Research Journal of Biotechnology

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Vol. 13(1) January 2018

Characterization and Expression Analysis of Nadh Dehydrogenase (Ubiquinone) Iron-Sulfur Protein-2 in Channa Striatus exposed to Hypoxia

Mohapatra S.D., Chakrapani V., Rasal K.D., Barman H.K. and Subudhi E.

The NADH dehydrogenase is the first and largest enzyme complex in the mitochondrial oxygen respiration chain. In the present study, we intended to investigate the role of NADH dehydrogenase during hypoxia stress in the Murrel, Channa Striatus. We have cloned and characterized full length cDNA of NADH dehydrogenase (Ubiquinone) iron-sulfur protein 2 (NDUFS2) of C. striatus which is known to be a hypoxia tolerant species that inhabits in the freshwater. The full-length cDNA of NDUFS2 contained an open reading frame of 1401bp encoding 467 amino acids. 5′-flanking region is 25bp, relative to start codon and 3′-UTR is 187bp. The conserved domain search analysis depicted the putative structural features of NADH dehydrogenase subunit D (77-466aa) and Respiratory-Chain NADH dehydrogenase 49Kd subunit (195-466aa). The NDUFS2 promoter was isolated bearing 1301 bp upstream of the transcription start site. The phylogenetic analysis depicted that NADH dehydrogenase of C. striatus was closely related to other fish species lives in low oxygen habitat. We have generated the 3D structure of NDUFS2 protein. We have also examined the mRNA expression under hypoxia stress. Significant increase in expression levels of NDUFS2 suggests a functional role of this gene in oxidative stress tolerance and adaptation.

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Molecular characterization of Rosa damascena Mill. growing in Taif and Almadinah using ISSR and SSR markers

- Aldhebiani Amal Y., Al Saud Najlaa S., Yaslam Weam A. and Hassan Sabah M.

Rose belongs to the great family Rosaceae. The genus Rosa L. belongs to the economically important family Rosaceae. In Saudi Arabia, there are some species belong to Rosa L., one of which is Rosa damascena Mill. Rosa damascena is one of the hybrid species that has been for long time considered as an original species. The most popular cities in growing roses in Saudi Arabia are Taif and Almadinah. According to local people, Taif rose is mainly used for oil and water extraction but Almadinah rose is mainly used for ornamentation and drinking with tea. In addition, the rose of Almadinah city is harvested throughout the year, but less in the winter. Whereas, Taif rose is harvested in a certain period in the spring between March and April. This study aimed to investigate if both Roses are derived from one original plant and to find some genetically related information using Inter-Simple Sequence Repeats (ISSR) and simple sequence repeats (SSR). The nine ISSR and the three SSR loci used in the current study, which are very simple, fast and cost effective, have been successfully discriminated between both Taif and Almadinah Rose.

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Acute Toxicity and Antihypertensive Effects of Artemisia afra and Leonotis leonurus in Spontaneously hypertensive rats

Tata Charlotte Mungho, Gwebu Ephraim Tobela, Aremu Olukayode Olasunkanmi, Sewani-Rusike Constance Rufaro, Oyedeji Adebola Omowumi, Oyedeji Opeopluwa Oyehan and Nkeh-Chungag Benedicta Ngwenchi

Acute toxicity and antihypertensive effects of hydroethanolic extracts of Artemisia afra and Leonotis leonurus were studied in Swiss albino mice and spontaneously hypertensive rats (SHR) respectively. Phytochemical screening was determined by colorimetric techniques. Lorke's method for acute toxicity testing was carried out in two phases: in phase I three groups of mice (n = 3) were treated with 10, 100 or 1000 mg/kg of the extracts while in phase II mice were treated with 1600, 2900 or 5000 mg/kg of the extracts. Blood pressure, heart rate, blood flow and blood volume were measured using a non-invasive tail cuff method before treatment and 2, 4, 6, 8 and 24 hrs after treatment. Phytochemical screening revealed the presence of phenols, terpenoids, flavonoids, glycosides, tannins, steroids, triterpenoids and saponins. Both plant extracts were non-toxic with LD50 values greater than 5000 mg/kg. Artemesia afra extracts had its greatest (p<0.01) antihypertensive effects at 2 and 4 hrs post treatment while the effects of Leonotis leonurus were weak at best. The antihypertensive effects of A. affra and L. leonurus were significantly higher (p<0.01) than the effects of furosemide 24 hrs post treatment. Results from this study suggest that even though A. afra and L. leonurus are used for hypertension treatment in South African traditional medicine, the former displayed better antihypertensive effects compared to the latter in SHR.

