Use of Green Fluorescent Protein (GFP) Vector in Classical Restriction Enzyme-based Cloning Methods of Gateway Cloning System

Generating of an expression clone that can produce the pharmaceutical proteins in an efficient and soluble form at high levels is considered as an important step in pharmaceutical industry. Recombination-based cloning could be a quick and efficient way for generating expression vectors. Thus, both efficient and robust subcloning is vital for the construction of gene expression vectors. In this study, we used the traditional restriction enzyme-based cloning methods for generation of expression-ready clones by the most well-known commercial cloning technologies, Gateway

Mice fetus liver tissue spectroscopic alterations following concomitant administration of Metronidazole & Miconazole

Introduction: Metronidazole (MET) and Miconazole (MIC) are antiprotozoal and imidazole antifungal agents, respectively. The aim of this study was to assess the usefulness of Fourier Transform Infrared micro-spectroscopy (FTIR-MSP) for discriminating and detecting spectral changes of mouse fetus liver tissue after mother’s exposure to concomitant use of MET and MIC. Methods and Results: The control group received only normal diet and the exposure groups received MET, MIC, and concomitant MET and MIC. The pregnant mice were anesthetized and their fetuses were surgically removed on gestation day 15. Fixative slides without any staining were put on KBr disc with a 1mm thick for IR micro spectroscopy. All information obtained from the spectra were analyzed using routine FTIR software. Based on our results, MET and MIC, alone and in combination together, have created some changes in the mice fetus liver biomolecules. Conclusions: FTIR biospectroscopy application in teratology is very challenging, but it has been accepted as a reliable technique; however, it is necessary to do more investigations

Processing on recognition of FTIR-MSP alteration of Heart tissue during mice fetal life

Introduction: Understanding of fetus development is one of the most complicated with a great impact on Teratology. FTIR-MSP is among the most useful spectroscopy technique for biological and cellular application. In this project, various statistical calculations were used for recognition and discrimination of heart tissue spectra during 9.5-17.5 days of mice fetal life. Method and Results: The mice fetuses were dissected on day 9.5-17.5 of gestation and then fixed by fixative solution. Tissue sections (10 µm) were used for FTIR-MSP measurement in the wavenumber region of 4000-400 cm-1. Spectra were preceded by baseline correction, smoothing, deconvolution and 2nd derivatisation. PCA, ANN and SVM have been used to find the most relevant modifications in during fetus development. PCA with adjusting data mass and seven selected major PCs have been used to find the most relevant modifications in different steps of mice fetus heart tissue development and also BP-FF ANN and SVM classifications could diagnose different steps of development up to 96.3% and 92.59% respectively. Conclusions: PCA, ANN and SVM methods could classify and discriminate the FTIR spectroscopic data and can be as a new potential tool for the teratogenic investigations

Isolation and characterization of novel phage displayed scFv antibody for human tumor necrosis factor alpha and molecular docking analysis of their interactions

Introduction: Tumor necrosis factor alpha (TNF-α) expression amplifies to excess amounts in several disorders such as rheumatoid arthritis and psoriasis. Although, Anti-TNF biologics have revolutionized the treatment of these autoimmune diseases, formation of anti-drug antibodies (ADA) has dramatically affected their use. The next generation antibodies (e.g. Fab, scFv) have not only reduced resulted immunogenicity, but also proved several benefits including better tumor penetration and more rapid blood clearance.This study highlights the use of phage display for identification of human single chain fragment antibody against disulfide-bonded TNF-α using phage display technology. Methods and Results: Using affinity selection procedures in this study, a scFv antibody clone was isolated from naïve Tomlinson I phage display library that specifically recognizes and binds to TNF-α. The TNF-α recombinant protein was expressed in genetically engineered Escherichia coli SHuffle® T7 Express, for the first time, which is able to express disulfide-bonded recombinant proteins into their correctly folded states. Conclusions: ELISA-based affinity characterization results indicated that the isolated novel 29.2 kDa scFv binds TNF-α with suitable affinity. In silico homology modeling study using ‘ModWeb’ as well as molecular docking study using Hex program confirmed the scFv and TNF-α interactions with a scFv-TNF- α binding energy of around -593 kj/mol which is well in agreement with our ELSIA results. The cloned scFv antibody may potentially be useful for research and therapeutic applications in the future

Chemopreventive effect of quince (Cydonia oblonga Mill.) fruit extract on hepatocellular carcinoma induced by diethylnitrosamine in rats

Introduction: Hepatocellular carcinoma (HCC) or primary liver cancer is one of the most prevalent and deadliest cancers, which has been increasing greatly worldwide. Diethylnitrosamine (DEN) is a well-known environmental toxin and potent hepatocarcinogenic dialkylnitrosoamine present in air, water, and in a number of foodstuffs. In the present study, we evaluated preventive effect of aqueous extract of quince (Cydonia oblonga Mill.) fruit (ACO) against DEN-induced hepatocellular carcinoma (HCC) in rats. Methods and Results: The model of hepatocellular carcinoma was induced by a single intraperitoneal injection of DEN (200 mg/kg) as an initiator that after two weeks followed by daily oral administration of 2-acetylaminofluorene (30 mg/kg) as a promoter for two weeks. Quince-treated rats were pretreated with ACO intragastrically at three different doses two weeks prior to DEN injection. The marked reduction of serum biomarkers of liver damage and cancer, including alfa-fetoprotein (AFP), gamma glutamyl transpeptidase (GGT), alanine transaminase (ALT), and aspartate transaminase (AST) were observed in ACO supplemented animals as compared with HCC rats at the end of the experiment. Moreover, the quince extract exhibited in vivo antioxidant activity by elevating glutathione (GSH) contents as well as preventing lipid peroxidation in the liver tissues of DEN-treated rats. The relative weight of liver was also reduced in quince-treaded rats as a prognostic marker in HCC. Conclusions: Our results clearly demonstrated that quince has a chemopreventive effect against HCC in rats and can be proposed as a promising candidate for the prevention of DEN-induced hepatocarcinogenesis. &nbsp

