Research Journal of Biotechnology

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Research Journal of Biotechnology

Optimization of PCR Reaction System and Selection of cpDNA Noncoding Primers for Sinomenium acutum

Ying He, Jin-jing Teng, Xi-yao Zeng, Hua Yang, Ping Qiu and Xing-yao Xiong

In this study, the main factors affecting the chloroplast DNA (cpDNA) PCR reaction system for Sinomenium acutum were optimized by single factor experiment which established the optimal cpDNA-PCR reaction system for Sinomenium acutum as follows: 2.5 μL of 10×PCR buffer, 40 ng of DNA template, 0.4 μL of Taq DNA polymerase (1.0 U), 2.5 μL of Mg2+ (2.5 mmol·L-1), 3 μL of dNTPs (0.3 mmol·L-1) and 0.5 μL of each primer (0.2 μmol·L-1), they were mixed and topped up to 25 μL with ddH2O. The optimal reaction system was used to screen out 4 pairs of universal primers of cpDNA noncoding regions, which were atpI-atpH, trnQ-5'rps16, trnH-psbA and trnL-trnF.

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Immobilization of Polyphenol Oxidase from Princess Tree Leaf and its applications in Dye Decolorization

- Arabaci G., Usluoglu A. and Cesko C.

Textile industries effluents have become a major source of water pollution all over the world. There are many types of wastewater treatment processes such as chemical, physical and biotechnological processes. Unluckily, chemical and physical conventional wastewater treatment processes result in inadequate dye removal and the formation of dangerous byproducts, resulting in high cost and dense energy requirements. In recent years, biotechnologically processes are of great interest in removing dye contaminants from aqueous solutions. In this work, Polyphenol oxidase (PPO) enzyme was extracted and partially purified from princess tree leaves (Paulownia tomentosa). Then, it was immobilized on calcium alginate beads. The free and immobilized PPO enzymes were applied for the decolorization of metal complex and acid dyes. The metal complex and acid dyes in water were mixed with free and immobilized princess tree PPO for one hour. The percent of decolorization was determined by using untreated dye mixture. Immobilized PPO from princess tree leaf was considerably more active in decolorzation of the dyes than the free PPO. Our results suggested that the immobilized princess tree PPO could be useful for removing artificial dyes from industrial wastes.

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Isolation and characterization of phenol degrading bacteria Stenotrophomonas sp. SKC_BP54

Kasbekar Santosh, Pal Rajesh Ramavadh and Shanthakumar S.P.

Bioremediation of phenol by microbial cultures is a widely accepted approach for mitigation of this pollutant from environment. Herein, four phenol-degrading bacterial species were isolated from the oil-effluent contaminated soil samples of Kalmeshwar area in Nagpur, Maharashtra, India. The cultures were evaluated for the feasibility of practical bioremediation of industrial wastewater laden with aromatics such as phenol. All the four isolates identified as Stenotrophomonas sp. (SKC_BP54), Pandoraea sp. (SKC_BP7), Pseudomonas geniculata (SKC_BI20) and Pseudomonas geniculata (SKC_BI20) showed complete phenol removal from media in 72 h; however, SKC_BP54 proved to be most efficient and thus was used for subsequent biodegradation studies. Shake flask experiments were carried out for several initial phenol concentrations of 500 mg/L, 1000 mg/L, 1500 mg/L and 2000 mg/L. SKC_BP54 showed most efficient phenol degradation kinetics, removing phenol with the rate of 15.15 mg/L/h for initial phenol concentration of 1000 mg/L. Furthermore, optimum pH and temperature for bioremediation activity of SKC_BP54 were found to be pH 7 and 30 0C respectively. Owing to tolerance towards high phenol concentration, Stenotrophomonas sp. SKC_BP54 could prove to be a great future prospect for phenol bioremediation strategies.

