Research Journal of Biotechnology

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Population genetic structure analysis of the root maggot, Bradysia odoriphaga (Diptera: Sciaridae) based on COI markers

Wenxiang Li, Juan Lv, Haixia Yan, Wen Xie, Qingjun Wu, Shaoli Wang, Baoyun Xu, Guoshu Wei and Youjun Zhang

Page No: 1-9

Abstract: The chive midge, Bradysia odoriphaga Yang and Zhang (Diptera: Sciaridae) with a wide host range caused huge economic losses especially in Chinese chives of China in recent years. We identified 29 polymorphic sites and 23 haplotypes based on the genetic structure of mitochondrial cytochrome c oxidase subunit I gene (mtCOI) from 17 Bradysia odoriphaga populations of China. The total haplotype diversity (hd), nucleotide diversity (Pi) and average number of nucleotide differences (K) were 0.25085, 0.00065 and 0.44181 respectively. Tajima's D tests between AHBZ, HBBD and TJWQ were statistically significant, suggesting that there might have been a population expansion in recent history.

Values of the population pairwise fixation index (FST) ranged from 0 to 0.6053 with the highest value between SDZQ and LNSY. Genetic differentiation was not seen among SD populations. The haplotype parsimony network showed that haplotype 1 (H1) was shared by all populations and a low level of gene flow occurred between populations HBBD, AHBZ and TJWQ, but there was no gene flow among the other populations. The data obtained in this study was the first report on genetic variation and phylogeography of B. odoriphaga and will be relevant for integrated control measures of this pest.

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Recognition of bifunctional aptamers for SMMC-7721 and Bel-7404 liver carcinoma cells

Shihua Zhang

Page No: 10-24

Abstract: Developing bifunctional aptamers for human hepatic carcinoma (HCC) cells of SMMC-7721 and Bel-7404 with high affinity and specificity is important in early diagnosis and advanced targeted therapies for HCC. This study is a report on pattern recognition models for bifunctional aptamers possessing wide variety of applications. Three molecular descriptors used for the classification model were calculated from amino acid sequences that are translated from DNA aptamer sequences with DNAMAN software.

The classification model is a logical regression equation developed by applying binary logical regression analysis from the training set of 178 aptamer sequences. Then the model was validated with the test set of 89 aptamer sequences. The classification model has prediction accuracies, sensitivity and specificity for both training set and test set over 90%. Therefore, it is feasible to calculate molecular descriptors from amino acid sequence translated from DNA aptamer sequences in order to develop pattern recognition model for bifunctional aptamers of HCC SMMC-7721 and HCC Bel-7404.

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Genetic diversity among culturable marine biofilm forming bacteria isolated from different test panels

Bragadeeswaran S. and Sri Kumaran N.

Page No: 25-33

Abstract: In the present study, 29 biofilm forming-bacteria were isolated from aluminum, fiber glass and wood panels deployed in Vellar estuary, Parangipettai, Southeast coast of India. Among the 29 strains, Proteobacteria was found to be dominant (52%) compared to Firmicutes (26%), Actinobacteria (18%) and Bacteroidetes (4%). Five morphologically different biofilm-forming bacteria from each of the 29 strains were sequenced and submitted to the NCBI with an average length of 1483 bp. Further, these 5 sequences were analysed for phylogenetic relationships with 21 marine biofilm sequences extracted via FASTA format from NCBI. Maximum Likelihood (ML), Maximum Parsimony (MP) analysis, genetic distances with standard error, nucleotide contents and percentage of the 16S rDNA sequences of the marine biofilm forming bacteria were also studied.

Bacillus sp. SBS10 showed 100% similarity with Bacillus pumilus. Bacillus cereus strain JBS10 shows 88% of similarity with Bacillus sp. and Bacillus flexus. Proteus sp. SBP10 shows 100% similarity with Proteus mirabilis. Vibrio sp. SBP sequence shows a maximum identity of 100% with Vibrio parahaemolyticus strain SB. The result clearly suggests that only biochemical and physiological tests in conjunction with 16S rDNA analysis can yield a realistic picture of the evolutionary relationships of diverse groups of bacteria.

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Measurement of Glucose Uptake Potential of Pluchea lanceolata, Alhagi pseudalhagi, Caesalpinia bonduc in L-6 Myotube

Sachan Anupam K., Rao Ch. V. and Sachan Nikhil K.

