Integrated subtractive genomics and structure-based approach to unravel the therapeutic drug target of Leishmania species.

Leishmaniasis is a complex vector-borne disease caused by intracellular protozoan parasites of the Leishmania genus. It presents a significant public health challenge in tropical and subtropical regions globally. As resistance to treatment increases, managing and controlling Leishmaniasis becomes more challenging, necessitating innovative approaches. To address this challenge, our study utilized subtractive genomics and structure-based approaches to identify common drug targets and combat antimicrobial resistance (AMR) across five Leishmania species strains. The subtractive genomics approach unraveled Glutamate Dehydrogenase (GDH) as a promising drug target for treating Leishmania infections. The investigation considered established methodologies observed in analogous studies, orthologous group, and druggability tests. Multiple sequence alignment revealed conserved sequences in GDH, while phylogenetic tree analysis provided insights into the evolutionary origin and close relationships of GDH across Leishmania species. Conserved sequences in GDH along with its function in pathogenicity provided insights into the close relationships of GDH across Leishmania species. Using a structure-based approach, our study showed the molecular interactions between GDH and three ligands-Bithionol, GW5074, and Hexachlorophene-through molecular docking and 100 ns molecular dynamics (MD) simulations. GW5074 exhibited a significant affinity for GDH, as indicated by stable RMSD values, a more compact conformation, and a higher number of hydrogen bonds than Bithionol. MMPBSA analysis confirmed the superior binding energy of the GW5074-GDH complex, emphasizing its potential as a potent ligand for drug development. This comprehensive analysis identified GW5074 as a promising candidate for inhibiting GDH activities in Leishmania species, contributing to the development of effective therapeutics against Leishmania infections.

Identification of Peptidoglycan Glycosyltransferase FtsI as a Potential Drug Target against Salmonella Enteritidis and Salmonella Typhimurium Serovars Through Subtractive Genomics, Molecular Docking and Molecular Dynamics Simulation Approaches.

INTRODUCTION: Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium are among the main causative agents of nontyphoidal Salmonella infections, imposing a significant global health burden. The emergence of antibiotic resistance in these pathogens underscores the need for innovative therapeutic strategies. OBJECTIVE: To identify proteins as potential drug targets against Salmonella Enteritidis and Salmonella Typhimurium serovars using In silico approaches. METHODS: In this study, a subtractive genomics approach was employed to identify potential drug targets. The whole proteome of Salmonella Enteritidis PT4 and Salmonella Typhimurium (D23580), containing 393 and 478 proteins, respectively, was analyzed through subtractive genomics to identify human homologous proteins of the pathogen and also the proteins linked to shared metabolic pathways of pathogen and its host. RESULTS: Subsequent analysis revealed 19 common essential proteins shared by both strains. To ensure hostspecificity, we identified 10 non-homologous proteins absent in humans. Among these proteins, peptidoglycan glycosyltransferase FtsI was pivotal, participating in pathogen-specific pathways and making it a promising drug target. Molecular docking highlighted two potential compounds, Balsamenonon A and 3,3',4',7-Tetrahydroxyflavylium, with strong binding affinities with FtsI. A 100 ns molecular dynamics simulation having 10,000 frames substantiated the strong binding affinity and demonstrated the enduring stability of the predicted compounds at the docked site. CONCLUSION: The findings in this study provide the foundation for drug development strategies against Salmonella infections, which can contribute to the prospective development of natural and cost-effective drugs targeting Salmonella Enteritidis and Salmonella Typhimurium.

Rapid Tissue Perfusion Using Sacrificial Percolation of Anisotropic Networks.

Tissue engineering has long sought to rapidly generate perfusable vascularized tissues with vessel sizes spanning those seen in humans. Current techniques such as biological 3D printing (top-down) and cellular self-assembly (bottom-up) are resource intensive and have not overcome the inherent tradeoff between vessel resolution and assembly time, limiting their utility and scalability for engineering tissues. We present a flexible and scalable technique termed SPAN - Sacrificial Percolation of Anisotropic Networks, where a network of perfusable channels is created throughout a tissue in minutes, irrespective of its size. Conduits with length scales spanning arterioles to capillaries are generated using pipettable alginate fibers that interconnect above a percolation density threshold and are then degraded within constructs of arbitrary size and shape. SPAN is readily used within common tissue engineering processes, can be used to generate endothelial cell-lined vasculature in a multi-cell type construct, and paves the way for rapid assembly of perfusable tissues.