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Fingerprinting Rice Hybrids using Restorer Gene Linked SSR Markers

Debbarma Mampi, Dutta Deka Sharmila and Sarma Debojit

We used ten rice hybrids and their parental lines for fingerprinting with ten SSR markers linked to fertility restorer (Rf) genes. Seven markers were found polymorphic among the 21 rice genotypes amplifying 20 alleles with an average of 2.86 alleles per marker. RM 228 was a diagnostic marker for identification of six rice hybrids. RM490, RM1108 and RM6344 clearly distinguished the hybrids - IR68888A/Chilarai, IR68888A/Kolong and IR68888A/IR 36. The average PIC value of 0.51 indicated the presence of much genetic diversity among the hybrids and their parental lines. UPGMA based clustering separated the 21 genotypes into nine major clusters at a threshold similarity coefficient of 0.16. At similarity value of 0.5, all the hybrids got differentiated from their parents indicating that SSR markers linked to Rf genes could be successfully employed for hybrid identification.

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Rapid detection of hemolysin gene of Klebsiella pneumoniae by Loop-Mediated Isothermal Amplification (LAMP)

Rathinasabapathi Pasupathi

Klebsiella pneumoniae is a wide-spread nosocomial pathogen that causes a broad range of diseases including pneumonia. In the current study, a loop-mediated isothermal amplification (LAMP) method was developed for a rapid and sensitive detection of K. pneumoniae by targeting hemolysin gene. The supplementation of additives such as dimethyl sulfoxide and betaine enhanced the efficiency of DNA amplification. The LAMP amplified the target gene with four specific primers in 45 min at 65oC in a simple water bath. All the strains other than K. pneumoniae were found negative for LAMP detection which indicates a high specificity of LAMP reaction. The method was found to be sensitive up to one femtogram of genomic DNA which is 1000 times more than that of PCR. The rapidness and specificity of LAMP assay advocate the potential of the LAMP for early detection of K. pneumoniae in clinical and environmental samples.

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In Vitro Plant Regeneration from Cotyledonary Node of Diploknema butyracea

Kunwar Arti, Thakur Ajay and Rather M. Maqbool

An efficient plant regeneration protocol was described for Diploknema butyracea (Roxb.) Lam. using whole cotyledonary node as an explant. Seeds were surface sterilized with 0.3% (w/v) mercuric chloride for 15 minutes and germinated (85.7 %) in growth regulator free MS medium. Maximum multiplication rate (3.00) was obtained in treatment by 13.32 µM BAP + 2.69 µM NAA in MS medium. Regenerated multiple shoots were excised and cultured on half-strength MS medium containing different concentrations of IBA, NAA and IAA for root induction. Best root development was obtained on half-strength MS media with 4.92 µM IBA which were 85.7 % for root induction, 8.6 for average root number and 2.73 cm for root length. Regenerated plantlets with well developed shoots and roots were acclimatized successfully.

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Screening of patients with Gilbert’s Syndrome from South India identifies UGT1A1*28 as the most predominant mutation in UGT1A1 gene

Dhanya Ramakrishna Iyer and Madasamy Parani

Bilirubin is a by-product of heme degradation which needs to be conjugated to water-soluble bilirubin glucuronides for excretion by the action of uridine diphosphate - glucuronosyltransferase. Gilbert’s Syndrome (GS) is a medical condition wherein bilirubin levels are elevated due to mutations in the uridine diphosphate-glucuronosyltransferase 1A1 gene (UGT1A1). However, bilirubin levels are also elevated in case of liver damage due to fatty liver, infections, haemolytic anaemia etc. While the former is harmless, the latter may require immediate medical attention. Therefore, in cases of elevated bilirubin content, it is very important to rule out the possibility of GS to avoid anxiety, unnecessary diagnostic tests and treatments. In the studies conducted in Kolkata and New Delhi, the UGT1A1*28 recessive mutation was reported to be the predominant genetic cause of GS. However, there is no report in the South Indian population which is genetically different from the North Indian population. In the current study, genetic testing of 11 GS patients showed that all of them had UGT1A1*28 mutation indicating that this mutation is common in South Indian population as well. Thus, genetic testing could be used as a screening tool for early diagnosis of GS to avoid anxiety and unnecessary clinical investigations and to help in genetic counselling.

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Anti-infertility activity of Cissus quadrangularis in male Wistar Rats

Santhosh Kumar R., Jayan Neethu, Das Susmita and Asha Devi S.

