Potential distribution of Biscogniauxia mediterranea and Obolarina persica causal agents of oak charcoal disease in Iran's Zagros forests.

In Iran, native oak species are under threat from episodes of Charcoal Disease, a decline syndrome driven by abiotic stressors (e.g. drought, elevated temperature) and biotic components, Biscogniauxia mediterranea (De Not.) Kuntze and Obolarina persica (M. Mirabolfathy). The outbreak is still ongoing and the country's largest ever recorded. Still, the factors driving its' epidemiology in time and space are poorly known and such knowledge is urgently needed to develop strategies to counteract the adverse effects. In this study, we developed a generic framework based on experimental, machine-learning algorithms and spatial analyses for landscape-level prediction of oak charcoal disease outbreaks. Extensive field surveys were conducted during 2013-2015 in eight provinces (more than 50 unique counties) in the Zagros ecoregion. Pathogenic fungi were isolated and characterized through morphological and molecular approaches, and their pathogenicity was assessed under controlled water stress regimes in the greenhouse. Further, we evaluated a set of 29 bioclimatic, environmental, and host layers in modeling for disease incidence data using four well-known machine learning algorithms including the Generalized Linear Model, Gradient Boosting Model, Random Forest model (RF), and Multivariate Adaptive Regression Splines implemented in MaxEnt software. Model validation statistics [Area Under the Curve (AUC), True Skill Statistics (TSS)], and Kappa index were used to evaluate the accuracy of each model. Models with a TSS above 0.65 were used to prepare an ensemble model. The results showed that among the different climate variables, precipitation and temperature (Bio18, Bio7, Bio8, and bio9) in the case of O. persica and similarly, gsl (growing season length TREELIM, highlighting the warming climate and the endophytic/pathogenic nature of the fungus) and precipitation in case of B. mediterranea are the most important influencing variables in disease modeling, while near-surface wind speed (sfcwind) is the least important variant. The RF algorithm generates the most robust predictions (ROC of 0.95; TSS of 0.77 and 0.79 for MP and OP, respectively). Theoretical analysis shows that the ensemble model (ROC of 0.95 and 0.96; TSS = 0.79 and 0.81 for MP and OP, respectively), can efficiently be used in the prediction of the charcoal disease spatiotemporal distribution. The oak mortality varied ranging from 2 to 14%. Wood-boring beetles association with diseased trees was determined at 20%. Results showed that water deficiency is a crucial component of the oak decline phenomenon in Iran. The Northern Zagros forests (Ilam, Lorestan, and Kermanshah provinces) along with the southern Zagros forests (Fars and Kohgilouyeh va-Boyer Ahmad provinces) among others are the most endangered areas of potential future pandemics of charcoal disease. Our findings will significantly improve our understanding of the current situation of the disease to pave the way against pathogenic agents in Iran.

An updated review on oral protein-based antigen vaccines efficiency and delivery approaches: a special attention to infectious diseases.

Various infectious agents affect human health via the oral entrance. The majority of pathogens lack approved vaccines. Oral vaccination is a convenient, safe and cost-effective approach with the potential of provoking mucosal and systemic immunity and maintaining individual satisfaction. However, vaccines should overcome the intricate environment of the gastrointestinal tract (GIT). Oral protein-based antigen vaccines (OPAVs) are easier to administer than injectable vaccines and do not require trained healthcare professionals. Additionally, the risk of needle-related injuries, pain, and discomfort is eliminated. However, OPAVs stability at environmental and GIT conditions should be considered to enhance their stability and facilitate their transport and storage. These vaccines elicit the local immunity, protecting GIT, genital tract and respiratory epithelial surfaces, where numerous pathogens penetrate the body. OPAVs can also be manipulated (such as using specific incorporated ligand and receptors) to elicit targeted immune response. However, low bioavailability of OPAVs necessitates development of proper protein carriers and formulations to enhance their stability and efficacy. There are several strategies to improve their efficacy or protective effects, such as incorporation of adjuvants, enzyme inhibitors, mucoadhesive or penetrating devices and permeation enhancers. Hence, efficient delivery of OPAVs into GIT require proper delivery systems mainly including smart target systems, probiotics, muco-adhesive carriers, lipid- and plant-based delivery systems and nano- and microparticles.

