LYZ2-SH3b as a novel and efficient enzybiotic against methicillin-resistant Staphylococcus aureus.

BACKGROUND: Enzybiotics are promising alternatives to conventional antibiotics for drug-resistant infections. Exolysins, as a class of enzybiotics, show antibacterial effects against methicillin-resistant Staphylococcus aureus (MRSA). This study evaluated a novel exolysin containing an SH3b domain for its antibacterial activity against MRSA. METHODS: This study designed a chimeric exolysin by fusing the Cell-binding domain (SH3b) from Lysostaphin with the lytic domain (LYZ2) from the gp61 enzyme. Subsequently, LYZ2-SH3b was cloned and expressed in Escherichia coli (E. coli). Finally, the antibacterial effects of LYZ2-SH3b compared with LYZ2 and vancomycin against reference and clinical isolates of MRSA were measured using the disc diffusion method, the minimal inhibitory concentration (MIC), and the minimal bactericidal concentration (MBC) assays. RESULTS: Analysis of bioinformatics showed that LYZ2-SH3b was stable, soluble, and non-allergenic. Protein purification was performed with a 0.8 mg/ml yield for LYZ2-SH3b. The plate lysis assay results indicated that, at the same concentrations, LYZ2-SH3b has a more inhibitory effect than LYZ2. The MICs of LYZ2 were 4 µg/mL (ATCC 43,300) and 8 µg/mL (clinical isolate ST239), whereas, for LYZ2-SH3b, they were 2 µg/mL (ATCC 43,300) and 4 µg/mL (clinical isolate ST239). This suggests a higher efficiency of LYZ2-SH3b compared to LYZ2. Furthermore, the MBCs of LYZ2 were 4 µg/mL (ATCC 43,300) and 8 µg/mL (clinical isolate ST239), whereas, for LYZ2-SH3b, they were 2 µg/mL (ATCC 43,300) and 4 µg/mL (clinical isolate ST239), thus confirming the superior lytic activity of LYZ2-SH3b over LYZ2. CONCLUSIONS: The study suggests that phage endolysins, such as LYZ2-SH3b, may represent a promising new approach to treating MRSA infections, particularly in cases where antibiotic resistance is a concern. But further studies are needed.

ß-Galactosidase: From its source and applications to its recombinant form.

Carbohydrate-active enzymes are a group of important enzymes playing a critical role in the degradation and synthesis of carbohydrates. Glycosidases can hydrolyze glycosides into oligosaccharides, polysaccharides, and glycoconjugates via a cost-effective approach. Lactase is an important member of ß-glycosidases found in higher plants, animals, and microorganisms. ß-Galactosidases can be used to degrade the milk lactose for making lactose-free milk, which is sweeter than regular milk and is suitable for lactose-intolerant people. ß-Galactosidase is employed by many food industries to degrade lactose and improve the digestibility, sweetness, solubility, and flavor of dairy products. ß-Galactosidase enzymes have various families and are applied in the food-processing industries such as hydrolyzed-milk products, whey, and galactooligosaccharides. Thus, this enzyme is a valuable protein which is now produced by recombinant technology. In this review, origins, structure, recombinant production, and critical modifications of ß-galactosidase for improving the production process are discussed. Since ß-galactosidase is a valuable enzyme in industry and health care, a study of its various aspects is important in industrial biotechnology and applied biochemistry.

Glucose oxidase: Applications, sources, and recombinant production.

Glucose oxidase is a subset of oxidoreductase enzymes that catalyzes the transfer of electrons from an oxidant to a reductant. Glucose oxidases use oxygen as an external electron acceptor that releases hydrogen peroxide (H2 O2 ). Glucose oxidase has many applications in commercial processes, including improving the color and taste, increasing the persistence of food materials, removing the glucose from the dried egg, and eliminating the oxygen from different juices and beverages. Moreover, glucose oxidase, along with catalase, is used in glucose testing kits (especially in biosensors) to detect and measure the presence of glucose in industrial and biological solutions (e.g., blood and urine specimens). Hence, glucose oxidase is a valuable enzyme in the industry and medical diagnostics. Therefore, evaluating the structure and function of glucose oxidase is crucial for modifying as well as improving its catalytic properties. Finding different sources of glucose oxidase is an effective way to find the type of enzyme with the desired catalysis. Besides, the recombinant production of glucose oxidase is the best approach to produce sufficient amounts of glucose oxidase for various uses. Accordingly, the study of various aspects of glucose oxidase in biotechnology and bioprocessing is crucial.

Development of a recombinant nucleocapsid protein-based ELISA for the detection of IgM and IgG antibodies to SARS-CoV-2.