Page No: 34-39

Abstract: The main objective of this work is to investigate in vitro antidiabetic activity of hydro-ethanolic extract of Pluchea lanceolata, Alhagi pseudalhagi and Caesalpinia bonduc. The selected plant material was subjected to the extract preparation by Soxhlet apparatus. During phytochemical screening, various kinds of phytochemicals were detected and then in vitro antidiabetic activity of these extracts was detected by measurement of glucose uptake in L6 cell lines.

The study reveals various kinds of phytoconstituents among the various plant extracts. The extracts of Caesalpinia bonduc and Pluchea lanceolata samples have shown some potential in glucose uptake activity when tested in L6 Myotubes. The results were compared with insulin amd metformin as standard drugs. Alhagi pseudalhagi did not show any glucose uptake activity. Probably the mode of action of Alhagi pseudalhagi is not the insulin sensitization or it may not act through glucose uptake mechanism.

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Effects of some factors on calves’ viability and growth in Simmental cattle

Neamt R.I., Ilie D.E., Saplacan S. and Cziszter L.T.

Page No: 40-46

Abstract: The aim of this study was to investigate the relationship between factors that influence calving ease and their effects on calves’ welfare. Knowledge on the influencing factors could prove relevance in setting up 'alarm' thresholds to aid animal welfare strategies. Multifactorial regression models were used to evaluate the influence of studied factors. Calving ease represented an important factor acting on calves’ viability under the effects of calves’ birth weight, type of births and cow’s pelvic area. Higher incidence of morbidity was recorded in twin vs. single calves [P<0.001].

Dystocia increased morbidity rate significantly [P<0.001]. The higher incidence of mortality was recorded in twins vs. singles [P<0.001] and between calves born from dystocic vs. eutocic calving [P<0.001]. Dystocia represented the main cause of morbidity and mortality in calves, being influenced by the calves’ birth weight [P<0.001] and cows’ pelvic area [P<0.001]. Cows experiencing dystocia and calving twins were narrower at ischia compared to those having eutocic [P≤0.05] and singles [P<0.001]. Viable calves were heavier at 90 days of age than morbid calves [P<0.001]. Assessing of calves’ welfare could be a rapid method for evaluation of the rearing system, also an opportunity to assess the ability of calves to accommodate to new environments.

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Optimization of ultrasound parameters on salt pickling of marinated eggs based on orthogonal experimental design

Wu Hui-qing, Ma Mei-hu, Jin Yong-guo and Wei Shuai

Page No: 47-55

Abstract: The effects of ultrasound on salt diffusion, hardness, chewiness, gumminess and color of marinated eggs were studied. The optimization of ultrasonic power, frequency and time was based on orthogonal experimental design. The results showed that with the increase of ultrasonic power and frequency, the salt content of the egg white protein gel increased significantly and the hardness, chewiness and gumminess of the protein gel significantly improved with the increase of L* and a* and decrease of b*. The three ultrasonic parameters affecting marinated eggs in a descending sequence was as follows, ultrasonic power, ultrasonic frequency, and ultrasound time.

The optimized condition for marinated eggs was obtained at an ultrasonic power of 400 W, an ultrasonic frequency of 60 kHz and an ultrasound time of 1.5 h. The properties of marinated eggs under the optimal condition were significantly higher than eggs prepared under normal conditions with 24 h pickling and they were as follows: salt content of 0.153%, hardness of 1697.36 g, chewiness of 1181.30 g and gumminess of 1142.19 g.

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Isolation and Identification of Endophytic Fungi from Seaweeds in Southern Thailand and their Antibacterial Activities against Some Antibiotic Resistance Bacteria

Amina Samae, Asadhawut Hiranrat, Sompong O-Thong, Preuttiporn Supaphon and Nugul Intrasungkha

Page No: 56-62

Abstract:This research was conducted by isolating the endophytic fungi from 8 seaweeds collecting from coastal areas of 4 southern provinces, Thailand. 98 fungal isolates were obtained. By using morphological and molecular approaches for identification, we found that the endophytic fungi belonged to 3 genera, 4 species and other 33 unidentified namely Penicillium spp., Rhizopus spp. and Aspergillus spp. (Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus and Aspergillus rhizopodus). However, we found their morphological differences among these fungal isolates indicating the diverse distinct morphotypes. Therefore 3 isolates: Aspergillus fumigates, Aspergillus niger and Aspergillus rhizopodus were selected for further molecular phylogenetic analysis using primers that focus on ribosomal DNA sequences including the internal transcribed spacers (ITS1 and ITS4) and β-Tubulin (Bt2a and Bt2b).

