Research Journal of Biotechnology

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Research Journal of Biotechnology

On Field Detection of White Spot Syndrome Virus (WSSV) infected Shrimp using Loop Mediated Isothermal Amplification

Devadas Samyukthaa and Pasupathi Rathinasabapathi*

White spot syndrome virus (WSSV) is a baculovirus associated with infections in cultivated shrimp resulting in high mortality within days of onset bringing in a need for early detection for economic importance. The objective of this study is to develop a Loop-mediated Isothermal Amplification (LAMP) method for sensitive, rapid and a less expensive detection of WSSV in Penaeus monodon (tiger shrimp). A set of four specific primers were designed targeting the WSSV vp28 (viral coat protein) gene. The target DNA was amplified as early as 30 mins of incubation at 65°C in a simple water bath by using Bst DNA polymerase large fragment which has strand displacement activity. A positive reaction was identified through the detection of LAMP products by green fluorescence under UV (ultraviolet) light with the help of Calcein. The sensitivity of LAMP is compared with that of PCR and could detect as low as 100 autogram where as PCR detect till 500 femtogram only. The high sensitivity, specificity, simplicity and rapidness make this method suitable for early detection of WSSV infection.

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Repair Effect of Sanchi-loaded Electrospun Artificial Skin Substitute on Damaged Skin in Rats

Zhou Jiaming1,Cui Xiuming1, Zhang Wenbin1 and Xu Feng2*

Artificial skin substitute made of biomaterials and traditional Chinese medicine without living cells is promising for damaged skin repair. The kind of skin substitute possesses excellent histocompatibility without any risk of virus or immunogenicity. The objective of this study was to evaluate the repair effect of a novel Sanchi (Panax Notoginseng (Burk) F.H.Chen) -loaded Electrospun artificial skin substitute (SASS) on damaged skin in rats. SASS was made of polylactic acid, gelatin and Panax Notoginseng by electrospinning method. Animal model of skin damage was established. Wound of 3cm × 3cm in the back of each SD rat was formed under anesthesia. After model establishment, the rats were randomized into two groups. In one group the damaged skin was covered with SASS and in other group with petrolatum gauze (PG). The wound healing was observed for 8 weeks. The tissue sample was drawn for histological examination on 2nd, 4th and 8th week. The results showed that all rats in both groups grew well, but the resistance to infection and prevention wound bleeding in SASS group were better than in PG cover group. Histological observation indicated that SASS could improve much tissue regeneration than PG. SASS material is soft and easy to cut, has a good ability of anti-tearing and adhesion to wound. Compared with PG, SASS is an excellent artificial substitute for skin damage repair.

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Rapid in vitro propagation of Dioscorea bulbifera through bulbils and nodal segments

Manjunatha B.K1., Vidya S.M2., Pradeepa2, Banu Shameem3, Taranum Ruhinaz3, Nalini S.M3., Sunitha B.J.3 and Nadig Smitha3

The rapid micropropagation protocol of yam species, Dioscorea bulbifera was carried out by using nodal and bulbil segments cultured on MS medium supplemented with different concentrations and combinations of cytokinins for in-vitro shoot induction. Highest frequency (100%) of shoots was induced in nodal and bulbil explants cultured on MS medium supplemented with kinetin (Kn). Mean number and length of shoots developed per nodal explants were 25.7  1.30 and 8.45  0.25 respectively compared with BAP (4mg/l) as 24.5  0.20 and 2.56 0.12. For bulbils mean number and length of shoots were 19.1  0.19 and 5.9 ± 0.11 for Kn (3mg/l) and 27.5  0.20 and 2.96  0.22 for BAP (4mg/l) respectively. Concentration of all cytokinins tested above the optimum level showed reduced frequency of shoot proliferation. An average of 25 shoots was induced per explants indicating the efficient proliferation of D.bulbifera. The shoots were then transferred to the MS half strength medium containing different concentrations (0.5-2.5mg/l) of auxins including NAA, IBA and IAA to test the efficiency of root induction. In the present study, NAA at the concentration of 0.5 mg/l induced 81.5% root initials and 80% of the plants were successfully survived. This technique offers an effective means of disease free plant propagation.

