Research Journal of Biotechnology

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Research Journal of Biotechnology

Acquired Multicellular- and Extra Cellular Matrix- mediated Resistance in a Three-dimensional Model of HT-29 Cancer Cells

Li Meiying and Feng Guanping

Here we established a three-dimensional (3D) model for HT-29 colorectal cancer cells using type I collagen as scaffolds and further evaluated the difference of cells through the 3D model and through monolayer culture (2D) with regards to their morphology and drug sensitivity. A couple of morphological differences were observed between the above two types of cancer cell. The cells by the 3D model could aggregate to form cellular spheroids and even produced microvilli on their surface whereas the cells by the 2D adhered to plate and lost their original shape. In addition, viabilities of both cells were observed to decrease upon exposure to butyric acid (BA) at various concentrations (2~40 mM) for 12, 24, 48 and 72 h, but the cell survival rates by 3D were significantly higher than those by 2D at the same drug concentrations. Also after BA treatment their cell apoptosis was investigated. Our flow cytometric analysis indicated that the cell apoptosis by 2D was extremely low at early stage but had high rates of apoptotic and necrotic and a clear dose-dependent relationship but for the cells by 3D was completely opposite to the result of the cells by 2D. In summary, our findings further demonstrated that the HT-29 colon cancer cells by the 3D showed a significant drug resistance in comparison to the cells by the 2D. Therefore, our research result showed that the 3D model can provide an effective in-vitro method to screen drugs for colon cancer treatment.

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Characterisation of Bioactive Metabolites from Endophytic Bacterial Isolate

Lal Moti, Tia Neelam, Verma Shiv Kr., Dutta Debashis and Das Mira Debnath

The aim of this study was to isolate and identify endophytic bacteria from stems and root of adhathoda beddomei and to evaluate their antibacterial and antifungal properties. The present investigation was carried out to analyse the morphological, physiological, biochemical and cultural characteristics diversity in the bacterial endophyte of two species of adhathoda beddomei, a total of 10 bacterial. Endophytes were isolated from tissues, stem, root. The antimicrobial and antifungal activity was tested against Escherichia coli. Pseudomonas aeroginosa, Staphylococcus aureus, Penicillium chrysogenum, Lactobacillus lactis, Bacillus subtilis and Candida albican. Minimum inhibitory concentration (MIC) of crude extract against performed microorganisms was determined. NMR, FTIR, element and mass analysis of fractionated crude extract were carried out for empirical formula and functional group.

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Development of novel locus specific SCAR (sequence characterized amplified region) marker for cotton (Gossypium spp.)

Kahodariya J. H., Sanghani A.O., Ramani H. R., Bambharolia R. P. and Vakharia D. N.

The fifteen cotton cultivars belonging to four species viz. G. hirsutum, G. herbaceum, G. arboretum and G. barbadense were screened through 21 RAPD primers for the development of unique band pattern. The RAPD primer OPB-11 amplified a unique band of around 900bp in all G. arboretum cultivars. This segment was cloned and sequenced in genetic analyzer. Alignment of forward and reverse sequence generated a 914bp segment and primer was designed which will give 297bp segment to confirm the presence of that in the screened cultivars. Then the sequence was submitted in NCBI genebank with the accession number HM 490146 and there were no matched sequences in any genebank, so it was considered as a novel gene sequence for cotton.

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Effect of 6-benzylaminopurine and α-naphthalene acetic acid on in vitro micropropagation of Musa sp. (ABB group) of banana cv. ‘Karpuravalli’

Shalini S. and Deepa Sankar P.

Banana cv. Karpuravalli (ABB), an economically important Indian variety was evaluated for its in vitro response to different combinations and concentrations of 6-BAP (2, 3 and 4 mg/l) and NAA (1 and 2 mg/l) added to Murashige and Skoog (MS) basal media, as there are only very few reports on the successful micropropagation of this variety. MS medium supplemented with 3mg/l 6-BAP and 1mg/l NAA was found to be the best for shoot proliferation among the different concentrations tested. The mean survival rate for cultured explants was observed to be 67%. The highest number of mean shoots generated per explant was 18.0 and the highest mean height of shoots and mean pseudo-stem diameter were 13.42 ± 0.69 cm and 2.77 ± 0.17 cm respectively in media supplemented with 3mg/l 6-BAP and 1mg/l NAA after seventh subculture. Microscopic root hair study showed dense root hairs in plantlets supplemented with 3mg/l 6-BAP and 1mg/l NAA. However highest concentration of 6-BAP (4mg/l) resulted in abnormal shoots with reduced leaf length and formation of clusters. The in vitro regenerated plants were successfully hardened and transferred to field condition.

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Classification of mammographic image abnormalities based on emo and LS-SVM techniques

Vimalkumar M.N., Helenprabha K. and Surendar A.