Cissus quadrangularis commonly known as bone setter is considered as a native plant to many tropical countries and is being used for its medicinal value from the ancient period itself. On chronic exposure to lead acetate, it can lead to infertility in both humans and animals through the mechanism of oxidative stress. The prime aim in conducting this study was to evaluate the anti-infertility activity of the Ethanolic extract of the stem of the herb C. quadrangularis on lead acetate induced damage in the reproductive system of male wistar rats. During experimental study, the animals were categorized into 4 groups: control, negative control, 300mg/kg and 500mg/kg dosage of the plant extract given for 28 days. Lead acetate was induced in the animals except control. The animals were scarified on 29th day and processed for sperm and antioxidant enzymatic assays. Sperm morphology and sperm cell count were almost normal in the animals treated with co-administration of the extract of C. quadrangularis and lead acetate when compared with the control. The results obtained by performing the antioxidant assays in the tissue homogenate were promising for the objective of the study indicating the beneficial role of the ethanolic extract of the plant stem in restoring the defects caused by lead acetate.

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De novo assembly of complete genome sequence of Bacillus subtilis from a probiotic capsule using Illumina sequencing

Natarajan Purushothaman and Madasamy Parani

Bacillus subtilis is an endospore-producing, rod-shaped, gram-positive bacterium was used as an oral probiotic along with other probiotic strains. The comprehensive analysis of this probiotic bacterium’s complete genome is necessary to gain insight into its molecular physiology which is involved in the host interaction and to predict its safety and probiotic effects in humans. Here, we present the complete genome sequence of B. subtilis isolated from a probiotic capsule (BIFILAC®) using Illumina sequencing. The complete genome sequence was de novo assembled using only paired-end reads and the gaps were filled using manual alignment methods followed by validation using Sanger sequencing. The complete genome of B. subtilis with a single circular chromosome consisted of 4,057,487 bp with 4199 genes; there was no plasmid genome in the total DNA. The complete genome assembly and annotation of B. subtilis TO-A with information on genes for antibiotic resistance, virulence factors and other genomic features will be an invaluable resource for further study of this probiotic bacterium.

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Acidic electrolyzed water, hydrogen peroxide, ozone water and sodium hypochlorite influence quality, shelf life and antimicrobial efficacy of cherry tomatoes

Mohammad Zahirul Islam, Mahmuda Akter Mele, Khalid Abdallah Hussein and Ho-Min Kang

To maintain the quality and shelf life of cherry tomatoes, microbial decontamination is essential. The objective of this study was to determine the effect of acidic electrolyzed water (AcEW), hydrogen peroxide (H2O2), ozone water (O3) and sodium hypochlorite (NaClO) on quality, shelf life and microbial decontamination of cherry tomatoes. Water was used as control. AcEW, H2O2, O3 and NaClO were sprayed one day prior to harvest. Afterwards, tomato fruits were stored at 5 ˚C to determine their quality, shelf life and microbial activity. Respiration (p ≤ 0.001) and ethylene production (p ≤ 0.01) were significantly lower in O3 -treated tomato fruits compared to those in other treatments. The lowest fresh weight loss and the longest shelf life 20 days (p ≤ 0.05) were found for O3 -treated tomato fruits. Their fungal incidence rate (p ≤ 0.001) and microbial activity were the lowest. However, fruits firmness 13.36N (p ≤ 0.01) was significantly retained for O3 -treated tomato fruits. Color development, lycopene content and fructose and glucose contents suppressed by O3 -treatment might have prolonged the shelf life of tomato fruits. Based on comprehensive results of physiological, physicochemical and microbiological analysis, O3 -treated tomato fruits prolonged shelf life and maintained their quality at 5˚C. Therefore, O3 -treatment might have great potential to be used for long time storage of cherry tomatoes.

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Response Reaction on Composition Changes of Cell Membrane Fatty Acid of Two Lactobacillus acidophilus Strains on the condition of Acid Stress

Zhao Ruixiang, Xia Lulu, Ran Junjian, Yuan Zheng, Niu Shengyang and Liang Xinhong