Genome-level therapeutic targets identification and chimeric Vaccine designing against the Blastomyces dermatitidis.

Blastomyces dermatitidis is a thermally dimorphic fungus that can cause serious and sometimes fatal infections, including blastomycosis. After spore inhalation, a pulmonary infection develops, which can be asymptomatic and have lethal effects, such as acute respiratory distress syndrome. Its most common extra-pulmonary sites are the central nervous system, bones, skin, and genito-urinary systems. Currently, no vaccine has been approved by the FDA to prevent this infection. In the study, a peptide-based vaccine was developed against blastomycosis by using subtractive proteomics and reverse vaccinology approaches. It focuses on mining the whole genome of B. dermatitidis, identifying potential therapeutic targets, and pinpointing potential epitopes for both B- and T-cells that are immunogenic, non-allergenic, non-toxic, and highly antigenic. Multi-epitope constructs were generated by incorporating appropriate linker sequences. A linker (EAAAK) was also added to incorporate an adjuvant sequence to increase immunological potential. The addition of adjuvants and linkers ultimately resulted in the formation of a vaccine construct in which the number of amino acids was 243 and the molecular weight was 26.18 kDa. The designed antigenic and non-allergenic vaccine constructs showed suitable physicochemical properties. The vaccine's structures were predicted, and further analysis verified their interactions with the human TLR-4 receptor through protein-protein docking. Additionally, MD simulation showed a potent interaction between prioritized vaccine-receptor complexes. Immune simulation predicted that the final vaccine injections resulted in significant immune responses for the T- and B-cell immune responses. Moreover, in silico cloning ensured a high expression possibility of the lead vaccine in the E. coli (K12) vector. This study offers an initiative for the development of effective vaccines against B. dermatitidis; however, it is necessary to validate the designed vaccine's immunogenicity experimentally.

Evaluation of the effect of thermocycling on the trueness and precision of digitally fabricated complete denture bases.

BACKGROUND: While many denture base materials are currently available on the market, little data exists regarding their dimensional stability after exposure to the oral environment. This study aimed to evaluate the effect of thermocycling on the trueness and precision of milled, 3-dimensional (3D)-printed, and conventional digitally fabricated complete denture bases (CDBs). METHODS: A completely edentulous maxillary stone model was scanned to generate a standard tessellation language (STL) file; this was imported into metal-milling-machine software (Redon Hybrid CAD-CAM metal milling machine, Redon, Turkey) to produce a metal model for fabricating 30 CDBs. These were divided into three groups (n = 10 in each) according to the construction technique: group 1, CAD-CAM milled CDBs; group 2, 3D-printed CDBs; and group 3, conventional compression molded CDBs. All CDBs were scanned after fabrication and evaluated before and after thermocycling using superimposition. The data were analyzed using a one-way ANOVA, Tukey's post hoc test, and a paired t-test. RESULTS: The level of trueness between the CAD-CAM milled, 3D-printed, and compression molded CDBs showed significant differences before and after thermocycling (P < 0.05). Group 1 showed the highest degree of trueness before and after thermocycling, group 3 exhibited a higher degree of trueness than group 2 before thermocycling, and group 2 had a higher degree of trueness than group 3 after thermocycling. There was a significant difference in the precision for each CDB type before and after thermocycling (P < 0.05). CONCLUSION: The trueness of the CAD-CAM milling system in complete denture (CD) fabrication is superior to that of the 3D printing and conventional compression molding systems before and after thermocycling. Thermocycling had a significant effect on the precision of all CDB types. The compression molding system in CD construction is the most negatively affected via thermocycling with regard to the measures of trueness and precision. CLINICAL TRIAL NUMBER: Not applicable, no human participants were involved.

To Reconstruct or Discard: A Comparison of Additive and Subtractive Charge Sharing Correction Algorithms at High and Low X-ray Fluxes.

