Design of an oral vaccine using Lactococcus lactis against brucellosis: an in vitro and in vivo study.

Brucellosis is regarded as one of the world's most severe zoonotic diseases. This study aimed to investigate the possibility of using recombinant Lactococcus lactis (L. lactis) as a live vector to produce recombinant Brucella abortus (B. abortus) Omp10. The gene sequences were obtained from GenBank. The proteins' immunogenicity was assessed using Vaxijen. After confirming the cloning of the Omp10 gene in the pNZ8148 vector by enzymatic digestion and PCR, transformation into L. lactis was done. SDS-PAGE and western blot methods evaluated omp10 protein expression. Mice received oral recombinant L. lactis vaccines. IgG antibodies against Omp10 were tested using ELISA. Real-time PCR and ELISA were used to analyze cytokine responses. Survival rate and histopathological changes were evaluated after the challenge. Omp10 was chosen for its 1.5524 antigenicity score. Enzymatic digestion and PCR identified a 381-bp gene fragment. A 10 kDa band indicated the success of L. lactis transformation. Mice administered the L. lactis-pNZ8148-Omp10-Usp45 vaccination 14 days after priming showed significantly higher Omp10-specific total IgG and IgG1 (P < 0.001) than the PBS control group. The mice who received the L. lactis-pNZ8148-Omp10-Usp45 and IRBA vaccines had significantly elevated levels of IFN-γ, TNFα, IL-4, and IL-10 in samples collected on days 14 and 28 (P < 0.001). Inflammatory response, morphological damage, alveolar edema, and lymphocyte infiltration were reduced in the target group. A recombinant L. lactis expressing the Omp10 protein was constructed as an oral Lactococcus-based vaccine and compared to live attenuated vaccines for future brucellosis investigations.

Effects of bromodomain and extra-terminal inhibitor JQ1 and interleukin-6 on breast cancer cells.

BACKGROUND: Bromodomain and extra-terminal (BET) proteins are recognized acetylated lysine of histone 4 and act as scaffolds to recruit many other proteins to promoters and enhancers of active genes, especially at the super-enhancers of key genes, driving the transcription process and have been identified as potential therapeutic targets in breast cancer. However, the efficacy of BET inhibitors such as JQ1 in breast cancer therapy is impeded by interleukin-6 (IL-6) through an as-yet-defined mechanism. METHODS AND RESULTS: We investigated the interplay between IL-6 and JQ1 in MCF-7 and MDA-MB-231 human breast cancer cells. The results demonstrate that the efficacy of JQ1 on the inhibition of cell growth and apoptosis was stronger in MDA-MB-231 cells than in MCF-7 cells. Further, MCF-7 cells, but not MDA-MB-231 cells, exhibited increased expression of CXCR4 following IL-6 treatment. JQ1 significantly reduced CXCR4 surface expression in both cell lines and diminished the effects of IL-6 pre-treatment on MCF-7 cells. While IL-6 suppressed the extension of breast cancer stem cells in MCF-7 cells, JQ1 impeded its inhibitory effect. In MCF-7 cells JQ1 increased the number of senescent cells in a time-dependent manner. CONCLUSION: Analysis of gene expression indicated that JQ1 and IL-6 synergistically increase SNAIL expression and decrease c-MYC expression in MCF-7 cells. So, the BET proteins are promising, novel therapeutic targets in late-stage breast cancers. BET inhibitors similar to JQ1 show promise as therapeutic candidates for breast cancers, especially when triple-negative breast cancer cells are increased and/or tumor-promoting factors like IL-6 exist in the tumor microenvironment.

Brucella abortus antigen omp25 vaccines: Development and targeting based on Lactococcus lactis.