Finding and engineering the newly found bacterial superoxide dismutase enzyme to increase its thermostability and decrease the immunogenicity: a computational and experimental research.

Superoxide dismutase (SOD) is one of the most important antioxidant enzymes that can reduce oxidative stress in the cell environment. Nowadays, bacterial sources of enzyme are commercially applicable in the cosmetics and pharmaceutical industries, but the allergenic effect of proteins from non-human sources has been mentioned as disadvantage of these kinds of enzymes. In this study, to find the suitable bacterial SOD candidate for decreasing immunogenicity, the sequences of five thermophilic bacteria were selected as reference species. Then, linear and conformational B-cell epitopes of the SOD were analyzed by different servers. The stability and immunogenicity of mutant positions were also evaluated. The mutant gene was inserted into the pET-23a expression vector and transformed into E. Coli BL21 (DE3) for expression of the recombinant enzyme. Afterward, the expression of the mutant enzyme was evaluated by SDS-PAGE analysis and the recombinant enzyme activity was assessed. Anoxybacillus gonensis was selected as a reasonable SOD source according to BLAST search, physicochemical properties analysis, and prediction of allergenic features. Regarding our results, five residues including E84, E142, K144, G147, and M148 were predicted as candidates for mutagenesis. Finally, the K144A was chosen as the final modification due to the increase in the stability of the enzyme and decreased immunogenicity of the enzyme as well. The enzyme activity was 240 U/ml at room temperature. Alternation in K144 to alanine caused increased stability of the enzyme. In silico studies confirmed non-antigenic protein after mutation.

In silico design and validation of a novel multi-epitope vaccine candidate against structural proteins of Chikungunya virus using comprehensive immunoinformatics analyses.

Chikungunya virus (CHIKV) is an emerging viral infectious agent with the potential of causing pandemic. There is neither a protective vaccine nor an approved drug against the virus. The aim of this study was design of a novel multi-epitope vaccine (MEV) candidate against the CHIKV structural proteins using comprehensive immunoinformatics and immune simulation analyses. In this study, using comprehensive immunoinformatics approaches, we developed a novel MEV candidate using the CHIKV structural proteins (E1, E2, 6 K, and E3). The polyprotein sequence was obtained from the UniProt Knowledgebase and saved in FASTA format. The helper and cytotoxic T lymphocytes (HTLs and CTLs respectively) and B cell epitopes were predicted. The toll-like receptor 4 (TLR4) agonist RS09 and PADRE epitope were employed as promising immunostimulatory adjuvant proteins. All vaccine components were fused using proper linkers. The MEV construct was checked in terms of antigenicity, allergenicity, immunogenicity, and physicochemical features. The docking of the MEV construct and the TLR4 and molecular dynamics (MD) simulation were also performed to assess the binding stability. The designed construct was non-allergen and was immunogen which efficiently stimulated immune responses using the proper synthetic adjuvant. The MEV candidate exhibited acceptable physicochemical features. Immune provocation included prediction of HTL, B cell, and CTL epitopes. The docking and MD simulation confirmed the stability of the docked TLR4-MEV complex. The high-level protein expression in the Escherichia coli (E. coli) host was observed through in silico cloning. The in vitro, in vivo, and clinical trial investigations are required to verify the findings of the current study.

Expression, purification and investigation of antibacterial activity of a novel hybrid peptide LL37/hBD-129 by applied comprehensive computational and experimental approaches.

Antibiotic-resistant pathogens have become a great universal health concern. Antimicrobial peptides (AMPs) are small amphipathic and cationic polypeptides with high therapeutic potential against various microorganisms containing drug-resistant strains. Two major groups of these peptides, which have antibacterial activity against Gram-positive and Gram-negative bacteria, antiviral activity, and even antifungal activity, are defensins and cathelicidins. Hybridization of various AMPs is an appropriate approach to achieving new fusion AMPs with high antibacterial activity but low cellular toxicity. In the current research, the amino-acid sequence of human cathelicidin LL-37 (2-31) and Human beta-defensin (hBD)-129 were combined, and the fusion protein was evaluated by bioinformatics tool. The designed AMP gene sequence was commercially synthesized and cloned in the pET-28a expression vector. The LL-37/hBD-129 fusion protein was expressed in E.coli BL21-gold (DE3). The expression of the recombinant protein was evaluated using the SDS-PAGE method. The LL37/hBD-129 was successfully expressed as a recombinant hybrid AMP in E.coli BL21-gold (DE3) strain. Purification of the expressed AMP was performed by Ni-NTA column affinity chromatography, and the purified AMP was validated using the Western blot technic. Finally, the antimicrobial activity of the fusion AMP against Staphylococcus aureus and Escherichia coli bacteria was assessed. Based on the in silico analysis and experimental evaluations, the fusion AMP showed a significant antimicrobial effect on E. coli and Staphylococcus aureus bacteria.

