Research Journal of Biotechnology

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Vol. 14(8) August 2019

Comparative analysis of prolonged Lipopolysaccharide exposure on BV-2 and Mouse Primary Microglia

Twayana Krishna Sundar and Palaniyandi Ravanan

Page No: 1-13

Abstract: Endotoxin preconditioned immune cells (monocytes/macrophages) exhibit significant reduction in inflammatory response to subsequent challenge. This general paradigm of immunosuppression termed as ‘Endotoxin tolerance’ has been observed in different pathologies. Analysis of morphological and secretional profiles of the cells in this context helps in better understanding the mechanistic basis of immunosuppression involved in such pathologies. Here we show that prolonged endotoxin exposure induces immunosuppression in both BV2 and primary microglia and it reverts back upon withdrawal of the stimuli and re-maintenance on normal media. Cells were cultured in LPS (50 ng/ml) containing media for six days with passage and re-exposure at third day (prolonged LPS exposed cells [PLECs]). PLECs were not sensitized by further stimuli exposure and showed dampened cytokines and chemokines expression (IL6, IL1β, TNFα and CCL2). Stimuli withdrawal and culture of PLECs in normal media reversed the immunosuppression (LPS withdrawal cells [LWCs]) and cells started responding to the stimuli similar to control cells (Never LPS exposed cells [NLECs]). Despite the immunosuppression, PLECs showed significantly higher phagocytosis activity inferred by engulfment of higher numbers of fluorescently labeled latex beads. Conclusively, our study highlights microglial plasticity (secretional and functional) during prolonged endotoxin exposure induced immunosuppression.

Investigation of Potential Use of Human Originated Lactobacillus fermentum strains for Food Applications

Meltem Asan-Ozusaglam and Ayse Gunyakti

Page No: 14-22

Abstract:This study aimed to investigate the potential use of two Lactobacillus fermentum (MA-7 and MA-8) strains originated from human breast milk as a natural preservative agent in food industry and industrial processes. For this purpose, antimicrobial activity was tested against different originated test microorganisms (clinical and food-borne human and fish pathogens) using well diffusion method. The strains were evaluated for their in vitro antioxidant activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and the reduction of the red Fe2+-ferrozine complex. Nisin resistance was determined spectrophotometrically and using well diffusion method. Also, sodium benzoate sensitivity was performed spectrophotometrically and cell counting. Enzyme activities were qualitatively determined using spot culture method. L. fermentum MA-7 and MA-8 strains showed anti-bacterial and anti-fungal activities against tested pathogenic microorganisms at a range of 2.17-5.90 mm.

The antioxidant activities of MA-7 and MA-8 strains were determined as 26.62% and 41.30% against DPPH free radicals. The strains were found to be resistant to 100 µg/mL concentrations of nisin uses as a natural bio-preservative in food industry. L. fermentum MA strains were also resistant to the sodium benzoate food preservative. Amylase enzyme activity was observed only in MA-7 while lichenase enzyme activity was observed in both strains. Therefore, the L. fermentum strains can be recommended to be used in food applications.

Molecular Phylogeny of Rice leaf folder (Cnaphalocrocis medinalis)

Mashhoor K., Ramesh N., Lazar K.V. and Shanas S.

Page No: 23-28

Abstract: The rice leaf folder C. medinalis is one of the major pests of paddy, widely distributed in rice growing areas in Asia, Oceania, North east Australia and Madagaskar. Leaf folders fold the leaf longitudinally and fasten the margin of the leaf with silk like thread. About eight species of pyralid moths fold or roll leaf of graminaceous plants. Among them, the Cnaphalocrocis medinalis, Marasmia patnalis and Marasmia exigua have only attained the pest status. There are three species of leaf folder prevalent in India of which, C. medinalis is the dominant species. Several outbreaks of C. medinalis have been reported from many Asian countries like India, China, Japan, Korea, Malaysia, Srilanka and Vietnam. In some cases, M. patnalis has been confused with C. medinalis. Understanding the genetic structures of the pest’s population may provide important biological information to design IPM strategies for the control of this pest.