Prevention of liver cancer by standardized extract of Melissa officinalis L. in a rat model of hepatocellular carcinoma: Its potential role as a chemopreventive agent

Introduction: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and the third most common cause of cancer-related death worldwide. Melissa officinalis L. (M. officinalis L.), known as lemon balm is a medicinal plant, which has a wide range of pharmacological properties. This study was aimed to assess the chemopreventive effect of aqueous extract of M. officinalis (AMO) against diethyl nitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. Methods and Results: The model of hepatocellular carcinoma was induced by a single intraperitoneal injection of DEN (200 mg/kg) as an initiator and after two weeks was followed by daily oral administration of 2-acetylaminofluorene (30 mg/kg) as a promoter for two weeks. Lemon balm-treated rats were pretreated with AMO intragastrically at three different doses two weeks prior to DEN injection. At the end of the experiment, the marked reduction of serum biomarkers of liver damage and cancer, including alfa-fetoprotein (AFP), gamma glutamyl transpeptidase (GGT), alanine transaminase (ALT), and aspartate transaminase (AST) were observed in AMO complemented rats compared to DEN-treated animals. Furthermore, the extract exhibited in vivo antioxidant activity by elevating GSH concentration and preventing lipid peroxidation in the liver tissues of HCC rats. The relative weight of liver was also reduced in lemon balm-treated rats as a prognostic marker in HCC. Conclusion: Our findings demonstrated that M. officinalis has a chemopreventive effect against HCC in rats and can be suggested as a potential agent for the prevention of primary liver cancer. &nbsp

Cancer chemoprevention by oleaster (Elaeagnus angustifoli L.) fruit extract in a model of hepatocellular carcinoma induced by diethylnitrosamine in rats

Hepatocellular carcinoma (HCC) is a frequent and fatal human cancer with poor diagnosis that accounts for over half a million deaths each year worldwide. Elaeagnus angustifolia L. known as oleaster has a wide range of pharmacological activities. This study aimed to investigate the chemopreventive effect of aqueous extract of E. angustifolia fruit (AEA) against diethylnitrosamine (DEN)-induced HCC in rats. HCC was induced in rats by a single injection of DEN (200 mg/kg) as an initiator. After two weeks, rats were orally administered 2-acetylaminofluorene or 2-AAF (30 mg/kg) as a promoter for two weeks. Oleaster-treated rats were orally pretreated with the increasing doses of AEA two weeks prior to DEN injection that continued until the end of the experiment. In the current study, a significant decrease in serum biomarkers of liver damage and cancer, including alfa-fetoprotein (AFP), gamma glutamyl transpeptidase (GGT), alanine transaminase (ALT), and aspartate transaminase (AST) was observed in AEA-treated rats when compared to HCC rats. Furthermore, the oleaster extract exhibited in vivo antioxidant activity by elevating reduced glutathione (GSH) contents as well as preventing lipid peroxidation in the liver tissues of DEN-treated rats. The relative weight of liver, a prognostic marker of HCC, was also reduced in oleaster-treated rats. To conclude, our results clearly demonstrated that oleaster fruit possesses a significant chemopreventive effect against primary liver cancer induced by DEN in rats. It can be suggested that the preventive activity of oleaster against hepatocarcinogenesis may be mediated through the antioxidant, anti-inflammation, and antimutagenic effects of the fruit

Antioxidant and chemopreventive effects of Asperugo procumbens in a rat model of hepatocellular carcinoma

Introduction: Hepatocellular carcinoma (HCC) cancer is the fifth most common malignancy, with 0.25–1 million new cases diagnosed annually worldwide. The objective of the present study was to evaluate the antioxidant and chemopreventive effects of aqueous extract of Asperugo procumbens L. (AAP) against diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. Methods and Results: The model of hepatocellular carcinoma was induced by a single intraperitoneal injection of DEN (200 mg/kg) as an initiator that after two weeks followed by daily oral administration of 2-acetylaminofluorene (30 mg/kg) as a promoter for two weeks. AAP-treated rats were pretreated with the extract intragastrically at three different doses two weeks prior to DEN injection. At the end of the experiment, the marked reduction of serum biomarkers of liver damage and cancer, including alfa-fetoprotein (AFP), gamma glutamyl transpeptidase (GGT), alanine transaminase (ALT), and aspartate transaminase (AST) were observed in AAP complemented rats as compared to DEN-treated animals. Besides, the extract exhibited in vivo antioxidant activity that evident by increasing GSH concentration along with lipid peroxidation prevention in the liver tissues of HCC animals. In addition, A. procumbens showed in vitro free radical scavenging activity that determined by 1, 1-Diphenyl-2-picryl hydroxyl (DPPH) antioxidant assay. The relative weight of liver was also reduced in AAP-treaded rats as a prognostic marker in HCC. Conclusions: Our results obviously confirmed that A. procumbens possesses a chemopreventive effect against primary liver cancer induced by DEN in rats as well as  in vitro and in vivo antioxidant activities