Effective X-ray photon-counting spectral imaging (x-CSI) detector design involves the optimisation of a wide range of parameters both regarding the sensor (e.g., material, thickness and pixel pitch) and electronics (e.g., signal-processing chain and count-triggering scheme). Our previous publications have looked at the role of pixel pitch, sensor thickness and a range of additive charge sharing correction algorithms (CSCAs), and in this work, we compare additive and subtractive CSCAs to identify the advantages and disadvantages. These CSCAs differ in their approach to dealing with charge sharing: additive approaches attempt to reconstruct the original event, whilst subtractive approaches discard the shared events. Each approach was simulated on data from a wide range of x-CSI detector designs (pixel pitches 100-600 µm, sensor thickness 1.5 mm) and X-ray fluxes (106-109 photons mm-2 s-1), and their performance was characterised in terms of absolute detection efficiency (ADE), absolute photopeak efficiency (APE), relative coincidence counts (RCC) and binned spectral efficiency (BSE). Differences between the two approaches were explained mechanistically in terms of the CSCA's effect on both charge sharing and pule pileup. At low X-ray fluxes, the two approaches perform similarly, but at higher fluxes, they differ in complex ways. Generally, additive CSCAs perform better on absolute metrics (ADE and APE), and subtractive CSCAs perform better on relative metrics (RCC and BSE). Which approach to use will, thus, depend on the expected operating flux and whether dose efficiency or spectral efficiency is more important for the application in mind.

From Proteome to Potential Drugs: Integration of Subtractive Proteomics and Ensemble Docking for Drug Repurposing against Pseudomonas aeruginosa RND Superfamily Proteins.

Pseudomonas aeruginosa (P. aeruginosa) poses a significant threat as a nosocomial pathogen due to its robust resistance mechanisms and virulence factors. This study integrates subtractive proteomics and ensemble docking to identify and characterize essential proteins in P. aeruginosa, aiming to discover therapeutic targets and repurpose commercial existing drugs. Using subtractive proteomics, we refined the dataset to discard redundant proteins and minimize potential cross-interactions with human proteins and the microbiome proteins. We identified 12 key proteins, including a histidine kinase and members of the RND efflux pump family, known for their roles in antibiotic resistance, virulence, and antigenicity. Predictive modeling of the three-dimensional structures of these RND proteins and subsequent molecular ensemble-docking simulations led to the identification of MK-3207, R-428, and Suramin as promising inhibitor candidates. These compounds demonstrated high binding affinities and effective inhibition across multiple metrics. Further refinement using non-covalent interaction index methods provided deeper insights into the electronic effects in protein-ligand interactions, with Suramin exhibiting superior binding energies, suggesting its broad-spectrum inhibitory potential. Our findings confirm the critical role of RND efflux pumps in antibiotic resistance and suggest that MK-3207, R-428, and Suramin could be effectively repurposed to target these proteins. This approach highlights the potential of drug repurposing as a viable strategy to combat P. aeruginosa infections.

Nanoindentation creep: The impact of water and artificial saliva storage on milled and 3D-printed resin composites.

PURPOSE: This study evaluated the effects of artificial saliva and distilled water on the nanoindentation creep of different 3D-printed and milled CAD-CAM resin composites. MATERIAL AND METHODS: Disk-shaped specimens were subtractively fabricated from polymer-infiltrated ceramic network (EN) and reinforced resin composite (B) and additively from resin composite (C) and hybrid resin composite (VS) using digital light processing (DLP). Specimens from each material were divided into two groups according to their storage conditions (artificial saliva or distilled water for 3 months). Creep was analyzed by nanoindentation testing. Statistical analysis was done using two-way ANOVA, one-way ANOVA, Bonferroni post hoc tests, and independent t-test (α = 0.05). RESULTS: The main effects of material and storage conditions, and their interaction were statistically significant on nanoindentation (p < 0.001). Storage condition had the greatest influence (partial eta squared ηP 2 = 0.370), followed by the material (ηP 2 = 0.359), and the interaction (ηP 2 = 0.329). The nanoindentation creep depths after artificial saliva storage ranged from 0.34 to 0.51 µm and from 0.50 to 0.87 µm after distilled water storage. One of the additively manufactured groups had higher nanoindentation creep depths in both storage conditions. CONCLUSIONS: All specimens showed comparable performance after artificial saliva storage, but increased nanoindentation creep after distilled water storage for 3 months. The subtractive CAD-CAM blocks showed superior dimensional stability in terms of nanoindentation creep depths in both storage conditions. Additively manufactured composite resins had lower dimensional stability than one of the subtractively manufactured composites, which was demonstrated as having higher creep deformation and maximum recovery. However, after artificial saliva storage, one of the additively manufactured resins had dimensional stability similar to that of subtractively manufactured.