Hence the present study has made an attempt to reveal the genetic structure of C. medinalis using Cytochrome oxidase subunit I partial sequence. The C. medinalis isolated from Kerala showed 100% similarity with that of Papua New Guinea. The C. medinalis isolated from Australia and China showed 0.22% divergence and C. medinalis isolated from Punjab showed 0.45% divergence with that of Kerala. The C. poeyalis showed 5.75%, C. trapezalis showed 7.86% and Marasmia sp. showed 6.25% divergence from C. medinalis isolated from Kerala.

A highly efficient method for micropropagation of Allium akaka subsp. akaka, an endangered medicinal plant

Arezo Pazoki, Mahboobeh Zare Mehrjerdi, Maryam Norouzi and Shirin Dianati Daylami

Page No: 29-33

Abstract: Allium akaka subsp. akaka, an endangered medicinal plant belongs to the Amaryllidaceae family and is native to Iran. This study was conducted to establish an efficient protocol for micropropagation of Allium akaka subsp. akaka. The influence of growth regulators on callus induction and shoot regeneration and also the effect of growth regulator and sucrose concentration on bulb production were studied. The results indicated that using the basal plate explant and B5 medium, the highest frequencies of callus induction (100%) were found at 0.5 mg/l benzyladenine (BA) with 1.5 or 2 mg/l 2,4-dichlorophenoxyaceticacid (2,4-D). The highest percentage of regeneration and number of shoots per culture were observed at 0.5 mg/l naphthaleneacetic acid (NAA) with 0.5 mg/l BA (83.3% and 36.5 respectively).

In continuation bulb production experiment on regenerated shoots with B5 medium was conducted; the results indicated that the production of bulb in the medium containing 6% sucrose without using growth regulators was 100%. The resulting protocol can be a method for the mass production and commercialization of this valuable plant.

Genotypic characterization and efficacy of phosphate solubilising bacteria in improving the crop yield of Zea mays

Dar Aijaz Ahmad and Bhat A.K.

Page No: 34-39

Abstract: The study aimed to explore and evaluate the various rhizospheres in Jammu region for the presence of phosphate-solubilizing bacteria to improve the crop yield in Zea mays. Preliminary investigations on the basis of the halo-zone formation showed the presence of eight PSB isolates with clearing zones varying from 0.95mm to 7.79mm. Phosphorus solubilizing efficiency determined was between 38.56% and 78.52%.

Out of eight isolates, three PSB isolates showed stable phosphatase activity which were further characterized for various biochemical tests and also subjected to the 16S rRNA sequence analysis and were identified as Bacillus pumilus (PSB1 and PSB4) and one as Bacillus atrophaeus (PSB7). These isolates were evaluated for their efficacy in promoting the dry matter yield of Maize (Zea mays) variety CM-135 in the pot experiment and showed a significant increase in dry matter yield as compared to the control. The cultures serve as promising candidates as biofertilizers and can help in crop growth as a cheap and environment friendly method.

DNA fingerprinting and molecular characterization of Brassica cultivars using RAPD markers

Sarker Md, Azam S.M., Islam Md, Parvin Shahanaz and Rahman Md

Page No: 40-44

Abstract:The aim of this study is to explore the genetic variation and diversity among mustard and rape seed genotypes using RAPD marker. The production of Brassica germplasm with a wider genetic base is essential for utilizing in the genetic improvement of mustard. PCR-based Random Amplified Polymorphic DNA (RAPD) technique is used for assessing genetic diversity and relationships among six Brassica cultivars viz., Safal, Sampad, Binasarisha-4, Binasarisha-5, Daulot and Rai-5. A total of nine RAPD primers are used for this study. Out of nine, three primers (OPA-02, OPB-01 and OPC-02) generated 33 distinct polymorphic bands. Each primer generated various banding pattern with an average of 11 scorable bands per primer. Among the three primers, the primer OPA-02 produced a maximum number of the band (14) and the other two primers OPB-01 and OPC-02 generated 10 and 9 bands respectively.