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Isolation, screening and characterization of cellulose producing Bacillus subtilis KG10 from virgin forest of Kovai Kutralam, Coimbatore, India

Ramalingam Kowsalya and Ramasamy Gurusamy*

Cellulose degradation for the renewable energy production is gaining importance for which untapped virgin forest of Kovai Kutralum, Coimbatore, Tamil Nadu, India was chosen for cellulase producing bacteria isolation. Altogether, 20 soil samples were collected, the pH of the collected soil samples ranged between 5.5 and 7.5. A total of 40 different isolates of bacteria were isolated using selective medium Cellulose-Congo red. Qualitative and quantitative screenings for cellulolytic activity of all the 40 isolates revealed that the isolate KG10 showed significant clear zone on cellulose agar and exhibited remarkable enzyme activity of 0.140 U/ml hence, the isolate KG10 was selected for further studies. The taxonomical position of the isolate KG10 was characterized using morphological, biochemical and molecular approach (i.e. 16S rRNA gene sequence analysis) and the results clearly revealed that the isolate KG 10 was Bacillus subtilis.

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Association of Arbuscular mycorrhizal fungi and other rhizosphere microbes with different medicinal plants

Althaf Hussain Sk. and Srinivas P.*

Occurrence of AM fungi in the rhizosphere soil was investigated in different medicinal plants growing in similar climatic conditions. Ten medicinal plant species Emblica officinalis, Aloe barbadensis, Smilax sp., Rauwolfia serpentine, Rauvolfia tetraphylla, Sapindus trifoliatus, Euphoria longan, Centella asiatica, Mimosa pudica and Trachyspermum copticum that were growing in Medicinal arbour of Department of Biotechnology Kakatiya university, Warangal were studied. The present study was carried out to study the interaction of ten different medicinal plants with AM fungi by spore population and root colonization. Huge variation was observed in the spore population and root colonization in spite of their growth in similar climatic conditions. A great variation was observed in other bacterial and fungal population also.

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Uptake and breakdown of lignin, lignin derivatives and dyes by a dimorphic fungus MVI.2011

Lipin Dev M. S. and Thankamani Vaidyanathan*

MVI.2011, a highly potent alkalophilic, non sporing soil fungus was characterized in detail with respect to utilisation of lignin and its derivatives as a major source of carbon for its growth. The fungus produced large colonies (20-40 mm dia.) in minimal media containing 0.1 % each of lignin, its derivatives veratrol, guaicol, veratraldehyde, aceto syringone, methoxy acetophenone and hydroxyl benzaldehyde and dyes (azur B and phenol red) at 25-28oC in 12-18hours varying in microscopy, colony morphology and pigmentation. MVI.2011 could metabolise lignins and dyes as carbon source in the presence of 1% glucose co substrate. In most of the lignin substrates, the isolate showed dimorphism and pleomorphism. In liquid media containing lignins, maximum biomass was obtained in methoxyacetophenone (48g/l) and minimum biomass in veratrol (29g/l). In substrates giving the highest biomass, the pH remained high between 8.8 and 8.5. Veratrol producing the lowest biomass showed a drop of pH from 8.5 to 6.8. The absorption maxima of all the products in the culture supernatant differed from that of the respective controls indicating metabolic break down of the substrates by MVI.2011. The 18SrRNA sequence proved MVI.2011 to be a novel “uncultured fungus” with Genbank Accession number JN606084.

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Prediction of Drug Target Proteins with Affinity Propagation Algorithm

Feng Yanghe1* and Wang Jiao T.2

Drug targets identification and validation is crucial in biomedical research. Most recently, lots of studies have been conducted on analysis of drug target features aiming at getting a better understanding of drug target features. But most of them were either based on strong biological hypothesis or study those targets separately. In this study we adopted affinity propagation (AP) algorithm to cluster the drug targets and predicted 123 potential drug targets.

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The induction of ferrous sulfate or sodium acetate on mRNA expression of carotenoid genes in Haematococcus pluvialis

Gao Zhengquan1, Meng Chunxiao1*, Gao Hongzheng1, Zhang Xiaowen2, Su Yuanfeng1 and Ye Naihao2*

Haematococcus pluvialis accumulates large amounts of astaxanthin under a variety of environmental stresses. In this study, transcriptional pattern of eight carotenoid genes involved in astaxanthin biosynthesis exposed to ferrous sulfate (1 and 2mM named Fe1 and Fe2) or sodium acetate (20 and 40mM named sa20 and sa40) was separately revealed using qRTPCR. Results showed that all eight carotenoid genes were up-regulated by ferrous sulfate or sodium acetate with different expression profiles. Ferrous sulfate induction had a greater effect on the transcriptional expression of ipi-1, pds, crtR-b and bkt in Fe1 and pds and bkt in Fe2 than on other genes and sodium acetate treatments had more influence on the transcriptional expression of ipi-1, ipi-2, psy, lyc and bkt in sa20 and ipi-1 and psy in sa40 than on other genes. Furthermore, astaxanthin biosynthesis in the presence of ferrous sulfate might be up-regulated by ipi-1, ipi- 2, pds, lyc, bkt and crtR-b at transcriptional level, psy at post-transcriptional level and by crtO at both transcriptional and post-transcriptional levels. Astaxanthin biosynthesis exposed to sodium acetate was upregulated mainly by ipi-1, ipi-2, psy, pds, crtR-b, bkt and crtO at post-transcriptional level and lyc at both transcriptional and post-transcriptional levels.

