Cutting Edge: Induced Loss of Rasgrp1 in Peripheral CD4+ T Cells of Conditional Rasgrp1-Deficient Mice Reveals an Essential Role for Rasgrp1 in TCR/CD28-Induced Ras-MAPK Signaling.

Ras guanine nucleotide-releasing protein 1 (Rasgrp1) is a Ras guanine nucleotide exchange factor that participates in the activation of the Ras-ERK signaling pathway in developing T cells and is required for efficient thymic T cell positive selection. However, the role of Rasgrp1 in mature peripheral T cells has not been definitively addressed, in part because peripheral T cells from constitutive Rasgrp1-deficient mice show an abnormal activated phenotype. In this study, we generated an inducible Rasgrp1-deficient mouse model to allow acute disruption of Rasgrp1 in peripheral CD4+ T cells in the context of normal T cell development. TCR/CD28-mediated activation of Ras-ERK signaling was blocked in Rasgrp1-deficient peripheral CD4+ T cells. Furthermore, Rasgrp1-deficient CD4+ T cells were unable to synthesize IL-2 and the high-affinity IL-2R and were unable to proliferate in response to TCR/CD28 stimulation. These findings highlight an essential function for Rasgrp1 for TCR/CD28-induced Ras-ERK activation in peripheral CD4+ T cells.

Putative new combination vaccine candidates identified by reverse vaccinology and genomic approaches to control enteric pathogens.

OBJECTIVES: The pathogenic microorganisms that cause intestinal diseases can significantly jeopardize people's health. Currently, there are no authorized treatments or vaccinations available to combat the germs responsible for intestinal disease. METHODS: Using immunoinformatics, we developed a potent multi-epitope Combination (combo) vaccine versus Salmonella and enterohemorrhagic E. coli. The B and T cell epitopes were identified by performing a conservancy assessment, population coverage analysis, physicochemical attributes assessment, and secondary and tertiary structure assessment of the chosen antigenic polypeptide. The selection process for vaccine development included using several bioinformatics tools and approaches to finally choose two linear B-cell epitopes, five CTL epitopes, and two HTL epitopes. RESULTS: The vaccine had strong immunogenicity, cytokine production, immunological properties, non-toxicity, non-allergenicity, stability, and potential efficacy against infections. Disulfide bonding, codon modification, and computational cloning were also used to enhance the stability and efficacy of expression in the host E. coli. The vaccine's structure has a strong affinity for the TLR4 ligand and is very durable, as shown by molecular docking and molecular modeling. The results of the immunological simulation demonstrated that both B and T cells had a heightened response to the vaccination component. CONCLUSIONS: The comprehensive in silico analysis reveals that the proposed vaccine will likely elicit a robust immune response against pathogenic bacteria that cause intestinal diseases. Therefore, it is a promising option for further experimental testing.

A novel chimeric vaccine containing multiple epitopes for simulating robust immune activation against Klebsiella pneumoniae.

BACKGROUND: Due to antibiotic resistance, the Klebsiella genus is linked to morbidity and death, necessitating the development of a universally protective vaccine against Klebsiella pathogens. METHODS: Core sequence analysis prioritized non-redundant host molecules and expected lipid bilayer peptides from fully sequenced Klebsiella genomes. These proteins were refined to identify epitopes, examining their immunogenicity, toxicity, solubility, and interaction with MHC alleles. Epitopes were linked to CPG ODN C274 via EAAAK, HEYGAEALERAG, and GGGS linkers to enhance immunological responses. The vaccine's tertiary structure was modelled and docked with MHC-I and MHC-II. RESULTS: Fifty-five proteins were recognized in the Vaxign collection as having remarkable features. Twenty-three proteins with potential pathogenicity were then identified. Eight options for vaccines emerged after the immunogenicity of proteins was examined. The best antigens were three proteins: MrkD, Iron-regulated lipid membrane polypeptides, and RmpA. These compounds were selected for their sensitivity. The structural protein sequences of K. pneumoniae were utilized to identify seven CTL epitopes, seven HTL epitopes, and seven LBL epitopes, respectively. The produced immunization displayed a stable contact with the receptors, based on molecular dynamic simulations lasting 250 nanoseconds. Intermolecular binding free energies also indicated the dominance of the van der Waals and electrostatic energies. CONCLUSION: In summary, the results of this study might help scientists develop a novel vaccine to prevent K. pneumoniae infections.

Oral administration of DNA alginate nanovaccine induced immune-protection against Helicobacter pylori in Balb/C mice.

