Probiotic Potential, Antiproliferative, and Proapoptotic Activities of Lactobacillus fermentum Isolated from Breast Milk.

Background: Breast milk is always the best choice for infant's nutrition due to its useful compounds such as immune cells and molecules, oligosaccharides, as well as bacteria and their metabolites. We identified and characterized the isolated strain from human breast milk in this study. Materials and Methods: A total of 20 lactating mothers aged 25 to 34 years were enrolled in our study. We collected the breast milk samples in sterile microtubes. 100 µl of each sample was spread on de Man-Rogosa-Sharpe (MRS) agar plates and incubated aerobically at 37°C for 48 hr. After identifying the isolated strain, initial tests (hemolysis inactivity and L-arginine hydrolysis, catalase), the acid tolerance, bile tolerance, and antibiotics susceptibility of the isolated strain were estimated. Furthermore, the antiproliferative and proapoptotic activities of heat-killed cells) HKC) and cell-free supernatant (CFS) of the strain on the HT-29 cell line were evaluated using MTT assay and flow cytometry analysis, respectively. Results: The isolated strain was Gram-positive, bacilli in shape, catalase-negative, non-hemolytic, and negative for L-arginine hydrolysis. By 16S rRNA gene sequencing, the isolated strain was Lactobacillus fermentum. According to MTT assay and flow cytometry results, the HKC and CFS of the isolated strain reduced the viability of the HT-29 cells. The total apoptosis induced in HT-29 cells by HKC and CFS was 65.98% and 70.1%, respectively. Conclusion: Our findings suggest that this strain, despite the properties of probiotic bacteria, has potential antiproliferative and proapoptotic capabilities.

Immunization of mice with chimeric protein-loaded aluminum hydroxide and selenium nanoparticles induces reduction of Brucella melitensis infection in mice.

Purpose: Due to the many problems with commercially available vaccines, the production of effective vaccines against brucellosis is a necessity. The aim of this study was to evaluate the immune responses caused by the chimeric protein consisting of trigger factor, Bp26, and Omp31 (TBO) along with aluminum hydroxide (AH/TBO) and selenium (Se/TBO) nanoparticles (NPs) as adjuvants in mouse model. Materials and Methods: Recombinant antigen expression was induced in Escherichia coli BL21 (DE3) bacteria using IPTG (isopropyl-d-1-thiogalactopyranoside). Purification and characterization of recombinant protein was conducted through NiFe3O4 NPs, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot. NP characteristics, including morphology and particle size, were measured in vitro. The recombinant TBO was loaded on to AH and Se NPs and were administered subcutaneously. After mice immunization, measurement of antibody titter and protection assay was performed. Results: The average sizes of AH and Se NPs were about 60 nm and 150 nm, respectively. The enzyme-linked immunosorbent assay results showed that the serum of mice immunized by subcutaneous injection with both nanovaccines produced significant immunoglobulin G (IgG) responses against the chimeric antigen. The results of TBO-specific IgG isotype (IgG2a/IgG1) analysis showed that both AH and Se NPs induced a type to T-helper immune response. In addition, the results of the challenge with the pathogenic strain of Brucella melitensis 16M showed that vaccinated mice with AH/TBO NPs indicated a higher reduction of bacterial culture than immunized mice with Se/TBO NPs and TBO alone. Conclusion: The results showed that AH NPs carrying chimeric antigen can be a promising vaccine candidate against brucellosis by producing protective immunity.

Effects of Venlafaxine on the Size of Brain and Expression of SHANK3, TUBB5 and DDC Genes in BALB/c Mice.

Objectives: A growing body of evidence has recently suggested that taking venlafaxine during pregnancy may be linked to increased risk of certain congenital defects. The study aimed to address the effects of venlafaxine use during pregnancy on the development of the brain in mice. Experimental design: Fourteen female BALB/c mice were randomly divided into two equally-sized groups: venlafaxine-treated and control. After mating, pregnant mice of venlafaxine-treated group were orally received the venlafaxine 35 mg/kg/day throughout pregnancy, while pregnant control mice did not receive any treatment. All pups were killed on postnatal day 21 and brain images were quantified using ImageJ software. The mRNA expression levels of SHANK3, TUBB5 and DDC of genes in pups' brain tissue samples were evaluated using quantitative real-time PCR method. Principal observations: The mean brain size of pups was significantly smaller in the venlafaxine-treated group than in the control group. Results showed that the mRNA expression levels of SHANK3 and TUBB5 was significantly downregulated in venlafaxine-treated mice compared to control group. Expression of DDC gene didn't showed significant differences between two groups. Conclusions: These results provide evidence that use of venlafaxine during pregnancy may affect the brain development in mice and altered the expression of SHANK3 and TUBB5 genes in brain tissue.