BACKGROUND: Most Brucella infections take place on mucosal membranes. Therefore, creating vaccinations delivered through the mucosa may be crucial for managing brucellosis. Consequently, we assessed the efficacy of a recombinant oral antigen delivery system based on Lactococcus lactis for Brucella abortus omp25 antigen. METHOD: Oral vaccinations with L. lactis transformed with pNZ8148 variants encoding for omp25 (pNZ8148:omp25) and free-pNZ8148 were administered to mice. On day 30, following immunization in animal groups, anti-omp25-specific IgG1 antibodies were assessed by the ELISA test. Additionally, nasal and bronchoalveolar lavages containing omp25-specific secretory IgA (sIgA) were analysed by ELISA. ELISA test and real-time PCR were also used to analyse cytokine responses up to 28 days following the last boost. In addition, the protective potential of L. lactis pNZ8148:omp25 vaccines was assessed in BALB/c mice by exposing them to the B. abortus strain. RESULTS: Based on the initial screening results, the omp25 protein was identified for immunogenicity because it had the maximum solubility and flexibility and antigenic values of 0.75. The produced plasmid was digested using KpnI and XbaI. By electrophoretic isolation of the digestion fragments at 786 bp, the omp25 gene, the successful production of the recombinant plasmid, was confirmed. Antigen expression at the protein level revealed that the target group generated the 25 kDa-sized omp25 protein, but there was no protein expression in the control group. Fourteen days after priming, there was a considerable amount of omp25-specific IgG1 in the sera of mice vaccinated with pNZ8148-Usp45-omp25-L. lactis (p < 0.001 in target groups compared to the phosphate-buffered saline control group). IFN-γ and TNF-α levels were more significant in samples from mice that had been given the pNZ8148-Usp45-omp25-L. lactis and IRBA vaccinations, in samples taken on days 14 and 28, respectively (p < 0.001). The pNZ8148-Usp45-omp25-L. lactis and IRBA immunization groups had significantly greater IL-4 and IL-10 transcription levels than the other groups. The spleen portions from the pNZ8148-Usp45-omp25-L. lactis and IRIBA vac group had less extensive spleen injuries, alveolar oedema, lymphocyte infiltration and morphological damage due to the inflammatory process. CONCLUSION: Our study offers a novel method for using the food-grade, non-pathogenic and noncommercial bacterium L. lactis as a protein cell factory to produce the novel immunogenic fusion candidate romp25. This method offers an appealing new approach to assessing the cost-effective, safe, sustainable, simple pilot development of pharmaceutical products.

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.

Cell loaded hydrogel containing Ag-doped bioactive glass-ceramic nanoparticles as skin substitute: Antibacterial properties, immune response, and scarless cutaneous wound regeneration.

An ideal tissue-engineered dermal substitute should possess angiogenesis potential to promote wound healing, antibacterial activity to relieve the bacterial burden on skin, as well as sufficient porosity for air and moisture exchange. In light of this, a glass-ceramic (GC) has been incorporated into chitosan and gelatin electrospun nanofibers (240-360 nm), which MEFs were loaded on it for healing acceleration. The GC was doped with silver to improve the antibacterial activity. The bioactive nanofibrous scaffolds demonstrated antibacterial and superior antibiofilm activities against Gram-negative and Gram-positive bacteria. The nanofibrous scaffolds were biocompatible, hemocompatible, and promoted cell attachment and proliferation. Nanofibrous skin substitutes with or without Ag-doped GC nanoparticles did not induce an inflammatory response and attenuated LPS-induced interleukin-6 release by dendritic cells. The rate of biodegradation of the nanocomposite was similar to the rate of skin regeneration under in vivo conditions. Histopathological evaluation of full-thickness excisional wounds in BALB/c mice treated with mouse embryonic fibroblasts-loaded nanofibrous scaffolds showed enhanced angiogenesis, and collagen synthesis as well as regeneration of the sebaceous glands and hair follicles in vivo.

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.

Irisin, A Mediator of Muscle Crosstalk with Other Organs: From Metabolism Regulation to Protective and Regenerative Effects.

