Background and purpose: Lonidamine is a hexokinase II inhibitor, works as an anticancer molecule, and is extensively explored in clinical trials. Limited information prevails about the stability-indicating methods which could determine the forced degradation of lonidamine under stressed conditions. Hence, we report the use of a rapid, sensitive, reproducible, and highly accurate liquid chromatography and mass spectrometry method to analyze lonidamine degradation. Experimental approach: The Xbridge BEH shield reverse phase C18 column (2.5 μm, 4.6 × 75 mm) using isocratic 50:50 water: acetonitrile with 0.1% formic acid can detect lonidamine with help of mass spectrometer in tandem with an ultraviolet (UV) detector at 260 nm wavelength. Findings/ Results: A linear curve with r² > 0.99 was obtained for tandem liquid chromatography-mass spectrometry (LC-MS)-UV based detections. This study demonstrated (in the present set up of isocratic elution) that LC-MS based detection has a relatively high sensitivity (S/N (10 ng/mL): 220 and S/N (20 ng/mL): 945) and accuracy at lower detection and quantitation levels, respectively. In addition to developing the LC-MS method, we also report that the current method is stability-indicating and shows that lonidamine gets degraded over time under all three stress conditions; acidic, basic, and oxidative. Conclusion and implications: LC-MS based quantitation of lonidamine proved to be a better method compared to high-performance liquid chromatography (HPLC)-UV detections for mapping lonidamine degradation. This is the first report on the stability-indicating method for studying the forced degradation of lonidamine using LC-MS method.

Background and purpose: Irregularities of angiogenesis may participate in the pathogenesis of diabetes complications. Pramlintide is an amylin analogue administered for the treatment of type 1 and type 2 diabetes. The present investigation aimed at surveying the effect of pramlintide on angiogenesis-related markers in human umbilical vein endothelial cells (HUVECs). Experimental approach: The proliferation of cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) method. The effect of pramlintide on migration was estimated by Transwell® assay. in vitro evaluation of angiogenesis was performed by tube formation assay. The secretion of vascular endothelial growth factor (VEGF) to the supernatant of HUVECs was measured by an enzyme- linked immunosorbent assay (ELISA) kit. All experiments were performed in triplicate. Findings / Results: Pramlintide exhibited no inhibitory effect on HUVECs proliferation. It significantly increased cell migration at the concentration of 1 μg/mL. Pramlintide (1 μg/mL) also enhanced average tubules length, size, and the mean number of junctions. However, there was not any significant change in VEGF release from HUVECs. Conclusion and implications: Findings of this research revealed the effect of pramlintide on angiogenesis- related markers via enhancing migration and tubulogenesis in vitro, suggesting a worthwhile proposition for further clinical researches on improving vascular complications and healing of diabetic wounds.

Background and purpose: Tobacco etch virus (TEV) protease, a 27 KDa protein, consists of the catalytic domain of nuclear inclusion a (NIa) protein produced by Tobacco etch virus. Because of its unique sequence, TEV protease is used for purging fusion tags from proteins. It also has many advantages such as stability and activity in a board range of temperature and pH and overproduction in Escherichia coli and these benefits make this protease valuable. Despite all these benefits, TEV protease has problems like low solubility (less than 1 mg/mL). There are methods for enhancing protein solubility and in this study, the effect of additives during cell lysis was studied. Experimental approach: Eleven different additives that made twelve different lysis buffers were used and their effect on TEV protease solubility analyzed by Plackett-Burman and response surface methodology methods. Findings / Results: Three best effective additives on TEV solubility (L-proline, sodium selenite, and CuCl2) were selected according to software analysis and the best concentration of them was applied to optimize TEV protease solubility. Conclusion and implications: The obtained results provided the composition of an optimum solvent for obtaining soluble TEV protease.

Background and purpose: The prevalence of leishmaniasis is reported in more than 98 countries and Iran is one of the endemic areas. There is no vaccine for this disease and few effective drugs are available to treat it. Moreover, drug resistance to the disease is increasing. During the past decade, several in vitro and in vivo studies have been performed on dihydropyrimidine derivatives as antileishmanial agents. Experimental approach: In the present project, a few 6-methyl-4-aryl-N-aryl dihydropyrimidinone/thiones (A7-A11) and N-heteroaryl-3-(para-methoxy benzyl) amino but-enamides (A1-A6) were synthesized, structurally characterized, and finally subjected to in vitro anti-leishmanial effect against Leishmania major promastigotes. Findings / Results: Results of the study showed that compound A10, 4-(3-chlorophenyl)-6-methyl-N-phenyl- 2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide, exhibited superior anti-leishmanial effect with IC50 value of 52.67 μg/mL (more active than standard drug Glucantim® with IC50 71000 ± 390 μg/mL). Conclusion and implications: It was demonstrated that some dihydropyrimidine thiones were able to inhibit Leishmania major promastigotes. Structure-activity relationship evaluations indicated that more electron-poor rings such as isoxazole afforded higher activity within A1-A6 series and in these derivatives, N-benzothiazole rings reinforced anti-leishmanial activity concerning thiazole. It was also observed that higher anti-parasite activities of A10 and A11 concerning A7-A9 might be related to the incorporation of the sulfur atom into C2 position, replacement of N-thiazole carboxamide by N-phenyl carboxamide on C5 position of dihydropyrimidine ring, and also replacement of para with meta-substituted phenyls within C4 of dihydropyrimidine ring. The results may help unveil new 4-aryl-5-carboxamide dihydropyrimidines as potential anti-leishmanial agents and their further structural modification toward more potent derivatives.