Structural proteomics guided annotation of vaccine targets and designing of multi-epitopes vaccine to instigate adaptive immune response against Francisella tularensis.

Francisella tularensis can cause severe disease in humans via the respiratory or cutaneous routes and a case fatality ratio of up to 10 % is reported due to lack of proper antibiotic treatment, while F. novicida causes disease in severely immunocompromised individuals. Efforts are needed to develop effective vaccine candidates against Francisella species. Thus, in this study, a systematic computational work frame was used to deeply investigate the whole proteome of Francisella novicida containing 1728 proteins to develop vaccine against F. tularensis and related species. Whole-proteome analysis revealed that four proteins including (A0Q492) (A0Q7Y4), (A0Q4N4), and (A0Q5D9) are the suitable vaccine targets after the removal of homologous, paralogous and prediction of subcellular localization. These proteins were used to predict the T cell, B cell, and HTL epitopes which were joined together through suitable linkers to construct a multi-epitopes vaccine (MEVC). The MEVC was found to be highly immunogenic and non-allergenic while the physiochemical properties revealed the feasible expression and purification. Moreover, the molecular interaction of MEVC with TLR2, molecular simulation, and binding free energy analyses further validated the immune potential of the construct. According to Jcat analysis, the refined sequence demonstrates GC contents of 41.48 % and a CAI value of 1. The in-silico cloning and optimization process ensured compatibility with host codon usage, thereby facilitating efficient expression. Computational immune simulation studies underscored the capacity of MEVC to induce both primary and secondary immune responses. The conservation analysis further revealed that the selected epitopes exhibit 100 % conservation across different species and thus provides wider protection against Francisella.

Conventionally and digitally fabricated removable complete dentures: manufacturing accuracy, fracture resistance and repairability.

OBJECTIVES: Conventionally and digitally manufactured removable complete dentures with different dentition forms were examined for manufacturing accuracy (trueness, precision), fracture forces under torsional loading and subsequent repairability. METHODS: A total of 90 mandibular prostheses were manufactured. Ten were made using the injection molding technique and finished with prefabricated teeth. 40 bases each, were manufactured subtractively and additively. Digitally the prosthesis' dental arch was divided either into two quadrants or three sextants, or kept as full arch. Afterwards, ten additive and subtractive bases were finished with prefabricated teeth and ten of each with milled quadrants, sextants and full arches. After manufacturing, all specimens were rescanned for accuracy comparisons using the Root Mean Square (RMS). Lastly, all specimens were tested to failure under torsional loading. RESULTS: Conventionally manufactured dentures showed the greatest deviation in accuracy. The type of base manufacturing did not determine the fracture resistance of the prostheses. The dentition form had a significant influence. While prefabricated teeth (86.01 ± 19.76 N) and quadrants (77.89 ± 9.58 N) showed a low fracture resistance, sextants (139.12 ± 21.41 N) and full arches (141.05 ± 17.14 N) achieved the highest fracture forces. Subtractive bases with prefabricated teeth or quadrants were assessed to be repairable, digital dentures with full arch were assessed as not repairable. SIGNIFICANCE: The presented testing set-up is suitable to determine the fracture behavior of dentures rather than of standards. With the possibility of digital design and individual manufacturing, dentures' mechanical stability can be significantly increased, especially with suitable dentition forms.

Natural products can be potential inhibitors of metalloproteinase II from Bacteroides fragilis to intervene colorectal cancer.

Bacteroides fragilis, a gram negative and obligate anaerobe bacterium, is a member of normal gut microbiota and facilitates many essential roles being performed in human body in normal circumstances specifically in Gastrointestinal or GI tract. Sometimes, due to genetics, epigenetics, and environmental factors, Bacteroides fragilis and their protein(s) start interacting with intestinal epithelium thus damaging the lining leading to colorectal cancers (CRC). To identify these protein(s), we incorporated a novel subtractive proteomics approach in the study. Metalloproteinase II (MPII), a Bacteroides fragilis toxin (bft), was investigated for its virulence and unique pathways to demonstrate its specificity and uniqueness in pathogenicity followed by molecular docking against a set of small drug-like natural molecules to discover potential inhibitors against the toxin. All these identified inhibitor-like molecules were analyzed for their ADMET calculations and detailed physiochemical properties to predict their druggability, GI absorption, blood brain barrier and skin permeation, and others. Resultantly, a total of ten compounds with the least binding energies were obtained and were subjected to protein-compound interaction analysis. Interaction analysis revealed the most common ligand-interacting residues in MPII are His 345, Glu 346, His 339, Gly 310, Tyr 341, Pro 340, Asp 187, Phe 309, Lys 307, Ile 185, Thr 308, and Pro 184. Therefore, top three compounds complexed with MPII having best binding energies were selected in order to analyze their trajectories. RMSD, RMSF, Rg and MMPBSA analysis revealed that all compounds showed good binding and keeping the complex stable and compact throughout the simulation time in addition to all properties and qualities of being a potential inhibitor against MPII.

