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.

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.

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.

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.

The neuroprotective effect of BSA-based nanocurcumin against 6-OHDA-induced cell death in SH-SY5Y cells.

OBJECTIVE: Parkinson's disease (PD) is regarded as the second most common neurodegenerative disease affecting elderly population. There is a tendency toward finding natural cures to suppress the initiation and progression of this disease. Some epidemiological studies indicated lower incidence of PD in populations that consume curry. Curcumin, as the main ingredient of turmeric, has been supposed to have a protective role against PD progression. However, low bioavailability of curcumin is still a challenge in evaluation of the therapeutic potential of this substance. In this study, we aimed to produce a BSA-based nanocurcumin to assess its effect on 6-hydroxydopamine (6-OHDA)-induced death and Akt signaling disruption in SH-SY5Y cells. MATERIALS AND METHODS: BSA-based nanocurcumin was produced using desolvation method. Human neuroblastoma cells were treated with OHDA with/without different doses of nanocurcumin and MTT test was used to assess their viability besides observing cells morphological changes. The protective doses of nanocurcumine were chosen according to MTT results and western blot studies were done to assess p-Akt/t-Akt ratio. RESULTS: 6-OHDA exposure led to decreased cell viability, while nanocurcumin at doses of 400 and 500 nM prevented cell death. Moreover, this nanoformulation of curcumin restored p-Akt/t-Akt decrement induced by 6-OHDA. The protective effect of BSA-based nanocurcumin was estimated to be at least 4 time higher than that of natural curcumin according to the MTT results. CONCLUSION: It seems that BSA-based nanocurcumin can be regarded as a potent substitute for natural curcumin in protecting SH-SY5Y cell as a cellular model of PD.

Curcumin-Loaded BSA Nanoparticles Protect More Efficiently Than Natural Curcumin Against Scopolamine-Induced Memory Retrieval Deficit.

INTRODUCTION: There is evidence indicating that the rate of AD is lower in curry consuming populations. Then, there is an effort to elucidate if curcumin -as the main ingredient of turmeric-might affect the process of AD. However, in clinical trials of AD, a six-month curcumin treatment failed to show any progress, which might be attributable to its low bioavailability. In this line, a recent human study revealed that a more bioavailable solid lipid curcumin enhances cognition in aged adults. By the application of Bovine Serum Albumin (BSA), the current study aimed at converting curcumin to nano sizes and assessing its protective effects against scopolamine-induced passive avoidance memory retrieval deficit. METHODS: Nanocurcumin was prepared via dissolution method. Male NMRI mice (20-25 g body weight) were used. The effective doses of nanocurcumin were selected according to the initial pilot test. The mice were treated with nanocurcumin 15 or 20 mg/kg/p.o or distilled water for 10 days. The animals were habituated and trained in passive avoidance apparatus on the day 10. The retention test was performed 24 hours later. Scopolamine (1 mg/kg/i.p.) or saline was injected 30 minutes before memory retention trial. RESULTS: The findings indicated that nanocurcumin in doses 15 or 20 mg/kg/p.o prevented the retrieval deficit induced by scopolamine while natural curcumin in its equivalent doses did not have such an effect. Furthermore, nanocurcumin by itself improved memory retention comparing with the control group. CONCLUSION: These findings implied that the potential anti-amnesic effects of curcumin might be observed by producing and using its nanoformulation form.

CdS nanocrystals/graphene oxide-AuNPs based electrochemiluminescence immunosensor in sensitive quantification of a cancer biomarker: p53.

An ultrahigh sensitive, simple and reliable Electrochemiluminescence (ECL) immunosensor for selective quantification of p53 protein was designed according to the enhancement effects of AuNPs on ECL emission of CdS nanocrystals (CdS NCs). CdS NCs were immobilized on the glassy carbon electrode and AuNPs introduced to the process through formation of a sandwich-type immunocomplex between first anti-p53/p53/ secondary anti-p53. ECL of CdS NCs firstly evoked the SPR of AuNPs which in return amplified the CdS NCs ECL intensity. By using graphene oxide in immunosensor fabrication procedure, and attaching more AuNPs on the surface of the electrode, the ECL intensity was further increased resulting in much higher sensitivity. After applying the optimum conditions, the linear range of the developed immunosensor was found between 20 and 1000 fg/ml with a calculated limit of detection of 4 fg/ml. Moreover, the interference, reproducibility and storage stability studies of the immunosensor were investigated. Finally, immunosensor's authenticity was evaluated by detecting the p53 protein in human spikes which offers it as a potential in early detection of cancer, monitoring the cancer progress and clinical prognosis.

