Search results for: enzyme assay

Authors: Kornkanok Noulta, Pornsri Pakeyangkoon, Stephen T. Dubas, Pomthong Malakul, Manit Nithithanakul

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In recent years, interest in the development of material for tissue engineering application has increased considerably. Poly(High Internal Phase Emulsion) (PolyHIPE) foam is a material that is good candidate for used in tissue engineering application due to its 3D structure and highly porous with interconnected pore. The PolyHIPE was prepared from poly (styrene/ethylene glycol dimethacrylate) through high internal phase emulsion polymerization technique and loaded with hydroxyapatite (HA) to improve biocompatibility. To further increase hydrophilicity of the obtained polyHIPE, layer-by-layer polyelectrolyte multilayers (PEM) technique was used. A surface property of polyHIPE was characterized by contact angle measurement. Morphology and pore size was observed by scanning electron microscope (SEM). The cell viability was revealed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay technique.

Keywords: polyelectrolyte multilayer thin film, high internal phase emulsion, polyhipe foam, scaffold, tissue engineering

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Authors: Apisaraporn Baicharoen, Prapasiri Pongprayoon

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Betaine aldehyde dehydrogenase 2 (BADH2) is an enzyme that inhibits the accumulation of 2-acetyl-1-pyrroline (2AP), a potent flavor compound in rice fragrance. BADH2 contains three domains (NAD-binding, substrate-binding, and oligomerization domains). It catalyzes the oxidation of amino aldehydes. The lack of BADH2 results in the formation of 2AP and consequently an increase in rice fragrance. To date, inadequate data on BADH2 structure and function are available. An insight into the nature of BADH2 can serve as one of key starting points for the production of high quality fragrant rice. In this study, we therefore constructed the homology model of BADH2 and employed 500-ns Molecular Dynamics simulations (MD) to primarily understand the structural and dynamic properties of BADH2. Initially, Ramachandran plot confirms the good quality of modeled protein structure. Principle Component Analysis (PCA) was also calculated to capture the protein dynamics. Among 3 domains, the results show that NAD binding site is found to be more flexible. Moreover, interactions from key amino acids (N162, E260, C294, and Y419) that are crucial for function are investigated.

Keywords: betaine aldehyde dehydrogenase 2, fragrant rice, homology modeling, molecular dynamics simulations

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Authors: Abdelbary Prince, Said Moussa, Hyungkyu Kim, Eman Gouda, Jin Han

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We compared the dynamic alterations of mitochondrial proteome of control, ischemia-reperfusion (IR) and ischemic preconditioned (IPC) rabbit hearts. Using 2-DE, we identified 29 mitochondrial proteins that were differentially expressed in the IR heart compared with the control and IPC hearts. For two of the spots, the expression patterns were confirmed by Western blotting analysis. These proteins included succinate dehydrogenase complex, Acyl-CoA dehydrogenase, carnitine acetyltransferase, dihydrolipoamide dehydrogenase, Atpase, ATP synthase, dihydrolipoamide succinyltransferase, ubiquinol-cytochrome c reductase, translation elongation factor, acyl-CoA dehydrogenase, actin alpha, succinyl-CoA Ligase, dihydrolipoamide S-succinyltransferase, citrate synthase, acetyl-Coenzyme A dehydrogenase, creatine kinase, isocitrate dehydrogenase, pyruvate dehydrogenase, prohibitin, NADH dehydrogenase (ubiquinone) Fe-S protein, enoyl Coenzyme A hydratase, superoxide dismutase [Mn], and 24-kDa subunit of complex I. Interestingly, most of these proteins are associated with the mitochondrial respiratory chain, antioxidant enzyme system, and energy metabolism. The results provide clues as to the cardioprotective mechanism of ischemic preconditioning at the protein level and may serve as potential biomarkers for detection of ischemia-induced cardiac injury.

Keywords: ischemic preconditioning, mitochondria, proteome, cardioprotection

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Authors: Harun Rashid, Keerthi S. S. Atapaththu, Takashi Asaeda

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Cesium (Cs) induced growth and stress response of Nitella were studied after exposure to four concentration of the metal; i.e. 0 (control), 0.001, 0.01, and 0.1 ppm Cs in growth media. Each treatment with three replicates were randomly allocated to 12 glass beakers in a complete randomize design and the experiment was continued for 30 days. At the end of the experiment, shoot length, cesium content, total chlorophyll, and plant stress response were compared. Anti-oxidant enzyme activities (peroxidase, catalase, and ascorbic peroxidase) and the concentration of H2O2 were measured to check plant stress. The longest shoot was found in control treatment (0 ppm Cs) and the shoot length of plants exposed to 0.001 ppm was statistically similar to that of control. Concentration of cesium in plants grown at 0.001, 0.01, and 0.1 ppm were significantly higher than those in control treatments. The antioxidant enzymes activities of plants exposed to cesium were significantly higher than those grown without any Cs (control). An elevated level of H2O2 concentration was also observed in former groups of plants. Further, the reduction in chlorophyll concentration and chlorophyll fluorescence in response to cesium exposure indicated the chronically damaged photosynthetic efficiency in cesium stressed Nitella.

