Research Journal of Biotechnology

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Glucose and low temperature enhanced plasmid stability for increasing expression of Nipah virus glycoprotein in Escherichia coli

Gan Khai Swan, Tan Chon Seng, Othman Rofina Yasmin and Harikrishna Jennifer Ann*

Escherichia coli (E. coli) provides an attractive system for the production of viral proteins for study and for biotechnological uses such as vaccine development. However, the expression of some viral genes may interfere with the survival of recombinant E. coli cells leading to bacterial death. Manipulation of culture conditions may alleviate some of these problems and extend the use of E. coli for recalcitrant proteins. Nipah virus (NiV) is a zoonotic virus resulting in several fatalities each year and for which no human vaccine is currently available. In this study, a gene sequence encoding the extra-cellular domain of NiV envelope attachment glycoprotein, a potential candidate for a vaccine for NiV, was ligated into a bacterial expression plasmid vector, pRSET A. Under standard growing conditions, E. coli containing the expression plasmid showed extremely slow growth. However, reduction of the culture growth temperature and inclusion of glucose in the medium during the growth phase improved the plasmid stability as measured by percentage of plasmid-carrying cells by more than 2-fold. Incorporation of these conditions into a two stage-cultivation strategy increased expression of the NiV glycoprotein by over 14-fold.

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Hepatoprotective activity of aqueous extract of Phyllanthus niruri in CCl4 induced liver toxicity - in vivo study

Pandian Manonmani, Marimuthu Ramar*, Natesan Geetha, Mariadhas Valan Arasu, Rajagopal Raskin Erusan and Justin Jerlin Sowmiya

In rural India, Phyllanthus niruri is mainly used as the traditional medicine for the treatment of liver disorders. The present study was aimed to evaluate the efficacy of Phyllanthus niruri on the induction of oxidative hepatic damage by carbon tetrachloride (CCl4) in male Swiss albino mice. Single dose of 0.2 ml/kg of CCl4 along with vehicle alternatively for 15 days and 100 mg /kg of Phyllanthus niruri plant extract was orally administered for 20 days. Liver damage was assessed by the estimation of serum activities of alanine aminotransferase (ALT) and asparate aminotransferase (AST).

Results indicated a significant increase in the level of liver enzymes like ALT, AST, ALP and bilirubin content 273.6 ± 2.40, 98.60 ± 2.13, 248.6 ± 4.87 U/L and 6.5 ± 0.16 μmol/L in CCl4 intoxicated animals when compared with that of the control group of mice 120.8 ± 2.87, 42.73 ± 1.98, 173.5 ± 3.24 U/L and 0.6 ± 0.02 μmol/L respectively. Haematological investigations (RBC count, PVC, Hb, WBC count and Platelet count) did not reveal any significant changes among the different groups whereas histological examination confirmed that the liver hepatocytes arranged in one cell thickness indicated the curative effect of cystic fibrosis, a mild inflammatory change and greater area of regeneration. The reduction in body weight was minimal and liver enlargement was also less compared to the animals in toxic control group. Overall results indicated that the aqueous extract of P. niruri possesses hepatoprotective effects which can be used in treatment of liver diseases.

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Carbapenem-resistant Acinetobacter baumannii complex bacteraemia in elderly patients in northern Taiwan

Chow S. F., Sheu D. C.* and Liu C. P.*

The elderly patients (aged ≥ 65y) among general population have increased in Taiwan. The aim of this study is to identify the risk factors for attributable mortality among elderly patients with carbapenem-resistant Acinetobacter baumannii (CRAB) complex bacteraemia. A retrospective study of patients with CRAB complex bacteraemia at a medical center in northern Taiwan from 2009 to 2013 was performed. Variables such as clinical characteristics, antibiotics susceptibilities, and outcomes were compared between elderly patients (aged ≥ 65y) and the other patients (aged < 65y). Risk factors for attributable mortality (67/121, 55.37%) among elderly patients included APACHE II score > 25 (OR 6.702, p = 0.001), respiratory tract source (OR 4.416, p = 0.026) and cefepime (OR 13.203, p = 0.023). Our findings show at the hospital in northern Taiwan, elderly patients (aged ≥ 65y) with CRAB complex bacteraemia had a different epidemiology than the other patients (aged < 65y). Elderly patients with an APACHE II score > 25, respiratory tract source and the use of cefepime had associated with mortality. Infection control measures, especially for respiratory tract source and early adequate treatment may be helpful in the survival of elderly patients (aged ≥ 65y).