Breast cancer is a cancer originated from breast tissue. Signs of breast cancer may include a lump in the breast, a change in breast shape, dimpling of the skin, fluid coming from the nipple or a red scaly patch of skin. In this study, an EMO method was used for segmenting an image into multiple threshold levels using its mean and variance values. During the segmentation process, it classifies the pixels into two or more groups depending upon their intensity levels and a threshold value. The Electro-magnetism-Like algorithm (EMO) is an evolutionary method which mimics the attraction-repulsion mechanism among charges to evolve the members of a population. LS-SVM technique is used to guarantee the effectiveness of the hybrid algorithm by searching for the optimal values of the classifier. The proposed algorithm was compared with different classifier algorithms which were applied on the same database. The experimental results showed the superiority of the proposed algorithm that could achieve a classification accuracy of 97.75%.

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Evaluation of Medicinal Plant Extracts against LOVO and MDA-MB-231 Adenocarcinoma Cell Lines

AL-Zharani Mohammed, Abutaha Nael, Nasr Fahd A., Dekhil Hafedh and Wadaan Muhammad A.

Medicinal plants are considered a promising alternative for the treatment of cancer. Plants were extracted with different solvents using Soxhlet apparatus. The cytotoxicity of the extracts was evaluated on human colon adenocarcinoma cell line (LoVo) and human breast adenocarcinoma cell line (MDA-MB-231) using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Lactate dehydrogenase (LDH) assays. Induction of apoptosis was studied using fluorescent staining (Hoechst 33258). A significant reduction of cell was observed in dose dependent manner. Hexane and ethyl acetate extract of Rutagraveolens and chloroform extract of Convolvulus arvensis induced 50% death at the concentration ranging between 100-200μg/mL, however, the other extracts induced 50% death at the concentration ranging between 225-700μg/mL. Alternation in the chromatin condensation was observed in LoVo cells treated with most cytotoxic extracts. In conclusion, the present data revealed that R. graveolens and C. arvensis may inhibit the proliferation of colon and breast cancer cells and induce apoptosis.

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Melioidosis Database (MDB): A comprehensive web-based resource on Melioidosis and gene protein repository for Burkholderia pseudomallei

Swetha Rayapadi G., Ramaiah Sudha, Sekar Kanagaraj and Anbarasu Anand

Melioidosis caused by Burkholderia pseudomallei (B. pseudomallei), is a predominant infectious disease in tropical countries. Melioidosis awareness is surprisingly low among physicians and health care workers in endemic regions in spite of its higher prevalence. In recent decades, there is a significant interest in comparing the B. pseudomallei genomes of different strains as there is a rapid increase in the number of whole genome sequences from B. pseudomallei strains. Thus, it is imperative to provide organism specific annotations to the scientific community exploring on B. pseudomallei. This prompted us to develop a database exclusively for melioidosis and thus, we have developed the “Melioidosis database” (MDB). Various tools have been deployed in MDB to compare gene and protein sequences of B. pseudomallei strains. The MDB aids in the analysis of phenotypic variations also to perform comprehensive proteomics and genomics studies. The database can be accessed at and it will be updated regularly.

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An improved method for rapid isolation and quantification of Rhizoctonia solani from mungbean rhizospheric soil

Bashyal Bishnu Maya, Kharayat Bhupendra Singh, Kumar Jagat, Dubey Sunil Chandra and Aggarwal Rashmi

Rhizoctonia solani causes web blight/wet root rot disease of mungbean and is also the major constraint to mungbean crop production in India. An improved procedure for quantitative estimation of propagules of Rhizoctonia solani in soil was developed. Maximum number of colonies were observed in Ko and Hora medium (10) followed by water agar (8) following serial dilution after sieving through 0.150 mm sieve. The highly positive correlation (r = 0.96) was observed between disease incidence (%) and number of cfu/g of soil. In the q-PCR analysis for quantification and monitoring of R. solani isolates, the maximum level of inoculum was observed in the rhizospheric soil of mungbean genotype Pusa Vishal at 21 days after sowing. Method can be utilized for the monitoring of soil borne population of R. solani under glass house and field conditions.

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Influence of Lipase in Swelling Behavior of Hydrogel, Morphological Properties of Hydrogels and Enzymatic Polymerization Reaction of PCL by using Lipase Immobilized Hydrogels

Didem Saloglu

Lipase immobilized superabsorbent hydogels were prepared by crosslinking chitosan with glutaraldehyde. In order to increase the water holding capacity of chitosan hydrogels, they were modified using lipase enzyme in different loading degrees and the optimum amount of enyzmes was determined. The swelling behaviours of chitosan hydrogel and lipase immobilized superabsorbent hydrogel were investigated by gravimetric measurements with different pH values and at different intervals of time. Lipase immobilized superabsorbent hydrogels were fully characterized with FT-IR, TGA, DSC, XRD and SEM analyses. The study aims at presenting the use and development of original catalytic systems based on lipases which are efficient for polyester synthesis and allowing the preparation of biomaterials based on chitosan hydrogels. For this, ε-caprolactone (ε-CL) polymerization catalyzed by immobilized Candida antarctica lipase B (CALB) onto superabsorbent hydrogel was carried out in the presence of gluteraldehyde to obtain biomaterials through polymer chains grafting and growth from the hydroxyl groups of the chitosan. Immobilized form of CALB onto superabsorbent hydrogel has been tested as catalytic systems and its efficiency has been discussed.