Coronavirus 2019 (COVID-19) is a global concern for public health. Thus, early and accurate diagnosis is a critical step in management of this infectious disease. Currently, RT-PCR is routine diagnosis test for COVID-19, but it has some limitations and false negative results. enzyme-linked immunosorbent assay (ELISA) against SARS-CoV-2 antigens seems to be an appropriate approach for serodiagnosis of COVID-19. In the current study, an ELISA system, using a recombinant nucleocapsid (N) protein, was developed for the detection of IgM and IgG antibodies to SARS-CoV-2. The related protein was expressed, purified, and used in an ELISA system. Sera samples (67) for COVID-19 patients, as well as sera samples from healthy volunteers (112), along with sera samples from non-COVID-19 patients were examined by the ELISA system. The expression and purity of the recombinant N protein were approved by SDS-PAGE and Western blotting. The sensitivity of ELISA system was 91.04 and 92.53% for the detection of IgG and IgM antibodies, respectively. Moreover, the specificity of the developed ELISA system for IgG and IgM were 98.21 and 97.32%, respectively. Our developed ELISA system showed satisfactory sensitivity and specificity for the detection of antiSARS-CoV-2 IgM and IgG antibodies and could be used as a complementary approach for proper diagnosis of COVID-19.

Specific Targeting of Recombinant Human Pancreatic Ribonuclease 1 using Gonadotropin-Releasing Hormone Targeting Peptide toward Gonadotropin-Releasing Hormone Receptor-Positive Cancer Cells.

Background: Targeted drug delivery is a novel method to specifically deliver anticancer therapeutics to tumor sites. Gonadotropin-releasing hormone (GnRH) is a decapeptide, and its target binding property has attracted attention as a means of targeted drug delivery. Human pancreatic ribonuclease 1 (hpRNase1) has been shown to exert anticancer properties, when fused to a targeting moiety. The goal of the present study was to add a GnRH targeting peptide to the N-terminus of hpRNase1 to specifically target GnRH receptor (GnRH-R) expressing cells. Methods: This in vitro study was conducted at Shiraz Institute for Cancer Research (Shiraz, Iran) in 2019. The coding sequence of GnRH and hpRNase1 were fused, and the chimeric protein together with non-fused hpRNase1 were produced in E. coli (BL21). The recombinant proteins were purified, and their biological activity was evaluated using MTT and apoptosis assays. Non-parametric Kruskal-Wallis tests with Dunn's post hoc tests were performed to determine the significant differences between the study groups. Results: GnRH-hpRNase1 chimeric protein specifically inhibited the proliferation of PC-3 (P=0.021), LNCaP (P=0.034), and AD-Gn (P=0.041) cells, while the growth of negative cells (AD-293) was not significantly affected (P=0.081). GnRH-hpRNase1 decreased the IC50 values more than non-fused hpRNase1, by approximately 26.5-fold (P=0.036) for PC-3 cells, and exerted its growth inhibitory effects through apoptosis induction. Conclusion: Fusion of GnRH to hpRNase1 structure produced an enzyme, which could specifically target tumor cells. This approach can be used to eliminate tumors that harbor GnRH-R.

In Silico Design and Evaluation of PRAME+FliCΔD2D3 as a New Breast Cancer Vaccine Candidate.

Background: The most prevalent cancer in women over the world is breast cancer. Immunotherapy is a promising method to effectively treat cancer patients. Among various immunotherapy methods, tumor antigens stimulate the immune system to eradicate cancer cells. Preferentially expressed antigen in melanoma (PRAME) is mainly overexpressed in breast cancer cells, and has no expression in normal tissues. FliCΔD2D3, as truncated flagellin (FliC), is an effective toll-like receptor 5 (TLR5) agonist with lower inflammatory responses. The objective of the present study was to utilize bioinformatics methods to design a chimeric protein against breast cancer. Methods: The physicochemical properties, solubility, and secondary structures of PRAME+FliCΔD2D3 were predicted using the tools ProtParam, Protein-sol, and GOR IV, respectively. The 3D structure of the chimeric protein was built using I-TASSER and refined with GalaxyRefine, RAMPAGE, and PROCHECK. ANTIGENpro and VaxiJen were used to evaluate protein antigenicity, and allergenicity was checked using AlgPred and Allergen FP. Major histocompatibility complex )MHC( and cytotoxic T-lymphocytes )CTL( binding peptides were predicted using HLApred and CTLpred. Finally, B-cell continuous and discontinuous epitopes were predicted using ABCpred and ElliPro, respectively. Results: The stability and solubility of PRAME+FliCΔD2D3 were analyzed, and its secondary and tertiary structures were predicted. The results showed that the derived peptides could bind to MHCs and CTLs. The designed chimeric protein possessed both linear and conformational epitopes with a high binding affinity to B-cell epitopes. Conclusion: PRAME+FliCΔD2D3 is a stable and soluble chimeric protein that can stimulate humoral and cellular immunity. The obtained results can be utilized for the development of an experimental vaccine against breast cancer.