BACKGROUND: Helicobacter pylori (H. Pylori), is an established causative factor for the development of gastric cancer and the induction of persistent stomach infections that may lead to peptic ulcers. In recent decades, several endeavours have been undertaken to develop a vaccine for H. pylori, although none have advanced to the clinical phase. The development of a successful H. pylori vaccine is hindered by particular challenges, such as the absence of secure mucosal vaccines to enhance local immune responses, the absence of identified antigens that are effective in vaccinations, and the absence of recognized indicators of protection. METHODS: The DNA vaccine was chemically cloned, and the cloning was verified using PCR and restriction enzyme digestion. The efficacy of the vaccination was investigated. The immunogenicity and immune-protective efficacy of the vaccination were assessed in BALB/c mice. This study demonstrated that administering a preventive Alginate/pCI-neo-UreH Nanovaccine directly into the stomach effectively triggered a robust immune response to protect against H. pylori infection in mice. RESULTS: The level of immune protection achieved with this nano vaccine was similar to that observed when using the widely accepted formalin-killed H. pylori Hel 305 as a positive control. The Alginate/pCI-neo-UreH Nanovaccine composition elicited significant mucosal and systemic antigen-specific antibody responses and strong intestinal and systemic Th1 responses. Moreover, the activation of IL-17R signaling is necessary for the defensive Th1 immune responses in the intestines triggered by Alginate/pCI-neo-UreH. CONCLUSION: Alginate/pCI-neo-UreH is a potential Nanovaccine for use in an oral vaccine versus H. pylori infection, according to our findings.

pDNA-tachyplesin treatment stimulates the immune system and increases the probability of apoptosis in MC4-L2 tumor cells.

Breast cancer is the most common cancer among women worldwide, and marine creatures are the most abundant reservoir of anticancer medicines. Tachyplesin peptides have shown antibacterial capabilities, but their potential to inhibit cancer growth and trigger cancer cell death has not been investigated. A synthetic tachyplesin nucleotide sequence was generated and inserted into the pcDNA3.1( +) Mammalian Expression Vector. PCR analysis and enzyme digesting procedures were used to evaluate the vectors' accuracy. The transfection efficiency of MCF-7 and MCF10-A cells was 57% and 65%, respectively. The proliferation of MCF-7 cancer cells was markedly suppressed. Administration of plasmid DNA (pDNA) combined with tachyplesin to mice with tumors did not cause any discernible morbidity or mortality throughout treatment. The final body weight curves revealed a significant reduction in weight among mice treated with pDNA/tachyplesin and tachyplesin at a dose of 100 µg/ml (18.4 ± 0.24 gr, P < 0.05; 11.4 ± 0.24 gr P < 0.01) compared to the control group treated with PBS (22 ± 0.31 gr). Animals treated with pDNA/tachyplesin and tachyplesin exhibited a higher percentage of CD4 + Foxp3 + Tregs, CD8 + Foxp3 + Tregs, and CD4 + and CD8 + T cell populations expressing CTLA-4 in their lymph nodes and spleen compared to the PBS group. The groups that received pDNA/tachyplesin exhibited a substantial upregulation in the expression levels of caspase-3, caspase-8, BAX, PI3K, STAT3, and JAK genes. The results offer new possibilities for treating cancer by targeting malignancies using pDNA/tachyplesin and activating the mTOR and NFκB signaling pathways.

A comprehensive investigation of the medicinal efficacy of antimicrobial fusion peptides expressed in probiotic bacteria for the treatment of pan drug-resistant (PDR) infections.

The present work aimed to examine the intracellular antibacterial efficacy of Recombinant Lactobacillus acidophilus/antimicrobial peptides (AMPs) Melittin and Alyteserin-1a, specifically targeting Gram-negative bacteria. The first assessment was to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Recombinant L. acidophilus/AMPs versus Gram-negative and Gram-positive bacteria. In addition, the researchers examined the in vitro viability and safety of AMPs generated by L. acidophilus. The experiments included exposing the AMPs to elevated temperatures, proteases, cationic salts at physiological levels, and specific pH settings. The safety aspect was evaluated using hemolytic analysis utilizing sheep erythrocytes; cytotoxicity assays employing cell lines, and experiments on beneficial gut lactobacilli. An experiment was done using a time-kill method to assess the intracellular antibacterial efficacy of Recombinant L. acidophilus/AMPs compared to pathogenic varieties in HEp-2 cells. Previous investigations have shown that the MBC levels of recombinant L. acidophilus/AMPs were consistently two to four times higher than the equivalent MIC values when evaluated versus Gram-negative bacteria. Furthermore, the stability of the Recombinant L. acidophilus/AMPs showed variability when exposed to elevated temperatures (70 and 90 â„ƒ), treated with protease enzymes (proteinase K, lysozyme), exposed to higher concentrations of physiological salts (150 mM NaCl and 2 mM MgCl2), and varying pH levels (ranging from 4.0 to 9.0). The recombinant L. acidophilus/AMPs are non-hemolytic towards sheep erythrocytes, exhibit little cytotoxicity in RAW 264.7 and HEp-2 cells, and are considered safe when compared to beneficial gut lactobacilli. The research examined the intracellular bacteriostatic effects of recombinant L. acidophilus/AMPs on Gram-negative bacteria inside HEp-2 cells. Nevertheless, no notable bactericidal impact was seen on Gram-positive bacteria (P > 0.05). The research shows that recombinant L. acidophilus/AMPs, namely (L. acidophilus/melittin/Alyteserin-1a) as the focus of the investigation, effectively eliminate Gram-negative bacteria. Therefore, more investigation is necessary to elaborate on these discoveries.