Lactobacillus acidophilus is important probiotic bacteria in human’s intestines with a strong resistance to acid. The response reaction on composition changes of cell membrane fatty acids of Lactobacillus acidophilus on acidic conditions of different pH was researched in this study. The composition of cell membrane fatty acids of strains Lactobacillus acidophilus Ind-1(La-XH1)and Lactobacillus acidophilus Lakcid (La-XH2) was determined using GC-MS method. Results showed that the species of cell membrane fatty acids of La-XH1 and La-XH2 have not changed under different acidic conditions, but the relative content of fatty acid has changed, especially C18:1 and C19:0 which have greater changes. The relative mass fraction of La-XH1 of C18:1 and C19:0 ranged from 50.12% to 53.91% and from 2.18% to 6.22% respectively and that of La-XH2 of C18:1 and C19:0 ranged from 45.62% to 48.14% and from 4.34% to 6.56% respectively. Meanwhile, the relative mass fraction of La-XH1 of total unsaturated fatty acids of bacteria cell membrane (C16:1,C18:1 and C18:2) rose from 67.9% to 70.69% and that of La-XH2 rose from 61.85% to 65.06%. The relative mass fraction of La-XH1 of total long chain fatty acids (C18:0,C19:0 and C20:0) rose from 6.35% to 7.78% and that of La-XH2 rose from 11.9% to 13.06%. However, the relative amount of short chain fatty acids (C14:0 and C16:0) of La-XH1 declined from 25.70% to 21.53% and that of La-XH2 declined from 26.25% to 21.88%.

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Macrocyclic Metal Complexes as potential Anti-Microbial Agents

Sharma Kavita and Singh D.P.

Some new tetra-dentate transition metal macrocyclic complexes having a general formula: [M(C16H12N8)X]X2 were synthesized by one pot reaction of diaminomaleonitrile, bi-acetyl and trivalent metal ions where M = Cr(III), Fe(III) and X = Cl-, NO3-, CH3COO-. The structures of complexes were established on the basis of various spectral data magnetic moment, elemental analysis and conductance measurement. The electronic absorption spectra revealed that complexes possessed penta coordinated square pyramidal geometry assuming the effectual symmetry to be C4v around the metal ions. All the synthesized complexes were evaluated for their antimicrobial potential against some pathogenic gram-positive and gram-negative bacterial and fungal strains. The complexes 1, 4 and 5 were found to show excellent anti-bacterial activity with an MIC 5.5µg/ml as compared to the stranded drug Ciprofloxacin. Further, the complexes 1, 2, 4 and 5 show significant antifungal activities with MIC 10µg/ml in reference to the standard drug Amphotericin A.

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A Review on Methods and Devices to determine Body Iron Status

Talik Noor Azrina, Yap Boon Kar, Lim Wai Feng, Prajindra A/l Sankar, Ahmed Nasser Ammar, Ahmed Mubarak Al-Haiqi and Lai Mei I.

Despite the importance of iron for human body, the lack of iron as well as excess iron will lead to various health issues. Iron deficiency is the most common nutritional deficiency in the world responsible for a staggering amount of ill health, lost productivity as well as premature death. Iron overload causes iron toxicity leading to organ dysfunction such as damage in liver, heart and central nervous system. Detecting iron status at early stage is of paramount important especially in poor countries that have limited access to sophisticated laboratory diagnosis. Real-time measurement is always a hurdle due to time consuming and costly equipment required. This review discusses the methods and devices used to determine the body iron status. It also explores the perspective for the development of low-cost body iron reader.

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The crucial role of R-genes/RGAs in host-microbial interactions and plant immunity

Anand C. Reddy, Ponnam Naresh and Lakshmana Reddy D.C.

Plant diseases cause enormous damage to crop yields and are a threat to livelihood and economy of the farmer. Application of pesticides to control diseases is not eco-friendly and causes severe damage to the environment and human health. Therefore, identification of disease resistance genes (R-genes) will help in the development of resistant varieties/hybrids. The R-genes isolated so far can be classified into three different classes such as (i) NBS-LRR (nucleotide binding site-leucine rich repeats) type, (ii) R-genes containing LRR proteins and lacking NBS-domain and (iii) Other R-genes (Oth-R) encoding proteins lacking LRR and/NBS. Most of the R-genes/RGAs (resistant gene analogs) isolated from various crop species belong to NBS-LRR class of R-genes and are present mostly in clusters. Knowledge on involvement of R-genes/RGAs in plant immune system is moderate, but significant improvements have been made recently in understanding host-microbe interactions better through new technologies like Next Generation Sequencing (NGS). In this review, we focus on and summarize different types/structure of R-genes/RGAs and their role in host-microbe interaction mechanisms followed by downstream signalling pathways of R-gene induced resistance.

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