Background and purpose: Natural products are used to improve the damage caused by harmful reagents in various pathological situations. This study investigated the effect of grape sap as a natural product with antioxidant properties on follicle cell proliferation in bleomycin (as a chemotherapy agent with toxic effects on hair growth) treated rats skin. Experimental approach: The bleomycin treated rats were administrated grape sap. Wingless/integrated (wnt) and β-catenin gene expression as follicle proliferative markers were evaluated using real-time polymerase chain reaction. Furthermore, histological factors and total antioxidant capacity were evaluated. Findings / Results: The data showed that, grape sap increased the number of anagenic hair follicle in grape sap (100 mg/kg) group (P < 0.001), sebaceous glands (P < 0.001), blood vessel density (P < 0.001), and hair growth length (P < 0.001). Also, wnt and β-catenin gene expression was elevated. The data showed that wnt and β-catenin gene expression were elevated in grape sap treated animals versus bleomycin group (P < 0.01 and 0.001, respectively). Conclusion and implications: Our finding showed that grape sap can be effective in increasing hair growth a gains bleomycin toxic effects on skin hair growth.

Background and purpose: Articular cartilage defects aren’t repaired by itself. Numerous studies have been conducted in the area of cartilage tissue engineering and some of them considered herbal products. An attempt was made in this study to compare the effects of pomegranate fruit extract (PFE), avocado/soybean unsaponifiable (ASU), and their equal proportional mixture on the chondrogenesis of human adipose-derived stem cells (hADSCs). Experimental approach: PFE was prepared through the percolation method. ASU powder was dissolved in ethanol at 10 μg/mL concentration and was sterilized. The hADSCs first were isolated, expanded in monolayer culture and identified, and next seeded on fibrin scaffolds. The hADSCs/fibrin scaffolds were divided into 4 groups of control, ASU, PFE, and PFE+ ASU and subjected to in vitro induction for 2 weeks. The control group received chondrogenic medium, other groups received chondrogenic medium plus ASU, PFE, or PFE + ASU, respectively. The MTT assay was performed for cell viability evaluation, real-time polymerase chain reaction for expression of cartilage genes, and the toluidine blue, safranin-O, and immunohistochemistry for staining of the constructs. Findings / Results: Cell viability, cartilage genes expression, matrix staining density, and collagen II protein levels in PFE samples were significantly higher than those of the other groups (P < 0.05). Histological assessments revealed more chondrogenic centers (P < 0.05) in the PFE group compared to the other groups. Conclusion and implications: In this study, it was revealed that PFE can be considered as an induction factor for future chondrogenic studies.

Background and purpose: The aim of this study was to evaluate the in vitro and in vivo anti-Toxoplasma gondii (T. gondii) effect of 5-oxo-hexahydroquinoline compounds. Moreover, molecular docking study of the compounds into the active site of enoyl-acyl carrier protein reductase (ENR) as a necessary enzyme for the vitality of apicoplast was carried out. Experimental approach: A number of 5-oxo-hexahydoquinoline derivatives (Z1-Z4) were synthesized. The T. gondii tachyzoites of RH strain were treated by different concentrations (1-64 μg/mL) of the compounds. The viability of the encountered parasites with compounds was assessed using flow cytometry and propidium iodide (PI) staining. Due to the high mortality effect of Z3 and Z4 in vitro, their chemotherapy effect was assessed by inoculation of tachyzoites to four BALB/c mice groups (n = 5), followed by the gavage of various concentrations of the compounds to the mice. Molecular docking was done to study the binding affinity of the synthesized 5-oxo-hexahydroquinolines into ENR enzyme active site byusing AutoDock Vina® software. Docking was performed by a Lamarckian Genetic Algorithm with 100 runs. Findings / Results: Flow cytometry assay results indicated compounds Z3 and Z4 had relevant mortality effect on parasite tachyzoites. Besides, in vivo experiments were also performed and a partial increase of mice longevity between control and experiment groups was recorded. Molecular docking of Z3 and Z4 in the binding site of ENR enzyme indicated that the compounds were well accommodated within the binding site. Therefore, it could be suggested that these compounds may exert their anti-T. gondii activity through the inhibition of the ENR enzyme. Conclusion and implications: Compounds Z3 and Z4 are good leads in order to develop better anti-T. gondii agents as they demonstrated both in vitro and in vivo inhibitory effects on tachyzoites viability and infection. Further studies on altering the route of administration along with additional pharmacokinetics evaluations are needed to improve the anti-T. gondii impacts of 5-oxo-hexahydroquinoline compounds.