Prognostic significance of CHCHD2P9 and ZNF204P in breast cancer: exploring their expression patterns and associations with malignancy-related genes.

BACKGROUND: Non-coding RNAs (ncRNAs) have a crucial impact on diverse cellular processes, influencing the progression of breast cancer (BC). The objective of this study was to identify novel ncRNAs in BC with potential effects on patient survival and disease progression. METHODS: We utilized the cancer genome atlas data to identify ncRNAs associated with BC pathogenesis. We explored the association between these ncRNA expressions and survival rates. A risk model was developed using candidate ncRNA expression and beta coefficients obtained from a multivariate Cox regression analysis. Co-expression networks were constructed to determine potential relationships between these ncRNAs and molecular pathways. For validation, we employed BC samples and the RT-qPCR method. RESULTS: Our findings revealed a noteworthy increase in the expression of AC093850.2 and CHCHD2P9 in BC, which was correlated with a poor prognosis. In contrast, ADAMTS9-AS1 and ZNF204P displayed significant downregulation and were associated with a favorable prognosis. The risk model, incorporating these four ncRNAs, robustly predicted patient survival. The co-expression network showed an effective association between levels of AC093850.2, CHCHD2P9, ADAMTS9-AS1, and ZNF204P and genes involved in pathways like metastasis, angiogenesis, metabolism, and DNA repair. The RT-qPCR results verified notable alterations in the expression of CHCHD2P9 and ZNF204P in BC samples. Pan-cancer analyses revealed alterations in the expression of these two ncRNAs across various cancer types. CONCLUSION: This study presents a groundbreaking discovery, highlighting the substantial dysregulation of CHCHD2P9 and ZNF204P in BC and other cancers, with implications for patient survival.

Chitosan-LeoA-DNA Nanoparticles Promoted the Efficacy of Novel LeoA-DNA Vaccination on Mice Against Helicobacter pylori.

More than half of the world's population is infected with Helicobacter pylori (H. pylori), which may lead to chronic gastritis, peptic ulcers, and stomach cancer. LeoA, a conserved antigen of H. pylori, aids in preventing this infection by triggering specific CD3+ T-cell responses. In this study, recombinant plasmids containing the LeoA gene of H. pylori are created and conjugated with chitosan nanoparticle (CSNP) to immunize BALB/c mice against the H. pylori infection. We used the online Vaxign tool to analyze the genomes of five distinct strains of H. pylori, and we chose the outer membrane as a prospective vaccine candidate. Afterward, the proteins' immunogenicity was evaluated. The DNA vaccine was constructed and then encapsulated in CSNPs. The effectiveness of the vaccine's immunoprotective effects was evaluated in BALB/c mice. Purified activated splenic CD3+ T cells are used to test the anticancer effects in vitro. Nanovaccines had apparent spherical forms, were small (mean size, 150-250 nm), and positively charged (41.3 ± 3.11 mV). A consistently delayed release pattern and an entrapment efficiency (73.35 ± 3.48%) could be established. Compared to the non-encapsulated DNA vaccine, vaccinated BALB/c mice produced higher amounts of LeoA-specific IgG in plasma and TNF-α in splenocyte lysate. Moreover, BALB/c mice inoculated with nanovaccine demonstrated considerable immunity (87.5%) against the H. pylori challenge and reduced stomach injury and bacterial burdens in the stomach. The immunological state in individuals with GC with chronic infection with H. pylori is mimicked by the H. pylori DNA nanovaccines by inducing a shift from Th1 to Th2 in the response. In vitro human GC cell development is inhibited by activated CD3+ T lymphocytes. According to our findings, the H. pylori vaccine-activated CD3+ has potential immunotherapeutic benefits.