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Identification and quantification of quercetin glycosides present in different color onions (Allium cepa L.)

Seo Jeong Min, Arasu Mariadhas Valan, Kwak Jung-Ho, Kim Sunggil, Yoo Dae-seok, Yoon Moo Kyoung* and Kim Sun-Ju*

We identified and quantified quercetin 7, 4’-diglucoside (m/z 625), quercetin 3-glucoside (m/z 463), quercetin 4′-glucoside (m/z 463) and quercetin (m/z 301) using LC (ESI)-MS/MS and HPLC analysis. Quercetin glycosides contents varied amongst the different varieties of onion. The content of total quercetin glycosides was predominantly in first scaly leaves of all the varieties and drastically reduced. The total content of glycosides was mean 46.44 mg/g dry weight (DW), and quercetin 4′-glucoside was the predominant compound with mean 23.1 mg/g DW followed by quercetin 7,4′-diglucoside (11.20 mg/g DW) and quercetin 3-glucoside (3.68 mg/g DW) respectively. The highest quercetin content (32.81 mg/g DW) was found in first SL of chartreuse and second SL has higher quercetin content (9.88) than other cultivars. A distinct variation in the contents of quercetin glycosides was detected between the outer and inner layer. Thus, the variations of quercetin glycosides in the colorful onions were significant.

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Molecular diversity of different potassium solubilizing bacterial species of Pseudomonas through RAPD

Gore N. S.*, Navale A. M. and Ghutukade K. S.

In the present work, bacterial isolates which are able to solubilize rock K mineral powder such as Micas were obtained from 104 rhizosphere soil samples of selected district places of Western Maharashtra. On the basis of screening of potassium solubilizers, only 10 isolates were found efficient to solubilize potassium. The potassium solubilizing bacterial isolates were designated as KSB.

Based on the cultural, morphological and biochemical tests, the selected bacteria were identified as six were Pseudomonas fluorescens, two were Pseudomonas putida and Bacillus megaterium. Out of the 10 isolates, 8 were found Pseudomonas sp. which were further selected for analyzing molecular diversity within the group. Polymerase chain reaction (PCR) based technique, taking the random amplified polymorphic DNA (RAPD) has been applied for assessing genomic variability among the selected KSB isolates. The genomic DNA of 8 Pseudomonas sp. were subjected to PCR amplification using 20 random oligonucleotide primers (RBA 1 to RBA 20) showing 221 polymorphic bands. RAPD fingerprinting of Pseudomonas spp. revealed polymorphism in 97.54%.

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Identification of salt-responsive transcripts in Kodo millet (Paspalum scrobiculatum L)

Siddappa N. and Devaraj Varadahally R.*

Salinity stress is one of the major limiting factors reducing crop yield worldwide. Identification of salt tolerance genes from tolerant plants would help to transform susceptible plants. Kodo millet is known to be highly drought and salt tolerant crop as ascertained by antioxidants and antioxidant enzymes levels. Responses to abiotic stress like salinity are mediated by multiple gene products. In an effort to gain an insight into nature of genes expressed/repressed during applied salt stress, cDNA library was constructed from 300 mM NaCl- stressed seedlings. 15 ESTs differentially expressed under salinity stress were characterized by DDRT-PCR and their expression profile was assessed by real time RT-PCR. The expression levels indicated over expression of 11 ESTs and down regulation of 4 ESTs. Although their homologues were from diverse plant sources, 8 of these showed greater homology to foxtail millet.