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Microbial biotransformation of anticancer drug Abiraterone acetate using Cunninghamella elegans

Geetha Subramanian and Shanthakumari Dhanarajan

Cunninghamella microbe is an effective in vitro model for the production of drug metabolites which is important for metabolic studies and in drug development. In this study, microbial screening and biotransformation of prostate anticancer drug Abiraterone acetate was performed using Cunninghamella species and the extracted crude compounds were analyzed by MASS Spectra and HPLC. Major metabolite was purified by preparative TLC and characterized by MASS,1 HNMR and13 CNMR spectra. Out of the seven strains, only Cunninghamella elegans NCIM 690 was able to transform the Abiraterone acetate. Using HPLC analysis, almost 98% of Abiraterone acetate metabolic conversion was observed. Known human metabolites with molecular weights of 347.9 and 526.2 were identified from MASS analysis. The purified metabolite M1 was confirmed as Δ4-Abiraterone by MASS (m/z 347.5), 1HNMR and 13CNMR. This study confirms that Cunninghamella elegans NCIM 690 is the best in vitro model for microbial biotransformation of Abiraterone acetate to its metabolite Δ4-Abiraterone, a known human metabolite, which can be used as a reference standard for the pharmacological/toxicological/clinical studies.

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Supplementation of Lactobacillus brevis for Growth Promotion of the Freshwater Prawn Macrobrachium rosenbergii Post Larvae and identification of Gut Microflora through 16s rDNA

Madhayan Karthik and Periyakali Saravana Bhavan

The probiotic effect of Lactobacillus brevis (MTCC 4463) was studied on promotion of survival and growth, enhancement of contents of biochemical constituents and activities of digestive enzymes and establishment of its colony in the gut of Macrobrachium rosenbergii post-larvae (PL). Four groups of PL (1.62±0.08 cm length and 0.06±0.01 g weight), each consisting of 35 PL were maintained with 25 L of ground water and fed ad libitum with 0.5, 1.0 and 1.5% of L. brevis incorporated artificial diets respectively for a period of 90 days. Diet without incorporation of L. brevis was served as control. L. brevis was found to be alive in the feed even on day-15 after it was formulated. On day-90, survival, nutritional indices (weight gain, specific growth rate, food conversion ratio and protein efficiency ratio), activities of digestive enzymes (protease, amylase and lipase), concentrations of basic biochemical constituents (total protein, amino acid, carbohydrate and lipid), levels of essential amino acids and fatty acids were found to be significantly (P<0.05) increased when compared with control. These effects were found to be the best with 1% of L. brevis incorporated diet fed PL, better with 0.5% L. brevis and good with 1.5% L. brevis when compared to control. The staining intensity of 59, 52, 43, 37, 29, 27, 24, 21, 20, 18, 17, 15 and 14 kDa polypeptide bands were found to be increased in the experimental PL when compared with control. The biochemical confirmation tests revealed presence of Escherichia coli, Pseudomonas spp., Streptococcus spp. and Klebsiella pneumoniae in the gut of control PL whereas in the gut of experimental PL, in addition to the presence of E. coli, Pseudomonas spp. and Streptococcus spp., Lactobacillus spp., Bacillus spp. and Clostridium butyricum were detected, but K. pneumonia was excluded. The MTTC supplied L. brevis (MTCC 4463) when subjected to subculture showed that it contains 1516 bp DNA, which exactly matched with L. brevis sequence data available with NCBI data base. The established microbe sequence (1506 bp DNA) in the gut of control PL was 97% matched with Serratia sp. The sequence (1537 bp DNA) of dominant microbe established in the gut of M. rosenbergii PL fed with L. brevis incorporated feed was 99% matched with Lactobacillus sp. Therefore, in this study, incorporated probiotic colony establishment was confirmed up to genus level only. As L. brevis (1%) incorporated diet produced better survival and growth, it can be recommended as feed additive for sustainable production of Macrobrachium.