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Pairwise Sequence Alignment using Bee Colony Optimization on Compressed Biological Database

Kunthavai A.1* and Vasantharathna S.

Sequence alignment is a bioinformatics application that determines the degree of similarity between nucleotide sequences. Alignment reads query sequence from the user and makes an alignment against large genomic sequences and locate targets. Existing tools, BLAST and WU-BLAST, employ heuristics to improve the speed of the search, such heuristics sometimes miss targets. Considering the rapid growth of database sizes, this problem demands ever-growing computation resources and remains as a computational challenge. In this study, Bee Colony Optimization has been applied to find pairwise sequence alignment by preprocessing the size of the database based on longest common subsequence (LCS). Time complexity to find LCS using HashIndex algorithm is O (2 + γ) where ‘γ’ is the time taken to access the pattern. Space complexity is also reduced using BioDBMPHF Tool. IBCO Aligner obtains same results compared to that traditional algorithm in approximately half the time taken by them.

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Nucleotide sequence variability in exon 4 of prolactin locus in Murrah buffaloes (Bubalus bubalis)

Verma Archana

Prolactin plays an important role in mammary gland development, milk secretion and expression of milk protein genes. This is a candidate gene and a potential genetic marker for production traits in dairy animals. Therefore, present study aimed to screen buffalo herd maintained at National Dairy Research Institute, Karnal for the genetic polymorphism of the PRL gene using PCR–RFLP and PCR-SSCP analysis for future use of genetic variants as markers for animal selection. Genomic DNA was isolated from 150 lactating Murrah buffaloes maintained at National Dairy Research Institute, Karnal. Polymerase chain reaction (PCR) was set up to amplify exon 4 of prolactin locus using specific primers. PCR-RFLP analysis was carried out by digestion of 294 bp amplicon with Rsa I restriction enzyme. The animals included in the study exhibited monomorphism. PCR-SSCP resulted in two band patterns. DNA sequencing revealed nucleotide sequence variation at a total of six positions: two in intron 3, three in exon 4 and one in intron 4 regions. All were silent mutations except G8477T resulting in amino acid substitution from glycine to cysteine at 53rd position. Identification of a specific DNA marker and its association with production traits may be utilized in developing specific breeding programmes for animal selection.

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Epigenetic modifications as prognostic marker in cancer

Pamnani Mamta, Sinha Puja and Sachan Manisha*

DNA methylation as most predominant epigenetic modification is an important regulator of gene expression and for last few years it has been a topic of considerable interest for its role in carcinogenesis. Analysis of DNA methylation as epigenetic marks can facilitate the development of very accurate biomarkers for detection and diagnosis as well as prognosis of outcomes. Alterations in DNA methylation are common in a variety of tumors as well as in development. The most common outcome is hypermethylation which represses transcription of the promoter regions of tumor suppressor genes leading to gene silencing. However, global hypomethylation has also been recognized as a cause of oncogenesis. New information concerning the mechanism of DNA methylation and its regulation by variety of proteins and enzymes has made this event even more exciting. As methylation occurs early and can be detected in body fluids, it may be of potential use in early detection of tumors and for determining the prognosis. Many DNA demethylaing agents as well as small interference RNA are used for the treatment of variety of tumors.

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Harnessing PGPR from rhizosphere of prevalent medicinal plants in tribal areas of Central India

Singh Vasudha, Sharma Shivesh* and Shukla Keshav Prasad

Tribal region of India is a natural, invaluable store house of indigenous medicinal plant diversity of great importance for mankind. Protection of traditional knowledge of these tribal community is very important as overexploitation of natural resources due to increase in population may lead to extinction of important medicinal plant species and with careful management, a sustainable balance can be achieved. Some of the medicinal plants are known to grow only in their indigenous regions and it is very difficult to increase their population. Plant growth promoting rhizobacteria (PGPR) have been known to be applied to a wide range of agricultural crops for the purpose of growth enhancement and harvest yields. Similarly medicinal plants forming association with various microorganisms can be established as biofertilizer and biocontrol agents. Hence, it is very important to identify and characterize rhizospheric microorganisms associated with medicinal plants. The relationship between the soil microbial diversity and quality of medicinal plant has not been fully understood and it will be very helpful to investigate the quality of genuine authentic crude drug derived from these medicinal plants as well as to increase their productivity.

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