Keywords: antioxidant enzymes, cesium, growth, Nitella, oxidative stress

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Authors: Ali Mashinchian Moradi, Mahmood Sinaei

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Study on the biomarkers to assess health status of marine ecosystems has an important value in biomonitoring of marine environment. Accordingly, accumulation of polycyclic aromatic hydrocarbons in sediment, water and tissues (liver and gill) of mudskipper (Boleophthalmus dussmieri) and some physiological responses like lysosomal membrane change in haemocytes and the Glutathione-S Transferase (GST) activity in the liver were measured in mudskippers. Samples were collected from five sites along the noth western cost of the Persian Gulf. PAHs concentration was measured by HPLC method. The activity of GST enzyme was analysed by spectrophotometric method. Total PAH concentration in coastal seawater, sediments, liver and gill tissues ranged between 0.80-18.34 ug/L, 113.550-3384.34 ng/g dw, 3.99-46.64 ng/g dw and 3.11-17.This study showed that PAH concentrations in this region are not higher than available standards. The findings revile that lysosomal membrane destabilization and liver GST activities are highly sensitive to PAHs in mudskipper, B. dussumieri. Sediment PAH concentrations were strongly correlated with biomarkers, indicating PAHs were biologically available to fish. Thus, mudskipper perceived to be good sentinel organism for PAH pollution biomonitoring.

Keywords: PAHs, biomarker, mudskipper, Persian Gulf

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Authors: Paul Awolade, Parvesh Singh

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The global threat of pathogenic resistance to available therapeutic agents has become a menace to clinical practice, public health and man’s existence inconsequential. This has therefore led to an exigency in the development of new molecular scaffolds with profound activity profiles. In this vein, a versatile synthetic tool for accessing new molecules by incorporating two or more pharmacophores into a single entity with the unique ability to be recognized by multiple receptors hence leading to an improved bioactivity, known as molecular hybridization, has been explored with tremendous success. Accordingly, aware of the similarity in pharmacological activity spectrum of quinoline and 1,2,3-triazole pharmacophores such as; anti-Alzheimer, anticancer, anti-HIV, antimalarial and antimicrobial to mention but a few, the present study sets out to synthesize hybrids of quinoline and 1,2,3-triazole. The hybrids were accessed via click chemistry using copper catalysed azide-alkyne 1,3-dipolar cycloaddition reaction. All synthesized compounds were evaluated for their pharmaco-potential in an antimicrobial assay out of which the 3-OH derivative emerged as the most active with MIC value of 4 μg/mL against Cryptococcus neoformans; a value superior to standard Fluconazole and comparable to Amphotericin B. Structures of synthesized hybrids were elucidated using appropriate spectroscopic techniques (1H, 13C and 2D NMR, FT-IR and HRMS).

Keywords: bioisostere, click chemistry, molecular hybridization, quinoline, 1, 2, 3-triazole

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Authors: M. N. Hanina, M. Hairul Shahril, I. Ismatul Nurul Asyikin, A. K. Abdul Jalil, M. R. Salina, M. R. Maryam, M. Rosfarizan

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The extracellular proteins secreted by bacteria may be increased in stressful surroundings, such as in the presence of antibiotics. It appears that many antibiotics, when used at low concentrations, have in common the ability to activate or repress gene transcription, which is distinct from their inhibitory effect. There have been comparatively few studies on the potential of antibiotics as a specific chemical signal that can trigger a variety of biological functions. Therefore, this study was carried out to determine the effect of Streptomycin Sulfate in regulating extracellular proteins secreted by Bacillus subtilis ATCC21332. Results of Microdilution assay showed that the Minimum Inhibition Concentration (MIC) of Streptomycin Sulfate on B. subtilis ATCC21332 was 2.5 mg/ml. The bacteria cells were then exposed to Streptomycin Sulfate at concentration of 0.01 MIC before being further incubated for 48h to 72 h. The extracellular proteins secreted were then isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile revealed that three additional bands with approximate sizes of 30 kDa, 22 kDa and 23 kDa were appeared for the treated bacteria with Streptomycin Sulfate. Thus, B. subtilis ATCC21332 in stressful condition with the presence of Streptomycin Sulfate at low concentration could induce the extracellular proteins secretion.

Keywords: Bacillus subtilis ATCC21332, streptomycin sulfate, extracellular proteins, antibiotics

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Authors: Srinivasarao Kondaparla, Utsab Debnath, Awakash Soni, Vasantha Rao Dola, Manish Sinha, Kumkum Kumkum Srivastava, Sunil K. Puri, Seturam B. Katti

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In a focused exploration, we have designed synthesized and biologically evaluated chiral conjugated new chloroquine (CQ) analogs with substituted piperazines as antimalarial agents. In vitro as well as in vivo studies revealed that compound 7c showed potent activity [for in vitro IC₅₀= 56.98nM (3D7), 97.76nM (K1); for in vivo (up to at the dose of 12.5 mg/kg); SI = 3510] as a new lead of antimalarial agent. Other compounds 6b, 6d, 7d, 7h, 8c, 8d, 9a, and 9c are also showing moderate activity against CQ-sensitive (3D7) strain and superior activity against resistant (K1) strain of P. falciparum. Furthermore, we have carried out docking and 3D-QSAR studies of all in-house data sets (168 molecules) of chiral CQ analogs to explain the structure activity relationships (SAR). Our new findings specified the significance of H-bond interaction with the side chain of heme for biological activity. In addition, the 3D-QSAR study against 3D7 strain indicated the favorable and unfavorable sites of CQ analogs for incorporating steric, hydrophobic and electropositive groups to improve the antimalarial activity.