Upregulation of EPSTI1/Drp1/AKT1 Signaling Pathways Using pDNA/Melittin Against Breast Cancer.

This study investigates the role of genes related to breast cancer in apoptosis control. A melittin nucleic acid sequence was synthesized and introduced into a pcDNA3.1(+) Mammalian Expression Plasmid. The cloning accuracy was assessed using PCR testing and enzyme digestion techniques. The vectors were transfected into cells using LipofectamineTM2000. The transfection efficacy of MCF-7 and 4T1 cells was evaluated using fluorescence and bright-field imaging. Pure melittin produced from bee venom had a notable hemolytic impact, with lower hemolytic activity levels than the positive control, Triton X-100. The growth rate of 4T1 and MCF-7 cancer cells was significantly inhibited. The apoptosis rates were 8.54%, 46.20%, and 78.82% for free pDNA, melittin, and pDNA-melittin, respectively. The C-pDNA/Melittin-treated group showed a statistically significant reduction in cancer factors compared to the control group. The treated tumors exhibited significant necrosis and late apoptosis, with a prevalence ranging from about 5% to 10% of the lesions. After exposure to pDNA-melittin, there was no significant increase in transcription levels of caspase-3, caspase-8, BCRA1, BAX, Drp1, AKT1, and EPSTI1 genes in the normal non-cancerous groups. The findings provide novel opportunities for the therapeutic targeting of malignancies via melittin and the stimulation of the EPSTI1/Drp1/AKT1 signaling cascades.

Putative novel outer membrane antigens multi-epitope DNA vaccine candidates identified by Immunoinformatic approaches to control Acinetobacter baumannii.

Multi-epitope polypeptide vaccines, a fusion protein, often have a string-of-beads system composed of various specific peptide epitopes, potential adjuvants, and linkers. When choosing the sequence of various segments and linkers, many alternatives are available. These variables can influence the vaccine's effectiveness through their effects on physicochemical properties and polypeptide tertiary structure.The most conserved antigens were discovered using BLASTn. To forecast the proteins' subcellular distribution, PSORTb 3.0.2 was used. Vaxign was used for the preliminary screening and antigenicity assessment. Protein solubility was also predicted using the ccSOL omics. Using PRED-TMBB, it was anticipated that the protein would localize across membranes. The IEDB and BepiPred-2.0 databases were used to predict the immunogenicity of B cell epitopes. A multi-epitope construct was developed and analyzed to evaluate. Twenty epitopes from A. baumannii's outer membrane protein (omp) were included in the vaccination. TLR4 agonist explosibility was investigated. The physicochemical characteristics, secondary and tertiary structures, and B-cell epitopes of vaccine constructs were assessed. Additionally, docking and MD experiments were used to examine the relationship between TLR4 and its agonist.Thirteen antigens were discovered, and eight of the 13 chosen proteins were predicted to be surface proteins. The 34 kDa outer membrane protein, Omp38, Omp W, CarO, putative porin, OmpA, were chosen as having the right antigenicity (≥0.5). FhuE and CdiA were eliminated from further study because of their low antigenicity. The vaccine design was developed by combining the most effective 10 B-cell and 10 MHC-I/MHCII combined coverage epitopes. The molecular formula of the vaccine was determined to be C1718H2615N507O630S17. The vaccine form has a molecular weight of 40,996.70 Da and 47 negatively charged residues (Asp + Glu), whereas 28 positively charged residues (Arg + Lys). The estimated half-life was 7.2 hours (mammalian reticulocytes, in vitro), > 20 hours (yeast, in vivo) and > 10 hours (Escherichia coli, in vivo) for the vaccine. The multi-epitope vaccine insertion is carried via the expression vector pcDNA3.1 (+).The multi-epitope vaccine may stimulate humoral and cellular immune responses, according to our findings, and it may be a candidate for an A. baumannii vaccine.

Use of immunoinformatics and the simulation approach to identify Helicobacter pylori epitopes to design a multi-epitope subunit vaccine for B- and T-cells.