Nucleic acid-based vaccine platforms against the coronavirus disease 19 (COVID-19).

The coronavirus disease 2019 (COVID-19) pandemic has infected 673,010,496 patients and caused the death of 6,854,959 cases globally until today. Enormous efforts have been made to develop fundamentally different COVID-19 vaccine platforms. Nucleic acid-based vaccines consisting of mRNA and DNA vaccines (third-generation vaccines) have been promising in terms of rapid and convenient production and efficient provocation of immune responses against the COVID-19. Several DNA-based (ZyCoV-D, INO-4800, AG0302-COVID19, and GX-19N) and mRNA-based (BNT162b2, mRNA-1273, and ARCoV) approved vaccine platforms have been utilized for the COVID-19 prevention. mRNA vaccines are at the forefront of all platforms for COVID-19 prevention. However, these vaccines have lower stability, while DNA vaccines are needed with higher doses to stimulate the immune responses. Intracellular delivery of nucleic acid-based vaccines and their adverse events needs further research. Considering re-emergence of the COVID-19 variants of concern, vaccine reassessment and the development of polyvalent vaccines, or pan-coronavirus strategies, is essential for effective infection prevention.

Production of a Novel Multi-Epitope Peptide Vaccine against Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is one of the prevalent cancers in the world with a high recurrence rate. In recent years, different researches have focused on designing efficient multi-epitope peptide vaccines against HCC. In designing these vaccines, over-expressed antigens in HCC patients, such as α- fetoprotein (AFP) and glypican-3 (GPC-3), have been employed. In our previous study, a multi-epitope peptide vaccine for HCC was designed by in-silico methods. The designed vaccine construct included the AFP, GPC-3, and aspartyl-ß-hydroxylase (ASPH) as CytoLoxic T cell Lymphocytes (CTL), one epitope from Tetanus Toxin Fragment C (TTFrC) as Helper T cell Lymphocytes (HTL), and a segment of microbial heat shock protein (HSP70) peptide407-426 as an adjuvant. All the mentioned parts were connected by appropriate linkers. The aim of this study is the production of the designed vaccine. Methods: This research is experimental and was carried out in Fasa, Iran, in 2017. The designed vaccine construct gene was transformed to the Escherchia coli BL21 (DE3) strain and expressed in different isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentrations (0.6 and 1 mM), times (4, 6, 8, 16 hours), and temperatures (25 and 37 °C). Then, the expressed protein was analyzed by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot methods. Results: The best conditions for protein expression were obtained in the Super Optimal Broth (SOB) medium at 37 °C after the induction of expression by 1 mM IPTG for six hour. Conclusion: The recombinant HCC vaccine was produced with a proper concentration.

Recent achievements in nano-based technologies for ocular disease diagnosis and treatment, review and update.

Ocular drug delivery is one of the most challenging endeavors among the various available drug delivery systems. Despite having suitable drugs for the treatment of ophthalmic disease, we have not yet succeeded in achieving a proper drug delivery approach with the least adverse effects. Nanotechnology offers great opportunities to overwhelm the restrictions of common ocular delivery systems, including low therapeutic effects and adverse effects because of invasive surgery or systemic exposure. The present review is dedicated to highlighting and updating the recent achievements of nano-based technologies for ocular disease diagnosis and treatment. While further effort remains, the progress illustrated here might pave the way to new and very useful ocular nanomedicines.

WITHDRAWN: Nucleic Acid-Based Vaccines Platform Against Covid-19 Pandemic

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Design, expression, and purification of a multi-epitope vaccine against Helicobacter Pylori based on Melittin as an adjuvant.

