Current Pharmaceutical Biotechnology - Volume 20, Issue 6, 2019

Tuberculosis (TB) is an infectious disease that mainly affects the lungs and spreads to other organs of the body through the haematogenous route. It is one of the ten major causes of mortality worldwide. India has the highest incidence of new- and multidrug-resistant (MDR) - TB cases in the world. Bacille Calmette-Guerin (BCG) is the vaccine commonly available against TB. BCG does offer some protection against serious forms of TB in childhood but its protective effect wanes with age. Many new innovative strategies are being trailed for the development of effective and potent vaccines like mucosal- and epitope-based vaccines, which may replace BCG or boost BCG responses. The use of nanotechnology for diagnosis and treatment of TB is also in the pipeline along with many other vaccines, which are under clinical trials. Further, in-silico models were developed for finding new drug targets and designing drugs against Mycobacterium tuberculosis (Mtb). These models offer the benefit of computational experiments which are easy, inexpensive and give quick results. This review will focus on the available treatments and new approaches to develop potent vaccines for the treatment of TB.

Background: In this study, local sustainable enzyme sources involving excised digestive juice of African land snail and yeast were utilized to achieve the simultaneous saccharification (SSF) and fermentation (SSF) of plantain pseudostem (PPS) waste, and afterwards their effects on terpenoids using gas chromatography coupled to a flame ionization detector (GC-FID), were examined. Methods: The most abundant terpenoids were found in the order α-pinene > borneol > camphor > humulene > β-caryophellene, while the least in abundance were cis ocimene (8.78x10-6 mg/100g), and cyperene (1.81x10-5 mg/100g). The application of exclusive fermentation and SSF respectively elevated azuluene by 95.46 and 99.6%, while pinene-2-ol was elevated by 83.02 and 98.57%, respectively. Results: Both exclusive fermentation and SSF had no effect on myrcene, cyperene, ethyl cinnamate, germacrene b, valencene, beta selinene, aromadendrene, and taraxerol, while the degree of degradation of some of the terpenoids by both processes was respectively as follows; gama muurolene (100%), β-caryophyllene (97.60 and 93.14%), α-terpinenyl acetate (91.95 and 83.16%), geranyl acetate (94.81 and 43.87%), and terpinen-4-ol (94.40 and 57.00%). Conclusion: The findings of this study encourage the imminent application of simultaneous saccharification and fermentation for the enhancement of bioactivities of terpenoids.

Background: Zinc oxide nanoparticles (ZnO NPs) are increasingly utilized in both industrial and medical applications. Therefore, the study was aimed to investigate the effect of green nanoparticle complex (green tea extract/zinc oxide nanoparticles complex, GTE/ZnO NPs) on oxidative stress induced by monosodium glutamate (MSG) on the liver of rats. Methods: Wistar male rats (n=64) weighing between 200-250 g were divided randomly into eight groups: control group was given physiological saline (1 mg/kg), two groups were treated with two different doses of MSG (MSG-LD, MSG-HD; 6 and 17.5 mg/Kg, respectively), GTE was given 1 mg/mL, 5th group was treated with ZnO NPs and 6th group was treated with GTE/ZnO NPs complex while, 7th and 8th groups were treated with MSG-LD + GTE/ZnO NPs complex and MSG-HD + GTE/ZnO NPs complex, respectively. All substances were given orally for 30 consecutive days. At the end of the study, the liver was homogenized for measurement of the oxidative stress status and anti-inflammatory biomarkers as well as histological and transmission alternations. Results: Results showed that the antioxidant enzymes activity and glutathione level were significantly decreased in MSG groups than control in a dose-dependent manner. Conversely, the malondialdehyde and inflammatory cytokines levels were significantly increased in MSG groups than the control group. The liver indicated no evidence of alteration in oxidative status, anti-inflammatory and morphological parameters in GTE, ZnO NPs and GTE/ZnO NPs complex groups. Conclusion: In conclusion, MSG at both doses caused oxidative stress and inflammation on liver after 28 days of exposure that supported histological analysis and transmission view of hepatic parenchyma. GTE/ZnO NPs act as partial hepato-protective against MSG.

Background: Heparin is an essential drug used as an anticoagulant. Access to raw material suitable for heparin extraction is critical for creating a viable business opportunity. In Saudi Arabia, large amounts of raw material with potential for heparin extraction are wasted. Objective: To extract heparin and low-molecular-weight heparin (LMWH) from the camel lung, and measure its potency and activity. Methods: Heparin preparation included three steps: extraction, electrophoretic identification, and activity measurement. Fresh lung tissue (100 g) was minced and homogenized in a blender. Crude heparin extracts were prepared using Charles’s or Volpi’s method with slight modifications. Heparin was purified by electrophoresis using high-purity agarose gels in barium acetate buffer. The heparin activity of purified samples was assayed spectrophotometrically using commercial heparin kits. Results: Charles’s and Volpi’s extraction methods were simple and easy to establish. The yield was 90 mg crude heparin per 100 g of camel lung tissue following Volpi’s extraction protocol, whereas Charles’s method did not yield any heparin. The separation of heparin and LMWH by gel electrophoresis resulted in sharp and clear product bands using material prepared according to Volpi’s method. The heparin preparation had an anti-factor Xa activity of 37 IU/mg, indicating weak potency. Conclusion: Preparation of active heparin from camel lung tissue is a technology applicable in manufacturing. Further method development is needed to increase heparin purity and potency.