Evaluation of the two polymorphisms rs1801133 in MTHFR and rs10811661 in CDKN2A/B in breast cancer.

The 5,10-Methylenetetrahydrofolate reductase (MTHFR) was the rate-limiting enzyme in the methyl cycle, which was encoded by the MTHFR gene. MTHFR played a key role in homocysteine plasma level and was associated with the risk of breast cancer. The cyclin-dependent kinase (CDK) inhibitor (CDKN2A/B) was the tumor suppressor in the cell cycle regulation. The single-nucleotide polymorphism was thought to be associated with the predisposition of breast cancer and in subsequent immune response in different populations. The current study was conducted on a peripheral blood sample of 100 Iranian women with breast carcinoma and 142 cancer-free healthy female volunteers. The TaqMan real-time polymerase chain reaction technique was applied for genotyping of participants. The correlation of both variants and demographic data were investigated with the risk of breast cancer. Our data showed that the MTHFR allele T and TT genotype had the higher prevalence in patients (P < 0.0001) than the control group. The frequency of risk C allele into the CDKN2A/B rs10811661 was 72%. The correlations of menarche and underlying hormonal disorder with the risk of breast cancer were investigated; also our results showed that the menopause status was statistically significant between patients and controls (P = 0.036). Our investigations demonstrated that the MTHFR rs180113 and CDKN2A/B rs10811661 had a significant correlation with the elevated risk of breast cancer and they might be potentially valuable to apply as a prognostic factor for individual health care.

Seminal plasma proteomics as putative biomarkers for male infertility diagnosis.

Male infertility represents a significant global public health issue that is currently emerging as a prominent research focus. Presently, laboratories adhere to the guidelines outlined by the World Health Organization (WHO) manuals for conducting routine semen analysis to diagnose male infertility. However, the accuracy of results in predicting sperm quality and fertility is limited because some individuals with a normal semen analysis report, an unremarkable medical history, and a physical examination may still experience infertility. As a result, the importance of employing more advanced techniques to investigate sperm function and male fertility in the treatment of male infertility and/or subfertility becomes apparent. The standard test for evaluating human semen has been improved by more complex tests that look at things like reactive oxygen species (ROS) levels, total antioxidant capacity (TAC), sperm DNA fragmentation levels, DNA compaction, apoptosis, genetic testing, and the presence and location of anti-sperm antibodies. Recent discoveries of novel biomarkers have significantly enriched our understanding of male fertility. Moreover, the notable biological diversity among samples obtained from the same individual complicates the efficacy of routine semen analysis. Therefore, unraveling the molecular mechanisms involved in fertilization is pivotal in expanding our understanding of factors contributing to male infertility. By understanding how these proteins work and what role they play in sperm activity, we can look at the expression profile in men who can't have children to find diagnostic biomarkers. This review examines the various sperm and seminal plasma proteins associated with infertility, as well as proteins that are either deficient or exhibit aberrant expression, potentially contributing to male infertility causes.

Finding Appropriate Signal Peptides for Secretory Production of Recombinant Glucarpidase: An In SilicoMethod.

BACKGROUND: Methotrexate (MTX) is a general chemotherapeutic agent utilized to treat a variety of malignancies, woefully, its high doses can cause nephrotoxicity and subsequent defect in the process of MTX excretion. The recombinant form of glucarpidase is produced by engineered E. coli and is a confirmed choice to overcoming this problem. OBJECTIVE: In the present study, in silico analyses were performed to select suitable SPs for the secretion of recombinant glucarpidase in E. coli. METHODS: The signal peptide website and UniProt database were employed to collect the SPs and protein sequences. In the next step, SignalP-5.0 helped us to predict the SPs and the position of cleavage sites. Moreover, physicochemical properties and solubility were evaluated using Prot- Param and Protein-sol online software, and finally, ProtCompB was used to predict the final subcellular localization. RESULTS: Luckily, all SPs could form soluble fusion proteins. At last, it was found that PPB and TIBA could translocate the glucarpidase into the extracellular compartment. CONCLUSION: This study showed that there are only 2 applicable SPs for the extracellular translocation of glucarpidase. Although the findings were remarkable with high degrees of accuracy and precision based on the utilization of bioinformatics analyses, additional experimental assessments are required to confirm and validate it. Recent patents revealed several inventions related to the clinical aspects of vaccine peptides against human disorders.