Association of hsa-miR-5571-5p Expression with Clinicopathological Factors besides Identification of Its Hub Target Genes and Key Pathway in Breast Cancer.

Background: miRNAs are small non-coding RNAs; regulate gene expression using RNA degradation or translation repression. Dysregulation of miRNAs is involved in the initiation and progression of many cancers. We aimed to determine the relationship between miR-5571-5p expression and clinical factors and regulatory mechanisms in breast cancer. Methods: Histopathologic sections approximately with 25 microns thick from FFPE tissues were achievement of Al-Zahra Hospital (Isfahan, Iran) in 2020-2021 years by Pathologist. miR-5571-5p expression, determined using real-time PCR. For miRNA target genes prediction, integrated miRNA target prediction tools, were used. Gene ontology and KEGG pathway analysis were accomplished to identify the biological function. A PPI network was constructed to display key target genes. For hub genes validation, GEPIA databases were used. Results: miR-5571-5p was upregulated in breast tumor tissues, and its increase was significantly related to a poor prognosis in breast cancer (P<0.0001). At first, 324 target genes were predicted, and then 110 genes with a decrease in expression were selected. GO analysis showed that genes were mainly enriched in the regulation of the ERBB2 and EGFR signaling pathway. KEGG pathway analysis suggested that downregulated genes were enriched in glioma, the ErbB signaling pathway, and breast cancer. Finally, the ten hub genes (EGF, PIK3R1, SOS1, PTEN, SHC1, CBLB, LIFR, LEP, PDE1C, and NT5C2) were detected from the PPI network. Conclusion: miR-5571-5p up-regulation is associated with breast cancer progression and worse survival. The current study identified ten genes associated with breast cancer, which might help to provide candidate targets for the treatment.

Association of SOD2 rs2758339, rs5746136 and rs2842980 polymorphisms with increased risk of breast cancer: a haplotype-based case-control study.

BACKGROUND: A growing body of evidence indicates that oxidative stress, high levels of reactive oxygen species (ROS), is implicated in the pathogenesis of breast cancer (BC). Superoxide dismutase (SOD2), a mitochondria-resident antioxidant enzyme, protects cells from ROS by catalytically converting the superoxide radicals into less reactive species. OBJECTIVE: We aimed to investigate whether SOD2 rs2758339, rs5746136 and rs2842980 polymorphisms are associated with increased risk of BC. METHODS: A total of 100 patients with BC and 100 healthy controls were enrolled in the study. We used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for genotyping the SOD2 single-nucleotide polymorphisms (SNPs). Under co-dominant, dominant and recessive inheritance models, the genotypic and allelic associations of SOD2 SNPs with susceptibility to BC were evaluated using logistic regression analysis. The haplotype analysis was performed on the SOD2 SNPs to determine their combined effect on the BC risk. RESULTS: We found that SOD2 rs5746136 was significantly associated with decreased risk of developing BC in co-dominant and dominant inheritance models (P < 0.05). The SOD2 rs5746136 T allele confers an apparent protective effect against breast carcinogenesis (OR: 1.956; 95% CI 1.312-2.916; P < 0.0001). The SOD2 rs5746136/rs2842980 combined genotypes (CT/AA, CT/AT and TT/AA) were significantly more frequent in healthy subjects compared to BC patients (P < 0.05). The CTA and ACA haplotypes (rs2758339, rs5746136, rs2842980) were found to be a protective and a risk factor for BC, respectively. CONCLUSION: These data strongly suggest that SOD2 rs5746136 was significantly associated with reduced risk of BC, indicating its protective role in BC development.

Association of DROSHA rs6877842, rs642321 and rs10719 polymorphisms with increased susceptibility to breast cancer: A case-control study with genotype and haplotype analysis.