BACKGROUND: Helicobacter pylori cause a variety of gastric malignancies, gastric ulcers, and cause erosive diseases. The extreme nature of the bacterium and the implantation of this bacterium protects it against designing a potent drug against it. Therefore, employing a precise and effective design for a more safe and stable antigenic vaccine against this pathogen can effectively control its associated infections. This study, aimed at improving the design of multiple subunit vaccines against H. pylori, adopts multiple immunoinformatics approaches in combination with other computational approaches. RESULTS: In this regard, 10 HTL, and 11 CTL epitopes were employed based on appropriate adopted MHC binding scores and c-terminal cut-off scores of 4 main selected proteins (APO, LeoA, IceA1, and IceA2). An adjuvant was added to the N end of the vaccine to achieve higher stability. For validation, immunogenicity and sensitization of physicochemical analyses were performed. The vaccine could be antigenic with significantly strong interactions with TOLK-2, 4, 5, and 9 receptors. The designed vaccine was subjected to Gromacs simulation and immune response prediction modelling that confirmed expression and immune-stimulating response efficiency. Besides, the designed vaccine showed better interactions with TLK-9. CONCLUSIONS: Based on our analyses, although the suggested vaccine could induce a clear response against H. pylori, precise laboratory validation is required to confirm its immunogenicity and safety status.

Immunization of BALB/c mice with BAB1-0278: An initial investigation of a novel potential vaccine for brucellosis based on Lactococcus Lactis vector.

The gram-negative intracellular bacterium Brucella abortus causes bovine brucellosis, a zoonotic disease that costs a lot of money. This work developed a vector vaccine against brucellosis utilizing recombinant L. lactis expressing Brucella outer membrane protein BAB1-0278. Gene sequences were obtained from GenBank. The proteins' immunogenicity was tested with Vaxijen. The target vector was converted into L. lactis after enzymatic digestion and PCR validated the BAB1-0278 gene cloning in the pNZ8148 vector. The target protein was extracted using a Ni-NTA column and confirmed using SDS-PAGE and western blot. After vaccination with the target vaccine, the expression of IgG subclasses was evaluated by the ELISA method. Cytokine production was also measured by the qPCR method in the small intestine and spleen. Lymphocyte proliferation and innate immune response (NLR, CRP, and PLR) were also assessed. Finally, after the challenge test, the spleen tissue was examined by H&E staining. BAB1-0278 was chosen because of its antigenicity score of 0.5614. A 237-bp gene fragment was discovered using enzymatic digestion and PCR. The presence of a 13 kDa protein band was confirmed by SDS-PAGE and western blot. In comparison to the PBS group, mice given the L. lactis-pNZ8148-BAB1-0278-Usp45 vaccine 14 days after priming had substantially greater levels of total IgG, IgG1, and IgG2a (P < 0.001). Also, the production of cytokines (IFN-γ, TNFα, IL-4, and IL-10) indicating cellular immunity increased compared to the control group (P < 0.001). The target group had a lower inflammatory response, morphological impairment, alveolar edema, and lymphocyte infiltration. An efficient probiotic-based oral brucellosis vaccination was created. These studies have proven that the recommended immunization gives the best protection, which supports its promotion.

Genome editing approaches with CRISPR/Cas9: the association of NOX4 expression in breast cancer patients and effectiveness evaluation of different strategies of CRISPR/Cas9 to knockout Nox4 in cancer cells.

BACKGROUND: The increasing prevalence of cancer detection necessitated practical strategies to deliver highly accurate, beneficial, and dependable processed information together with experimental results. We deleted the cancer biomarker NOX4 using three novel genetic knockout (KO) methods. Homology-directed repair (HDR), Dual allele HITI (Du-HITI) and CRISPR-excision were utilized in this study. METHODS: The predictive value of the NOX4 expression profile was assessed using a combined hazard ratio (HR) with a 95% confidence interval (CI). With a 95% confidence interval, a pooled odd ratio (OR) was used to calculate the relationship between NOX4 expression patterns and cancer metastasis. There were 1060 tumor patients in all sixteen research that made up this meta-analysis. To stop the NOX4 from being transcribed, we employed three different CRISPR/Cas9-mediated knockdown methods. The expression of RNA was assessed using RT-PCR. We employed the CCK-8 assay, colony formation assays, and the invasion transwell test for our experiments measuring cell proliferation and invasion. Using a sphere-formation test, the stemness was determined. Luciferase reporter tests were carried out to verify molecular adhesion. Utilizing RT-qPCR, MTT, and a colony formation assay, the functional effects of NOX4 genetic mutation in CRISPR-excision, CRISPR-HDR, and CRISPR du-HITI knockdown cell lines of breast cancer were verified. RESULTS: There were 1060 malignant tumors in the 16 studies that made up this meta-analysis. In the meta-analysis, higher NOX4 expression was linked to both a shorter overall survival rate (HR = 1.93, 95% CI 1.49-2.49, P < 0.001) and a higher percentage of lymph node metastases (OR = 3.22, 95% CI 2.18-4.29, P < 0.001). In breast carcinoma cells, it was discovered that NOX4 was overexpressed, and this increase was linked to a poor prognosis. The gain and loss-of-function assays showed enhanced NOX4 breast carcinoma cell proliferation, sphere-forming capacity, and tumor development. To activate transcription, the transcriptional factor E2F1 also attaches to the promoter region of the Nanog gene. The treatment group (NOX4 ablation) had substantially more significant levels of proapoptotic gene expression than the control group (P < 0.01). Additionally, compared to control cells, mutant cells expressed fewer antiapoptotic genes (P < 0.001). The du-HITI technique incorporated a reporter and a transcription termination marker into the two target alleles. Both donor vector preparation and cell selection were substantially simpler using this approach than with "CRISPR HDR" or "CRISPR excision." Furthermore, single-cell knockouts for both genotypes were created when this method was applied in the initial transfection experiment. CONCLUSIONS: The NOX4 Knockout cell lines generated in this research may be used for additional analytical studies to reveal the entire spectrum of NOX4 activities. The du-HITI method described in this study was easy to employ and could produce homozygous individuals who were knockout for a specific protein of interest.

