Designing a T-cell epitope-based vaccine using in silico approaches against the Sal k 1 allergen of Salsola kali plant.

Allergens originated from Salsola kali (Russian thistle) pollen grains are one of the most important sources of aeroallergens causing pollinosis in desert and semi-desert regions. T-cell epitope-based vaccines (TEV) are more effective among different therapeutic approaches developed to alleviate allergic diseases. The physicochemical properties, and B as well as T cell epitopes of Sal k 1 (a major allergen of S. kali) were predicted using immunoinformatic tools. A TEV was constructed using the linkers EAAAK, GPGPG and the most suitable CD4+ T cell epitopes. RS04 adjuvant was added as a TLR4 agonist to the amino (N) and carboxyl (C) terminus of the TEV protein. The secondary and tertiary structures, solubility, allergenicity, toxicity, stability, physicochemical properties, docking with immune receptors, BLASTp against the human and microbiota proteomes, and in silico cloning of the designed TEV were assessed using immunoinformatic analyses. Two CD4+ T cell epitopes of Sal k1 that had high affinity with different alleles of MHC-II were selected and used in the TEV. The molecular docking of the TEV with HLADRB1, and TLR4 showed TEV strong interactions and stable binding pose to these receptors. Moreover, the codon optimized TEV sequence was cloned between NcoI and XhoI restriction sites of pET-28a(+) expression plasmid. The designed TEV can be used as a promising candidate in allergen-specific immunotherapy against S. kali. Nonetheless, effectiveness of this vaccine should be validated through immunological bioassays.

Molecular Identification and Characterization of Hevein Antimicrobial Peptide Genes in Two-Row and Six-Row Cultivars of Barley (Hordeum vulgare L.).

Heveins are one of the most important groups of plant antimicrobial peptides. So far, various roles in plant growth and development and in response to biotic and abiotic stresses have reported for heveins. The present study aimed to identify and characterize the hevein genes in two-row and six-row cultivars of barley. In total, thirteen hevein genes were identified in the genome of two-row and six-row cultivars of barley. The identified heveins were identical in two-row and six-row cultivars of barley and showed a high similarity with heveins from other plant species. The hevein coding sequences produced open reading frames (ORFs) ranged from 342 to 1002 bp. Most of the identified hevein genes were intronless, and the others had only one intron. The hevein ORFs produced proteins ranged from 113 to 333 amino acids. Search for conserved functional domains showed CBD and LYZ domains in barley heveins. All barley heveins comprised extracellular signal peptides ranged from 19 to 35 amino acids. The phylogenetic analysis divided barley heveins into two groups. The promoter analysis showed regulatory elements with different frequencies between two-row and six-row cultivars. These cis-acting elements included elements related to growth and development, hormone response, and environmental stresses. The expression analysis showed high expression level of heveins in root and reproductive organs of both two-row and six-row cultivars. The expression analysis also showed that barley heveins is induced by both biotic and abiotic stresses. The results of antimicrobial activity prediction showed the highest antimicrobial activity in CBD domain of barley heveins. The findings of the current study can improve our knowledge about the role of hevein genes in plant and can be used for future studies.

Immunochemical Characterization of Salix alba (S. alba) Pollen Allergens and Evaluation of the Cross-Reactivity Pattern with Common Allergenic Pollen Grains.

BACKGROUND: Pollens, particularly tree and plant pollens, are one of the major causes of allergic respiratory diseases worldwide. Allergy to pollens of different species of Salix trees has been reported in various regions of the world. The most common type of Salix tree in Iran is white willow (Salix alba). OBJECTIVES: This study aimed to identify and determine the immunochemical characteristics of allergenic proteins in S. alba tree pollen extract using SDS-PAGE and IgE- immunoblotting methods. Moreover, the cross-reaction pattern of the specific IgE antibody of S. alba tree pollen proteins with pollen allergens of common allergenic trees, i.e., Populus nigra (P. nigra), Cupressus sempervirens (C. sempervirens), Pinus brutia (P. brutia) and Platanus orientalis (P. orientalis) in the region was investigated. METHODS: The reaction of allergenic proteins in S. alba pollen extract with specific IgE antibodies in patients' sera was investigated using SDS-PAGE and IgE-immunoblotting methods. The cross-reaction of specific IgE antibodies of the proteins present in S. alba pollen extract with pollen allergens of common allergenic trees in the region was investigated using ELISA and immunoblotting inhibition methods. In silico methods such as phylogenetic tree drawing and alignment of amino acid sequences were used to examine the evolutionary relationship and homology structure of common allergenic proteins (Panallergens) responsible for cross reactions. RESULTS: More than 11 protein bands binding to specific IgE antibodies in patients' sera with a molecular weight between 13 and 95 kDa were identified in the S. alba tree pollen extract. ELISA and immunoblotting inhibition results showed that P. nigra extract could inhibit the binding of IgE antibodies to S. alba pollen extract proteins to a greater extent than C. sempervirens, P. brutia, and P. orientalis tree extracts. In silico methods investigated the results of ELISA and immunoblotting inhibition methods. Moreover, a high structural homology and evolutionary relationship were observed between S. alba and P. nigra tree pollen panallergens. CONCLUSION: In this study, it was found that more than 80 % of the sensitive patients who were examined had specific IgE antibodies reacting with the approximately a 15 kDa-protein present in the S. alba pollen extract. Furthermore, the specific IgE-binding proteins found in the pollens of S. alba and P. nigra trees had relative structural homology, and it is likely that if recombinant forms are produced, they can be used for diagnostic and therapeutic purposes for both of the trees.

