determining-ciliary-function-membrane-impermeability-pseudostratified

Determining Ciliary Function and Membrane Impermeability of the Pseudostratified Lung Airway Epithelium

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified ...

University of North Dakota

In recent decades, the role played by extracellular vesicles in physiological and pathological processes has attracted attention. Extracellular vesicles are released by different types of cells and carry molecules that could become biomarkers for the diagnosis of diseases. Extracellular vesicles are also moldable tools for the controlled release of bioactive substances in clinical and therapeutic applications. However, one of the significant challenges when studying these exciting and versatile vesicles is the purification process, which presents significant difficulties in terms of lack of purity, yield, and reproducibility, reflected in unreliable data. Therefore, our objective in the present study was to compare the proteomic profile of serum-derived EVs purified using ExoQuick™ (Systems Biosciences), Total Isolation Kit (Life Technologies), Ultracentrifugation, and Ultrafiltration. Each technique utilized for purification has shown different concentrations and populations of purified particles. The results showed marked differences in distribution, size, and protein content, demonstrating the need to develop reproducible and reliable protocols to isolate extracellular vesicles for their clinical application.

Comparison of Four Purification Methods on Serum Extracellular Vesicle Recovery, Size Distribution, and Proteomics.

Dengue virus infection (DVI) is a mosquito-borne disease that can lead to serious morbidity and mortality. Dengue fever (DF) is a major public health concern that affects approximately 3.9 billion people each year globally. However, there is no vaccine or drug available to deal with DVI. Dengue virus consists of four distinct serotypes (DENV1-4), each raising a different immunological response. In the present study, we designed a tetravalent subunit multi-epitope vaccine, targeting proteins including the structural protein envelope domain III (EDIII), precursor membrane proteins (prM), and a non-structural protein (NS1) from each serotype by employing an immunoinformatic approach. Only conserved sequences obtained through a multiple sequence alignment were used for epitope mapping to ensure efficacy against all serotypes. The epitopes were shortlisted based on an IC50 value <50, antigenicity, allergenicity, and a toxicity analysis. In the final vaccine construct, overall, 11 B-cell epitopes, 10 HTL epitopes, and 10 CTL epitopes from EDIII, prM, and NS1 proteins targeting all serotypes were selected and joined via KK, AAY, and GGGS linkers, respectively. We incorporated a 45-amino-acid-long B-defensins adjuvant in the final vaccine construct for a better immunogenic response. The vaccine construct has an antigenic score of 0.79 via VaxiJen and is non-toxic and non-allergenic. Our refined vaccine structure has a Ramachandran score of 96.4%. The vaccine has shown stable interaction with TLR3, which has been validated by 50 ns of molecular dynamics (MD) simulation. Our findings propose that a designed multi-epitope vaccine has substantial potential to elicit a strong immune response against all dengue serotypes without causing any adverse effects. Furthermore, the proposed vaccine can be experimentally validated as a probable vaccine, suggesting it may serve as an effective preventative measure against dengue virus infection.

Development of a tetravalent subunit vaccine against dengue virus through a vaccinomics approach.

Shigella dysenteriae, is a Gram-negative bacterium that emerged as the second most significant cause of bacillary dysentery. Antibiotic treatment is vital in lowering Shigella infection rates, yet the growing global resistance to broad-spectrum antibiotics poses a significant challenge. The persistent multidrug resistance of S. dysenteriae complicates its management and control. Hence, there is an urgent requirement to discover novel therapeutic targets and potent medications to prevent and treat this disease. Therefore, the integration of bioinformatics methods such as subtractive and comparative analysis provides a pathway to compute the pan-genome of S. dysenteriae. In our study, we analysed a dataset comprising 27 whole genomes. The S. dysenteriae strain SD197 was used as the reference for determining the core genome. Initially, our focus was directed towards the identification of the proteome of the core genome. Moreover, several filters were applied to the core genome, including assessments for non-host homology, protein essentiality, and virulence, in order to prioritize potential drug targets. Among these targets were Integration host factor subunit alpha and Tyrosine recombinase XerC. Furthermore, four drug-like compounds showing potential inhibitory effects against both target proteins were identified. Subsequently, molecular docking analysis was conducted involving these targets and the compounds. This initial study provides the list of novel targets against S. dysenteriae. Conclusively, future in vitro investigations could validate our in-silico findings and uncover potential therapeutic drugs for combating bacillary dysentery infection.

An integrated in-silico approach for drug target identification in human pathogen Shigella dysenteriae.