The cultivar Sampad (Brassica rapa L.) close to the cultivar Safal (Brassica campestris L.) with the lowest genetic distance (0.0265) and the highest genetic distance (0.981) is found between Sampad and Rai-5 (Brassica juncea, L.). On the basis of the dendrogram, most diverse genotypes are identified that can be used in future for Brassica breeding program.

Optimization of spacing and refuge strategy for improved Cr1Ac expression level in Bt cotton

Perumalla Srikanth, Devasahayam Mercy, Maxton Ann and Masih Sam A.

Page No: 45-52

Abstract:Resistance in insects is a global problem from last decade after introducing of transgenic cotton. Refuge strategy is being used efficiently for delaying this insect resistance in plants. During the present study, field trials were conducted with different percentages of Bt cotton with different refuge nBt cotton of five treatments. The results revealed that 25%Bt cotton with 75% nBt cotton treatment retains highest Cry1Ac expression in lower leaves (0.923µg/gm) followed by upper leaves (0.853 µg/gm), sepals (1.300 µg/gm) and boll bracts (1.025µg/gm) respectively. Although it was also noticed that these Cry1Ac expression decreases consistently as concerned plant age increased during all treatments. The plant height of Bt cotton and number of sympodia at different days at 90, 120 and 150 days were recorded in T3 (25% Bt cotton with 75% nBt cotton) 96.167(cm), 97.000(cm), 122.833(cm) and T2 (75% Bt cotton with 25% nBt cotton) 9.733, 10.600 and 14.600.The highest number of bolls per plant (24),boll size (16.344), number of seeds per boll (22.067), seeds per plant (509.000), seed index (8.232) and lint index (13.058) were recorded in T2 (75 % Bt cotton with 25% nBt cotton).

Results from present investigation concluded that highest morphology and yield parameters depend upon number of Bt cotton and nBt cotton plants present in all treatments while T2(75% Bt cotton with 25% nBt cotton) combination showed best suitable suggestion for farmers to opt in their field for improved yield and as a good strategy to defend the resistance in insects.

Molecular identification of Boletus speciosus (Boletaceae) using nuclear ribosomal internal transcribed spacer region in Vietnam

Xuan Phuong Bui, Huu Thu Do, Syed Noor Muhammad Shah, Phuong Dai Nguyen Nguyen, Quynh Mai Pham, Hung Manh Nguyen, Minh Duc Dao, Le Minh Bui and Dinh Duy Vu

Page No: 53-67

Abstract: DNA barcoding is a useful tool for species identification using standardized genomic DNA fragments. In Vietnam there are 12 species of genus Boletus, some valued for food and medicine, whereas others are extremely poisonous. However, there is poor knowledge about the number of poisonous species. We used DNA barcodes (internal transcribed spacer (ITS) to explore Boletus speciosus and to investigate the current taxonomy of Boletus.

In the current study, ITS region showed high level of universality for PCR and sequencing. All samples from Vietnam have close relationship with B. speciosus (MLBS = 100%, BPP=99%). Genetic p-distances interspecific divergence within and among Boletus species were varied from 0% to 21.3%.

Studies on Expression of CCA1 Gene in Agrobacterium-mediated Genetically Transformed Indica Rice

Tara Nayan, Devi Anita, Meena and Chaudhury Ashok

Page No: 68-78

Abstract: In higher plants, circadian rhythms regulate various biological processes including metabolism, photosynthesis, stomata opening and closing, water uptake, onset of flowering, hypocotyls elongation, anticipation of environmental changes and thus help in adaptation. Studies in Arabidopsis thaliana have found that suppression of CCA1 gene - an important candidate of circadian clock rhythm may be a source of increased biomass and growth vigor. Keeping this in view, independent lines derived from Agrobacterium-mediated genetically transformed Indica rice variety HKR 126. T0 plants for up regulation and down regulation of CCA1 gene were produced for studying CCA1 gene expression.

