Designing a Secretory form of RTX-A as an Anticancer Toxin: An In Silico Approach.

BACKGROUND: Cancer is a leading cause of death and a significant public health issue worldwide. Standard treatment methods such as chemotherapy, radiotherapy, and surgery are only sometimes effective. Therefore, new therapeutic approaches are needed for cancer treatment. Sea anemone actinoporins are pore-forming toxins (PFTs) with membranolytic activities. RTX-A is a type of PFT that interacts with membrane phospholipids, resulting in pore formation. The synthesis of recombinant proteins in a secretory form has several advantages, including protein solubility and easy purification. In this study, we aimed to discover suitable signal peptides for producing RTX-A in Bacillus subtilis in a secretory form. METHODS: Signal peptides were selected from the Signal Peptide Web Server. The probability and secretion pathways of the selected signal peptides were evaluated using the SignalP server. ProtParam and Protein-sol were used to predict the physico-chemical properties and solubility. AlgPred was used to predict the allergenicity of RTX-A linked to suitable signal peptides. Non-allergenic, stable, and soluble signal peptides fused to proteins were chosen, and their secondary and tertiary structures were predicted using GOR IV and I-TASSER, respectively. The PROCHECK server performed the validation of 3D structures. RESULTS: According to bioinformatics analysis, the fusion forms of OSMY_ECOLI and MALE_ECOLI linked to RTX-A were identified as suitable signal peptides. The final proteins with signal peptides were stable, soluble, and non-allergenic for the human body. Moreover, they had appropriate secondary and tertiary structures. CONCLUSION: The signal above peptides appears ideal for rationalizing secretory and soluble RTX-A. Therefore, the signal peptides found in this study should be further investigated through experimental researches and patents.

The unconditioned fear response in vertebrates deficient in dystrophin.

Dystrophin loss due to mutations in the Duchenne muscular dystrophy (DMD) gene is associated with a wide spectrum of neurocognitive comorbidities, including an aberrant unconditioned fear response to stressful/threat stimuli. Dystrophin-deficient animal models of DMD demonstrate enhanced stress reactivity that manifests as sustained periods of immobility. When the threat is repetitive or severe in nature, dystrophinopathy phenotypes can be exacerbated and even cause sudden death. Thus, it is apparent that enhanced sensitivity to stressful/threat stimuli in dystrophin-deficient vertebrates is a legitimate cause of concern for patients with DMD that could impact neurocognition and pathophysiology. This review discusses our current understanding of the mechanisms and consequences of the hypersensitive fear response in preclinical models of DMD and the potential challenges facing clinical translatability.

The KLF14 Variant is Associated with Type 2 Diabetes and HbA1C Level.

The purpose of this study was to scan variants in coding region of Krȕppel like factor14 (KLF14) locus and assess association related to type 2 diabetes (T2D) in Iranian population. We sequenced the coding region of KLF14 to scan variants in case-sibling study (92 individuals with T2D and 92 healthy older siblings). To confirm, we analyzed rs76603546 association with T2D in a larger unrelated case-control study by PCR-RFLP (475 cases and 512 controls). We analyzed the association of rs76603546 with HbA1C, BMI, fat mass, waist circumference, fasting glucose, cholesterol and HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) using one-way ANOVA analysis. Also, association of genotypes with T2D adjusted for confounding variables was analyzed using logistic regression. HaploReg v 4.1 was used to predict rs76603546 possible function. Sequencing results analysis revealed the association of C allele of rs76603546, synonymous variant C>T, [OR 2.10 (1.38-3.20), P value < 0.001] and CC genotype of rs76603546 [OR 4.3 (1.79-10.23), P value = 0.001] with susceptibility to T2D. PCR-Restriction Fragment Length Polymorphism (RFLP) results analysis confirmed the association of rs76603546 with T2D [C allele, OR 1.91 (1.59-2.29), P value = 0.002, CC genotype, OR 3.27 (2.26-4.73), P value = 0.002 and TC genotype, OR 1.74 (1.31-2.31), P value = 0.001]. The CC genotype of rs76603546 is associated with HbA1C level (P value < 0.001) and BMI (P value = 0.02). After adjustment with confounding variables, we observed association of CC genotype with T2D [OR 2.542 (1.25-3.77), P value = 0.03]. Among over 220 SNPs, rs76603546 was associated with T2D, HbA1C and BMI in our study.

