Optimization of expression, extraction & purification of the N-terminal region of ipaD gene in Shigella dysenteriae by proteomics analysis

زمینه و هدف: باکتری شیگلا دیسانتری یکی از عوامل پاتوژن مهم است که علی‌رغم تلاش‌های چندین ساله برای تهیه واکسن علیه آن هنوز مطالعات گسترده پیرامون آن ادامه دارد. محصولات پلاسمید تهاجمی شیگلا (Ipa) نقش مهمی در تهاجم باکتری ایفا می‌کنند. پروتئین IpaD یکی از اعضای این خانواده است که به عنوان کاندید واکسن شیگلا مطرح می‌باشد. مطالعات متعدد بر روی این پروتئین نشان داده که ناحیه N- ترمینال آن نقش مهمی در فرآیند تهاجمی باکتری دارد. این مطالعه با هدف بهینه سازی بیان N- ترمینال ژن IpaD به منظور افزایش تولید پروتئین نو ترکیب انجام شد. روش بررسی: در این مطالعه تجربی-آزمایشگاهی باکتری E. coli BL21(DE3) حامل پلاسمید pET-28a که ژن ناحیه N- ترمینال IpaD در آن همسانه سازی شده بود جهت مطالعات مورد استفاده قرار گرفت. پس از کشت باکتری، تاثیر سه فاکتور زمان القا، دما و غلظت ماده القا کننده ایزو-پروپیل-تایوبتا دی گالاکتوپیرانوزید (IPTG) بر میزان بیان، با استفاده از ژل سدیم دو دسیل سولفات-پلی آکریل آمید (SDS-PAGE) به صورت کیفی بررسی گردید. با استفاده از تصاویر دو بعدی تهیه شده از ژل‌ها با کمک نرم افزار آنالیز ژل‌های دو بعدی بررسی کمی بیان پروتئین صورت پذیرفت. مراحل استخراج و تخلیص پروتئین نوترکیب با کمک روش شیب اوره آغاز و با عبور نمونه‌ها از ستون کروماتوگرافی پایان یافت. یافته‌ها: نتایج بر روی ژل‌های SDS-PAGE نشان داد که میزان تقریبا مشابهی از تولید پروتئین نوترکیب در زمان‌ القا، دما و غلظت های مختلف IPTG بیان وجود دارد، اما یافته‌های نرم افزاری نشان داد بهترین شرایط بیان ناحیه N- ترمینال پروتئین IpaD در وکتور pET-28a دمای 37 درجه سانتی‌گراد، غلظت 7/0 میلی مولار IPTG و زمان 3 ساعت بعد از القا می‌باشد. نتیجه‌گیری: بر اساس نتایج این مطالعـــه هر پروتئین بعد از فرآیند همسانه سازی شرایط بیان مخصوص به خود را دارا می‌باشد که شرایط دمایی و طول زمان القا سلول‌ها در مقدار تولید پروتئین موثرتر می‌باشند

A Comprehensive Overview of Hepatitis Virus Genotyping Methods

The identification and differentiation of the various genotypes of hepatitis viruses are of great importance and can contribute to both clinical procedures and scientific research. There is a wide range of typing methods, which can be mainly divided into phenotypic and genotypic methods. Here, we focused on the available genotyping methods for hepatitis virus typing and tried to review and categorize them. This study aimed to study various hepatitis virus genotyping methods. In this study, to obtain a comprehensive overview of hepatitis virus genotyping methods, a perfect search was performed using related keywords in major journals and databases. Information was extracted from articles, analyzed, categorized, and compared. In analyzing the various articles, genotyping methods were divided into three different categories: Sequencing methods, hybridization methods, and methods based on DNA binding patterns. Sequencing-based methods were cited as the gold standard and the accuracy of other methods was compared to them. Hybridization-based methods, which were also used in commercial kits, include several methods. DNA binding pattern-based methods were mainly based on PCR with genotype-specific primers or RFLP. Although molecular methods allow accurate, sensitive, and reproducible genotyping, there is still much room to be exploited using emerging methods

Antibacterial activity of ethyl acetate and aqueous extracts of Mentha longifolia L. and hydroalcoholic extract of Zataria multiflora Boiss. plants against important human pathogens