Effect of Arbuscular Mycorrhizal Fungi on Verticillium wilt development of olive trees caused by Verticillium dahliae

Hanane Boutaj, Abdelilah Meddich, Said Wahbi, Abdelmajid Moukhli, Zainab El Alaoui-Talibi, Allal Douira, Abdelkarim Filali-Maltouf and Cherkaoui El Modafar

Page No: 79-88

Abstract: This study reports the effect of Arbuscular Mycorrhizal Fungi (AMF) on olive plants to enhance its tolerance against Verticillium dahliae under greenhouse conditions. Inoculated plants with native mycorrhizal consortium (Rhizolive) or with Glomus irregulare pure strain were treated three months after mycorhization with V. dahliae. It was found that root colonization by Rhizolive or G. irregulare was similar. Results showed that disease severity and percentage of dead plants were significantly reduced in G. irregulare + V. dahliae (1.36 and 20% respectively) compared to Rhizolive + V. dahliae treatments (2.20 and 28.30% respectively).

Moreover, compared to controls, lower dwarfing index and leaf alteration index were shown in inoculated plants with Rhizolive + V. dahliae (-9.73% and 0.143 in 21st week); also, in inoculated plants with G. irregulare + V. dahliae (-2.96% and 0.200 in 21st week) treatments. Mycorrhization with Rhizolive or G. irregulare seemed to help olive plants to reduce the disease symptoms caused by V. dahliae.

Isolation, Identification and Susceptibility Testing of Potential Lipase producing Bacterial and Fungal Strains from soil

Joshi Swapnil S. and Jobanputra Arpana H.

Page No: 89-96

Abstract:Lipases are the enzymes that are generally used in the catalysis of the triacylglycerols to glycerol and free fatty acids. Microbial lipases are highly useful industrial enzymes and they represent more than 45% of total enzyme sales in enzyme market. This enzyme has wide application in processing of organic chemical, detergent formulations, manufacturing of paper, nutrition, cosmetics and processing in pharmaceutical. Due to the increasing demand of lipases, their stable production from microbial source is in high demand. In the present study, the isolation and screening of the lipase producing fungal and bacterial strains have been carried out on the different soil samples collected from different sites.

In total, thirteen bacterial strains were isolated. Out of these strains, one potent strain of Pseudomonas aeruginosa (SJ2) was further validated by 16S rRNA sequencing, biochemical analysis and antibiotic resistance studies. Moreover, six fungal strains were also isolated and screened from which one strain selected for the additional study was Aspergillus flavus SJ3. The identity of the fungal strain was confirmed by the 18S rRNA gene sequencing. The best performing isolates from our collection can be commercialized after production process optimization.

Production of industrial enzymes from the gut microflora of fishes and insects and assessment of their destaining ability

John Sonia, Grigary Sneha and Sara Sumam Sunil

Page No: 97-99

Abstract:A number of researches have been done to obtain enzymes economically from yeast, bacteria, plants and animals. But the enzymes obtained from bacteria are considered to be the most stable and the enzyme production from them can be increased by manipulating the environment and genes. About 3000 enzymes are known to us but only a few are exploited industrially. This study is mainly based on the enzyme production from gut microbiota of fishes and insects. The gut of fishes and insects contains a number of bacteria which are in a symbiotic relationship with fish and insect respectively.

The enzymes play a major role in digestion of food. These microbes produce several enzymes which are useful for fish and this may include enzymes like amylase, lipase, protease and cellulose. The present study aims at the production of enzymes such as amylase and cellulase from the microbes present in fish and insect gut and to investigate its commercial applications.

Biological activity of prodigiosin from Serratia rubidaea RAM_Alex

Metwally Rasha A., Abdelwahab Abeer E., El Sikaily Amany, El-Sersy Nermeen A., Ghozlan Hanan A. and Sabry Soraya A.