Keywords: piperazines, CQ-sensitive strain-3D7, in-vitro and in-vivo assay, docking, 3D-QSAR

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Authors: Nur Fathin Alia Che Wahab, Thirumulu Ponnuraj Kannan, Zuliani Mahmood, Ismail Ab. Rahman, Hanafi Ismail

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Locally produced Biphasic Calcium Phosphate (BCP) consists of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) which is a promising material for dentin and bone regeneration as well as in tissue engineering applications. The study was carried out to investigate the mutagenic effect of locally produced BCP using Ames test. Mutagenicity was evaluated with and without the addition of metabolic activation system (S9). This study was performed on Salmonella typhimurium TA98, TA102, TA1537, and TA1538 strains using preincubation assay method. The doses tested were 5000, 2500, 1250, 625, 313 µg/plate. Negative and positive controls were also included. The bacteria were incubated for 48 hours at 37 ± 0.5 °C. Then, the revertant colonies were counted. Data obtained were evaluated using non-statistical method. The mean number of revertant colonies in strains with and without S9 mix treated with locally produced BCP was less than double when compared to negative control for all the tested concentrations. The results from this study indicate that the locally produced BCP is non-mutagenic under the present test conditions.

Keywords: ames test, biphasic calcium phosphate, dentin regeneration, mutagenicity

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Authors: N. A. Kazemi Sefat, M. M. Mohammadi, J. Hadjati, S. Talebi, M. Ajami, H. Daneshvar

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Colorectal cancer metastases result in a significant number of cancer related deaths. Histone deacetylase (HDAC) inhibitors induce growth arrest and apoptosis in a variety of human cancer cells. Sodium butyrate (SB) is a short chain fatty acid, belongs to HDAC inhibitors which is released in the colonic lumen as a consequence of fiber fermentation. In this study, we are about to assess the effect of sodium butyrate on HCT116 human colorectal carcinoma cell line. The viability of cells was measured by microscopic morphologic study and MTT assay. After 48 hours, treatments more than 10 mM lead to cell injury in HCT116 by increasing cell granulation and decreasing cell adhesion (p>0.05). After 72 hours, treatments at 10 mM and more lead to significant cell injury (p<0.05). Our results may suggest that the gene expression which is contributed in cell proliferation and apoptosis has been changed under pressure of HDAC inhibition.

Keywords: colorectal cancer, sodium butyrate, cytotoxicity, MTT

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Authors: Sahira Nsayef Muslim, Israa M. S. Al-Kadmy, Nadheema Hammood Hussein, Alaa Naseer Mohammed Ali, Buthainah Mohammed Taha, Rayim Sabah Abbood, Sarah Naji Aziz

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A novel strain of Bacillus licheniformis isolated from spoilt cucumber and pepper samples have the ability to produce the chitosanase enzyme when grown on chitosan substrate. Chitosanase was purified to homogeneity with a recovery yield of 35.71% and 5.5 fold of purification by using ammonium sulfate at 45% saturation followed by ion exchange chromatography on DEAE-cellulose column and gel filtration chromatography on Sephadex G-100 column. The purified chitosanase inhibited the biofilm formation ability for all Gram-negative and Gram-positive biofilm-forming bacteria (biofilm producers) after using Congo Red agar and Microtiter plates methods. Highly antibiofilm of chitosanase recorded against Pseudomonas aeruginosa followed by Klebsiella pneumoniae with reduction of biofilm formation ratio to 22 and 29%, respectively compared with (100)% of control. Thus, chitosanase has promising benefit as antibiofilm agent against biofilm forming pathogenic bacteria and has promising application as alternative antibiofilm agents to combat the growing number of multidrug-resistant pathogen-associated infections, especially in situation where biofilms are involved.

Keywords: chitosanase, Bacillus licheniformis, vegetables, biofilm

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Authors: Melasurej C. Francisco, Sophia D. Rusit

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Old age is a record of one’s own life; this is the crucial phase for most. However, there are individuals who believe that old people retain self-efficacy and self-worth throughout their existence. Geriatric institutions focus on the health of elderly, in which they have been supported with medicines and therapies by clinician thus, indicating that these may suffice physical, emotional, and mental health of the elderly. This study focuses on (1) Describing the level or degree of self-efficacy; (2) Recognizing the extent of self-worth; (3) Determining the significant relationship between self-efficacy and self-worth. It is a mixed method design. A combination of correlational research and in-depth interview. Purposive sampling technique was used to select participants, considering that this assay focused on elderly in geriatric institutions, it follows that respondents and participants are at least sixty years of age and must be living inside the institution. 121 senior citizens took part in this study. Scores from both General Self-Efficacy Scale (GSE) and Rosenberg Self-Esteem Scale (RSES) showed varying levels of self-efficacy and self-worth. SE had μ=28.099, σ=6.6262, σ²=43.9067 while; SW had μ=14.9669, σ=5.3789, σ²28.9322 which denotes that rₒbₜ (121)=0.3164 is higher than rcᵢₜ which is 0.150. Although this exhibits the positive moderate correlation between SE and SW, the relationship between variables is weak. Likewise, the pᵥₐₗᵤₑ (pᵥₐₗᵤₑ=0.000406) is lower than the significance level alpha=0.01, thus, rejecting the null hypothesis, and accepting the alternative hypothesis.