Genome-wide identification and expression profiling of snakin/GASA genes under drought stress in barley (Hordeum vulgare L.).

Gibberellic Acid-Stimulated Arabidopsis (GASA) proteins are present in various plants and have a role in plant growth, stress responses, and hormone crosstalk. GASA coding sequences in barley were discovered in this study. We then investigated gene and protein structure, physicochemical characteristics, evolutionary and phylogenetic relationships, promoter region, post-translational modification, and in silico gene expression. Finally, real-time quantitative PCR (RT-qPCR) was used to examine the expression of GASA genes in root and shoot tissues under drought stress. We found 11 GASA genes spread across six of seven chromosomes in the barley genome. A conserved GASA domain and 12-cysteine residues at the C-terminus were included in the proteins. All GASA genes contained secretory signal peptides. The GASA genes in Hordeum vulgare (HvGASA) have been classified into three subfamilies based on evolutionary analysis. According to synteny analyses, segmental duplications are significant in forming the GASA gene family. According to the cis-elements analyses, GASA genes may be induced by a variety of phytohormones and stresses. Tissue-specific expression analysis indicated that GASA genes had varied expression patterns in different tissues. Contrary to common perception, the expression study of GASA genes under biotic and abiotic stresses revealed that GASA genes are more induced by abiotic stresses than biotic stresses. The qPCR confirmed the response of GASA genes to abiotic stresses and showed different expression patterns of these genes under drought stress. Overall, these results can improve our knowledge about the function of GASA genes and provide data for future researches. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03545-8.

Genome-wide evaluation of transcriptomic responses of human tissues to smoke: A systems biology study.

The harmful compounds in various sources of smoke threaten human health. So far, many studies have investigated the effects of compounds of smoke on transcriptome changes in different human tissues. However, no study has been conducted on the effects of these compounds on transcriptome changes in different human tissues simultaneously. Hence, the present study was conducted to identify smoke-related genes (SRGs) and their response mechanisms to smoke in various human cells and tissues using systems biology based methods. A total of 6,484 SRGs were identified in the studied tissues, among which 4,095 SRGs were up-regulated and 2,389 SRGs were down-regulated. Totally, 459 SRGs were smoke-related transcription factors (SRTFs). Gene regulatory network analysis showed that the studied cells and tissues have different gene regulation and responses to compounds of smoke. The comparison of different tissues revealed no common SRG among the all studied tissues. However, the CYP1B1 gene was common among seven cells and tissues, and had the same expression trend. Network analysis showed that the CYP1B1 is a hub gene among SRGs in various cells and tissues. To the best of our knowledge, for the first time, our results showed that compounds of smoke induce and increase the expression of CYP1B1 key gene in all target and non-target tissues of human. Moreover, despite the specific characteristics of CYP1B1 gene and its identical expression trend in target and non-target tissues, it can be used as a biomarker for diagnosis and prognosis.

Molecular and immunochemical characterization of Pop n 2: A new allergen of Populus nigra pollen.

BACKGROUND: Pollen is one of the most common allergens that cause respiratory allergies worldwide. Pollen grains from poplars have been reported as important sources of pollinosis in many countries. OBJECTIVE: The aim of the present study was to determine the molecular and immunochemical characterization of Pop n 2, a novel allergen of Populus nigra (P nigra) pollen extract. METHODS: In this study, the pollen extract of P nigra was analysed by SDS-PAGE, and the allergenic profile was determined by IgE immunoblotting and specific ELISA using the sera of twenty allergic patients. The coding sequence of Pop n 2 was cloned and expressed in the Escherichia coli BL21 (DE3) using plasmid the pET-21b (+). Finally, the expressed recombinant Pop n 2 was purified by affinity chromatography. RESULTS: Pop n 2 belongs to the profilin family with a molecular weight of approximately 14 kDa. Pop n 2 is the most IgE-reactive protein (about 65%) in the P nigra pollen extract. The cDNA sequencing results indicated an open reading frame 396 bp that encodes 131 amino acid residues. The results of ELISA and Immunoblotting assays showed that recombinant Pop n 2 could react with the IgE antibody in patients' sera, like its natural counterpart. CONCLUSION: Our data revealed that Pop n 2 is a significant allergen in the P nigra pollen extract. Moreover, we observed that the recombinant Pop n 2 produced by the pET-21b (+) vector in the E colisystem acts as its natural counterpart.

