lose most of the amino acids and might not perform the proper function if the surveillance non-sense-mediated decay mechanism was skipped. Molecular dynamics analysis showed varied trajectories of wild-type and mutant DNM1 proteins in terms of root mean square deviation, root mean square fluctuation and radius of gyration. Similarly, RT-qPCR revealed a substantial reduction of the DNM1 gene in the index patient. Our finding further confirms the association of homozygous, loss-of-function variants in DNM1 associated with DEE type 31B. The study expands the genotypic and phenotypic spectrum of pathogenic DNM1 variants related to DNM1-associated pathogenesis.