Unveiling a Comprehensive Multi-epitope Subunit Vaccine Strategy Against Salmonella subsp. enterica: Bridging Core, Subtractive Proteomics, and Immunoinformatics.

Salmonella subsp. enterica (SE) presents a significant global health challenge in both developed and developing countries. Current SE vaccines have limitations, targeting specific strains and demonstrating moderate efficacy in adults, while also being unsuitable for young children and often unaffordable in regions with lower income levels where the disease is prevalent. To address these challenges, this study employed a computational approach integrating core proteomics, subtractive proteomics, and immunoinformatics to develop a universal SE vaccine and identify potential drug targets. Analysis of the core proteome of 185 SE strains revealed 1964 conserved proteins. Subtractive proteomics identified 9 proteins as potential vaccine candidates and 41 as novel drug targets. Using reverse vaccinology-based immunoinformatics, four multi-epitope-based subunit vaccine constructs (MESVCs) were designed, aiming to stimulate cytotoxic T lymphocyte, helper T lymphocyte, and linear B lymphocyte responses. These constructs underwent comprehensive evaluations for antigenicity, immunogenicity, toxicity, hydropathicity, and physicochemical properties. Predictive modeling, refinement, and validation were conducted to determine the secondary and tertiary structures of the SE-MESVCs, followed by docking studies with MHC-I, MHC-II, and TLR4 receptors. Molecular docking assessments showed favorable binding with all three receptors, with SE-MESVC-4 exhibiting the most promising binding energy. Molecular dynamics simulations confirmed the binding affinity and stability of SE-MESVC-4 with the TLR4/MD2 complex. Additionally, codon optimization and in silico cloning verified the efficient translation and successful expression of SE-MESVC-4 in Escherichia coli (E. coli) str. K12. Subsequent in silico immune simulation evaluated the efficacy of SE-MESVC-4 in triggering an effective immune response. These results suggest that SE-MESVC-4 may induce both humoral and cellular immune responses, making it a potential candidate for an effective SE vaccine. However, further experimental investigations are necessary to validate the immunogenicity and efficacy of SE-MESVC-4, bringing us closer to effectively combating SE infections.

Trueness and fit of complete-arch implant-supported frameworks in new-generation additively and subtractively manufactured polymers: An in-vitro study.

BACKGROUND: There is limited knowledge on the fabrication trueness and fit of additively or subtractively manufactured complete-arch implant-supported frameworks in recently introduced polymers. PURPOSE: To evaluate the trueness and marginal fit of additively or subtractively manufactured polymer-based complete-arch implant-supported frameworks, comparing with those of strength gradient zirconia frameworks. MATERIALS AND METHODS: A typodont model with 4 implants (left first molar (abutment 1), left canine (abutment 2), right canine (abutment 3), and right first molar (abutment 4)) was digitized (ATOS Core 80 5MP) and an implant-supported complete-arch framework was designed. This design file was used to fabricate frameworks from 5 different materials: strength gradient zirconia (SM-ZR), high impact polymer composite (SM-CR), nanographene-reinforced PMMA (SM-GR), PMMA (SM-PM), and additively manufactured temporary resin (AM) (n = 10). These frameworks were digitized and each scan file was virtually segmented into 4 regions (abutments, occlusal, overall without occlusal, and overall). The surface deviations at these regions, and linear and interimplant distance deviations were evaluated (Geomagic Control X). Marginal gaps were evaluated according to triple-scan protocol after seating frameworks on the model with the 1-screw test. Data were statistically analyzed (α = 0.05). RESULTS: Surface deviations of all regions differed among tested materials (p ≤ 0.001). AM frameworks mostly had surface deviations that were similar to or lower than those of other materials (p ≤ 0.031), except for the occlusal surface, where it mostly had higher deviations (p ≤ 0.013). Abutment 4 of SM-CR had higher linear deviations than abutment 2 (p = 0.025), and material type did not affect the linear deviations within abutments (p ≥ 0.171). Interimplant distance deviations differed within and among materials (p ≤ 0.017), except for those between abutments 1 and 2 among materials (p = 0.387). Marginal gaps of subtractively manufactured materials differed among abutments, while those of abutments 3 and 4 differed among materials (p ≤ 0.003). AM frameworks mostly had lower marginal gaps at abutments 3 and 4 (p ≤ 0.048). CONCLUSIONS: Although there was no clear trend among tested materials for measured deviations, marginal gaps of additively manufactured resin were mostly lower than those of subtractively manufactured materials and did not differ among abutment sites. Nevertheless, the differences in measured deviations among materials were small and marginal gaps were within the previously reported acceptability thresholds.