The protective effect of Zataria multiflora Boiss. hydroalcoholic extract on TNF-α production, oxidative stress, and insulin level in streptozotocin-induced diabetic rats.

OBJECTIVE: Oxidative stress leads to reactive oxygen species (ROS) overproduction, which causes tissue injury in diabetic patients. The aim of this study was to evaluate the effects of Zataria multiflora extract on TNF-α, oxidative stress products, and insulin levels as well as lipid profile in diabetic rats. MATERIALS AND METHODS: Rats were randomly divided into 6 groups of 10 animals. Diabetes was induced by a single injection of streptozotocin (STZ). Control and diabetic control rats orally received 1 mL/day of normal saline, whereas the other three groups received 250, 500 and 1000 mg/kg/day of Z. multiflora extract, and one non-diabetic group orally received 1000 mg/kg/day Z. multiflora extract, for 28 days. At the end of the treatment course, rats were anesthetized and their serum samples were analyzed for TNF-α, malondialdehyde (MDA), super oxide dismutase (SOD), total antioxidant capacity (TAC), lipid profile, total plasma protein, blood glucose, insulin, and liver enzymes levels. RESULTS: Our results showed that cholesterol, LDL, TG, MDA and TNF-α levels decreased, but HDL, SOD, TAC, and total protein increased significantly in the diabetic group receiving 1000 mg/kg Z. multiflora compared to the diabetic control group (P<0.05). Moreover, blood glucose level was significantly reduced following administration of different concentrations of Z. multiflora. Liver sections of diabetic rats treated with Z. multiflora 1000 mg/kg showed normal hepatocytes and restoration of liver architecture. CONCLUSION: Z. multiflora extract ameliorated oxidative stress, TNF-α serum level, lipid abnormality, blood glucose, and liver damage in rats with diabetes mellitus.

CEA Plasmid as Therapeutic DNA Vaccination against Colorectal Cancer.

BACKGROUND: Human colorectal cancer cells overexpress carcinoembryonic antigen (CEA). CEA is a glycoprotein which has shown to be a promising vaccine target for immunotherapy against colorectal cancer. OBJECTIVES: To design a DNA vaccine harboring CEA antigen and evaluate its effect on inducing immunity against colorectal cancer cells in tumor bearing mice. METHODS: In the first step the coding sequence of the CEA was cloned into the pcDNA3.1 vector. The mice were injected with the vaccine construct and the immune responses were monitored during the experiment period. The specific IgG anti-CEA, IFN-γ, IL-2 and IL-4 were measured by ELISA and levels of IFN-γ was detected by ELISpot assay. The lymphocyte proliferation was assessed using a 5-bromo-2-deoxyuridine (BrdU) cell proliferation assay kit. RESULTS: Immunization of the mice with the CEA plasmid resulted in stimulation of CEA-specific T cell and antibody responses. The serum level of specific IgG antibodies against CEA was increased in immunized mice. Moreover, the injection of CEA plasmid led to the stimulation of T-helper-1 by increase in the secretion of IFN-γ, IL-2 and lymphocyte proliferation response. CONCLUSION: As the CEA DNA vaccine displayed encouraging antitumor effects, therefore, we suggest that it can be a potential therapeutic modality for colorectal cancer and is worthy of further investigation.

Hydatid Cyst Surgeries in Patients Referred to Hospitals in East Azerbaijan Province during 2009-2011.

BACKGROUND: Hydatidosis, as the most important zoonotic parasitic disease in Iran, has posed many health and economic losses. This study was conducted to investigate the demographic characteristics of hydatid cyst surgeries in hospitals of East Azerbaijan Province, Northwest of Iran. METHODS: Demographic characteristics of all patients with hydatid cyst surgery in hospitals of the province, during 2009-2011 were gathered including age, gender, occupation, number and location of the cyst, clinical symptoms, place of residence and history of contact with dog. They were extracted from reports of health center and were analyzed using STATA 11 software. RESULTS: Out of 52 hydatid cyst surgeries, 27 cases were females. Mean age of patients was 38.3 yr. Liver was reported as the most involved organ. The most clinical symptoms were abdominal and liver pain. Housewives comprised the most victims of the disease. Forty seven percent of patients had one cyst and 59% had the history of contact with dog. The majority of the patients were living in rural areas. CONCLUSION: Due to the high costs of diagnosis and treatment of hydatidosis, collecting data on the prevalence and transmission of the disease as well as on vulnerable groups seems to be essential as the first step in controlling and preventing the disease.