Keywords: elderly, geriatric, self-efficacy, self-worth

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Authors: Seyedeh Nasim Mirbahari, Taha Azad, Mehdi Totonchi

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Oncolytic viruses, which only replicate in tumor cells, are being extensively studied for their use in cancer therapy. A particular virus known as the vaccinia virus, a member of the poxvirus family, has demonstrated oncolytic abilities glioma. Treating Glioma with traditional methods such as chemotherapy and radiotherapy is quite challenging. Even though oncolytic viruses have shown immense potential in cancer treatment, their effectiveness in glioblastoma treatment is still low. Therefore, there is a need to improve and optimize immunotherapies for better results. In this study, we have designed oncoVV-TT, which can more effectively target tumor cells while minimizing replication in normal cells by replacing the thymidine kinase gene with a luc-p2a-GFP gene expression cassette. Human glioblastoma cell line U251 MG, rat glioblastoma cell line C6, and non-tumor cell line HFF were plated at 105 cells in a 12-well plates in 2 mL of DMEM-F2 medium with 10% FBS added to each well. Then incubated at 37°C. After 16 hours, the cells were treated with oncoVV-TT at an MOI of 0.01, 0.1 and left in the incubator for a further 24, 48, 72 and 96 hours. Viral replication assay, fluorescence imaging and viability tests, including trypan blue and crystal violet, were conducted to evaluate the cytotoxic effect of oncoVV-TT. The finding shows that oncoVV-TT had significantly higher cytotoxic activity and proliferation rates in tumor cells in a dose and time-dependent manner, with the strongest effect observed in U251 MG. To conclude, oncoVV-TT has the potential to be a promising oncolytic virus for cancer treatment, with a more cytotoxic effect in human glioblastoma cells versus rat glioma cells. To assess the effectiveness of vaccinia virus-mediated viral therapy, we have tested U251mg and C6 tumor cell lines taken from human and rat gliomas, respectively. The study evaluated oncoVV-TT's ability to replicate and lyse cells and analyzed the survival rates of the tested cell lines when treated with different doses of oncoVV-TT. Additionally, we compared the sensitivity of human and mouse glioma cell lines to the oncolytic vaccinia virus. All experiments regarding viruses were conducted under biosafety level 2. We engineered a Vaccinia-based oncolytic virus called oncoVV-TT to replicate specifically in tumor cells. To propagate the oncoVV-TT virus, HeLa cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 10 MOI virus was added. After 48 h, cells were harvested by scraping, and viruses were collected by 3 sequential freezing and thawing cycles followed by removal of cell debris by centrifugation (1500 rpm, 5 min). The supernatant was stored at −80 ◦C for the following experiments. To measure the replication of the virus in Hela, cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 5 MOI virus or equal dilution of PBS was added. At the treatment time of 0 h, 24 h, 48 h, 72 h and 96 h, the viral titers were determined under the fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). Fluorescence intensity was quantified using the imagej software according to the manufacturer’s protocol. For the isolation of single-virus clones, HeLa cells seeded in six-well plates (5×105 cells/well). After 24 h (100% confluent), the cells were infected with a 10-fold dilution series of TianTan green fluorescent protein (GFP)virus and incubated for 4 h. To examine the cytotoxic effect of oncoVV-TT virus ofn U251mg and C6 cell, trypan blue and crystal violet assay was used.

Keywords: oncolytic virus, immune therapy, glioma, vaccinia virus

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Authors: Olga V. Chervyakova, Elmira T. Tailakova, Vitaliy M. Strochkov, Kulyaisan T. Sultankulova, Nurlan T. Sandybayev, Lev G. Nemchinov, Rosemarie W. Hammond

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Sheep pox is a highly contagious infection that OIE regards to be one of the most dangerous animal diseases. It causes enormous economic losses because of death and slaughter of infected animals, lower productivity, cost of veterinary and sanitary as well as quarantine measures. To control spread of sheep pox infection the attenuated vaccines are widely used in the Republic of Kazakhstan and other Former Soviet Union countries. In spite of high efficiency of live vaccines, the possible presence of the residual virulence, potential genetic instability restricts their use in disease-free areas that leads to necessity to exploit new approaches in vaccine development involving recombinant DNA technology. Vaccines on the basis of recombinant proteins are the newest generation of prophylactic preparations. The main advantage of these vaccines is their low reactogenicity and this fact makes them widely used in medical and veterinary practice for vaccination of humans and farm animals. The objective of the study is to produce recombinant immunogenic proteins for development of the high-performance means for sheep pox prophylaxis. The SPV proteins were chosen for their homology with the known immunogenic vaccinia virus proteins. Assay of nucleotide and amino acid sequences of the target SPV protein genes. It has been shown that four proteins SPPV060 (ortholog L1), SPPV074 (ortholog H3), SPPV122 (ortholog A33) and SPPV141 (ortholog B5) possess transmembrane domains at N- or C-terminus while in amino acid sequences of SPPV095 (ortholog А 4) and SPPV117 (ortholog А 27) proteins these domains were absent. On the basis of these findings the primers were constructed. Target genes were amplified and subsequently cloned into the expression vector рЕТ26b(+) or рЕТ28b(+). Six constructions (pSPPV060ΔТМ, pSPPV074ΔТМ, pSPPV095, pSPPV117, pSPPV122ΔТМ and pSPPV141ΔТМ) were obtained for expression of the SPV genes under control of T7 promoter in Escherichia coli. To purify and detect recombinant proteins the amino acid sequences were modified by adding six histidine molecules at C-terminus. Induction of gene expression by IPTG was resulted in production of the proteins with molecular weights corresponding to the estimated values for SPPV060, SPPV074, SPPV095, SPPV117, SPPV122 and SPPV141, i.e. 22, 30, 20, 19, 17 and 22 kDa respectively. Optimal protocol of expression for each gene that ensures high yield of the recombinant protein was identified. Assay of cellular lysates by western blotting confirmed expression of the target proteins. Recombinant proteins bind specifically with antibodies to polyhistidine. Moreover all produced proteins are specifically recognized by the serum from experimentally SPV-infected sheep. The recombinant proteins SPPV060, SPPV074, SPPV117, SPPV122 and SPPV141 were also shown to induce formation of antibodies with virus-neutralizing activity. The results of the research will help to develop a new-generation high-performance means for specific sheep pox prophylaxis that is one of key moments in animal health protection. The research was conducted under the International project ISTC # K-1704 “Development of methods to construct recombinant prophylactic means for sheep pox with use of transgenic plants” and under the Grant Project RK MES G.2015/0115RK01983 "Recombinant vaccine for sheep pox prophylaxis".