Background and purpose: Granulocyte colony-stimulating factor (G-CSF) is routinely used in combination with chemotherapy to battle neutropenia. However, studies suggest that this chemokine may increase the risk of metastasis and malignancy in many cancers. To counteract the adverse effects of G-CSF in cancer, antibodies have been used to block its action. However, antibodies are large and complex molecules which makes their production expensive. Thus in this study, we aim to construct different structure variants of the G-CSF receptor containing different domains and select the best variant that prevents the adverse actions of this chemokine. These novel structures are smaller than antibodies and easier to produce. Experimental approach: Different domains of the G-CSF receptor were designed and cloned into the pET28a expression vector. These recombinant receptor subunits were then expressed in Escherichia coli and purified using standard affinity chromatography techniques. Interaction of recombinant receptor subunits with G-CSF was assessed using enzyme-linked immunosorbent assay and NFS60 cells. Findings / Results: Two recombinant receptor subunits containing D1 + D2 + D3 domains and D2 domain showed the strongest inhibitory activity to G-CSF. Conclusion and implications: These novel recombinant receptor variants could be candidates for further studies in the development of novel therapeutics.

Background and purpose: Ovarian cancer is the deadliest cancer in women. The main challenge in the inhibition of ovarian cancer cells is chemo-resistance. Seeking to overcome this issue, several strategies have been suggested, including the administration of natural products. Grape seed extract (GSE) is a good source of polyphenols and its anticancer effects have been reported by many studies. In this study we aimed to evaluate the effects of GSE on OVCAR-3, a chemo-resistant ovarian cancer line. Experimental approach: OVCAR-3 cells were treated with GSE (71 μg/mL) for 24 and 48 h. Cell viability and cell apoptosis were measured by MTT and flow cytometry. The real-time polymerase chain reaction was used to determine the expression of genes involved in the cell cycle (PTEN, DACT1, AKT, MTOR, GSK3B, C-MYC, CCND1, and CDK4) and apoptosis (BAX, BCl2, CASP3, 8 and 9). The expression of CASP3 protein was evaluated by the CASP3 assay. Findings / Results: The results showed that treatment of OVCAR-3 cells with GSE, increased the expression level of PTEN and DACT1 tumor suppressor genes, as well as apoptotic genes, CASP3, 8, and 9 (P < 0.001). Also, the induction of tumor suppressor genes expression was associated with an increase in the expression of BAX/BCL2 gene ratio as pro- and anti-apoptotic genes. The expression of the genes involved in the cell cycle, CCND1 and CDK4, was inhibited (P < 0.001). The results indicated that GSE induced cell apoptosis in a time-dependent manner (P < 0.001). Also, the GSE treatment resulted in the CASP3 protein expression (P < 0.001). Conclusion and implications: According to the results of this study, GSE may exert anti-tumorigenic effects on chemo-resistant OVCAR-3 ovarian cancer cells which might be mediated by the expression of tumor suppressor genes that interact with cell signaling pathways, cell cycle, and cell apoptosis. Hence, the consumption of GSE extract during chemotherapy may overcome part of chemo-resistance in ovarian cancer.

Background and purpose: Polysaccharide sulfate is a major active phytochemical constituent of Caulerpa racemosa, whereas the Eleutherine americana (Aubl) Merr has antioxidant properties. The aim of this research was to investigate the combined effect of polysaccharide sulfate that was isolated from C. racemosa and E. americana on the macrophage activity. Experimental approach: The phenolic contents and antioxidant activities of E. americana extracts in water and various ethanol concentrations were studied using the Folin-Ciocalteu and 2,2-diphenyl-1-picryl-hydrazyl- hydrate (DPPH) methods, respectively. Polysaccharide sulfate was isolated from C. racemosa by precipitation method. To assess the macrophage activity, mice were treated orally for 14 days with either a combination of polysaccharide sulfate and E. americana 96% ethanol extract at a specific ratio or with each extract alone. Macrophages were isolated and the phagocytic activity was measured by assessing the ability of the macrophages to phagocytose latex particles and nitric oxide (NO) levels were assessed using a colorimetric assay. Findings / Results: The E. americana crude extract in water exhibited the highest yield (13.04%), compared with the extract in 96% ethanol, which had the highest phenolic content (6.37 ± 0.16 mg/g gallic acid equivalent) and the strongest antioxidant activity (IC50, 22.63 ± 1.09 μg/mL). The combination of extracts, when both extracts were administered at 65:65 mg/kg BW, resulted in the highest increases in phagocytosis activity (62.73 ± 5.77%) and NO levels (16.43 ± 1.37 μmol/L). Conclusion and implications: The results of this study confirmed the non-specific immunostimulant properties of the combination of polysaccharide sulfate and E. americana and justified their use in traditional medicine. The observed increase in macrophage activity appeared to be correlated with the increased ability of mice to fight infection.