Physical exercise is a therapeutic strategy for some systemic and non-systemic complications. Various processes or factors like myokines are involved in an exercise course. Irisin is produced in skeletal muscle during exercise, and its effects resemble many exercise effects. Besides the systemic effects of muscle-derived irisin, this peptide is produced in different tissues. Numerous studies have investigated the underlying molecular mechanisms of irisin effects. Despite some controversies, most studies have demonstrated the improvement of metabolic-related complications and immunomodulatory or regenerative mechanisms in correlation with the circulating level of this peptide or after in vivo/in vitro treatments that have introduced it as a peptide with therapeutic value. This review describes the similarities and differences of the effects in various tissues and their correlation with the most prevalent tissue-related complication to present a view for the mechanism(s) of function, efficacy, and safety of this peptide in each tissue as an exercise effector and endocrine peptide.

Silencing of α-N-acetylgalactosaminidase in the gastric cancer cells amplified cell death and attenuated migration, while the multidrug resistance remained unchanged.

Although the elevated level of the α-N-acetylgalactosaminidase enzyme (encoded by the NAGA gene) is a well-recognized feature of cancer cells; little research works have been undertaken on the cancer malignancy mechanisms. The effects of NAGA gene downregulation on cancer cells' features such as drug resistance, impaired programmed cell death, and migration were analyzed in this study. The cells grew exponentially with a doubling time of 30 h in an optimal condition. Toxicity of daunorubicin chemotherapy drug on NAGA-transfected EPG85.257RDB cells was evaluated in comparison to control cells and no significant change was recorded. Quantitative transcript analyses and protein levels revealed that the MDR1 pump almost remained unchanged during the study. Moreover, the NAGA gene downregulation enhanced the late apoptosis rate in EPG85.257RDB cells at 24 h posttransfection. The investigated expression level of genes and proteins involved in the TNFR2 signaling pathway, related to cancer cell apoptosis, showed considerable alterations after NAGA silencing as well. MAP3K14 and CASP3 genes were downregulated while IL6, RELA, and TRAF2 experienced an upregulation. Also, NAGA silencing generally diminished the migration ability of EPG85.257RDB cells and the MMP1 gene (as a critical gene in metastasis) expression decreased significantly. The expression of the p-FAK protein, which is located in the downstream of the α2 ß1 integrin signaling pathway, was reduced likewise. It could be concluded that despite drug resistance, NAGA silencing resulted in augmentative and regressive effects on cell death and migration.

Remyelination in PNS and CNS: current and upcoming cellular and molecular strategies to treat disabling neuropathies.

Myelin is a lipid-rich nerve cover that consists of glial cell's plasmalemma layers and accelerates signal conduction. Axon-myelin contact is a source for many developmental and regenerative signals of myelination. Intra- or extracellular factors including both enhancers and inhibitors are other factors affecting the myelination process. Myelin damages are observed in several congenital and hereditary diseases, physicochemical conditions, infections, or traumatic insults, and remyelination is known as an intrinsic response to injuries. Here we discuss some molecular events and conditions involved in de- and remyelination and compare the phenomena of remyelination in CNS and PNS. We have explained applying some of these molecular events in myelin restoration. Finally, the current and upcoming treatment strategies for myelin restoration are explained in three groups of immunotherapy, endogenous regeneration enhancement, and cell therapy to give a better insight for finding the more effective rehabilitation strategies considering the underlying molecular events of a lesion formation and its current condition.

Irisin injection mimics exercise effects on the brain proteome.

Physical inactivity can endanger human health and increase the incidence of neurodegenerative disease. Exercise has tremendous beneficial effects on brain health and cognitive function, especially in older adults. It also improves brain-related outcomes in depression, epilepsy and neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease. Irisin is a mediator of the beneficial effects of exercise. This study aimed to assess the proteome alterations in adult male National Maritime Research Institute (NMRI) mice brain tissue upon three different conditions including endurance exercise, resistance exercise and irisin injection. Quantification of irisin levels in blood was performed using irisin-ELISA Kit. Quantification and identification of proteins via two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)/MS showed the alteration of at least 21 proteins due to different treatments. Cellular pathway analysis revealed common beneficial effects of sole irisin treatment and different exercise procedures suggesting the capability of irisin injection to substitute the exercise when physical activity is not possible.

Muscles proteome analysis; irisin administration mimics some molecular effects of exercise in quadriceps muscle.