Design and evaluation of scFv-RTX-A as a novel immunotoxin for breast cancer treatment: an in silico approach.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer (BC) patients. Hence, immunotherapy is a proper treatment option for HER2-positive BC patients. Accumulating evidence has indicated that immunotoxin therapy is a novel approach to improve the potency of targeted therapy. Immunotoxins are antibodies or antibody fragments coupled with a toxin. We designed an immunotoxin. The physicochemical properties were evaluated using ProtParam servers and secondary structure was examined by PROSO II and GORV. Using I-TASSER, a 3D model was built and refined by GalaxyRefine. The model was validated using PROCHECK and RAMPAGE. To predict immunotoxin allergenicity and mRNA stability, AlgPred server and RNAfold were used. Furthermore, the immunotoxin and HER2 were docked by ZDOCK. The scFv+RTX-A could be a non-allergenic and stable chimeric protein, and the secondary structure of its components did not alter, and this protein had a proper 3D structure that might have stable mRNA structure which could bind to HER2. Given the fact that the designed immunotoxin was a non-allergenic and stable chimeric protein and that it could bind with high affinity to HER2 receptors, we proposed that this chimeric protein could be a useful candidate for HER-2 positive BC patients.

Altered expression patterns of complement factor H and miR-146a genes in acute-chronic phases in experimental autoimmune encephalomyelitis mouse.

Considerable advances have been made in identification of the involvement of immune modulators in diseases. There is growing evidence on the role of complement pathway in pathogenesis and course of multiple sclerosis (MS). Moreover, it has been recognized that microRNAs (miRNAs) play an essential role in modulation and development of immune response in the central nervous system. We aimed to investigate the expression profile of complement factor H (CFH) and miR-146a genes in experimental autoimmune encephalomyelitis (EAE) mouse model of MS to detect the possible roles of CFH and miR-146a as biomarkers of MS disease stats. Expression of CFH and miR-146a genes in liver and brain tissues of EAE mice was measured in acute and chronic phases of disease compared to matched controls using real-time polymerase chain reaction. In the liver, increased expression of CFH gene was observed in the chronic phase compared to the acute phase. However, no significant difference was observed between acute and chronic phase mice with normal mice, while miR-146a expression was significantly decreased in livers of EAE mice in chronic group compared to acute and control groups. The expression of CFH gene in brain had a significant decrease in acute and chronic phases compared to healthy mice. Taken together, these observations indicate probable implication of complement system and miR-146a in course of immune-related diseases and reveal more facts about the pathogenesis of MS. However, further work is needed to determine protein levels of CFH and other possible targets of miR-146a in serum and cerebrospinal fluid of MS patients.

Association between p53 codon 72 (Arg72Pro) polymorphism and primary open-angle glaucoma in Iranian patients.

BACKGROUND: Glaucomatous neuropathy is a type of cell death due to apoptosis. The p53 gene is one of the regulatory genes of apoptosis. Recently, the association between the p53 gene encoding for proline at codon 72 and primary open-angle glaucoma (POAG) has been studied in some ethnic groups. This study is the first association analysis of POAG and p53 codon 72 polymorphism in Iranian patients. METHODS: A cohort of 65 unrelated patients with POAG (age range from 12-62 years, mean ± SD of 40.16 ± 17.51 years) and 65 unrelated control subjects (without glaucoma, age range of 14-63 years, mean ± SD of 35.64 ± 13.61 years) were selected. In Iranian POAG patients and normal healthy controls, the p53 codon 72 polymorphism in exon 4 was amplified using polymerase chain reaction. The amplified DNA fragments were digested with the BstUI restriction enzyme, and the digestion patterns were used to identify the alleles for the polymorphic site. RESULTS: Comparisons revealed significant differences in allele and genotype frequencies of Pro72Arg between POAG patients and control group. A higher risk of POAG was associated with allele Pro (OR = 2.1, 95% CI = 1.2-3.4) and genotype Pro/Pro (OR = 3.9, 95% CI = 0.13-12.7). CONCLUSION: The p53 Pro72 allele was more frequent in Iranian POAG patients than in the control group (P<0.05). The present findings show that the individuals with the Pro/Pro genotype may be more likely to develop POAG. However, additional studies are necessary to confirm this association.