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A Missing Piece of CRISPR/Cas9 – Detection of No Indels in the T0 Generation Plants from Targeted Mutagenesis of NtSUT3 in Nicotiana tabacum

Abiramavalli M., Usha B., Ganesan Govindan and Sivaprakash Ramalingam

CRISPR/Cas9 is a very promising gene editing technique that is based on an RNA-guided endonuclease. CRISPR is Clustered Regularly Interspaced Short Palindromic Repeats found in bacteria and archaea as a defence mechanism against invading phages whereas Cas9 is an endonuclease guided by a short guide RNA (sgRNA). It is used to make a precise double stranded break (DSB) in targeted sites of the gene of interest. Though the CRISPR/cas9 system has evolved as a most successful genome engineering tool adopted in various cells and organisms, it has its own pitfalls. Factors including guide RNA design, target site selection, activity of Cas9, method of delivery, probable off target effects and repair mechanisms of the host organism affect its specificity and efficiency. In our study we intended to make a DSB in a sucrose transporter gene (NtSUT3) of Nicotiana tabacum with the CRISPR/Cas9 system, we expected the nick would be repaired by non-homologous end joining. Although, integration of Cas9 was confirmed in T0 generation plants, there was no incidence of insertions or deletions found at the target site. We assume that the reason for this could be because we overlooked a few important key points in the experiment design.

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Effective method of Ri plasmid transformation for hairy root induction in kalmegh

Mahobia Arpita and Jha Zenu

Medicinal and aromatic plants (MAPs) are gaining recognition worldwide as a source of raw material for pharmaceuticals and traditional medicine system. Kalmegh (Andrographis paniculata) is one of the well-known medicinal plant which has importance because of its broad range of uses in ayurvedic, unani and other traditional medicine system. It has principle constituent andrographolide which helps to cure various ailments i.e. for hepatoprotection, diabetes, fever, cancer etc. Andrographolide in leaf is 2 % in normal regenerated plant which is not sufficient to meet the demands commercially. Hence, kalmegh was subjected under in vitro condition for production of hairy roots with enhanced andrographolide content. Apical meristem was cultured on half strength MS medium (HMS) with treatment combination of NAA 1mg/L and acetosyringone. The Agrobacterium rhizogenes MTCC 532 strain which has root inducing (Ri) plasmid was used for inducing hairy roots by simple pricking the explant with drop method of infection. This method of transformation is found to be very effective with 58 % transformation in media with acetosyringone added in concentration of 400µM. Polymerase Chain Reaction analysis showed amplification of the rolC gene in hairy roots indicating the transformation event. This report successfully demonstrates an effective method for agrobacterium mediated transformation for hairy root induction.

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NAC Transcription Factor differentially regulated by Abiotic Stresses and Salicylic Acid in the Mangrove Plant Avicennia marina (Forsk.) Vierh.

Murugesan Ashok Kumar, Govindan Ganesan, Subramanian Sankara Rama H. M. and Parida Ajay

NAC (NAM, ATAF1/2 and CUC2) proteins are plant specific transcription factors involved in various biological processes including growth, development and stress response. Expression profiling of NAC transcription factors in response to stress in many plants has been well-documented. In the present study, we have analysed the expression profile of AmNAC1 in response to drought and cold in addition to salt, using quantitative real-time PCR. Furthermore, the expression pattern of AmNAC1 in response to salicylic acid, an important biotic stress signal, was also investigated. Expression analysis revealed that AmNAC1 transcript was strongly induced in the roots of Avicennia marina after 7 and 10 days of salt (NaCl) treatment. Under drought stress, AmNAC1 transcript accumulated 7-fold higher than control plants on the 10th day of treatment and the expression remained stable after 2 days of post recovery stress. Interestingly, AmNAC1 transcript showed more than 30-fold higher expression compared to untreated plants after 2 days of cold stress and gradually reduced when the treatment extended for a longer time. However, the salicylic acid (1mM and 10mM) treatment reduced the accumulation of AmNAC1 transcript after 24h, 48h and 72h, suggesting a negative correlation between salicylic acid signalling and AmNAC1 expression in Avicennia marina. Our results clearly indicate common transcript level regulation of AmNAC1 under abiotic stress and a possible existence of a biotic response independent of salicylic acid signaling in Avicennia marina.