Page No: 100-104

Abstract:Marine bacteria have not been examined as extensively as land bacteria. The marine environment in particular has attracted much attention as a source of natural product discovery. A red pigmented bacterial strain isolated from clam samples of Temsah Lake at Ismailia, Egypt was identified as Serratia rubidaea RAM_Alex. The strain produces the red pigment prodigiosin. The aim of the present study was to evaluate the potentiality of prodigiosin produced by the bacterial strain as an anticancer, anti-hepatitis C virus (HCV), antioxidant and antibacterial agent. As anticancer agent, prodigiosin showed inhibition on CaCo-2, MCF-7 and HelaS3 cell lines; but no activity was observed towards HepG2 cell line.

This pigment showed antioxidant activity confirmed by DPPH reduction method and succeeded in the inhibition of HCV replication at a concentration of 100µg/ml. Prodigiosin had antagonistic effect against Escherichia coli, Aeromonas hydrophila, Staphylococcus aureus, Streptococcus faecalis and Candida albicans.

In Vitro Cytotoxicity Study of Green Synthesized Copper Nanoparticles

Shobha G., Sagar S., Shashidhara K.S., Vishwaprakash Mahadimane and Ananda S.

Page No: 105-111

Abstract:In our earlier study, we reported the green synthesis of copper nanoparticles with antimicrobial activity. Here we report the cytotoxicity and genotoxicity potency of the green synthesized copper nanoparticles on cancer cells MCF-7. The different cell lines experimental results in our study have shown that the cells morphology changed significantly for cancer lines MCF-7 upon treatment with copper nanoparticles and IC50 dose for MCF-7 cells was found to be 1.71 μg ml−1 by MTT assay. At this concentration, copper nanoparticles had shown no cytotoxicity effect on normal cell line (3T3L1). The dual staining (Acridine orange/Ethidium Bromide) showed various degrees of accumulation of nanoparticles by apoptotic tumour cells with increasing concentration of copper nanoparticles.

The comet assay and DNA fragmentation assay by gel electrophoresis on cancer cell line MCF-7 showed the chromosomal condensation and fragmentation of DNA after treatment with the copper nanoparticles.

Partial Purification of Phytase and Mannanase from Lactobacillus plantarum and Kinetic Determination of the Features of the L. plantarum immobilized onto the Magnetite Florisil Nanoparticle

Çelenkli Cem, Uçar Sevda, Kotan Recep, Nadaroğlu Hayrunisa and Dikbaş Neslihan

Page No: 112-119

Abstract:In this study, production of the phytase and mannanase was carried out in a natural medium which includes corn (C) and wheat (W) using free and immobilized L. plantarum bacterial strains. Accordingly, test bacteria were inoculated into media containing C (10, 20, 30, 40 gr) and W (10, 15, 20 gr) at different ratios and left to incubate at the growth conditions pH 6.0-6.5 and 350C. First, -NH2 group was attached to the support material using APTES. Then, the sub-branch was formed using glutaraldehyde and the resulting Schiff was reduced with sodium borohydride to make the basic compound stable. Activated support material nano florisil was made magnetic with Fe3O4 compound and used for the immobilization of the bacteria. It was determined that approximately 50% of the prepared bacteria immobilized to the nanoparticles of magnetite florisil.

The production of phytase and mannanase was performed with both free microorganisms and microorganisms attached to the matrix phytase and mannanase production by L. plantarum using corn and wheat natural media increased by 10-15% compared to those of the free isolates. In light of these findings, it was concluded that magnetite florisil nanoparticles can be used in many areas especially food and agriculture.

Biosynthesis of Silver Nanoparticles from various Microbial and Green resources-A Review

Singh Anshu, Shekhar Sujata and Shrivastava J.N.