Our results suggest that Aspergillus rhizopodus which were only one species found to be dominant fungal endophytes showing the highest inhibitory activities against 4 pathogenic bacteria namely Staphylococcus aureus ATCC 25923, Salmonella weltevreden (ESBL), Escherichia coli ATCC 25922 and Staphylococcus aureus (Methicillin resistant Staphylococcus aureus; MRSA). Therefore A. rhizopodus isolate has been selected for further study of chemical type and structure of their antibacterial metabolites using the sequential solvent extract method with ethyl acetate and methanol. The crude extracts were later tested for the inhibitory effect against those antibiotic resistance bacteria. It was found that only ethyl acetate extraction interestedly inhibited against some antibacterial resistance bacterial strains and has been showing the highest inhibitory activities against 4 isolates: S.aureus ATCC 25923, S. Weltevreden (ESBL), E.coli ATCC 25922 and S. aureus (MRSA) with minimal inhibitory concentration (MIC) values of 3.90, 3.90, 7.81 and 15.63 µg/ml compared to the MIC values of standard antibiotic respectively.

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Molecular and morphological assessment of Papaya (Carica papaya)

Rout Gyana Ranjan, Palei Suvalaxmi and Dash Dilip Kumar

Page No: 63-70

Abstract: The study was undertaken to assess the genetic diversity on the basis of morphological and molecular characterization among 12 popular papaya varieties of India. Both morphological, agronomical traits and ISSR marker were studied across 12 popular papaya varieties to characterize the genetic assessment. The morphological attributes like plant height, stem girth, days to first flowering, flowering height, days to first fruiting, first fruiting height, days to first harvest, fruiting length, number of fruits per plant, fruit size and shape and yield per plant showed significant variation among the genotype. Out of total 134 alleles, a total of 89 polymorphic and 45 monomorphic alleles were detected.

Primer USB-811 showed the best marker for identification and diversity estimation of Indian papaya varieties followed by USB-839, USB-835, USB-840, USB-841and UBC-864 primers. On the basis of UPGMA cluster analysis, the 12 varieties were classified into two clusters with a similarity coefficient of 59%. The genotype pair ‘Sunrise Solo’ and ‘Arka Surya’ showed the maximum similarity (0.89) among the 12 papaya genotypes. The morphological attributes are closely resemblance with molecular analysis. This study offered a rapid and reliable method for the estimation of variability between different popular papaya varieties which could be utilized by the breeders for further improvement of Papaya.

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Comparative study of antioxidant activity of some medicinal plants belonging to Lamiaceae and Apiaceae from Al-Baha region, Saudi Arabia

Alshehri M.H., Aldhebiani A.Y. and Hussein R.M.

Page No: 71-78

Abstract: The flora of Saudi Arabia includes enormous numbers of endemic species representing row materials for different applications. Due to its vegetation prosperity, Al-Baha region is one of the most important regions in Saudi Arabia besides, Asir, Hijaz, Al-Taif and the western mountainous area of the kingdom. In this work, the antioxidant potential of ten plant samples from Lamiaceae and Apiaceae was studied using different solvents (ethanol, hexane and water) with respect to their total water-soluble antioxidant and lipid-soluble antioxidant activity (TLAC and TWAC), exhibiting that from Lamiaceae, the aqueous extract of sage plant samples (Salvia officinalis) had the highest total and strong water-soluble antioxidant (TWAC: 408.531 ±5.378 µmol L-ascorbic acid/g; TWAC37: 23.52 ±0.785 µmol L-ascorbic acid/g).

The ethanolic extract of mint plant samples (Mentha piperita) had the highest total lipid-soluble antioxidant capacity (TLAC: 269.129 ±11.069 µmol α-tocopherol/g) while the sage ethanolic extract revealed the highest TLAC37 (35.443 ±0.524 µmol α-tocopherol/g). The TLAC of tarragon (Menthe longifolia) hexanic extract was the highest (75.925 ±0.349 µmol α-tocopherol/g), therefore, the sage hexanic extract demonstrated the highest TLAC37 (4.016 ±0.009 µmol α-tocopherol/g). Lamiaceae plant samples were superior in comparison with Apiaceae plant material in all studied parameters. Anise, fennel and cumin were good sources of TWAC, TWAC37, TLAC and TLAC37.