Keywords: prophylactic preparation, recombinant protein, sheep pox virus, subunit vaccine

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Authors: Aussara Panya, Nunghathai Sawasdee, Mutita Junking, Chatchawan Srisawat, Kiattawee Choowongkomon, Pa-Thai Yenchitsomanus

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Dengue virus (DENV) infection is a global public health problem with approximately 100 million infected cases a year. Presently, there is no approved vaccine or effective drug available; therefore, the development of anti-DENV drug is urgently needed. The clinical reports revealing the positive association between the disease severity and viral titer has been reported previously suggesting that the anti-DENV drug therapy can possibly ameliorate the disease severity. Although several anti-DENV agents showed inhibitory activities against DENV infection, to date none of them accomplishes clinical use in the patients. The surface envelope (E) protein of DENV is critical for the viral entry step, which includes attachment and membrane fusion; thus, the blocking of envelope protein is an attractive strategy for anti-DENV drug development. To search the safe anti-DENV agent, this study aimed to search for novel peptide inhibitors to counter DENV infection through the targeting of E protein using a structure-based in silico design. Two selected strategies has been used including to identify the peptide inhibitor which interfere the membrane fusion process whereby the hydrophobic pocket on the E protein was the target, the destabilization of virion structure organization through the disruption of the interaction between the envelope and membrane proteins, respectively. The molecular docking technique has been used in the first strategy to search for the peptide inhibitors that specifically bind to the hydrophobic pocket. The second strategy, the peptide inhibitor has been designed to mimic the ectodomain portion of membrane protein to disrupt the protein-protein interaction. The designed peptides were tested for the effects on cell viability to measure the toxic to peptide to the cells and their inhibitory assay to inhibit the DENV infection in Vero cells. Furthermore, their antiviral effects on viral replication, intracellular protein level and viral production have been observed by using the qPCR, cell-based flavivirus immunodetection and immunofluorescence assay. None of tested peptides showed the significant effect on cell viability. The small peptide inhibitors achieved from molecular docking, Glu-Phe (EF), effectively inhibited DENV infection in cell culture system. Its most potential effect was observed for DENV2 with a half maximal inhibition concentration (IC50) of 96 μM, but it partially inhibited other serotypes. Treatment of EF at 200 µM on infected cells also significantly reduced the viral genome and protein to 83.47% and 84.15%, respectively, corresponding to the reduction of infected cell numbers. An additional approach was carried out by using peptide mimicking membrane (M) protein, namely MLH40. Treatment of MLH40 caused the reduction of foci formation in four individual DENV serotype (DENV1-4) with IC50 of 24-31 μM. Further characterization suggested that the MLH40 specifically blocked viral attachment to host membrane, and treatment with 100 μM could diminish 80% of viral attachment. In summary, targeting the hydrophobic pocket and M-binding site on the E protein by using the peptide inhibitors could inhibit DENV infection. The results provide proof of-concept for the development of antiviral therapeutic peptide inhibitors to counter DENV infection through the use of a structure-based design targeting conserved viral protein.

Keywords: dengue virus, dengue virus infection, drug design, peptide inhibitor

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Authors: Patience Orobosa Olajide

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Five hydrocarbon degrading bacterial strains isolated from contaminated environment were investigated with respect to polyhydroxybutyrate (PHB) biosynthesis. Screening for bioplastic production was done on assay mineral salts agar medium containing 0.2% poly (3-hydroxybutyrate) as the sole carbon source. Two of the test bacteria were positive for PHB biosynthesis and were identified based on gram staining, biochemical tests, 16S rRNA gene sequence analysis as Pseudomonas aeruginosa and Bacillus licheniformis which grew at 37 and up to 65 °C respectively, thus suggesting the later to be thermotolerant. In this study, the effects of different carbon and nitrogen sources on PHB production in these strains were investigated. Maximum PHB production was obtained in 48 hr for the two strains and amounted to yields of 72.86 and 62.22 percentages for Bacillus licheniformis and Pseudomonas aeruginosa respectively. In these strains, glycine was the most efficient carbon sources for the production of PHB compared with other carbon (glucose, lactose, sucrose, Arabinose) and nitrogen (L- glycine, L-cysteine, DL-Tryptophan, and Potassium Nitrate) sources. The screening of microbial strains for industrial PHB production should be based on several factors including the cell’s capability to mineralize an inexpensive substrate, rate of growth and the extent of polymer accumulation.

Keywords: bacteria, poly-3-hydroxybutyrate (PHB), hydrocarbon, thermotolerant

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Authors: Ozlem Temiz-Arpaci, Meryem Tasci, Fatma Sezer Senol, İlkay Erdogan Orhan

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Alzheimer’s disease (AD), a neurodegenerative disorder characterized by a progressive deterioration of memory and cognition, occurs more frequently in elderly people. Current treatment approaches in this disease with the major therapeutic strategy are based on the AChE and BChE inhibition. On the other hand, tyrosinase inhibition has become a target for the treatment of Parkinson’s disease (PD) since this enzyme may play a role in neuromelanin formation in the human brain and could be critical in the formation of dopamine neurotoxicity associated with neurodegeneration linked to PD. Also benzoxazoles are structural isosteres of natural nucleotides that can interact with biopolymers so that benzoxazoles showed a lot of different biological activities. In this study, a series of 2,5-disubstituted-benzoxazole derivatives were synthesized and were evaluated as possible inhibitors of acetylcholinesterase (AChE) / butyrylcholinesterase (BChE) and tyrosinase. The results demonstrated that the compounds exhibited a weak spectrum of AChE / BChE inhibitory activity ranging between 3.92% - 54.32% except compound 8 which showed no activity against AChE and compound 4 which showed no activity against BChE at the specified molar concentrations. Also, the compounds indicated lower than tyrosinase inhibitory activity of ranging between 8.14% - 22.90% to that of reference (kojic acid).