Background: EDIL3 is an extracellular matrix protein that plays a key role in angiogenesis. Changes in the pattern of its expression also affect cellular processes and the tumor microenvironment. Elevated level of EDIL3 is considered an unfavorable prognostic marker of survival. Objective: The aim of this study was to evaluate the changes in EDIL3 expression in endometrial cancer at various degrees of its differentiation (G1-G3) and to discuss its potential role as a molecular diagnostic marker and therapeutic target. Methods: The study group consisted of 45 patients with endometrial cancer: G1, 17; G2, 15; G3, 13. The control group (C) included 15 patients without neoplastic changes. The expression of EDIL3 was assessed using immunohistochemistry. Statistical analysis was performed using the Statistica 12 PL software (p<0.05). Results: Analysis of EDIL3 expression showed that the average optical density of the reaction product in G1 reached 130% of the control, while the values in G2 and G3 were 153% and 158%, respectively. Regardless of the endometrial cancer grade, an increase in EDIL3 level was observed compared to the control. Conclusion: In our study, we demonstrated overexpression of EDIL3 protein in endometrial cancer. Differences in expression between degrees of tumor differentiation suggest the potential of using changes in EDIL3 level as a new complementary diagnostic marker and target for anti-angiogenic therapy.

Background: Pullulan can partially or completely replace starch or fat, thanks to which it can be used for the production of dietetic food. It allows you to maintain the desired consistency, and increases the viscosity of the product. Objective: Therefore, an attempt was made to produce yogurts with pullulan addition and determine the effect of its presence on the number of LAB, pH change, titratable acidity, and sensory quality of yogurts during storage at 4°C for 28 days. Methods: The effect of addition of 0; 1.0 and 2.0 % w/v pullulan as a yogurt ingredient on the changes in pH, acidity, LAB number, and sensory quality of yogurt during storage at 4°C for 28 days was examined. Results: Pullulan did not affect yogurt pH, but the addition of 2.0 % w/v pullulan increased the acidity of yogurt as compared to that of the control yogurt. A statistically significantly higher total number of Lactobacillus, by approximately 1 logarithmic cycle, was found in yogurts with pullulan than in the control yogurt on the 28th day of storage. The yogurt with 1.0% w/v pullulan addition showed better sensory characteristics than that with 2.0% pullulan w/v addition. Conclusion: The conducted research proved that the presence of pullulan in the culture medium stimulates the growth of selected lactic acid bacteria and influences their fermentation capacity. The use of 1.0 % w/v pullulan during the production of yogurts allowed to obtain a sensorically acceptable product, it had a protective effect on the number of lactic acid bacteria during 28 days of refrigerated storage of yogurt.

Background: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. Objectives: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. Methods: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Results: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. Conclusion: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

Background: Lower human salivary aldehyde dehydrogenase (hsALDH) activity increases the risk of aldehyde mediated pathogenesis including oral cancer. Alliin, the bioactive compound of garlic, exhibits many beneficial health effects. Objective: To study the effect of alliin on hsALDH activity. Methods: Enzyme kinetics was performed to study the effect of alliin on the activity of hsALDH. Different biophysical techniques were employed for structural and binding studies. Docking analysis was done to predict the binding region and the type of binding forces. Results: Alliin enhanced the dehydrogenase activity of the enzyme. It slightly reduced the Km and significantly enhanced the Vmax value. At 1 μM alliin concentration, the initial reaction rate increased by about two times. Further, it enhanced the hsALDH esterase activity. Biophysical studies indicated a strong complex formation between the enzyme and alliin (binding constant, Kb: 2.35 ± 0.14 x 103 M-1). It changes the secondary structure of hsALDH. Molecular docking study indicated that alliin interacts to the enzyme near the substrate binding region involving some active site residues that are evolutionary conserved. There was a slight increase in the nucleophilicity of active site cysteine in the presence of alliin. Ligand efficiency metrics values indicate that alliin is an efficient ligand for the enzyme. Conclusion: Alliin activates the catalytic activity of the enzyme. Hence, consumption of alliincontaining garlic preparations or alliin supplements and use of alliin in pure form may lower aldehyde related pathogenesis including oral carcinogenesis.

Background: The study aimed to investigate the effects of the active ingredient, nimodipine, on chondrocyte proliferation and extracellular matrix (ECM) structures in cartilage tissue cells. Methods: Chondrocyte cultures were prepared from tissues resected via surgical operations. Nimodipine was then applied to these cultures and molecular analysis was performed. The data obtained were statistically calculated. Results: Both, the results of the (3-(4,5 dimethylthiazol2-yl)-2,5-diphenyltetrazolium (MTT) assay and the fluorescence microscope analysis [a membrane permeability test carried out with acridine orange/ propidium iodide staining (AO/PI)] confirmed that the active ingredient, nimodipine, negatively affects the cell cultures. Conclusion: Nimodipine was reported to suppress cellular proliferation; chondroadherin (CHAD) and hypoxia-inducible factor-1 alpha (HIF-1α) expression thus decreased by 2.4 and 1.7 times, respectively, at 24 hrs when compared to the control group (p < 0.05). Furthermore, type II collagen (COL2A1) expression was not detected (p < 0.05). The risk that a drug prescribed by a clinician in an innocuous manner to treat a patient by relieving the symptoms of a disease may affect the proliferation, differentiation, and viability of other cells and/or tissues at the molecular level, beyond its known side effects or adverse events, should not be forgotten.