Reverse Vaccinology Approach to Identify Novel and Immunogenic Targets against Streptococcus gordonii.

Streptococcus gordonii is a gram-positive, mutualistic bacterium found in the human body. It is found in the oral cavity, upper respiratory tract, and intestines, and presents a serious clinical problem because it can lead to opportunistic infections in individuals with weakened immune systems. Streptococci are the most prevalent inhabitants of oral microbial communities, and are typical oral commensals found in the human oral cavity. These streptococci, along with many other oral microbes, produce multispecies biofilms that can attach to salivary pellicle components and other oral bacteria via adhesin proteins expressed on the cell surface. Antibiotics are effective against this bacterium, but resistance against antibodies is increasing. Therefore, a more effective treatment is needed. Vaccines offer a promising method for preventing this issue. This study generated a multi-epitope vaccine against Streptococcus gordonii by targeting the completely sequenced proteomes of five strains. The vaccine targets are identified using a pangenome and subtractive proteomic approach. In the present study, 13 complete strains out of 91 strains of S. gordonii are selected. The pangenomics results revealed that out of 2835 pan genes, 1225 are core genes. Out of these 1225 core genes, 643 identified as non-homologous proteins by subtractive proteomics. A total of 20 essential proteins are predicted from non-homologous proteins. Among these 20 essential proteins, only five are identified as surface proteins. The vaccine construct is designed based on selected B- and T-cell epitopes of the antigenic proteins with the help of linkers and adjuvants. The designed vaccine is docked against TLR2. The expression of the protein is determined using in silico gene cloning. Findings concluded that Vaccine I with adjuvant shows higher interactions with TLR2, suggesting that the vaccine has the ability to induce a humoral and cell-mediated response to treat and prevent infection; this makes it promising as a vaccine against infectious diseases caused by S. gordonii. Furthermore, validation of the vaccine construct is required by in vitro and in vivo trials to check its actual potency and safety for use to prevent infectious diseases caused by S. gordonii.

Immunoinformatics and Reverse Vaccinology Approach for the Identification of Potential Vaccine Candidates against Vandammella animalimors.

Vandammella animalimorsus is a Gram-negative and non-motile bacterium typically transmitted to humans through direct contact with the saliva of infected animals, primarily through biting, scratches, or licks on fractured skin. The absence of a confirmed post-exposure treatment of V. animalimorsus bacterium highlights the imperative for developing an effective vaccine. We intended to determine potential vaccine candidates and paradigm a chimeric vaccine against V. animalimorsus by accessible public data analysis of the strain by utilizing reverse vaccinology. By subtractive genomics, five outer membranes were prioritized as potential vaccine candidates out of 2590 proteins. Based on the instability index and transmembrane helices, a multidrug transporter protein with locus ID A0A2A2AHJ4 was designated as a potential candidate for vaccine construct. Sixteen immunodominant epitopes were retrieved by utilizing the Immune Epitope Database. The epitope encodes the strong binding affinity, nonallergenic properties, non-toxicity, high antigenicity scores, and high solubility revealing the more appropriate vaccine construct. By utilizing appropriate linkers and adjuvants alongside a suitable adjuvant molecule, the epitopes were integrated into a chimeric vaccine to enhance immunogenicity, successfully eliciting both adaptive and innate immune responses. Moreover, the promising physicochemical features, the binding confirmation of the vaccine to the major innate immune receptor TLR-4, and molecular dynamics simulations of the designed vaccine have revealed the promising potential of the selected candidate. The integration of computational methods and omics data has demonstrated significant advantages in discovering novel vaccine targets and mitigating vaccine failure rates during clinical trials in recent years.