Keywords: AChE and BChE inhibition, Alzheimer’s disease, benzoxazoles, tyrosinase inhibition

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Authors: Rafat M. Mohareb

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Several D-ring alkylated estrone analogues display exceptionally high affinity for estrogen receptors. In particular, compounds in which an E-ring is formed are known to be involved in the inhibition of steroidogenic enzymes. Such compounds also have an effect on steroid dehydrogenase activity and the ability to inhibit the detrimental action of the steroid sulfatase enzyme. Generally, E-ring extended steroids have been accessed by modification of the C17-ketone in the D-ring by either arylimine or oximino formation, addition of a carbon nucleophile or hydrazone formation. Other approaches have included ketone reduction, silyl enol ether formation or ring-closing metathesis (giving five- or six-membered E-rings). Chemical modification of the steroid D-ring provides a way to alter the functional groups, sizes and stereochemistry of the D-ring, and numerous structure-activity relationships have been established by such synthetic alterations. Steroids bearing heterocycles fused to the D-ring of the steroid nucleus have been of pharmaceutical interest. In the present paper, we report on the efficient synthesis of estrone possessing pyran, pyrimidine and thiazole ring systems. This study focused on the synthesis and biochemical evaluation of newly synthesized heterocyclic compounds which were then subjected through inhibitory evaluations towards human cancer and normal cell lines.

Keywords: estrone, heterocyclization, cytotoxicity, biomedicine

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Authors: Supakan Rattanakon, Jakkrapan Boonpimon, Akkaragiat Bhuangsaeng, Aphiwat Ratriphruek

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Rice bran oil (RBO) has been found to lower the level of serum cholesterol, has antioxidant and anti-carcinogenic property, and attenuate allergic inflammation. These properties of RBO are due to antioxidant compositions, especially, phenolic compounds. The higher amount of these active compounds in RBO, the greater value of RBO is. Thermal process of rice bran before solvent RBO extraction has been found to have a higher phenolic contents. Therefore, the purpose of this study is to using different heating methods on rice bran before the solvent extraction. Then, % yield of RBO, total phenolic content (TPC), and antioxidant property of two white Thai rice; KDML105 and RD6 were determined. The Folin-Ciocalteu colorimetric assay was used to determine TPC and scavenging of free radicals (DPPH) was used to determine antioxidant property expressed as EC50. The result showed that thermal process did not increase % yield of RBO but increase the TPC with 1.41 mg gallic acid equivalent (GAEmg-1). The highest TPC was found in KDML105 by using sonicator. The highest antioxidant activity was found in RD6 using autoclave. The EC50 of RBO was 0.04 mg/mL. Further study should be performed on different pretreatments to increase the TPC and antioxidant property.

Keywords: antioxidant, rice bran oil, total phenol content, white rice

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Authors: Ahlam Inayatullah Badrul Munir, M. Husaini A. Rahman, Mohd Sukri Hassan

Abstract:

Real-time PCR is a molecular biology technique that is currently being widely used for halal services to differentiating between porcine and bovine DNA. The useful of technique become very important for student or workers (who works in the laboratory) to learn how the technique could be run smoothly without fail. Same concept with conventional PCR, real-time PCR also needed DNA template, primer, enzyme polymerase, dNTP, and buffer. The difference is in real-time PCR, have additional component namely fluorescent dye. The most common use of fluorescent dye in real-time PCR is SYBR green. The purpose of this study was to find out how sensitive, specific and efficient real-time PCR technique was combined with SYBR green method and specific primers of CYT b. The results showed that real-time PCR technique using SYBR Green, capable of detecting porcine and bovine DNA concentrations up to 0.0001 µl/ng. The level of efficiency for both types of DNA was 91% (90-110). Not only that in specific primer CYT b bovine primer could detect only bovine DNA, and porcine primer could detect only porcine primer. So, from the study could be concluded that real-time PCR technique that was combined with specific primer CYT b and SYBR green method, was sensitive, specific and efficient to detect porcine and bovine DNA.

Keywords: sensitivity, specificity, efficiency, real-time PCR, SYBR green, Cytochrome b, porcine DNA, bovine DNA

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Authors: Satya Eswari Jujjavarapu, Swasti Dhagat, Lata Upadhyay, Reecha Sahu

Abstract:

Silver nanoparticles have a broad range of antimicrobial and antifungal properties ranging from soaps, pastes to sterilization and drug delivery systems. These can be synthesized by physical, chemical and biological methods; biological methods being the most popular owing to their non-toxic nature and reduced energy requirements. Microbial surfactants, produced on the microbial cell surface or excreted extracellularly are an alternative to synthetic surfactants for the production of silver nanoparticles. Hence, they are also called as green molecules. Microbial lipopeptide surfactants (biosurfactant) exhibit anti-tumor and anti-microbial properties and can be used as drug delivery agents. In this study, biosurfactant was synthesized by using a strain of acillus subtilis. The biosurfactant thus produced was analysed by emulsification assay, oil spilling test, and haemolytic test. Biosurfactant-mediated silver nanoparticles were synthesised by microwave irradiation of the culture supernatant and further characterized by UV–vis spectroscopy for a range of 400-600 nm. The UV–vis spectra showed a surface plasmon resonance vibration band at 410 nm corresponding to the peak of silver nanoparticles.