 has been recognized as the second most prevalent pathogen associated with diarrhea that contains blood, contributing to 12.9% of reported cases, and it is additionally responsible for approximately 200,000 deaths each year. Currently, there is no  licensed vaccine. Multidrug resistance in all  spp. is a growing concern. Current vaccines, such as O-polysaccharide (OPS) conjugates, are in clinical trials but are ineffective in children but protective in adults. Thus, innovative treatments and vaccines are needed to combat antibiotic resistance. In this study, we used immuno-informatics to design a new multiepitope vaccine and identified  strain SD197's membrane protein targets using  methods. The target protein was prioritized using membrane protein topology analysis to find membrane proteins. B and T-cell epitopes were predicted for vaccine formulation. The epitopes were shortlisted based on an IC50 value <50, antigenicity, allergenicity, and a toxicity analysis. In the final vaccine construct, a total of 8 B-cell epitopes, 12 MHC Class I epitopes, and 7 MHC Class II epitopes were identified for the Lipopolysaccharide export system permease protein LptF. Additionally, 17 MHC Class I epitopes and 14 MHC Class II epitopes were predicted for the Lipoprotein-releasing ABC transporter permease subunit LolE. These epitopes were selected and linked via KK, AAY, and GGGS linkers, respectively. To enhance the immunogenic response, RGD (arginine-glycine-aspartate) adjuvant was incorporated into the final vaccine construct. The refined vaccine structure exhibits a Ramachandran score of 91.5% and demonstrates stable interaction with TLR4. Normal Mode Analysis (NMA) reveals low eigenvalues (3.925996e-07), indicating steady and flexible molecular mobility of docked complexes. Codon optimization was carried out in an effective microbial expression system of the  K12 strain using the recombinant plasmid pET-28a (+). Finally, the entire  analysis suggests that the suggested vaccine may induce a significant immune response against , making it a promising option for additional experimental trials.

Designing a multi-epitope vaccine against  using immuno-informatics approach.

 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  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  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  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 .

Reverse Vaccinology Approach to Identify Novel and Immunogenic Targets against .

Yersinia enterocolitica, a foodborne pathogen, has emerged as a significant public health concern due to its increased prevalence and multidrug resistance. This study employed reverse vaccinology to identify novel vaccine candidates against Y. enterocolitica through comprehensive in silico analyses. The core genome's conserved protein translocase subunit SecY was selected as the target, and potential B-cell, MHC class I, and MHC class II epitopes were mapped. 3B-cell epitopes, 3 MHCI and 11 MHCII epitopes were acquired. A multi-epitope vaccine construct was designed by incorporating the identified epitopes, TLR4 Agonist was used as adjuvants to enhance the immunogenic response. EAAAK, CPGPG and AYY linkers were used to form a vaccine construct, followed by extensive computational evaluations. The vaccine exhibited desirable physicochemical properties, stable secondary and tertiary structures as evaluated by PDBSum and trRosetta. Moreover, favorable interactions with the human Toll-like receptor 4 (TLR4) was observed by ClusPro. Population coverage analysis estimated the vaccine's applicability across 99.74 % in diverse populations. In addition, molecular dynamics simulations and normal mode analysis confirmed the vaccine's structural stability and dynamics in a simulated biological environment. Furthermore, codon optimization and in silico cloning facilitated the evaluation of the vaccine's expression potential in E. coli and pET-28a was used a recombinant plasmid. This study provides a promising foundation for the development of an efficacious vaccine against Y. enterocolitica infections.

In silico vaccine design for Yersinia enterocolitica: A comprehensive approach to enhanced immunogenicity, efficacy and protection.

The development of genome technology has opened new possibilities for comparative primate genomics. Non-human primates share approximately 98% genome similarity and provides vital information into the genetic similarities and variances among species utilized as disease models. DNA study links unique genetic variations to common facial attributes such as nose and eyes. This is because of higher adaptation and improved cognitive skills over these sensual areas. Non-protein coding sequences represent approximately 85% of the human DNA under evolutionary restrictions, and the primary part of this is the cis regulatory regions. In this study, a total of 103 tissue specific human enhancers were finalized with help of VISTA Enhancer Browser that showed distinctive expression in the facial tissues and their orthologs were collected. A total of 43 out of 190 transcription factors from TRANSFAC were seen as binding in both Human and Non-Human primate enhancers. It was observed that factor binding sites of 7 of the 43 transcription factors were exclusively gained in the human eye and nose enhancers (Oct, Pax, Sox, MyoD, Foxd3, cRel and Gata). Furthermore, we performed molecular docking through PyMol; DNA & Protein (pdb) structures were modelled by SCFBio & SWISSMODEL respectively to observe interactions of the transcription factors, either by placing the contact surface of the protein exclusively to identify the DNA, to enable a representation to gain information about identification and genetic expression.

Genetic Insights into Facial Variation and Craniofacial Development: Unraveling the Interplay of Genes, Expression Patterns, and Evolutionary Significance.

Article	Year
Comparison of Four Purification Methods on Serum Extracellular Vesicle Recovery, Size Distribution, and Proteomics. Proteomes\| PubMed ID: 37606419	2023
Development of a tetravalent subunit vaccine against dengue virus through a vaccinomics approach. Frontiers in immunology\| PubMed ID: 38045699	2023
An integrated in-silico approach for drug target identification in human pathogen Shigella dysenteriae. PloS one\| PubMed ID: 38753867	2024
Designing a multi-epitope vaccine against using immuno-informatics approach. Frontiers in genetics\| PubMed ID: 38826807	2024
Reverse Vaccinology Approach to Identify Novel and Immunogenic Targets against . Biology\| PubMed ID: 39056703	2024
In silico vaccine design for Yersinia enterocolitica: A comprehensive approach to enhanced immunogenicity, efficacy and protection. International immunopharmacology\| PubMed ID: 39369465	2024
Genetic Insights into Facial Variation and Craniofacial Development: Unraveling the Interplay of Genes, Expression Patterns, and Evolutionary Significance. Molecular biotechnology\| PubMed ID: 39724323	2024

Muhammad Faheem

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