Vol. 10(9) September 2015
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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