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Mechanism of Bactericidal Action of Biosynthesized Silver Nanoparticles

Senthil B., Rajasekar A. and Devasena T.

In the present study, silver nanoparticles were synthesized by a simple, eco-friendly, cheap, non-toxic and sustainable method. The formation of the AgNPs was confirmed by UV-Vis spectroscopy in which the maximum absorption was observed at 421nm. The synthesized AgNPs were characterized using FTIR, XRD and HRTEM. The FTIR spectroscopy confirms the interactions between the phytochemical components of the extract and AgNPs in which the possible bio-molecules responsible for the reduction were identified. Highly crystalline FCC structured AgNPs were confirmed from the peaks obtained in the XRD analysis. HRTEM showed dispersed spherical AgNPs with an average diameter of 20nm. Remarkably, the synthesized AgNPs exhibit superior antibacterial activity against K. Pneumonias and P. Aeruginosa gram-negative bacteria. The protein leakage from the AgNPs K. Pneumonias and P. Aeruginosa treated cells reveals the damage of cell membrane by the formation of pits like structure which may make cells inviable. The stressing of the cell structure and disruption of the cell wall were also observed using SEM analysis. Our results show that the synthesized AgNPs can be a superior candidate for effective bactericidal activity and also can be applied various the biomedical applications.

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Interaction between Gluconacetobacter diazotrophicus NFGM5 and Maize (Zea mays L.) by pot culture experiment

Rao Hema C. and Savalgi V.P.

A pot culture experiment was conducted during 2015–16 in green house at the Department of Agricultural Microbiology, University of Agricultural Sciences, Dharwad to study the influence of five efficient Gluconacetobacter diazotrophicus isolates on the growth and nitrogen uptake of maize at different levels of recommended nitrogen (RDN). Based on the lab studies, five multifunctional isolates (NFGS25, NFGS26, NFGM5, NFGM6 and NFGP7) were selected for the pot experiment along with the reference strains G.diazotrophocus (MTCC no.1224) and Azospirillum ACD 15. At 30 DAS (days after sowing), the growth parameters such as plant height (52.17 cm), stem girth (3.53 cm), chlorophyll content (32.5), root length (60 cm), dry matter production of shoot (3.5 g per plant) and root (0.57 g per plant) of maize were maximum in the treatment inoculated with NFGM5 at 75 % RDN level which was on par with other treatment inoculated with NFGM5 at 100 % RDN level. A similar trend was observed at 60 DAS. The per cent shoot nitrogen content was maximum in the treatment inoculated with, NFGM5 and NFGS25 at 75 % RDN (0.37 %). At 100 % RDN, NFGM5 recorded higher shoot nitrogen of 0.27 % as compared to NFGS25 (0.23 %) at 30 DAS. The nitrogen uptake was significantly higher in the treatment NFGM5 at 75 %RDN (8.65 g plant-1) followed by NFGS25 (7.73 g plant-1). The population of G.diazotrophicus was maximum in the treatment inoculated with NFGM5 at 75 % RDN (9.8 x 104 CFU g-1) followed by NFGS25 (3.5 x 104 CFU g-1) at 60 DAS. The two native isolates of G.diazotrophicus NFGM5 and NFGS25 proved efficient in nitrogen uptake and all the growth parameters in the pot culture experiment. These cultures were confirmed by the molecular characterization using 16S rDNA method and further they were recommended for the field trial.

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Association of Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G and Apolipoprotein E polymorphisms with risk of Myocardial Infarction in Indian Tamil Population

Nair Sahana, Budamagunta Vivekananda, Meenakshisundaram Abinaya, Hemapriyanka S., Prabhakaran Pournima, B. Suruthi Abirami, Devi A. and ArulJothi K.N.