Keywords: biosurfactant, Bacillus subtilis, silver nano particle, lipopeptide

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Authors: Mahmoud Mabrok, Channarong Rodkhum

Abstract:

Flavobacterium columanre is one of the devastating pathogen that causes noticeable economic losses in freshwater cultured fish. Like other filamentous bacteria, F. columanre tends to aggregate and fluctuate to all kind of media, thus revealing obstacles in recognition of its colonies. Since the molecular typing is the only fundamental tool for rapid and precise detection of this pathgen. The present study developed a species-specific PCR assay based on cslA unique gene of F. columnare. The cslA gene sequences of 13 F. columnare, strains retrieved from gene bank database, were aligned to identify a conserved homologous segment prior to primers design. The new primers yielded amplicons of 287 bp from F. columnare strains but not from relevant or other pathogens, unlike to other published set that showed no specificity and cross-reactivity with F. indicum. The primers were sensitive and detected as few as 7 CFUs of bacteria and 3 pg of gDNA template. The sensitivity was reduced ten times when using tissue samples. These primers precisely defined all field isolates in a double-blind study, proposing their applicable use for field detection.

Keywords: Columnaris infection, cslA gene, Flavobacterium columnare, PCR

Procedia PDF Downloads 127

Authors: Shahid-ul-Islam, Faqeer Mohammad

Abstract:

The present study was conducted to investigate the effect of annatto, teak and flame of the forest natural dyes on color, fastness, and antimicrobial property of protein based textile substrate. The color strength (K/S) of wool samples at various concentrations of dyes were analysed using a Reflective Spectrophotometer. The antimicrobial activity of natural dyes before and after application on wool was tested against common human pathogens Escherichia coli, Staphylococcus aureus, and Candida albicans, by using micro-broth dilution method, disc diffusion assay and growth curve studies. The structural morphology of natural protein fibre (wool) was investigated by Scanning Electron Microscopy (SEM). Annatto and teak natural dyes proved very effective in inhibiting the microbial growth in solution phase and after application on wool and resulted in a broad beautiful spectrum of colors with exceptional fastness properties. The results encourage the search and exploitation of new plant species as source of dyes to replace toxic synthetic antimicrobial agents currently used in textile industry.

Keywords: annatto, antimicrobial agents, natural dyes, green textiles

Procedia PDF Downloads 318

Authors: Sana Abbasa, Shahzad Bhattiab, Nadir Khan

Abstract:

Sargassum siliquastrum is brown seaweed with traditional claims for some medicinal properties. This research was done to investigate the methanol extract of S. siliquastrum for antiproliferative activity against human cervical cancer cell line, HeLa and its mode of cell death. From methylene blue assay, S. siliquastrum exhibited antiproliferative activity on HeLa cells with IC50 of 3.87 µg/ml without affecting non-malignant cells. Phase contrast microscopy indicated the confluency reduction in HeLa cells and changes on the cell shape. Nuclear staining with Hoechst 33258 displayed the formation of apoptotic bodies and fragmented nuclei. S. siliquastrum also induced early apoptosis event in HeLa cells as confirmed by FITC-Annexin V/propidium iodide staining by flow cytometry analysis. Cell cycle analysis indicated growth arrest of HeLa cells at G1/S phase. Protein study by flow cytometry indicated the increment of p53, slight increase of Bax and unchanged level of Bcl-2. In conclusion, S. siliquastrum demonstrated an antiproliferative activity in HeLa cell by inducing G1/S cell cycle arrest via p53-mediated pathway.

Keywords: sargassum siliquastrum, cervical cancer, P53, antiproleferation

Procedia PDF Downloads 631

Authors: Drashti G. Daraji, Rosa E. Moo-Puc, Hitesh D. Patel

Abstract:

Substituted 2-Thioimidazole analogues have been synthesized and confirmed by advanced spectroscopic techniques. Among them, ten compounds have been selected and evaluated for their in vitro anti-cancer activity at the National Cancer Institute (NCI) for testing against a panel of 60 different human tumor cell lines derived from nine neoplastic cancer types. Furthermore, synthesized compounds were tested for their in vitro antiprotozoal activity, and none of them exhibited significant potency against antiprotozoans. It was observed that the tested all compounds seem effective on the UACC-62 melanoma cancer cell line as compared to other cancer cell lines and also exhibited the least potent in the Non-Small Cell Lung Cancer cell line in one-dose screening. In silico studies of these derivatives were carried out by molecular docking techniques and Absorption, Distribution, Metabolism, and Excretion (ADME) using Schrödinger software to find potent B-Raf kinase inhibitor (PDB ID: 3OG7). All the compounds have been performed for docking study; Compound D4 has a good docking score for melanoma cancer as compared with other.

Keywords: anticancer activity, cancer cell line, 2-thio imidazole, one-dose assay, molecular docking

Procedia PDF Downloads 143

Authors: Bidya Dhar Sahu, Meghana Koneru, R. Shyam Sunder, Ramakrishna Sistla

Abstract:

Environmental and occupational exposure to hexavalent chromium [Cr(VI)] via textile manufacture, metallurgy, spray paints, stainless steel industries, drinking water containing chromium are often known to cause acute renal injury in humans and animals. Nephrotoxicity is the major effect of chromium poisoning. In the present study, we investigated the potential renoprotective effect and underlying mechanisms of carvedilol using rat model of potassium dichromate (K2Cr2O7)-induced nephrotoxicity. Exploration of the underlying mechanisms of carvedilol revealed that carvedilol attenuated nuclear translocation and DNA binding activity of NF-κB (p65), restored antioxidant and mitochondrial respiratory enzyme activities and attenuated apoptosis related protein expressions in kidney tissues. The serum levels of TNF-α, the renal iNOS and myeloperoxidase activity were significantly decreased in carvedilol pre-treated K2Cr2O7-induced nephrotoxic rats. These results were further supported and confirmed by histological findings. In conclusion, the findings of the present study demonstrated that carvedilol is an effective chemoprotectant against K2Cr2O7-induced nephrotoxicity in rats.