Intraperitoneal immunization with Urease loaded N-trimethyl Chitosan nanoparticles elicits high protection against Brucella melitensis and Brucella abortus infections.

Brucella (B) species are brucellosis causative agents, a worldwide zoonotic illness causing Malta fever in humans and abortion in domestic animals. In this work, we evaluated the vaccine potential of Trimethyl chitosan (TMC) nanoparticles formulation of Urease (TMC/Urease) against brucellosis. TMC/Urease nanoparticles and urease without any adjuvant were separately administered both orally and intraperitoneally. Intraperitoneal (i.p.) administration of urease alone as well as oral administration of both TMC/Urease nanoparticles and urease alone, elicited low titers of specific immunoglobulin G (IgG), while i.p. immunization with TMC/Urease nanoparticles induced high specific IgG production levels. As it was indicated by the cytokine assay and the antibody isotypes, i.p. immunization by urease alone, and TMC/Urease nanoparticles induced a mixed Th1-Th2 immune response, whereas oral administration of both urease alone and TMC/Urease nanoparticles induced a mixed Th1-Th17 immune response. In lymphocyte proliferation assay, spleen cells from i.p.-vaccinated mice with TMC/Urease nanoparticles showed a strong recall proliferative response. Vaccinated animals were challenged with virulent strains of B. melitensis and B. abortus. I.p. vaccination with TMC/Urease nanoparticles resulted in a high degree of protection. Altogether, our results indicated that TMC nanoparticles are a potent delivery system for i.p.-administered Brucella antigens.

Using gold nanoparticles in diagnosis and treatment of melanoma cancer.

Several studies have been devoted to clear functionalization of gold nanoparticles (AuNPs) in different fields such as cellular and molecular biology, microbiology, immunology and physiology. In line with the high diagnostic value of AuNPs, its therapeutic application has been intensively developed in tumour therapy, in recent years. One of the best clinical applications of AuNPs is its use in targeted delivery of anti-cancer drugs. Recent studies have focused on the application of AuNPs to treat melanoma - a malignant neoplasm sourced from melanocytes skin cells - with poor prognosis in advanced stages. Furthermore, early diagnosis can be successfully achieved through utilizing this technique even at early stages with localized distribution. Herein, this study details the previous researches focusing on the use of AuNPs as a novel diagnostic and therapeutic option in management of melanoma.

Oral immunization of mice with Omp31-loaded N-trimethyl chitosan nanoparticles induces high protection against Brucella melitensis infection.

Brucellosis is a group of closely associated zoonotic bacterial illnesses caused by members of the genus Brucella. B. melitensis Omp31 is a promising candidate for a subunit vaccine against brucellosis. This study surveyed the immunogenicity of Omp31 alone and with incomplete Freund's adjuvant (Omp31-IFA) and N-trimethyl chitosan (TMC/Omp31) nanoparticles (NPs), as well as the effect of Omp31 immunization route on immunological responses and protection. After expression and purification, the recombinant Omp31 (rOmp31) was loaded onto TMC NPs by ionic gelation. The particle size, loading efficiency and in vitro release of the NPs were examined. Omp31-IFA was administered intraperitoneally, while TMC/Omp31 NPs were administered orally and intraperitoneally. According to the antibody subclasses and cytokine profile, intraperitoneal immunization by Omp31-IFA and TMC/Omp31 NPs induced T helper 1 (Th1) and Th1-Th2 immune responses, respectively. On the other hand, oral immunization with TMC/Omp31 NPs elicited a mixed Th1-Th17 immune response. Data obtained from the cell proliferation assay showed that vaccination with Omp31 stimulated a vigorous antigen-specific cell proliferative response, which could be further increased after oral immunization with TMC/Omp31 NPs. Vaccinated groups of mice when challenged with B. melitensis 16M were found to be significantly protected in the orally administered group in comparison with the intraperitoneally immunized mice. Results of this study indicated that the reason for high protection after oral vaccination can be via elicited Th17 response.

Subcutaneous immunization with a novel immunogenic candidate (urease) confers protection against Brucella abortus and Brucella melitensis infections.