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Computational identification of salinity responsive microRNAs in contrasting genotypes of Finger millet (Eleusinecoracana L.)

Jagadeesh Selvam, Hifzur Rahman N., Senthil N., Manikanda Boopathi N. and Raveendran M.*

Globally, salinity causes huge losses to crop production and it has been the focus of intense research to unlock the complex mechanisms involved in salinity tolerance in plants. Recently, microRNAs (miRNAs) have been shown to play pivotal roles in modulating plant’s adaptation to salinity stress. The present study was aimed at identifying salinity stress-responsive miRNAs in Finger millet with a view towards mining novel candidate genes/pathways for improving salinity tolerance in crop plants. RNA-Seq data obtained from control and salinity stressed leaf tissues of two contrasting Finger millet genotypes viz., CO 12 (susceptible) and Trichy 1 (tolerant) were used for mining salinity responsive miRNAs in Finger millet. Forty-eight conserved/known miRNAs belonging to 23different miRNA families and 35 novel miRNAs were identified in Finger millet using appropriate computational tools.

Data analysis resulted in the identification of conserved miRNAs exhibiting significant salinity responsive differential expression in Finger millet. miRNAs viz., miR160, miR162, miR169, miR894 and miR2914 showed more than 10 fold up-regulation in the tolerant Trichy 1 during salinity than the susceptible CO 12. One of the miRNA namely, miR159 was found to be 25 fold less abundant in the tolerant Trichy 1 during salinity stress which was predicted to target key salinity tolerance related genes viz., MYB transcription factor(s) and calmodulin-related calcium sensor protein. This study also resulted in the identification of novel salinity responsive miRNAs in Finger millet and their putative targets. Our work provides the first insight into the salinity responsive miRNAs in Finger millet, thereby expanding our knowledge on potential salt-stress regulated genes.

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DNA for Species Identification in Leather: Fraud detection and endangered species protection

Merheb Maxime*, Vaiedelich Stéphane, Maniguet Thierry and Hänni Catherine

In this study, we focused on species identification in leather. Based on optimized extraction method combined with a panel of ultra-specific primers targeting short mitochondrial DNA fragments, we have developed a complete and efficient method that allows molecular identification of species origin in leather. Despite great advances in the fields of forensic, species identification in processed animal products is still very limited, not only because of classic problems as the tiny amount of DNA and/or DNA degradation, but also because of the critical problem of PCR inhibitors which are co-extracted with DNA molecules. In perspective, this study could be applied to detect frauds in particular in leather market and/or protect endangered species.

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Mucor indicus: the novel bioconversion producer of L-phenylalanine into 2-phenylethanol

Song F.F. and Xiang W. L.*

The novel trait of dimorphic Mucor indicus to produce 2-phenylethanol (2-PE) from L-phenylalanine (L-Phe) by yeast-like cells was first reported. In the L-Phe bioconversion, the spore inoculation level and glucose concentration had an important impact on the cell morphological developing to filamentous or yeast-like and thus affected on the 2-PE production. In the potato dextrose broth with 80 g l-1 glucose supplement, about 2.0 × 106 spore ml-1 inoculation and 6 g l-1 L-Phe were added at 36 h cultivation, M. indicus had synthesized 1.48 g l-1 2-PE by yeast-like after 108 h with 0.32 g g-1 bioconversion rate, which appeared promising for industries.

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Double and Triple Gel Stocks Method in Pulsed Field Gel Electrophoresis

Unal Senel

Although pulsed-field gel electrophoresis (PFGE) is a very valuable and gold standard method in molecular typing of bacteria-based epidemic diseases, its main disadvantages are as follows; it is time-consuming, it enables simultaneous working on a very limited samples and it requires experienced personnel. This study aims to work on 2-3 times more samples during the single gel system protocol simultaneously with the application of double and triple gel system to current PFGE system. In order to ensure the perfection of DNA patterns observed in gels to be assessed as those in single system and to prevent the contamination of these DNA patterns; intermediate lamellas manufactured from 13x14 cm polystyrene (PS), acrylonitrile-butadiene-styrene (ABS) and acetyl cellulose were used.