Anti-allergic effects of vitamin E in allergic diseases: An updated review.

Allergic diseases are caused by the immune system's response to innocent antigens called allergens. Recent decades have seen a significant increase in the prevalence of allergic diseases worldwide, which has imposed various socio-economic effects in different countries. Various factors, including genetic factors, industrialization, improved hygiene, and climate change contribute to the development of allergic diseases in many parts of the world. Moreover, changes in lifestyle and diet habits play pivotal roles in the prevalence of allergic diseases. Dietary changes caused by decreased intake of antioxidants such as vitamin E lead to the generation of oxidative stress, which is central to the development of allergic diseases. It has been reported in many articles that oxidative stress diverts immune responses to the cells associated with the pathogenesis of allergic diseases. The aim of this short review was to summarize current knowledge about the anti-allergic properties of vitamin E.

Identification of common key regulators in rat hepatocyte cell lines under exposure of different pesticides.

Pesticides exposure can have harmful effects on human health. The liver is the most common organ of pesticides toxicity due to its major metabolic activity. The molecular mechanism of pesticides effect is complex and is controlled by gene regulatory networks. All components of regulatory networks are controlled by transcription factors and other regulatory elements. Therefore, identification of key regulators through system biology approaches and high-throughput techniques can help to provide comprehensive insights into molecular mechanisms of the pesticide effect. In the current study, a microarray data-set was used to potentially identify molecular mechanisms that regulate gene expression profile of rat hepatocyte cell lines in response to pesticides exposure. Results showed that the number of differentially expressed genes (DEGs) and differentially expressed transcription factors (DE-TFs) were dramatically different among pesticides tested. Results also revealed 205 common DEGs and 11 DE-TFs among pesticides tested. Additionally, we found that six DE-TFs (CREB1, CTNNB1, PPARG, SP1, SRF and STAT3) had the highest number of interactions with other DEGs and acted as the key regulatory genes. The results of this study revealed regulator genes that have the key functions in response to pesticides toxicity in rat liver, which can provide the basis for future studies. Furthermore, these regulatory genes can be used as toxicity biomarkers to improve diagnosis and prognosis.

Molecular cloning and characterization of six defensin genes from lentil plant (Lens culinaris L.).

Six full-length gene and cDNA sequences of defensin were identified from Lens culinaris L. plant. The identified genes and cDNAs were different in length and their coding sequences contained Knot1 functional domain. Phylogenetic analysis classified the identified defensins into two subfamilies. All defensin genes contained only one intron and had extracellular signal peptides. Secondary structures of identified defensins were completely composed of alpha helix and beta strand. Presence of conserved Cys amino acids and disulfide bridges, interaction with defense and signaling proteins and antimicrobial activity were other common features of these peptides. The identified defensins displayed differential expression pattern in the various tissues. The highest expression level of defensins was observed in seed, pod, and root tissues. Defensin 4 was significantly expressed in all examined tissues, whereas the other defensins were only expressed in some tissues. Also, in the fungal and wounding treatments, lentil defensins showed different expression pattern. Defensin 1 was up-regulated in both fungal and wounding treatments. Defensin 4 showed decreased expression level in both fungal and wounding treatments. Defensins 2 and 6 were up-regulated in wounding and fungal treatments, respectively. In this study, for the first time, six defensin genes were isolated and characterized from lentil. Our results highlighted the role of defensins in lentil plant that can be used for future studies.

A Systematic In Silico Analysis of the Legionellaceae Family for Identification of Novel Drug Target Candidates.