Helicobacter Pylori, a Gram-negative bacterium in the human stomach, causes adenocarcinoma and MALT (mucosa-associated lymphoid tissue) lymphoma in addition to infection and gastric ulcer. With regard to Helicobacter Pylori prevalence rate and widespread, producing an effective vaccine against this bacterium appears reasonable and necessary. Today, vaccine design by immunoinformatics is a promising solution in vaccine field. In the present study, potential immunodominant CD4⁺ T cell epitopes of UreB, HpaA, and NapA antigens were selected with a focus on IFN-γ secretion inducing ability. After joining the selected epitopes with KK and GPGPG linkers, sequence of Melittin, the major active protein of honey bee venom, was put in C-terminal by DPRVPSS linker as adjuvant. After reverse translation and codon optimization, the designed vaccine was cloned into pET-23a vector. The final construct was estimated as antigenic (71 & 74%) and non-allergenic with molecular weight of 36.785KD. The instability index (II) and codon frequency distribution were predicted to be 26.5 and 92%, respectively. The pET-23a vector transformed to the E.coli BL21 (DE3) strain. The evaluation of expression by SDS-PAGE analysis showed that the optimized expression is in SOB medium 8 h after induction by 0.5 mM IPTG. Finally, purification was performed by Ni-NTA affinity chromatography and Western blot analysis validated the purified protein. Future research is needed to investigate the designed vaccine efficiency against H. pylori, and also it's potential as a gastric cancer-preventive candidate.

A New Approach for Cancer Immunotherapy Based on the Cancer Stem Cell Antigens Properties.

BACKGROUND: Cancer stem cells (CSCs) are a rare population of tumor cells, which play an important role in tumor initiation, progression, and maintenance. The concept that cancer cells arise from stem cells was presented about 150 years ago. Recently, this hypothesis was renewed considering the heterogeneity of tumor cells. CSCs are resistant to chemo- and radio-therapy. Therefore, targeting CSCs could be a novel and effective strategy to struggle with tumor cells. OBJECTIVE: In this mini-review, we highlight that different immunotherapeutic approaches can be used to target cancer cells and eradicate different tumor cells. The most important targets are specific markers recognized on the CSC surface as CSC antigens such as CD44, CD133, Aldehyde Dehydrogenase (ALDH), and SOX family members. This article emphasizes recent advances in CSCs in cancer therapy. RESULTS: Our results present that CSC antigens play an important role in tumor initiation, especially in the cells that are resistant to chemo- and radiotherapy agents. Therefore, they are ideal targets for cancer immunotherapy, for instance, in developing different types of cancer vaccines or antibodies against tumor cells. CONCLUSION: The current studies related to cancer immunotherapy through targeting the CSC antigens based on their properties are briefly summarized. Altogether, CSC antigens can be efficiently targeted to treat cancer patients.

Harnessing Bioinformatics for Designing a Novel Multiepitope Peptide Vaccine Against Breast Cancer.

Breast cancer (BC) remains as one of the important causes of cancer deaths among women globally. Therefore, finding an effective treatment for BC is really needed. Cancer immunotherapy, as an emerging field, has a notable role in BC therapy. Peptide vaccines possess an outstanding role among different strategies in cancer immunotherapy. In vaccine design for cancer, induction of cellular and humoral immune responses should be considered. In the current study, cytolytic T lymphocytes (CTL) epitopes were evoked from human epidermal growth factor receptor (HER2), mucin 1 protein (MUC1), and heparanase antigenic proteins; and helper T lymphocytes (HTL) epitopes were determined from survivin protein by various immunoinformatics servers. Furthermore, our vaccine peptide contains several linear and conformational B cell epitopes that can induce humoral immunity. In order to elicit broad cellular and humoral immune responses, Por B protein from Neisseria meningitides, which is one of the toll like receptor 2 (TLR2) agonists, was utilized as an adjuvant in the vaccine construct. The designed peptide vaccine contains the extracellular domain of murine ULBP-like transcript 1 (MULT1), which binds to a natural killer group 2 member D (NKG2D) receptor with a high affinity and has a key role in triggering the innate immune response. All the mentioned segments were fused together by functional and structural amino acid linkers. Taken together, we project that our vaccine construct can potentially induce cellular, humoral, and innate immune responses in BC patients.