Keywords: apoptosis, carvedilol, inflammation, potassium dichromate-induced nephrotoxicity, applied pharmacology

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Authors: H. Aksas, S. Boughrara, K. Louhab

Abstract:

The goal of this study was the use of life cycle assessment (LCA) methodology to assess the environmental impact of pharmaceutical product (four kinds of syrup form and tree kinds of pomade form), which are produced in one leader manufactory in Algeria town that is SAIDAL Company. The impacts generated have evaluated using SimpaPro7.1 with CML92 Method for syrup form and EPD 2007 for pomade form. All impacts evaluated have compared between them, with determination of the compound contributing to each impacts in each case. Data needed to conduct Life Cycle Inventory (LCI) came from this factory, by the collection of theoretical data near the responsible technicians and engineers of the company, the practical data are resulting from the assay of pharmaceutical liquid, obtained at the laboratories of the university. This data represent different raw material imported from European and Asian country necessarily to formulate the drug. Energy used is coming from Algerian resource for the input. Outputs are the result of effluent analysis of this factory with different form (liquid, solid and gas form). All this data (input and output) represent the ecobalance.

Keywords: pharmaceutical product, drug residues, LCA methodology, environmental impacts

Procedia PDF Downloads 246

Authors: Purnima Rawat, Divya Vohora, Sarika Gupta, Farhan J. Ahmad, Sushama Talegaonkar

Abstract:

Nanoparticles (NPs) that target bone tissue were developed using PLGA–mPEG (poly(lactic-co-glycolic-acid)–polyethylene glycol) diblock copolymers by using pamidronate as a bone-targeting moieties. These NPs are expected to enable the transport of hydrophilic drugs. The NP was prepared by in situ polymerization method, and their in- vitro characteristics were evaluated using dynamic light scattering, transmission electron microscopy (TEM) and in phosphate-buffered solution. The bone targeting potential of the NP was also evaluated on in-vitro pre-osteoblast MCT3E1 cell line using ALP activity, degree of mineralization and RT-PCR assay. The average particle size of the NP was 101.6 ± 3.7nm, zeta potential values were negative (-25±0.34mV) of the formulations and the entrapment efficiency was 93± 3.1 % obtained. The moiety of the PLGA–mPEG–pamidronate NPs exhibited the best apatite mineral binding ability in-vitro MCT3E1 pre-osteoblast cell line. Our results suggested that the developed nanoparticles may use as a delivery system for Pamidronate in bone repair and regeneration, warranting further evaluation of the treatment of bone disease.

Keywords: nanoparticle, pamidronate, in-situ polymerization, osteoblast

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Authors: Ashish Kumar Jain, Deepak Mishra

Abstract:

The objective of the present investigation was to prepare and evaluate a vesicular dual drug delivery system for effective management of mucosal ulcer. Inner encapsulating and Double liposomes were prepared by glass bead and reverse phase evaporation method respectively. The formulation consisted of inner liposomes bearing Ranitidine Bismuth Citrate (RBC) and outer liposomes encapsulating Amoxicillin trihydrate (AMOX). The optimized inner liposomes and double liposomes were extensively characterized for vesicle size, morphology, zeta potential, vesicles count, entrapment efficiency and in vitro drug release. In vitro, the double liposomes demonstrated a sustained release of AMOX and RBC viz 91.4±1.8% and 77.2±2.1% respectively at the end of 72 hr. Furthermore binding specificity and targeting propensity toward H. pylori (SKP-56) was confirmed by agglutination and in situ adherence assay. Reduction of the absolute alcohol induced ulcerogenic index from 3.01 ± 0.25 to 0.31 ± 0.09 and 100% H. pylori clearance rate was observed. These results suggested that double liposomes are potential vector for the development of dual drug delivery for effective treatment of H. pylori-associated peptic ulcer.

Keywords: double liposomes, H. pylori targeting, PE liposomes, glass-beads method, peptic ulcers

Procedia PDF Downloads 448

Authors: Izdihar Ismail, Alona C. Linatoc, Maryati Mohamed

Abstract:

The tropical rainforest of Malaysia is known for its rich biological diversity and high endemicity. The potential for these forests to hold the cure for many diseases and illnesses is high and much is yet to be discovered. This study explores the richness of the tropical rainforest of Endau-Rompin National Park in Johor, Malaysia in search of plants traditionally used by the indigenous people in the treatment of malaria and malaria-like symptoms. Seven species of plants were evaluated and tested for antiplasmodial activities. Different plant parts were subjected to methanolic and aqueous extractions. A total of 24 extracts were evaluated by histidine-rich protein II (HRP2) assay against K1 strain of Plasmodium falciparum chloroquine-resistant. Ten extracts showed significant inhibition of the growth of P. falciparum. Phytochemical screening of the same extracts revealed the presence of alkaloids, flavonoids, terpenoids and anthraquinones. This study affirms that tropical rainforests may still hold undiscovered cures for many diseases and illnesses that have inflicted millions of people worldwide. The species studied herein have not known to have been studied elsewhere before.

Keywords: Endau-Rompin, malaria, Malaysia, tropical rainforest, traditional knowledge

Procedia PDF Downloads 272