AbstractObjectiveTo determine the potential antibacterial activity of ethyl acetate and aqueous extracts from Mentha longifolia L. (M. longifolia) and hydroalcoholic extract of Zataria multiflora Boiss. (Z. multiflora) against important human pathogens.MethodsPseudomonas aeruginosa, Shigella dysenteriae, Klebsiella pneumoniae (K. pneumonia), Enterobacter cloacae, Salmonella typhi, Proteus mirabilis, Serratia marcescens, Bacillus cereus, Staphylococcus saprophyticus and Staphylococcus aureus were kinds of pathogenic bacteria to determine the antibacterial effect of aqueous and ethyl acetate extracts of M. longifolia and hydroalcoholic extract of Z. multiflora using broth microdiluation method.ResultsThe lowest minimum inhibitory concentration and minimum bactericidal concentration values for K. pneumonia and Pseudomonas aeruginosa (1.25 and 2.5 mg/mL) were observed by the hydroalcoholic extract of Z. multiflora and the lowest minimum inhibitory concentration and minimum bactericidal concentration values for K. pneumonia and Serratia marcescens (2.5 and 5 mg/mL) were observed by the aqueous extracts of M. longifolia.ConclusionsIn conclusion, it seems that Z. multiflora and M. longifolia extracts could inhibit the growth of all of the mentioned bacteria

Cloning and Expression of N-terminal Region of IpaD from Shigella dysenteriae in E. coli

Genus Shigella is one of the important members of the family enterobacteriacae. There are numerous antigens in Shigella carrying by a 220 kb plasmid. Among them, IpaD is the key virulence factor of S. flexneri. Apart from having effectors function that is essential for host cell invasion and intracellular survival, this protein also controls the secretion and translocation of other effector proteins into eukaryotic host cells. In the present study, we have cloned and expressed the ipaD in E. coli. The ipaD gene was amplified by PCR. Prokaryote expression vector pET-28a(+)- ipaD was constructed, and used to transform E. coli BL21DE3 plySs. The expression of recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot were used to determine immunoreactivity of IpaD-His by a rabbit monoclonal antibodies against his-tag. SDS-PAGE demonstrated that the constructed prokaryotic expression efficiently produced IpaD at the 1 mmol/L of IPTG. IpaD protein was able to react with the rabbit monoclonal antibody against His-tag.  IpaD is essential for Shigella spp invasion. N-terminal region is most significant functional fragment of IpaD. Purification of IpaD from the wild type of Shigella is difficult furthermore profound study on a specific domain on the N-terminal of IpaD by using the wild type of purified IpaD is not feasible.

Association between COVID-19 and Gastrointestinal Manifestations and Available Treatment Options

Background & Objective: Corona virus disease 2019 (COVID-19) is an emerging outbreak which has involved almost all of the countries of the world now. While the main symptoms of the disease are known to be respiratory symptoms like coughing and shortness of breath, extrapulmonary symptoms have also been reported in many cases of COVID-19. Gastrointestinal (GI) manifestations including diarrhea, nausea and vomiting, abdominal pain and liver injury are amongst the most common extrapulmonary symptoms in COVID-19 patients. Materials & Method: We used Scopus, PubMed, and Google scholar databases for this review. The last search was run on November 21, 2020 Results: Liver injury is mostly accompanied by an elevation in AST and ALT levels and a slight increase in serum billirubin levels that is observed in approximately 14.8-53.1% of COVID-19 patients.1-29% of COVID-19 patients present nausea and vomiting and 2 to 10% develop diarrhea. Abdominal pain is seen in about 2.2-6% of COVID-19 patients and most frequently seen in severely ill patients. Conclusion: Diarrhea, nausea, and vomiting and liver injury are the most common GI symptoms in COVID-19 patients while abdominal pain is not pretty common. There are no medications of proven efficacy to treat COVID-19 or its GI manifestations so far

Surface engineering of solid supports; Cellulose, Nitrocellulose and Nylon to

increase the efficiency of antibody immobilization in diagnostic system

Immunotoxins Immunotherapy against Hepatocellular Carcinoma: A Promising Prospect

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. Therefore, fighting against such cancer is reasonable. Chemotherapy drugs are sometimes inefficient and often accompanied by undesirable side effects for patients. On the other hand, the emergence of chemoresistant HCC emphasizes the need for a new high-efficiency treatment strategy. Immunotoxins are armed and rigorous targeting agents that can purposefully kill cancer cells. Unlike traditional chemotherapeutics, immunotoxins because of targeted toxicity, insignificant cross-resistance, easy production, and other favorable properties can be ideal candidates against HCC. In this review, the characteristics of proper HCC-specific biomarkers for immunotoxin targeting were dissected. After that, the first to last immunotoxins developed for the treatment of liver cancer were discussed. So, by reviewing the strengths and weaknesses of these immunotoxins, we attempted to provide keynotes for designing an optimal immunotoxin against HCC

Investigating the effect of Curcumin on Long Noncoding RNAs NUTM2A-AS1 and HCG18 expression changes in Hepatocellular Carcinoma (HCC): Curcumin on Hepatocellular Carcinoma (HCC)