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Enhancing the Sensitivity of ELISA using Monoclonal and Polyclonal antibody to rWbSXP-1 Antigen

Simon Chandramohan, Charles Jason, Mahalakshmi Natarajan, Desai Pradip K. and Kaliraj Perumal

Page No: 79-88

Abstract: Earlier, we developed an ELISA based method for the detection of circulating filarial antigen using monoclonal (1F6H3) and polyclonal antibody to WbSXP-1 antigen (method I). Here, an attempt was made to enhance the sensitivity of this method with different reagent combinations which detected antigen up to 0.24ng/ml by method II, as against the 125ng/ml by method I. This also increased the optical density values in microfilaremic clinical samples up to 2.034± 0.106 by method II against 0.792± 0.055 by method I. There was 10% increase in number of samples showing antigen positive for Endemic Normal and Chronic Pathology by method II.

The field application of Method II was evaluated using 4700 blood samples collected on microscopic slides. A total of171 (3.64%) were found to have antigenemia, of which 168 samples were reported as endemic normal probably having occult filarial infection (MF - ve) and three were microfilaraemic positive by conventional staining method.

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Evaluation of antibacterial activity by antifungal Bacillus strains

Shrivastava Amita Agrawal Ruchi and Singhal Pradeep Kumar

Page No: 89-94

Abstract:The antifungal strain Brevibacillus brevis B87 inhibited growth of all the tested human bacterial pathogens, viz. Escherichia coli (MTCC 77), Micrococcus luteus (MTCC 4300), Pseudomonas aeruginosa (MTCC 741), Salmonella enterica (MTCC 3218) and Staphylococcus aureus (MTCC 7443). Out of its secondary metabolites eluted by column chromatography, the fraction 1 recorded the maximum inhibition of all the test pathogens.

GC-MS profile of the fraction 1 revealed the presence of 3 classes each of alkanes and esters and a single class each of benzene, fatty acid and ketone with the highest concentration of 1-butyl 2-(8-methylnonyl) phthalate. These volatile compounds may be exploited in novel drug formulation.

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Scope of in vitro culture targeting genetic transformation in sugarcane

Tripathy Swapan K.

Page No: 95-100

Abstract:Sugarcane is highly responsive to callusing and regeneration. Number of protocols are now available for rapid and efficient induction of somatic embryos. Besides, the crop is an excellent material for profuse microtillering. Therefore, production of transgenics can be a better alternative to improve quality traits and resistance to biotic and abiotic stresses. In this pursuit, the author presented a detailed review of the status of in vitro culture amenable for genetic transformation in sugarcane.

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Genetics and molecular markers for resistance to major soil borne pathogens in chilli (Capsicum annuum L.)

Ponnam Naresh, Kumari Meenu, Acharya G.C., Anand C. Reddy and Lakshmana Reddy D.C.

Page No: 101-105

Abstract:Three major soil borne pathogens namely Phytophthora capsaici Leon causing root rot, bacterium Ralastonia solanacearum causing bacterial wilt and root- knot nematodes (Meloidogyne spp.) are pervasive across the major pepper growing parts of world mainly in the areas of repeated cultivation and poly houses impeding production as these pathogens are very difficult/ impossible to control. Repeated application of chemicals is in practice among the farmers and chemical management often leads to environmental pollution and the presence of pesticide residues in the fruits thus raises the concern of food safety and reduces the export potential. Recently grafting strategy using resistant root stock has been proposed mainly for protected cultivation. Resistance breeding through incorporating the resistant genes/QTLs is the best advocated strategy to circumvent these devastating soil-borne diseases which aim at developing a variety/root stock having combined and durable resistance.

Marker assisted backcross breeding is the best method which ensures precise transfer of the resistant genes/QTLs of interest from the donors to the recurrent parent with accelerated recurrent parent genome which requires thorough knowledge on the molecular marker available for fore ground selection. This review mainly emphasized on the molecular markers available for breeding peppers for combined resistance to soil borne pathogens.

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