The family Legionellaceae consists of Gram-negative bacteria that are widely distributed in aquatic environments around the world. This family consists of a single genus, Legionella, that is recognized as an important cause of community-acquired pneumonia and hospital-acquired pneumonia. Legionella consists of intracellular pathogens, thus cellular pharmacokinetic and pharmacodynamic properties of an antibiotic against these bacteria as well as uptake and subcellular distribution into macrophages should be considered for a successful outcome of disease. Treatment strategies for Legionella infection require a combination of multiple antibiotics. Hence, because of the possible development of resistance to the drugs during therapy, a new alternative targeted therapy is yielding promising results. In this study, a comprehensive in silico target identification pipeline was performed on members of the family Legionellaceae to identify the best targets. Using a homology-based computational pipeline method, new drug targets were identified. Of 4,358 analyzed proteins, 18 proteins, including proteins involved in metabolism (amino acid, energy, and lipid metabolisms), cellular transport, cell division, and cell motility, were selected as the final putative drug targets. These proteins play an important role in the survival and propagation of Legionella infection. In conclusion, homology-based methods could improve the identification of novel drug targets and the drug discovery process, which can potentially be effective for the prevention and treatment of Legionella infections.

Genome-Wide Identification of Potential Drug Target in Enterobacteriaceae Family: A Homology-Based Method.

The Enterobacteriaceae is a large family of Gram-negative, facultative anaerobic, non-spore forming rod-shaped bacteria that includes harmless and pathogenic organisms. The emergence and development of drug resistance in Enterobacteriaceae is complicating the treatment of serious infections. The aim of this study is to predict and characterize putative drug targets in Enterobacteriaceae family employing a homology-based computational method. The final putative drug targets were qualitatively characterized via cellular function prediction, subcellular localization prediction, broad-spectrum, and druggability analyses. Of 6,327 analyzed proteins, 35 proteins were selected as final putative drug targets in Enterobacteriaceae family. These putative drug targets were involved in different vital pathways like metabolism, biosynthesis of macromolecule, and cell division. Predicted drug targets were also localized in the cytoplasm and cytoplasmic membrane of the pathogen that acts as antimicrobial or vaccine targets. Of 35 drug targets, 5 targets were druggable and 30 targets were not druggable and were predicted as novel drug targets, which should be further evaluated to develop new antimicrobial. Thirteen drug targets were considered as broad-spectrum targets. It is expected that results of our study could facilitate the production of novel antibacterial for efficient treatment of infections caused by Enterobacteriaceae pathogens.

Expression of recombinant Arabian camel lactoferricin-related peptide in Pichia pastoris and its antimicrobial identification.

BACKGROUND: Lactoferricin (LFcin) is a strong cationic peptide released from the N-terminus of lactoferrin by gastric pepsin digestion. LFcin has some important properties, including high antimicrobial activity. To date, lactoferricins have been isolated and characterised from various animal species, but not from camel. The aim of this study was to characterise and express recombinant camel lactoferricin (LFcinC) in Pichia pastoris and investigate its antimicrobial activity. RESULTS: After methanol induction, LFcinC was expressed and secreted into a culture broth medium and the results determined by concentrated supernatant culture medium showed high antimicrobial activity against the following microorganisms: Escherichia coli PTCC 1330 (ATCC 8739), Staphylococcus aureus PTCC 1112 (ATCC 6538), Pseudomonas aeruginosa PTCC 1074 (ATCC 9027), Bacillus subtilis PTCC 1023 (ATCC 6633), and Candida albicans PTCC 5027 (ATCC 10231). Thermal stability was clarified with antibacterial activity against Escherichia coli PTCC 1330 (ATCC 8739). CONCLUSION: Results confirmed that camel lactoferricin had suitable antimicrobial activity and its production by Pichia pastoris can be used for recombinant production.

In silico investigation of lactoferrin protein characterizations for the prediction of anti-microbial properties.

Lactoferrin (Lf) is an iron-binding multi-functional glycoprotein which has numerous physiological functions such as iron transportation, anti-microbial activity and immune response. In this study, different in silico approaches were exploited to investigate Lf protein properties in a number of mammalian species. Results showed that the iron-binding site, DNA and RNA-binding sites, signal peptides and transferrin motifs in the Lf structure were highly conserved. Examined sequences showed three conserved motifs which were repeated twice in the Lf structure, demonstrating ancient duplication events in its gene. Also, results suggest that the functional domains in mammalian Lf proteins are Zinc finger, Tubulin/FtsZ, GTPase, α/ß hydrolase and Zinc knuckle. The potential site for nucleic acid binding and the major DNA and RNA- binding sites in this protein were found in the lactoferricin (Lfc) fragment. Due to its high positive charge, Lf is able to bind a large number of compounds. Our analysis also revealed that the interactions between Lf and ITLN1, LYZ, CSN2, and CD14 proteins played an important role in the protective activities of Lf. Analysis for the prediction of secondary structures indicated that high amounts of α-helix, ß-strand and ß-sheet were present in Lf. The high degree of conservation among mammalian Lf proteins indicates that there is a close relationship between these proteins, reflecting their important role.