Because of health-promoting effects, the adaptation of skeletal muscles to exercise is considered a therapeutic strategy for metabolic complications and musculoskeletal disabilities. Myokines display many beneficial effects of different exercise modalities. Among them, irisin is known as a systemic effector that positively influences several organs. There are a few studies about the effects of irisin on skeletal muscles, and irisin prosperities need to be well-defined for being an exercise mimetic. To aim this purpose, we assessed the proteome profile of mouse skeletal muscle after eight weeks of irisin injection comparing to resistance and endurance exercise treated groups. In the current study, two-dimensional gel electrophoresis was used to evaluate the protein content of the quadriceps muscle. The results were analyzed with Image Master 2D Platinum V6 software. Differentially expressed proteins were characterized by mass spectrometry (MALDI TOF/TOF) and interpreted using protein data banks and co-expression network. Irisin increases cellular ATP content by driving its overproduction through glycolysis and oxidative phosphorylation similar to two exercise protocols and as a specific property, decreases ATP consumption through creatine kinase downregulation. It also improves the microstructural properties of quadriceps muscle by increasing fiber proteins and might induce cellular proliferation and differentiation. Network analysis of differentially expressed proteins also revealed the co-expression of Irisin precursor with structural and metabolic-related proteins. The protein alterations after irisin administration display the potential of this myokine to mimic some molecular effects of exercise, suggesting it a promising candidate to improve muscle metabolism and structure.

Comparative proteome analyses highlight several exercise-like responses of mouse sciatic nerve after IP injection of irisin.

Many beneficial effects of exercise on the nervous system are mediated by hormone (growth factor)/receptor signaling. Considering the accumulating evidence on the similarity of some beneficial effects, irisin can be a proposed effector of exercise; however, the mechanism underlying these effects remains largely unknown. More evidence on the mechanism of action might reveal its potential as a treatment strategy to substitute exercise recovery protocols for nerve injuries in physically disabled patients. To evaluate the underlying mechanism of irisin involvement in nerve adaptation and exerting beneficial effects, we studied the proteome profile alteration of mouse sciatic nerve after irisin administration. We also compared it with two 8-week protocols of resistance exercise and endurance exercise. The results indicate that irisin contributes to the regulation of nerve metabolism via overexpression of Ckm and ATP5j2 proteins. Irisin administration may improve sciatic nerve function by maintaining the architecture, enhancing axonal transport, and promoting synapse plasticity through increased structural and regulatory proteins and NO production. We also showed that irisin has the potential to induce neurotrophic support on the sciatic nerve by maintaining cell redox homeostasis, and responses to oxidative stress via the upregulation of disulfide-isomerase and superoxide dismutase enzymes. Comparing with exercise groups, these effects are somewhat exercise-like responses. These data suggest that irisin can be a promising therapeutic candidate for specific targeting of defects in peripheral neuropathies and nerve injuries as an alternative for physical therapy.

The Effects of 8-Week Resistance and Endurance Trainings on Bone Strength Compared to Irisin Injection Protocol in Mice.

BACKGROUND: Osteoporosis is a prevalent elderly complication that is characterized by decreased bone mineral density and increased fracture risk because of dysregulation in bone mineralization and resorption. Physical activity can enhance bone strength by exerting mechanical forces and myokines. Irisin is a myokine that is increased following physical exercise and can affect bones. In this study, 8 weeks of resistance and endurance exercises are applied in mice compared to irisin injection to assess the contribution of the protocols and this myokine to bone strength. MATERIALS AND METHODS: Thirty-five male NMRI mice were separated into five groups; control, placebo, irisin injection, resistance exercise, and endurance exercise. 8-week of exercise protocols and irisin injection protocol (100 µg/kg/week) was applied. Plasma irisin concentration and bone strength were measured using enzyme-linked immunoassay and 3-point bending assay, respectively. Statistical analyses were done through one-way ANOVA and Tukey test, and P < 0.05 was considered the significant difference. RESULTS: Serum irisin concentration and bone strength in resistance exercise and irisin-injected groups were significantly higher than control and placebo groups (P < 0.0001). Serum irisin concentration, but not bone strength, of the endurance exercise group was also significantly higher than control and placebo groups (P < 0.0001) but lower than resistance and irisin-injected groups. CONCLUSION: Resistance exercise and irisin injection, but not endurance exercise, are likely to be effective in increasing bone strength. There may be a threshold for plasma irisin level to affect bones which the applied protocols of irisin injection and resistance exercise but not endurance exercise can reach.