The results obtained from the double gel system including polystyrene (PS), acrylonitrile-butadiene-styrene(ABS) are similar with the original gel system without sacrificing quality. However the desired results could not be obtained from the triple gel system in which the intermediate lamella made of acetyl cellulose was used.

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Effect of Lactic acid Fermentation on Cognitive Activities of Aronia merlanocarpa Elliot

Kim Nam Young, Ryu Jung Man and Lee Hyeon Yong*

The extract of the fermented A. melanocarpa by 70% ethanol (FEE) showed higher DPPH free radical scavenging ability of 70.36 than 65.59% of the extract by 70% ethanol only (EE) as a control at 1.0 mgml-1. The FEE also showed better acetylcholinesterase inhibition than the EE: 63.21% vs 46.25% in adding 1.0 mgml-1 of the extracts. For in vivo assessment of cognitive functions, Morris water maze test, the latency time of the mouse escape was observed as 65 seconds and 79 seconds in feeding the FEE and EE extracts, respectively. The FEE also had better passive avoidance test results as 109 seconds in feeding the FEE while 53 seconds in the EE. Both in vitro and in vivo results can clearly demonstrate that the FEE should have better cognitive activities that the EE.

For HPLC analysis of both extracts, the FEE contained 112.8 mgg-1 of higher amounts of Cyanidin-galactoside, a major anthocyanin of high antioxidant in A. melanocarpa than that of the EE, possibly by high elution of this active substance as a result of the growth of lactic acid bacteria. Moreover, the FEE also had 101.8 mgg-1 of very high amounts of total phenolic acids and this data would imply that high cognitive activities of the FEE were caused by strong antioxidant activities of the extract due to lactic acid fermentation process. In this work, it was first quantitatively proved that the cognitive activities of A. melanocarpa could be enhanced by Lactobacillus rhamnosus, lactic acid fermentation whose results were found to be closely associated with its high antioxidant activities.

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DNA Fingerprinting based Identification and Classification of Indica Rice (Oryza sativa L) to assess Genetic diversity via ISSR and RAPD markers

Singh Ashu* and Sengar R. S.

Genetic improvement mainly depends on the extent of genetic variability present in the population. The molecular marker is a useful tool for assessing genetic variations and resolving cultivar identities. The objective of this study was to evaluate the genetic divergence of 30 rice varieties (Basmati, Non Basmati, Aerobic) using 10 ISSR, RAPD markers each. The diversity or similarities and dissimilarities between all thirty rice varieties were calculated using 0 1 sheets. In RAPD analysis PIC values vary from 0.811(OPD-08) to 0.9925(OPF-13) with average of 0.9635 and resolving power varies from 1.32(OPJ-08) to 2.066(OPJ-13) with average of 1.8256. In ISSR analysis, PIC value ranged from 0.8791(ISSR6) to 0.9916(ISSR5) with an average value of 0.9482. The resolving power varies between 1.6(ISSR3) and 8.366(ISSR2) with an average value of 5.2708. The PIC values and Resolving power were calculated for individual primers.

The analysis indicated that ISSR expressed maximum resolving power of 8.336 and RAPD gave maximum PIC values of 0.9925. RAPD primer OPF-13 gave the maximum accessions coverage (depending on the value of PIC) in the rice genome. Out of 52 amplified bands, 49 bands were polymorphic and 3 bands were monomorphic. The cluster analysis using the two marker systems could distinguish the different genotypes. The dendogram generated on the principle of Unweighted Pair Wise Method using Arithemetic Average (UPGMA) was constructed by Jaccard’s Coefficient and the genotypes were grouped in to clusters. The dendogram developed for aroma and quality traits showed that the genotypes with common phylogeny and geographical orientation tend to cluster together, thus marker based molecular fingerprinting could serve as a sound basis in the identification of genetically distant accessions as well as in the duplicate sorting of the morphologically close accessions as the case is common in differentiating basmati and non basmati.

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