Sub-genomic RNAi-assisted strain evolution of filamentous fungi for enhanced protein production.

Genetic engineering at the genomic scale provides a rapid means to evolve microbes for desirable traits. However, in many filamentous fungi, such trials are daunted by low transformation efficiency. Differentially expressed genes under certain conditions may contain important regulatory factors. Accordingly, although manipulating these subsets of genes only can largely reduce the time and labor, engineering at such a sub-genomic level may also be able to improve the microbial performance. Herein, first using the industrially important cellulase-producing filamentous fungus Trichoderma reesei as a model organism, we constructed suppression subtractive hybridization (SSH) libraries enriched with differentially expressed genes under cellulase induction (MM-Avicel) and cellulase repression conditions (MM-Glucose). The libraries, in combination with RNA interference, enabled sub-genomic engineering of T. reesei for enhanced cellulase production. The ability of T. reesei to produce endoglucanase was improved by 2.8~3.3-fold. In addition, novel regulatory genes (tre49304, tre120391, and tre123541) were identified to affect cellulase expression in T. reesei. Iterative manipulation using the same strategy further increased the yield of endoglucanase activity to 75.6 U/mL, which was seven times as high as that of the wild type (10.8 U/mL). Moreover, using Humicola insolens as an example, such a sub-genomic RNAi-assisted strain evolution proved to be also useful in other industrially important filamentous fungi. H. insolens is a filamentous fungus commonly used to produce catalase, albeit with similarly low transformation efficiency and scarce knowledge underlying the regulation of catalase expression. By combining SSH and RNAi, a strain of H. insolens producing 28,500 ± 288 U/mL of catalase was obtained, which was 1.9 times as high as that of the parent strain.IMPORTANCEGenetic engineering at the genomic scale provides an unparalleled advantage in microbial strain improvement, which has previously been limited only to the organisms with high transformation efficiency such as Saccharomyces cerevisiae and Escherichia coli. Herein, using the filamentous fungus Trichoderma reesei as a model organism, we demonstrated that the advantage of suppression subtractive hybridization (SSH) to enrich differentially expressed genes and the convenience of RNA interference to manipulate a multitude of genes could be combined to overcome the inadequate transformation efficiency. With this sub-genomic evolution strategy, T. reesei could be iteratively engineered for higher cellulase production. Intriguingly, Humicola insolens, a fungus with even little knowledge in gene expression regulation, was also improved for catalase production. The same strategy may also be expanded to engineering other microorganisms for enhanced production of proteins, organic acids, and secondary metabolites.

Hybrid FFF/CNC: An open source hardware & software system.

This paper presents a low-cost milling system composed of spindle mountable on a multi tool 3D printer equipped with maxwell kinematic coupling (E3D "ToolChanger" in this article) as well as two open-source software solutions for implementing a hybrid FFF/CNC manufacturing process. The first solution is the use of a traditional CAM software (FreeCad) for machining programming through the development of a dedicated post-processor. The second is an automatic layer-by-layer hybridization enabled by the software "SuperSlicer". This method requires no machining knowledge but only allows contouring operations. Results of experiments show that the spindle presented in this work is capable of successfully carrying out a hybrid process that significantly improves the surface roughness parameters, with an improvement factor of 10 for most parameters. An uniformization of surface roughness parameters was also observed in the construction direction and in the deposition/machining direction. The layer-by-layer hybridization yields the better results in terms of surface roughness. This is because the reduced depth of cut (equivalent to a printed layer) minimizes stress and temperature rise, resulting in highly favorable cutting conditions.

Identification of therapeutic drug target of Shigella Flexneri serotype X through subtractive genomic approach and in-silico screening based on drug repurposing.