Cardiovascular disease CVD is a major threat in South India and yields high mortality rates. In this study, we investigated the association of apolipoprotein E (APOE) and the plasminogen activator inhibitor (PAI-1) polymorphism for myocardial infarction (MI) in the South Indian Tamil population. A total of 300 control and test group samples were analysed for genotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Selected populations followed the Hardy-Weinberg Equilibrium with polymorphisms showing an association with MI risk. Logistic regression analysis showed the ε4 allele to be the risk allele for APOE with three times the odds of MI compared to ε2 while ε3 had a neutral effect. The alleles seemed to have an interesting effect on lipid levels with ε4/ε4 eliciting the highest HDL values in the total population and ε2/ε2 eliciting the least. Moreover, the combination of ε4 and ε2 seems to increase total cholesterol (TC) while ε2/ε2 seems to lower it. The ε2 allele provides a high triglyceride (TG) level in the MI group. In the case of PAI-1, the 4G allele is most prevalent in this population and is noted for its risk allele while 5G seems to have a protective function. In addition, 5G/5G offers an elevated TG level for the test group while 4G/5G produces elevated HDL levels in the mainstream population.

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Production of Polyhydroxyalkanoates (PHAs) using Synthetic Biology and Metabolic Engineering Approaches

Rayasam Vijay and Kumar Tarika

Polyhydroxyalkanoates are polyesters that are biological in origin and are produced by micro-organisms through fermentation processes. Researchers have been attracted towards applications of these polyester compounds as an alternative to plastics as they are biodegradable, renewable, biocompatible and eco-friendly. However, presently these polymers have limitations because of lower rate of microbial production and high production cost. Production steps like the selection and improvement of strains, media development, downstream processing are being intensively researched in order to improve the overall process efficiency and finally to substitute the mainstream polymers with that of biodegradable polymers. This review article provides an insight into PHA biosynthesis, microbial strains that produce PHA, use of novel methods such as synthetic biology or morphology engineering to design new pathways in microorganisms and also use of plants by altering their metabolic pathways in a way to produce biopolymers.

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Fingerprinting of IGFBP3 and FecB genes as molecular markers and their association with productive and reproductive traits in livestock species

Qureshi Muhammad Iqbal, Mutwakil Mohamed H., El - Hanafy Amr A., Abou-Alsoud Mohamed, Sabir Jamal, El-Ashmaoui Hassan, Ramadan Hassan A. I. and Ahmed Mohamed Morsi M.

Enhancing prolificacy in the commercial flocks of sheep was formerly limited to breeding programs and selection strategies. However, recent advances in animal genetics have unlocked the prospects of utilising genomic alterations in major gene(s) associated with ovulation rate and litter size. The famous Booroola gene (FecB), initially identified in the Australian Merino sheep was primarily responsible for high fecundity and litter size. It also regulates other features like endocrinology, fatal and postnatal growth1-3. Insulin-like growth factor binding protein 3 (IGFBP3) also controls growth, development and metabolism in mammals. IGFBP3 binds the majority of insulin-like growth factors (IGFs) in circulation for regulation of its actions on growth and metabolism. This review mainly aims at exploring possibilities of exploiting both FecB and IGFBP3 genes as potential markers in various varieties of sheep and signifying their association with productive and reproductive traits 3,4.

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An Overview of Protease Inhibitors from Actinobacteria

Neethu Kamarudheen and Bhaskara Rao K.V.

Protease inhibitors are those key bio-molecules which impede and thus regulate proteases. Apart from the outlook of protease inhibitors as elements of enzyme and structure studies, they have been lately looked upon as potential pharmacological agents. They exert inhibition against several clinically challenging pathogenic proteases being pharmacologically significant. With the clinical and commercial success of protease inhibitors against AIDS and hypertension, the perspective towards proteases inhibitors have altered. In this mini review, we focus on the protease inhibitors from actinobacteria, a class of prolific secondary metabolite producers. The mini review explains about low molecular weight peptidomimetic proteases inhibitors extracted from the culture supernatant of actinobacteria which are targeted towards various classes of proteases that aid the onset and dissemination of pathological conditions. When almost entirely the production of protease inhibitors is attributed to Streptomyces sps, a couple of rare actinobacteria are also involved in production.

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