BACKGROUND: Multiple lines of evidence suggest that single nucleotide polymorphisms (SNPs) in genes encoding components of the microRNA processing machinery may underlie susceptibility to various human diseases, including cancer. OBJECTIVE: The present study aimed to investigate whether rs6877842, rs642321 and rs10719 SNPs of DROSHA, a key component of the miRNA biogenesis pathway, are associated with increased risk of breast cancer. METHODS: A total of 100 patients diagnosed with breast cancer and 100 healthy women were included. Following extraction of DNA, genotyping was performed by tetra primer- amplification refractory mutation system-PCR (T-ARMS-PCR) technique. Under the co-dominant, dominant and recessive inheritance models, the association between DROSHA SNPs and breast cancer risk was determined by logistic regression analysis. The association of DROSHA SNPs with patients' clinicopathological parameters was assessed. Also, haplotype analysis was performed to evaluate the combined effect of DROSHA SNPs on breast cancer risk. RESULTS: We observed a statistically significant association between DROSHA rs642321 polymorphism and breast cancer susceptibility (P < 0.05). Under the dominant inheritance model, DROSHA rs642321 polymorphism was significantly associated with increased risk of breast cancer (OR: 6.091; 95% CI: 3.291-11.26; P = 0.0001). Our findings demonstrated that DROSHA rs642321 T allele can contribute to the development of breast cancer (OR: 3.125; 95% CI: 1.984-4.923; P = 0.0001). We also found that GTC and GTT haplotypes conferred significant risk for breast cancer (OR: 2.367; 95% CI: 1.453-3.856; P = 0.0001 and OR: 7.944; 95% CI: 2.073-30.43; P = 0.0001, respectively). CONCLUSIONS: These results provide the first evidence that DROSHA rs642321 polymorphism is associated with increased risk of breast cancer. However, further studies are needed to firmly validate these findings.

Efficacy of Ficus carica leaf extract on morphological and molecular behavior of mice germ stem cells.

Infertility is one of the most prevalent health disorders in reproductive-age males and females. Ficus carica (Fc), an herbal plant, has been used traditionally for the treatment of different diseases such as infertility especially in Iranian folk medicine. This study examined the effects of Fc leaf extract on the proliferation of mice spermatogonial stem cells (SSCs). Phenolic, flavonoid content, major polyphenolic compounds and antioxidant activity of the extract was evaluated respectively by Folin-Ciocateu, aluminum chloride, HPLC and the FRAP and DPPH methods. Testicular cells of neonate mice were extracted and their identity was confirmed using cytokeratin for Sertoli and Oct-4, CDHI and PLZF for SSCs. Effects of Fc (0.0875, 0.175, 0.35, 0.71 and 1.42 mg/ml) was evaluated at third, 7th, 9th and 14th days of culture by colony assay. The expression of the Mvh, GFRα1 and Oct-4 genes and the viability and proliferation of cultured cells was assessed at the end of the culture period. The extract has a rich phenolic and flavonoid content such as Rutin, Psoralen, Bergapten and Caffeoylmalic acid using HPLC analysis. It also had a potent reducing and radical scavenging activity. Morphology of colonies was similar in all groups. Higher viability, proliferation, colony number and diameter of SSCs was seen in the presence of Fc leaf extract in a dose-dependent manner so that higher number and diameter of colonies were observed in two higher doses of 0.71 and 1.42 mg/ml, separately for each time point relative to other groups. The Mvh, Oct-4 and GFRα1 genes expression had no significant differences between groups. It seems that Fc leaf extract not only had no any cytotoxic effects on the viability and proliferation of SSCs but also support their stemness state. So, this culture system can be employed for enrichment of germ stem cells for use in clinical applications.

Evaluation of serotonin receptors (5HTR2A and 5HTR3A) mRNA expression changes in tumor of breast cancer patients.

Background: Several studies have proven the pattern of neurotransmitters, especially serotonin, in carcinogenesis and tumor development. Several studies have also shown that changes in serotonin receptors, especially 5HTR2A and 5HTR3A, can play an important role in incidence of cancers. This study was conducted to investigate changes in mRNA expression of 5HTR2A and 5HTR3A receptors in the breast tumor tissue compared to their marginal zone. Methods: In this study, tissue samples were obtained from 40 female patients with breast cancer. Entire RNA was obtained from the tissues and cDNA synthesis was performed. Finally, real ime PCR technique was performed to investigate the gene expression variation of both 5HTR2A and 5HTR3A. To analyze the results of real time PCR, both ΔΔCt and 2-ΔΔCt equations were used. All statistical analyses were performed using the SPSS 18 software and R-Studio 1.0.136. P values less than 0.05 (p<0.05) and 0.001 (p<0.001) were considered statistically significant. Results: The results showed increased expression of 5HTR2A and 5HTR3A genes in tumoral tissues of patients with breast cancer compared to their marginal tissues, where the 5HTR2A and 5HTR3A genes expression in tumor tissue was 3.12 and 3.24 times more than that of the marginal zone, respectively. Conclusion: The results indicated an increase in the mRNA expression of serotonin receptors (5HTR2A and 5HTR3A) in the tumor tissue compared to the marginal zone, which due to the mitogenic nature of these receptors, is likely to induce more proliferation of cancer cells.