Hepatocellular carcinoma (HCC) is a common tumor in liver tissue. The lack of molecular markers for the diagnosis and evaluation of treatment methods is noticeable despite the progression of HCC diagnostic and treatment methods; therefore, the present study aimed to find molecular markers with key roles in the initiation and progression of HCC and investigate the impact of curcumin on their expression. To this end, bioinformatics studies were conducted to candidate two long noncoding RNAs (lncRNAs) related to HCC, which interacted with the highest number of microRNAs (NUTM2A-AS1 and HCG18), and five microRNAs (hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-195-5p, and hsa-miR-424-5p), which interacted with most lncRNAs among candidate lncRNAs. Afterward, their expression was measured in hepG2 cancer cells and fibroblast cells treated with curcumin compared to the untreated samples. The expression of miRNAs and lncRNAs at half inhibition concentrations (IC50) of curcumin indicated that curcumin significantly increased the expression of miR-195, miR-15a/16, and miR-424 and reduced the expression of miR-15b-5P, NUTM2A-AS1 lncRNA, and HCG18 in comparison with a control group (P≤0.05). Obtained data showed that curcumin increases the expression of anti-cancer genes, miR-195, miR-15a/16, and miR-424, and decreases the activity of cancerous genes, miR-15b-5P, NUTM2A-AS1 lncRNA, and HCG18; therefore, it can be used as an anti-cancer agent in the treatment of HCC

Investigation of NSD2 Protein Expression Changes in Hepatocellular Carcinoma Cells After Treatment With Curcumin and Phthalates: Investigation of NSD2 Protein Expression Changes in HCC Cancer Cells

Background: Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide. The role of molecular markers in the development and progression of this cancer has been extensively studied. The overexpression of the 90-kilobase protein methyltransferase NSD2 (nuclear receptor binding SET domain- protein 2) is associated with tumor development and some types of cancers. This study aims to investigate the changes in NSD2 protein expression in HCC cells after treatment with curcumin and phthalates.Method: This study compared the NSD2 protein expression in HepG2 cancer cells and fibroblast cells that were either untreated or treated with the half-maximal inhibitory concentration (IC50%) of phthalates, curcumin, or their combination. The Western blot method and protein quantification were used to detect and determine NSD2 protein levels, and ImageJ software was used to analyze the desired bands.Results: Curcumin, phthalates, and their combination reduced the expression of NSD2 in HepG2 cancer cells and normal fibroblast cells compared to untreated cells (P<0.001). This decrease in expression was more significant in cells treated with both curcumin and phthalates than in treatment with curcumin or phthalates alone.Conclusion: The IC50% of curcumin, phthalates, and their combination can reduce NSD2 expression, where the effect of the combined form is greater. Therefore, the combination of phthalates and curcumin is recommended as a potential anti-cancer agent against HCC cells with an effect on reducing NSD2 expression

DT389-YP7, a Recombinant Immunotoxin against Glypican-3 That Inhibits Hepatocellular Cancer Cells: An In Vitro Study

Hepatocellular carcinoma (HCC) is one of the high-metastatic types of cancer, and metastasis occurs in one-third of patients with HCC. To maintain the effectiveness of drug compounds on cancer cells and minimize their side effects on normal cells, it is important to use new approaches for overcoming malignancies. Immunotoxins (ITs), an example of such a new approach, are protein-structured compounds consisting of toxic and binding moieties which can specifically bind to cancer cells and efficiently induce cell death. Here, we design and scrutinize a novel immunotoxin against an oncofetal marker on HCC cells. We applied a truncated diphtheria toxin (DT389) without binding domain as a toxin moiety to be fused with a humanized YP7 scFv against a high-expressed Glypican-3 (GPC3) antigen on the surface of HCC cells. Cytotoxic effects of this IT were investigated on HepG2 (GPC3+) and SkBr3 (GPC3−) cell lines as positive- and negative-expressed GPC3 antigens. The dissociation constant (Kd) was calculated 11.39 nM and 18.02 nM for IT and YP7 scfv, respectively, whereas only IT showed toxic effects on the HepG2 cell line, and decreased cell viability (IC50 = 848.2 ng/mL). Changing morphology (up to 85%), cell cycle arrest at G2 phase (up to 13%), increasing intracellular reactive oxygen species (ROSs) (up to 50%), inducing apoptosis (up to 38% for apoptosis and 23% for necrosis), and an almost complete inhibition of cell movement were other effects of immunotoxin treatment on HepG2 cells, not on SkBr3 cell line. These promising results reveal that this new recombinant immunotoxin can be considered as an option as an HCC inhibitor. However, more extensive studies are needed to accomplish this concept