Oral vaccination with novel Lactococcus lactis mucosal live vector-secreting Brucella lumazine synthase (BLS) protein induces humoral and cellular immune protection against Brucella abortus.

This work aimed to provide recombinant Lactococcus lactis as a potential live vector for the manufacture of recombinant Brucella abortus (rBLS-Usp45). The sequences of the genes were collected from the GenBank database. Using Vaxijen and ccSOL, the proteins' immunogenicity and solubility were evaluated. Mice were given oral vaccinations with recombinant L. lactis. Anti-BLS-specific IgG antibodies were measured by ELISA assay. Cytokine reactions were examined using real-time PCR and the ELISA technique. The BLS protein was chosen for immunogenicity based on the vaccinology screening findings since it had maximum solubility and antigenic values ​​of 99% and 0.75, respectively. The BLS gene, digested at 477 bp, was electrophoretically isolated to demonstrate that the recombinant plasmid was successfully produced. Protein-level antigen expression showed that the target group produced the 18 kDa-sized BLS protein, whereas the control group did not express any proteins. In the sera of mice given the L. lactis-pNZ8148-BLS-Usp45 vaccine 14 days after priming, there was a significant level of BLS-specific IgG1, IgG2a (P < 0.001) compared to the PBS control group. Vaccinated mice showed higher levels of IFN-γ, TNFα, IL-4, and IL-10 in samples obtained on days 14 and 28, after receiving the L. lactis-pNZ8148-BLS-Usp45 and IRBA vaccines (P < 0.001). The inflammatory reaction caused less severe spleen injuries, alveolar edema, lymphocyte infiltration, and morphological damage in the target group's spleen sections. Based on our findings, an oral or subunit-based vaccine against brucellosis might be developed using L. lactis-pNZ8148-BLS-Usp45 as a novel, promising, and safe alternative to the live attenuated vaccines now available.

CRISPR/Cas9-mediated LINC00511 knockout strategies, increased apoptosis of breast cancer cells via suppressing antiapoptotic genes.

BACKGROUND: The growing detection of long noncoding RNAs (lncRNAs) required the application of functional approaches in order to provide absolutely precise, conducive, and reliable processed information along with effective consequences. We utilized genetic knockout (KO) techniques to ablate the Long Intergenic Noncoding RNA 00,511 gene in several humans who suffered from breast cancer cells and at the end we analyzed and examined the results. RESULTS: The predictive relevance of LINC00511 expression pattern was measured by using a pooled hazard ratio (HR) with a 95% confidence interval (CI). The link among LINC00511 expression profiles and cancer metastasis was measured by using a pooled odds ratio (OR) with a 95% confidence interval. This meta- analysis was composed of fifteen studies which contained a total of 1040 tumor patients. We used three distinct CRISPR/Cas9-mediated knockdown techniques to prevent the LINC00511 lncRNA from being transcribed. RT-PCR was used to measure lncRNA and RNA expression. We used CCK-8, colony formation tests, and the invasion transwell test to measure cell proliferation and invasion. The stemness was measured by using a sphere-formation test. To validate molecular attachment, luciferase reporter assays were performed. The functional impacts of LINC00511 gene deletion in knockdown breast cancer cell lines were confirmed by using RT-qPCR, MTT, and a colony formation test. This meta-analysis was composed of 15 trials which contained a total of 1040 malignant tumors. Greater LINC00511 expression was ascribed to a lower overall survival (HR = 1.93, 95% CI 1.49-2.49, < P 0.001) and to an increased proportion of lymph node metastasis (OR = 3.07, 95% CI 2.23-4.23, P < 0.001) in the meta-analysis. It was found that the role of LINC00511 was overexpressed in breast cancer samples, and this overexpression was ascribed to a poor prognosis. The gain and loss-of-function tests demonstrated findings such as LINC00511 increased breast cancer cell proliferation, sphere-forming ability, and tumor growth. Additionally, the transcription factor E2F1 binds to the Nanog gene's promoter site to induce transcription. P57, P21, Prkca, MDM4, Map2k6, and FADD gene expression in the treatment group (LINC00511 deletion) was significantly higher than in the control group (P < 0.01). In addition, knockout cells had lower expression of BCL2 and surviving genes than control cells P < 0.001). In each of the two target alleles, the du-HITI approach introduced a reporter and a transcription termination signal. This strategy's donor vector preparation was significantly easier than "CRISPR HDR," and cell selection was likewise much easier than "CRISPR excision." Furthermore, when this approach was used in the initial transfection attempt, single-cell knockouts for both alleles were generated. CONCLUSIONS: The methods employed and described in this work could be extended to the production of LINC00511 knockout cell lines and, in theory, to the deletion of other lncRNAs to study their function.