Page No: 120-130

Abstract:One of the advanced techniques of material science is nanotechnology which includes the small sized nanoparticles that exhibit enhanced properties as compared to the bulk material. Due to the extremely small size, nanoparticles have large surface area relative to their volume. Wide range of biological systems ranging from bacteria to plants and mammals can be used for synthesizing nano-sized particles of interest in order to develop clean, non-toxic and eco-friendly technologies. Biological molecules as reducing agents and oxidizing agents are significantly safe over their chemical counterparts thus obviating the need of chemical reagents for synthesis. It has also become necessary to explore the properties of nano-rugs due to the reason that microbes are abruptly gaining resistance against the wide range of present day antibiotics.

Silver nanoparticles are being more attracted due to their therapeutic, bimolecular, detection and catalysis applications. Nanotechnology is a rapidly growing field with significant applications in various areas including the synthesis and applications of nano-materials. The present review explores the huge microbial and plant diversity to be utilized towards rapid and single step protocol preparatory method.

Emerging Trends of Non-Invasive Nano-Systems in Disease Detection through Saliva and Sweat

Khare Ruchi, Singh Pushpendra, Yasir Mohammad, Joseph Abey and Shrivastava Rahul

Page No: 131-136

Abstract:Biofluids contains a wide range of molecules and these molecules help us to reflect our body’s state by their physiochemical, biochemical properties and their concentration parameters. Initially, biofluids used for detection of diseases were blood, urine and many others, but now saliva and sweat can be exploited for the detection of diseases. Saliva and sweat are two different biofluids composed of different inorganic and organic ions and several other metabolites like lactate, glucose, Na+, Cl-. These metabolites and their byproducts serve as diagnostic tools. In saliva many proteins like, DNAs, RNAs, hormones and interleukins are present which can be used for the detection of diseases and the presence of such molecules confirms saliva as potential biofluid for diagnosis of heart diseases, cancer using nano detection strips.

Drug abuse can also be detected using these two biofluid because drug appears in sweat and saliva. The analysis is performed by comparing the concentration of these diagnostic tools present in sweat and saliva with the concentration level present in blood, serum or urine and based on the molecule concentration, several microfluidic devices are fabricated. This study provides a brief detail of biomarkers present in biofluid and the use of saliva and sweat for emerging non-invasive technique for detection of diseases.

Synthetic Biology: A World of Effectual Biomedicines

Sana Gulzar, Muhammad Adeel Baig, Saira Anwar, Aqsa Rashid, Kinza Abad, Hamza Hafeez, Fazeeha Amjad and Pervez Anwar

Page No: 137-149

Abstract:The paramount synthetic biology has come forth to comprehend the complex biological systems involving the implication of bioengineering principles. Recent achievements in this area have served along to construct and engineer the bacterial and viral devices thereby elucidating the novel strategies for the treatment of cancer on delivering the desired enzymatic sequences to the target sites. This acquirement has superseded the common dreadful ways of cancer treatment by targeting specifically the tumor cells. Biosynthetically developed biosensors comprised by sensitive components affix with ligands and transducer elements to activate or inhibit the retaliation of cells. The therapeutic drug monitoring is enormously contributed by nano biosensors. Synthetic biology is pre-eminently focused on the biosynthesis of inexpensive and potent bio drugs by interacting with microbial engineering. Biosynthetic based drugs produced until now are inclusive of Taxol (an anti-cancer drug against breast and lungs cancer), Artemisinin (anti-malarial drug) and Gamma-amino butylate (GABA) to boost up immune response.

Beside these biomedical applications, this discipline also finds its major role in the designing of synthetic gene networks which execute core cellular functions owing the formation of biodegradable scaffolds to regenerate the damaged tissues. Consequently, the chances of tissue rejection have been diminished. Supplementary living therapies aided by Synthetic biology have sorted out the issues of antibiotic resistance. This progress is primely supported by powerful bioinformatic tools to identify the best combinations of enzymes and pathways in living systems and produce the drugs more efficiently.