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Phage display library based antiviral screening: porcine respiratory and reproductive syndrome virus endoribonuclease is a target for antiviral screening

Jie Zhou, Jianhua Hu, Zixue Shi and Cheng Gao

Viral endoribonuclease plays a vital role in the physiological and biochemical activities of viral RNA. In our studies on PRRSV, we seek to screen candidate antiviral molecules that target this endoribonuclease. Through 5 rounds of phage-display screening of 12-ammino acid peptides, 4 peptides that show high affinity to the viral endoribonuclease were identified. In vitro experiments showed very high antiviral activity of all these 4 peptides. The E4 peptide (NIPIKPRPRLMK) showed the highest antiviral activity (IC50 = 39.21μM), yet the lowest cytotoxicity. E4 can directly bind to the endoribonuclease of PRRSV (NSP11) and reduce copy number of viral RNA by 23.68 folds. Through large-scale phage-display screening and following in vitro analysis, we proved that viral endoribonuclease is a potential target of antiviral drugs.

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Detection of Listeria monocytogenes and Brucella spp. in a Turkish home-made çeçil cheese by real-time PCR

Serap Kılıç Altun, Akın Yiğin, Mehmet Demirci, Aydın Daş and Sadık Serkan Aydın

Çeçil cheese is a kind of soft cheese made from raw cow's skimmed milk produced in Erzurum, Turkey. The aim of this study was to analyze the presence of Listeria monocytogenes, Brucella spp. and microbial composition in çeçil cheeses sold in the markets and bazaars of district in Erzurum. The presence of Brucella spp. and L.monocytogenes was investigated in a total of 104 fresh home-made çeçil cheese samples via real-time PCR. The prevalance of Brucella spp. and Listeria monocytogenes was found to be 2,9% and 16,3% respectively. Overall mean levels of colony counts were detected for different microorganisms: for aerobic microorganisms 7,74x103cfu/g, for psychrotrophic microorganisms 8,66x103 cfu/g, for yeast and mould 7,55xl03 cfu/g, for Enteroccoci 4,08xl03 cfu/g and for E.coli 1,80xl01 cfu/g. The study showed that home-made çeçil cheeses were found to be not enough hygienic and after consumption of these kind of cheeses contains foodborne pathogens such as L. monocytogenes and Brucella spp. and may cause serious outbreaks. Also, real-time PCR can be used for rapid identification of L.monocytogenes and Brucella spp. from Listeria spp and Brucella spp. positive colonies respectively. As a result, hygiene awareness should be increased to the consumption of products made from raw milk such as Çeçil cheese.

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The clinical application and efficacy of stent for bronchopleural fistula after pulmonary resection

Jin Jiang-Tao and Han Xin-Wei

Aim of study is to observe the therapeutic effect of a series of special airway stent to treat bronchial stump fistula on patients with bronchial fistula. Our team individually designed the plugged stents according to the location, size, stump length and angle of the orificium fistula of BPF, then placed them into the airway of patients who developed BPF after pneumonectomy under the guild of DSA equipment. According to the patient's condition to determine whether to carry bronchial fistula drainage tube or not, we calculated the post-operative changes of the residual cavity size, drainage and lung infection. In the next regular follow-up, the patient’s quality of life was assessed and the survival rate was recorded. Implantation of individual BPF-plugged stents can plug BPF effectively. The rate of pulmonary infection in BPF patients with drainage is lower than those without drainage. Patients of pulmonary lobectomy recover faster with a higher recover rate than those of pneumonectomy. Implantation of individual BPF-plugged stents is able to plug BPF effectively. Interventional treatment by implantation of BPF-plugged stents is safe, less invasive and effective with low cost and little suffering making this treatment deserves a wild clinical application.

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The isolation and mutagenic improvement of Aspergillus niger for production of Citric Acid

Erkmen O.

Aspergillus niger is used in the production of citric acid. The aim of this study was to isolate A. niger and to subject random mutagenesis by UV radiations, ethidium bromide and sodium azide for producing mutant A. niger strain capable of overproducing citric acid. A. niger strains were isolated from seventy-five mud samples. They were subjected to random mutagenesis by UV radiations, ethidium bromide and sodium azide to improve the citric acid production capacity. Their citric acid production capacity was tested in shake flask and fermenter studies. Mutant and wild strain were compared for their citric acid production ability, glucose utilization and yield. Some mutants produced more citric acid with higher consumption of glucose compared to parental strains. The mean glucose consumption and citric acid production among five A. niger strains during fermentation period are significantly different (P0.05). Of all mutant generated by UV exposure for 70 min produced higher concentration of citric acid. This study proved that citric acid production was increased with UV and chemicals mutated A. niger strains compared with wild strains. This mutational work has a potent A. niger strain for production of citric acid.

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