Allergen specific immunotherapy with plasmid DNA encoding OVA-immunodominant T cell epitope fused to Tregitope in a murine model of allergy.

BACKGROUND: Peptide-based immunotherapy (PIT) was introduced as an attractive approach in allergen-specific immunotherapy (AIT). However, PIT clinical trials have shown variable results, and immune response to peptides is not precisely predictable. On the other hand, induction of antigen-specific tolerance may be augmented when allergens are combined with the regulatory T cell epitope (Tregitope). This study aimed to evaluate the therapeutic administration of a plasmid DNA encoding Tregitope and ovalbumin (OVA) immunodominant epitope in the murine model of allergy. METHODS: Following the induction of allergic rhinitis by ovalbumin, vaccinated group received three doses of recombinant plasmid containing Signal peptide-Tregitope-OVA T cell epitope. After the final OVA challenge, clinical symptoms, histopathological changes, OVA-specific IgE level, and cytokine secretion pattern of spleen cells were examined. RESULTS: Our data are showing that AIT with the recombinant DNA vaccine significantly suppressed airway inflammation; reduced eosinophilic infiltration in the nasal mucosa; decreased expression level of IL-4 and IL-17 in spleen cells, while IFN-γ, IL-10, and TGF-ß expression were increased. Moreover, OVA-specific IgE levels were also decreased. CONCLUSION: These results suggest that Tregitope-immunodominant T cell epitope fusion can act as a safe and effective approach in DNA-based allergen-specific immunotherapy.

Expression and purification of recombinant alpha-toxin AnCra1 from the scorpion Androctonus crassicauda and its functional characterization on mammalian sodium channels.

BACKGROUND: Alpha-scorpion toxins with long-chain peptide and four disulfide bonds represent diverse pharmacological profiles for various subtypes of voltage-gated sodium channels. Obtaining the natural toxins are difficult and time-consuming process, which represents the major difficulty to interpreting analysis of their structural and functional properties. METHODS AND RESULTS: This study describes the toxin peptide and plasmid construct containing the gene coding for mammalian toxin AnCra1 from the scorpion Androctonus crassicauda venom. We have established genetic construction of fusion protein in pET32a + vector containing thioredoxin (Trx-tag), enterokinase cleavage site and 6xhistidine-tag for efficient expression in Escherichia coli strain RG2 (DE3). The soluble expressed peptide, then purified by Ni-NTA resin affinity chromatography and its purity was confirmed by reverse-phase HPLC and mass spectrometry (7433.54 Da.). The electrophysiological data showed that recombinant AnCra1 selectively inhibits the fast inactivation of hNav1.7 channel (EC50 = 136.7 ± 6.6 nM). CONCLUSIONS: Our findings demonstrate that the AnCra1 is structurally and functionally analogous to alpha excitatory toxins; furthermore, expression and purification of bioactive scorpion toxins in bacterial cells can be a practicable and efficient way to obtain a novel source of toxin peptides as tools to study the function and physiological responses of ion channels.

Changes in NOTCH1 gene and its regulatory circRNA, hsa_circ_0005986 expression pattern in human gastric adenocarcinoma and human normal fibroblast cell line following the exposure to extremely low frequency magnetic field.