In Silico Perspectives on the Prediction of the PLP's Epitopes involved in Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS). The main cause of the MS is yet to be revealed, but the most probable theory is based on the molecular mimicry that concludes some infections in the activation of T cells against brain auto-antigens that initiate the disease cascade. OBJECTIVES: The Purpose of this research is the prediction of the auto-antigen potency of the myelin proteolipid protein (PLP) in multiple sclerosis. MATERIALS AND METHODS: As there wasn't any tertiary structure of PLP available in the Protein Data Bank (PDB) and in order to characterize the structural properties of the protein, we modeled this protein using prediction servers. Meta prediction method, as a new perspective in silico, was performed to fi nd PLPs epitopes. For this purpose, several T cell epitope prediction web servers were used to predict PLPs epitopes against Human Leukocyte Antigens (HLA). The overlap regions, as were predicted by most web servers were selected as immunogenic epitopes and were subjected to the BLASTP against microorganisms. RESULTS: Three common regions, AA58-74, AA161-177, and AA238-254 were detected as immunodominant regions through meta-prediction. Investigating peptides with more than 50% similarity to that of candidate epitope AA58-74 in bacteria showed a similar peptide in bacteria (mainly consistent with that of clostridium and mycobacterium) and spike protein of Alphacoronavirus 1, Canine coronavirus, and Feline coronavirus. These results suggest that cross reaction of the immune system to PLP may have originated from a bacteria or viral infection, and therefore molecular mimicry might have an important role in the progression of MS. CONCLUSIONS: Through reliable and accurate prediction of the consensus epitopes, it is not necessary to synthesize all PLP fragments and examine their immunogenicity experimentally (in vitro). In this study, the best encephalitogenic antigens were predicted based on bioinformatics tools that may provide reliable results for researches in a shorter time and at a lower cost.

Association of New Putative Epitopes of Myelin Proteolipid Protein (58-74) with Pathogenesis of Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune disease in which auto-reactive T cells react with self-antigens expressed in the central nervous system (CNS). The main cause of MS is unknown. Nonetheless, the most probable theory is based on molecular mimicry, which suggests that some infections can activate T cells against brain auto-antigens like myelin proteolipid protein (PLP) and initiate the disease cascade. This study is conducted to evaluate the activatory effects of PLP58-74 on T lymphocytes and humoral immunity. PLP58-74 was considered as an immunodominant epitope candidate of PLP using bioinformatics tools. Patients and healthy individuals' peripheral blood mononuclear cells (PBMCs) were treated with PLP58-74 and its proliferative effects were evaluated through assessing proliferating cell nuclear antigen (PCNA) gene expression changes by real time PCR and immunocytochemistry assay. Finally, the rate of CD4+ and CD8+ T cells were assessed by flowcytometry. ELISA was also performed to measure anti PLP58-74 antibody in patients' serum. PLP58-74 induced proliferation in patients' PBMCs while it did not influence PBMCs of healthy individuals. CD4+ T cells were the main activated cells in reaction to PLP58-74 which increased from 22% to 39.91%. In addition, immune assay showed threefold increase in specific anti PLP58-74 IgG in patients compared to healthy controls. Results showed that PLP58-74 can stimulate CD4+ T cells and humoral immunity. Therefore it seems that the epitopes of some microorganisms mimicking PLP such as PLP58-74 might have a potential role in the initiation of MS.

Lipid production and mixotrophic growth features of cyanobacterial strains isolated from various aquatic sites.

The present study was conducted to determine the potential of five cyanobacteria strains isolated from aquatic zones to induce lipid production. The phylogenetic affiliation of the isolates was determined by 16S rRNA gene sequencing. Amongst the isolates, an efficient cyanobacterium, Synechococcus sp. HS01 showing maximal biomass and lipid productivity, was selected for further studies. In order to compare lipid productivity, the HS01 strain was grown in different media to screen potential significant culture ingredients and to evaluate mixotrophic cultivation. Mixotrophic cultivation of the strain using ostrich oil as a carbon source resulted in the best lipid productivity. GC analysis of fatty acid methyl esters of the selected cyanobacterial strain grown in media supplemented with ostrich oil showed a high content of C16 (palmitoleic acid and palmitic acid) and C18 (linoleic acid, oleic acid and linolenic acid) fatty acids of 42.7 and 42.8 %, respectively. Transmission electron micrographs showed that the HS01 cells exhibited an elongated rod-shaped appearance, either isolated, paired, linearly connected or in small clusters. According to initial experiments, ostrich oil, NaNO3 and NaCl were recognized as potential essential nutrients and selected for optimization of media with the goal of maximizing lipid productivity. A culture optimization technique using the response surface method demonstrated a maximum lipid productivity of 56.5 mg l(-1) day(-1). This value was 2.82-fold higher than that for the control, and was achieved in medium containing 1.12 g l(-1) NaNO3, 1 % (v/v) ostrich oil and 0.09 % (w/v) NaCl.