Brucellosis is a world prevalent endemic illness that is transmitted from domestic animals to humans. Brucella spp. exploits urease for survival in the harsh conditions of stomach during the gastrointestinal infection. In this study, we examined the immune response and the protection elicited by using recombinant Brucella urease (rUrease) vaccination in BALB/c mice. The urease gene was cloned in pET28a and the resulting recombinant protein was employed as subunit vaccine. Recombinant protein was administered subcutaneously and intraperitoneally. Dosage reduction was observed with subcutaneous (SC) vaccination when compared with intraperitoneal (IP) vaccination. rUrease induced mixed Th1-Th2 immune responses with high titers of specific IgG1 and IgG2a. In lymphocyte proliferation assay, splenocytes from IP and SC-vaccinated mice displayed a strong recall proliferative response with high amounts of IL-4, IL-12 and IFN-γ production. Vaccinated mice were challenged with virulent Brucella melitensis, B. abortus and B. suis. The SC vaccination route exhibited a higher degree of protection than IP vaccination (p value ≤ 0.05). Altogether, our results indicated that rUrease could be a useful antigen candidate for the development of subunit vaccines against brucellosis.

Survey of Omp19 immunogenicity against Brucella abortus and Brucella melitensis: influence of nanoparticulation versus traditional immunization.

Brucellosis is the most common zoonotic bacterial disease. Prevention of human brucellosis is achieved through pasteurization of dairy products, appropriate sanitation and vaccination of domestic animals against the Brucella species. B. abortus unlipidated 19 kDa outer membrane protein (U-Omp19) is a promising candidate for a subunit vaccine against brucellosis. This study investigates immunogenicity of Omp19 alone and with Freund's adjuvant (Omp19-IFA) and N-trimethyl chitosan (TMC/Omp19) nanoparticles, as well as the effect of Omp19 administration route on immunological responses and protection. The omp19 gene was expressed in E. coli BL21 (DE3). After purification, the recombinant Omp19 was loaded onto TMC nanoparticles by ionic gelation with tripolyphosphate. Particle size and loading efficiency of the nanoparticles were determined. Omp19-IFA was administered intraperitoneally while TMC/Omp19 nanoparticles were administered orally and intraperitoneally. The results indicated that intraperitoneal (i.p.) immunization by Omp19-IFA and TMC/Omp19 nanoparticles induced Th1 and Th2 immune responses, respectively, whereas oral immunization of TMC/Omp19 nanoparticles induced a mixed Th1/Th17 immune response. Moreover, oral immunization increased IgA levels in feces. Immunized mice were challenged with virulent B. melitensis 16 M and B. abortus 544. Oral immunization with TMC/Omp19 nanoparticles induced a remarkably high protection level against B. melitensis and B. abortus. The results showed that immunization route has a pivotal role in immune response polarization and protective efficiency of Omp19 antigen. Also, it was deduced that the higher protection level achieved through oral administration of TMC/Omp19 nanoparticles may be due to the elicited Th17 response.

Mitochondrial complex I gene variations; as a potential genetic risk factor in pathogenesis of multiple sclerosis.

BACKGROUND AND PURPOSE: Multiple sclerosis (MS) is an autoimmune-mediated inflammatory and debilitating disease of the central nervous system. Several investigations have suggested that the mitochondrial DNA encoded subunits of complex I gene variations are involved in the progression of MS. In this study, we investigated the possible association between mitochondrial complex I gene variations and MS in a Filipino population. MATERIAL AND METHODS: A total of 300 individuals were included in the present study, two-hundred patients with MS clinical symptoms, and one-hundred healthy subjects without MS clinical features. We amplified target genes of mtDNA using polymerase chain reaction technique (PCR), and sequenced these to evaluate mitochondrial complex I gene variations. RESULTS: We found nine variations (Nt 4216 T>C, Nt 5153 A>G, Nt 10142 C>T, Nt 11353 T>C, Nt 11935 T>C, Nt 12062 C>T, Nt 13042 G>A, Nt 13708 G>A and Nt 14179 G>A) in mtDNA-encoded complex I subunit genes. Our results showed that the prevalence of ND1, ND2, ND3, ND4 and ND5 gene variations was significantly higher in patients than in healthy controls (P<0.0001). Whereas, the frequency of Nt 14179 G>A variation in ND6 gene was significantly higher in the control group compared with the patients (P<0.0001). CONCLUSION: Taken together our data supports a strongly positive association between mitochondrial complex I gene variations and MS pathogenesis in a Filipino population.