The effect of an extremely low-frequency magnetic field (ELF-MFs) on the expression levels of NOTCH1 and its regulatory circular RNA (circ-RNA) in gastric cancer has not yet investigated. This study aimed to find the expression changes of NOTCH1 and its regulatory circ-RNA, hsa_circ_0005986, in human gastric adenocarcinoma cell line (AGS) and human normal fibroblast (Hu02) cells fallowing the exposure to discontinuously magnetic flux densities (MFDs) of 0.25, 0.5 ,1 and 2 millitesla (mT) for 18h in comparison to unexposed cells. In addition, the effect of various MFDs on viability of tumor and normal cells was investigated. The cell viability was evaluated by MTT assay. The relative expression of NOTCH1and hsa_circ_0005986 mRNAs was analyzed by quantitative Real-time PCR. The viability of tumor cells was decreased under the exposure of MFs, while the normal cells viability was increased. NOTCH1 was significantly down-regulated in AGS cells and up-regulated in Hu02 cells at all MFDs. The expression changes of NOTCH1 in tumor and  normal cells was depended to the MFD of MFs. According to our results, the tumor and normal cells show different behavior at the molecular level in various MFDs in terms of NOTCH1 and hsa_circ_0005986 expression level. Decrease in tumor cell survival following the exposure to ELF-MFs may be the result of decreased in the expression level of NOTCH1 and its Reg-circ-RNA. These magnetic field-reducing effects on cancer cell survival through the change on the expression of genes involved in the proliferation and progression of cancer can be a new key in cancer treatment.

In silico and in vitro effects of the I30T mutation on myelin protein zero instability in the cell membrane.

Charcot-Marie-Tooth (CMT) diseases are a heterogeneous group of genetic peripheral neuropathies caused by mutations in a variety of genes, which are involved in the development and maintenance of peripheral nerves. Myelin protein zero (MPZ) is expressed by Schwann cells, and MPZ mutations can lead to primarily demyelinating polyneuropathies including CMT type 1B. Different mutations demonstrate various forms of disease pathomechanisms, which may be beneficial in understanding the disease cellular pathology. Our molecular dynamics simulation study on the possible impacts of I30T mutation on the MPZ protein structure suggested a higher hydrophobicity and thus lower stability in the membranous structures. A study was also conducted to predict native/mutant MPZ interactions. To validate the results of the simulation study, the native and mutant forms of the MPZ protein were separately expressed in a cellular model, and the protein trafficking was chased down in a time course pattern. In vitro studies provided more evidence on the instability of the MPZ protein due to the mutation. In this study, qualitative and quantitative approaches were adopted to confirm the instability of mutant MPZ in cellular membranes.

Effect of the cagW-based gene vaccine on the immunologic properties of BALB/c mouse: an efficient candidate for Helicobacter pylori DNA vaccine.

BACKGROUND: Helicobacter pylori (H. pylori) infect more than half of the world population, and they cause different serious diseases such as gastric carcinomas. This study aims to design a vaccine on the basis of cagW against H. pylori infection. After pcDNA3.1 (+)-cagW-CS-NPs complex is produced, it will be administered into the muscles of healthy BALB/c mice in order to study the effect of this DNA vaccine on the interleukin status of mice, representing its effect on the immune system. After that, the results will be compared with the control groups comprising the administration of cagW-pCDNA3.1 (+) vaccine, the administration of chitosan and the administration of PBS in the muscles of mice. METHODS: The cagW gene of H. pylori was amplified by employing PCR, whose product was then cloned into the pcDNA3.1 (+) vector, and this cloning was confirmed by PCR and BamHI/EcoRV restriction enzyme digestion. CagW gene DNA vaccine was encapsulated in chitosan nanoparticles (pcDNA3.1 (+)-cagW-CS-NPs) using a complex coacervation method. The stability and in vitro expression of chitosan nanoparticles were studied by DNase I digestion and transfection, and the immune responses elicited in specific pathogen-free (SPF) mice by the pcDNA3.1 (+)-cagW-CS-NPs were evaluated. Apart from that, the protective potential pcDNA3.1 (+)-cagW-CS-NPs was evaluated by challenging with H. pylori. RESULTS: The pcDNA3.1 (+)-cagW-CS-NPs comprises cagW gene of H. pylori that is encapsulated in chitosan nanoparticles, produced with good morphology, high stability, a mean diameter of 117.7 nm, and a zeta potential of + 5.64 mV. Moreover, it was confirmed that chitosan encapsulation protects the DNA plasmid from DNase I digestion, and the immunofluorescence assay showed that the cagW gene could express in HDF cells and maintain good bioactivity at the same time. In comparison to the mice immunized with the control plasmid, in vivo immunization revealed that mice immunized with pcDNA3.1 (+)-cagW-NPs showed better immune responses and prolonged release of the plasmid DNA. CONCLUSIONS: This research proves chitosan-DNA nanoparticles as potent immunization candidates against H. pylori infection and paves the way for further developments in novel vaccines encapsulated in chitosan nanoparticles.