The relation between the ghrelin receptor and FOXP3 in bladder cancer.

Immune responses play an important role in the fate of bladder cancer tumors. Treg cells are immunosuppressive and down-regulate the proliferation of effector T cells, which favor tumor survival. Ghrelin is a hormone that stimulates release of growth hormone and anti-inflammatory response to cancer cells. Ghrelin also is a gastrointestinal hormone that regulates immune responses via the growth hormone secretagogue receptor (GHS-R1a). The relation among ghrelin, its receptor, and Treg cells that surround bladder tumors is not clear. We found that Foxp3+ T and GHS-R1a cells are increased significantly in bladder tumor tissues. Therefore, we suggest that ghrelin may increase the number of Treg cells in the tumor and suppress activity of the immune system against bladder cancer.

The functions of azurin of Pseudomonas aeruginosa and human mammaglobin-A on proapoptotic and cell cycle regulatory genes expression in the MCF-7 breast cancer cell line.

Azurin protein of Pseudomonas aeruginosa is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of azurin and human MAM-A genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of azurin and MAM-A genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of azurin and MAM-A genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant (p < 0.05) difference after transfection. The expression of BAK, FAS, and BAX genes in transformed cells compare with non-transformed and transformed MCF-7 by pBudCE4.1 were increased statistically significant (p < 0.05) increases. Although, the increase of SURVIVIN and P53 expressions in transformed cells were not statistically significant (p > 0.05). Co-expression of azurin and MAM-A genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of BAK, FAS, and BAX genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated.

Upregulation of Neuroprogenitor and Neural Markers via Enforced miR-124 and Growth Factor Treatment.

Previous studies have shown that miR-124 plays an important role in the development of auditory neurons, which are degenerated in the sensorineural hearing loss. However, whether the combined use of miR-124 and growth factors can increase the expression of neural related markers in human dental pulp stem cells has been remained unknown so far. In this study, human dental pulp stem cells were transfected with miR-124 following treatment with brain-derived neurotrophic factor or epidermal growth factor/basic fibroblast growth factor. The expression of some neural related markers (nestin, SOX2, ß-tubulin III, MAP2, and peripherin) was analyzed in two groups by qRT-PCR or immunofluorescence. Cellular treatment resulted in morphological changes including neurosphere-like colonies formation. Nestin and SOX2 were up-regulated, and MAP2 and peripherin were down-regulated in dental pulp stem cells transfected by miR-124 following treatment with brain-derived neurotrophic factor. Replacement of brain-derived neurotrophic factor with epidermal growth factor/ basic fibroblast growth factor resulted in the up-regulation of nestin, MAP2, peripherin, and ß-tubulin III and down-regulation of SOX2. The expression of SOX2 and nestin was also confirmed by immunofluorescence. The combination of miR-124 and growth factors would provide a promising starting point for upregulating the neural progenitor markers in human dental pulp stem cells.

Biodecomposition of Phenanthrene and Pyrene by a Genetically Engineered Escherichia coli.

BACKGROUND: Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach. OBJECTIVE: In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli. METHODS: Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil. RESULTS: The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p<0.05). Moreover, catalase test was positive while the oxidase tests were negative. CONCLUSION: These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

Differentiation of dental pulp stem cells into neuron-like cells.