Shigellosis, induced by Shigella flexneri, constitutes a significant health burden in developing nations, particularly impacting socioeconomically disadvantaged communities. Designated as the second most prevalent cause of diarrheal illness by the World Health Organization (WHO), it precipitates an estimated 212,000 fatalities annually. Within the spectrum of S. flexneri strains, serotype X is notably pervasive and resilient, yet its comprehensive characterization remains deficient. The present investigation endeavors to discern potential pharmacological targets and repurpose existing drug compounds against S. flexneri serotype X. Employing the framework of subtractive genomics, the study interrogates the reference genome of S. flexneri Serotype X (strain 2,002,017; UP000001884) to delineate its proteome into categories of non-homologous, non-paralogous, essential, virulent, and resistant constituents, thereby facilitating the identification of therapeutic targets. Subsequently, a screening of approximately 9000 compounds from the FDA library against the identified drug target aims to delineate efficacious agents for combating S. flexneri serotype X infections. The application of subtractive genomics methodology yields prognostic insights, unveiling non-paralogous proteins (n = 4122), non-homologues (n = 1803), essential (n = 1246), drug-like (n = 389), resistant (n = 167), alongside 42 virulent proteins within the reference proteome. This iterative process culminates in the identification of Serine O-acetyltransferase as a viable drug target. Subsequent virtual screening endeavors to unearth FDA-approved medicinal compounds capable of inhibiting Serine O-acetyltransferase. Noteworthy candidates such as DB12983, DB15085, DB16098, DB16185, and DB16262 emerge, exhibiting potential for mitigating S. flexneri Serotype X. Despite the auspicious findings, diligent scrutiny is imperative to ascertain the efficacy and safety profile of the proposed drug candidates vis-à-vis S. flexneri.

An integrated in silico approach for the identification of novel potential drug target and chimeric vaccine against Neisseria meningitides strain 331401 serogroup X by subtractive genomics and reverse vaccinology.

Neisseria meningitidis, commonly known as the meningococcus, leads to substantial illness and death among children and young adults globally, revealing as either epidemic or sporadic meningitis and/or septicemia. In this study, we have designed a novel peptide-based chimeric vaccine candidate against the N. meningitidis strain 331,401 serogroup X. Through rigorous analysis of subtractive genomics, two essential cytoplasmic proteins, namely UPI000012E8E0(UDP-3-O-acyl-GlcNAc deacetylase) and UPI0000ECF4A9(UDP-N-acetylglucosamine acyltransferase) emerged as potential drug targets. Additionally, using reverse vaccinology, the outer membrane protein UPI0001F4D537 (Membrane fusion protein MtrC) identified by subcellular localization and recognized for its known indispensable role in bacterial survival was identified as a novel chimeric vaccine target. Following a careful comparison of MHC-I, MHC-II, T-cell, and B-cell epitopes, three epitopes derived from UPI0001F4D537 were linked with three types of linkers-GGGS, EAAAK, and the essential PADRE-for vaccine construction. This resulted in eight distinct vaccine models (V1-V8). Among them V1 model was selected as the final vaccine construct. It exhibits exceptional immunogenicity, safety, and enhanced antigenicity, with 97.7 % of its residues in the Ramachandran plot's most favored region. Subsequently, the vaccine structure was docked with the TLR4/MD2 complex and six different HLA allele receptors using the HADDOCK server. The docking resulted in the lowest HADDOCK score of 39.3 ± 9.0 for TLR/MD2. Immune stimulation showed a strong immune response, including antibodies creation and the activation of B-cells, T Cytotoxic cells, T Helper cells, Natural Killer cells, and interleukins. Furthermore, the vaccine construct was successfully expressed in the Escherichia coli system by reverse transcription, optimization, and ligation in the pET-28a (+) vector for the expression study. The current study proposes V1 construct has the potential to elicit both cellular and humoral responses, crucial for the developing an epitope-based vaccine against N. meningitidis strain 331,401 serogroup X.

Design of a cryptococcus neoformans vaccine by subtractive proteomics combined with immunoinformatics.

The emergence of Cryptococcus neoformans has posed an undeniable burden to many regions worldwide, with its strains mainly entering the lungs through the respiratory tract and spreading throughout the body. Limitations of drug regimens, such as high costs and limited options, have directed our attention toward the promising field of vaccine development. In this study, the subtractive proteomics approach was employed to select target proteins from databases that can accurately cover serotypes A and D of the Cryptococcus neoformans. Further, two multi-epitope vaccines consisting of T and B cell epitopes were demonstrated that they have good structural stability and could bind with immune receptor to induce desired immune responses in silico. After further evaluation, these vaccines show the potential for large-scale production and applicability to the majority of the population of the world. In summary, these two vaccines have been theoretically proven to combat Cryptococcus neoformans infections, awaiting further experimental validation of their actual protective effects.