Background and objectives: With regard to their ease of harvest and common developmental origin, dental pulp stem cells (DPSCs) may act as a favorable source of stem cells in generation of nerves. Moreover; cellular migration and differentiation as well as survival, self-renewal, and proliferation of neuroprogenitor species require the presence of the central nervous system (CNS) mitogens including EGF and bFGF. Accordingly, the possibility of the induction of neuronal differentiation of DPSCs by EGF and bFGF was evaluated in the present study.Materials and methods: DPSCs were treated with 20 ng/ml EGF, 20 ng/ml bFGF, and 10 µg/ml heparin. In order to further induce the neuroprogenitor differentiation, DPSC-derived spheres were also incubated in serum-free media for three days. The resulting spheres were then cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS. The morphology of the cells and the expression of the differentiation markers were correspondingly analyzed by quantitative polymerase chain reaction (qPCR), western blotting, and immunofluorescence (IF).Results: The EGF/bFGF-treated DPSCs showed significant increase in the expression of the neuroprogenitor markers of Nestin and SRY (sex determining region Y)-box 2 (SOX2), 72 h after treatment. The up-regulation of Nestin and SOX2 induced by growth factors was confirmed using western blotting and IF. The cultures also yielded some neuron-like cells with a significant rise in Nestin, microtubule-associated protein 2 (MAP2), and Neurogenin 1 (Ngn1) transcript levels; compared with cells maintained in the control media (p < 0.05).Conclusion: DPSCs seemed to potentially differentiate into neuron-like cells under the herein-mentioned treatment conditions.

Ghrelin induces autophagy and CXCR4 expression via the SIRT1/AMPK axis in lymphoblastic leukemia cell lines.

T cell acute lymphoblastic leukemia (T-ALL) is one of the most frequent malignancies in children, and the CXCR4 receptor plays an important role in the metastasis of this malignancy. Ghrelin is a hormone with various functions including stimulation of the release of growth hormone and autophagy in cancer cells. Moreover, SIRT1 and AMPK (AMP-activated protein kinase) stimulate expression of proteins involved in autophagy. On the other hand, autophagic cell death can be an alternative target for cancer therapy, in the absence of apoptosis. The relationship between ghrelin and the SIRT1/AMPK axis and the resulting effects on autophagy, apoptosis, proliferation, and expression of CXCR4 and the ghrelin receptor (GHS-R1a), in Jurkat and Molt-4 human lymphoblastic cell lines was not previously clear. Here we demonstrate that SIRT1 expression is upregulated during the induction of autophagy by ghrelin, an effect that is inhibited by inactivation of SIRT1/AMPK axis. In addition, ghrelin can affect CXCR4 and GHS-R1a expression. In conclusion, this work reveals that ghrelin induces autophagy, invasion, and downregulation of ghrelin receptor expression via the SIRT1/AMPK axis in lymphoblastic cell lines. However, in these cell lines ghrelin-induced autophagy does not lead to cell death due to weak induction of apoptosis.

The expression of Cysteine-Rich Secretory Protein 2 (CRISP2) and miR-582-5p in seminal plasma fluid and spermatozoa of infertile men.

Cysteine-Rich Secretory Protein 2 (CRISP2) plays an important role in the morphology and motion of male ejaculated spermatozoa. The association of its expression with some miRNAs is also well known. The aim of this study was to determine the expression of CRISP2 and mir-582 in the seminal plasma fluid and spermatozoa of three groups of infertile men and the possible association of their expressions. In this experimental study, the expression of CRISP2 in seminal plasma fluid and spermatozoa of 17 men with asthenozoospermia, 15 men with teratozoospermia, 17 men with teratoasthenozoospermia, and 18 infertile individuals with normozoospermia were measured using western blotting. Then by using bioinformatics studies, miR-582-5p was nominated as a CRISP2-associated miRNA, and its expression was evaluated by means of Real-Time PCR. Comparison of expression of CRISP2 and miRNA-582 in the studied groups was analyzed by t-test and Mann-Whitney U test. The expression of CRISP2 showed a significant reduction in the spermatozoa and seminal plasma fluid of all three groups, (p < 0.05). MiR-582-5p expression significantly increased in teratozoospermia patients (<0.05), and significantly decreased in teratoasthenozoospermia patients (p < 0.05). Meanwhile, changes in the expression of miR-582-5p in teratoasthenozoospermia individuals was associated with a decrease in the expression of CRISP2, which could represent the potential role of miR-582-5p in regulation of CRISP2 expression in teratoasthenozoospermia individuals.