Promotion of Growth and Modulation of As- Induced Stress Ethylene in Maize by As- Tolerant ACC Deaminase Producing Bacteria

One of the major pollutants in the environment is arsenic (As). Due to the toxic effects of As to all organisms, its remediation is necessary. Conventional technologies used in the remediation of As contaminated soils are expensive and may even compromise the structure of the soil. An attractive alternative is phytoremediation, which is the use of plants which can take up the contaminant in their tissues. Plant growth promoting bacteria (PGPB) has been known to enhance growth of plants through several mechanisms such as phytohormone production, phosphate solubilization, siderophore production and 1-aminocyclopropane-1- carboxylate (ACC) deaminase production, which is an essential trait that aids plants especially under stress conditions such as As stress. Twenty one bacteria were isolated from As-contaminated soils in the vicinity of the Janghang Smelter in Chungnam Province, South Korea. These exhibited high tolerance to either arsenite (As III) or arsenate (As V) or both. Most of these isolates possess several plant growth promoting traits which can be potentially exploited to increase phytoremediation efficiency. Among the identified isolates is Pseudomonas sp. JS1215, which produces ACC deaminase, indole acetic acid (IAA), and siderophore. It also has the ability to solubilize phosphate. Inoculation of JS1215 significantly enhanced root and shoot length and biomass accumulation of maize under normal conditions. In the presence of As, particularly in lower As level, inoculation of JS1215 slightly increased root length and biomass. Ethylene increased with increasing As concentration, but was reduced by JS1215 inoculation. JS1215 can be a potential bioinoculant for increasing phytoremediation efficiency.

Effect of Euphorbia Pulcherrima Leaf and Inflorescence Extract on Various Cytomorphological Parameters of Aspergillus fumigatus

Microorganisms can be removed, inhibited or killed by physical agents, physical processes or chemical agents but they have their inherent disadvantages such as increased resistance against antibiotics etc. Since, plants have endless ability to synthesize aromatic substances which act as the master agents for plant defense mechanisms against microorganisms, insects and herbivores. Thus, secondary metabolites or phytochemicals obtained from plants can be used as agents of disease control nowadays. In the present study effect of different concentrations of acetone fraction of leaves and alcohol fraction of inflorescence of Euphorbia pulcherrima on various cytomorphological parameters i.e. cell number, mycelium width, conidial size, conidiophore size etc. of Aspergillus fumigatus has been studied. Change in mycelium/ hyphal cell width, conidium size, conidiophore size etc. was measured with the help of a previously calibrated oculometer. To study effect on morphology, fungal mycelium along with conidiophore and conidia were stained with cotton blue and mounted in lactophenol and observed microscopically. Inhibitory action of the acetone extract of Euphorbia pulcherrima leaf on growth of Aspergillus fumigatus was investigated. Control containing extract free medium supported profuse growth of the fungus. Although decrease in growth was observed even at 3.95μg/ml but significant inhibition of growth was started at7.81μg/ml concentration of the extract. Complete inhibition was observed at 15.62μg/ml and above. Microscopic examination revealed that at 3.95, 7.81 and 15.62μg/ml extract concentration hyphal cell width was found to be increased from 1.44μm in control to 3.86, 5.24 and 8.98 μm respectively giving a beaded appearance to the mycelium. Vesicle size was reduced from 24.78x20.08μm (control) to 11.34x10.06μm at 3.95μg/ml concentration. At 7.81 and 15.62μg/ml concentration no phialides and sterigmata were observed. Inhibitory action of the alcohol extract of inflorescence on the growth of Aspergillus fumigatus was also studied. Control containing extract free medium supported profuse growth of the fungus. Although decrease in growth was observed even at 3.95μg/ml but complete inhibition was observed at 62.5μg/ml and above. Microscopic examination revealed that hyphal cell width of Aspergillus fumigatus was found to be increased from 1.67μm in control to 5.84μm at MIC i.e. at 62.5μg/ml. Vesicle size was reduced from 44.76x 24.22μm (control) to 11.36x 6.80μm at 15.62μg/ml concentrations. At 31.25 μg/ml and 62.5μg/ml concentration no phialides and sterigmata was found. Spore germination was completely found to be inhibited at 3.95μg/ml concentration. Similarly 92.87% reduction in vesicle size was observed at 15.62μg/ml concentration. It is evident from the results that plant extracts inhibit fungal growth and this inhibition is concentration dependent. 

Re-Examination of Louis Pasteur’s S-Shaped Flask Experiment

No negative control nor control to prevent microbes from escaping was set when the S-shaped flask experiments were performed by Pasteur. Microscope was not used to observe the media in the flasks. Louis Pasteur’s S-shaped flask experiment was re-examined by using U-shaped flasks, modified S-shaped flasks and microscope. A mixture of microbes was isolated from the room air, from which one rod-shaped Bacillus species with proposed name Bacillus gaso-mobilis sp nov and one grape-shaped Staphylococcus species with proposed name of Staphylococcus gaso-mobilis sp nov were identified. Their penicillin and ampicillin resistant strains containing plasmids were isolated. These bacteria could change color, produce odor and automatically move in the air. They did not form colonies on solid media. They had a high suspension capacity in liquid media. Their light absorbance peaked at the wave length of 320 nm.  It was concluded that there were flaws with Louis Pasteur’s S-shaped flask experiments. 

Nile Red, an Alternative Fluorescence Method for Quantification of Neutral Lipids in Microalgae

According to biodiesel from microalgae is an attractive fuel for several reasons such as renewable, biodegradable and environmental friendly. Thus, this study, green microalgae Scenedesmus acutus PPNK1 isolated from natural water, was selected based on high growth rates, easy cultivation and high lipid content. The Nile red fluorescence method has been successfully applied to the determination of lipids in S. acutus PPNK1. The combination of the method to the lipid composition in algal cells showed the yellow fluorescence under fluorescent microscope. Interestingly, maximum cell numbers and biomass concentration were obtained at 5.44´107 cells/mL and 1.60 g/L when it was cultivated in BG-11 medium while in case of BG-11 with nitrogen deprivation (N 0.25 g/L), accumulated lipid content in cells (44.67%) was achieved that was higher than that found in case of BG-11 medium at about 2 times (22.63%).

Structure Based Computational Analysis and Molecular Phylogeny of C- Phycocyanin Gene from the Selected Cyanobacteria

Cyanobacteria play a vital role in the production of phycobiliproteins that includes phycocyanin and phycoerythrin pigments. Phycocyanin and related phycobiliproteins have wide variety of application that is used in the food, biotechnology and cosmetic industry because of their color, fluorescent and antioxidant properties. The present study is focused to understand the pigment at molecular level in the Cyanobacteria Oscillatoria terebriformis NTRI05 and Oscillatoria foreaui NTRI06. After extraction of genomic DNA, the amplification of C-Phycocyanin gene was done with the suitable primer PCβF and PCαR and the sequencing was performed. Structural and Phylogenetic analysis was attained using the sequence to develop a molecular model.

A Study of Fatty Acid Production in the Batch Reactor via the Carbohydrate Fermentation by C. butyricum

Carbohydrate can be used as a substrate that can be consumed by C. butyricum and converted to useful chemicals such as acetic and butyric acid. Influence of concentration and types of carbohydrate to cell growth, carbohydrate consumed, productivity and carbon balance have been explored. Batch reactor was selected in this study to avoid contamination due to simpler operation system. Glucose was preferred as first types of carbohydrate to be tested. Six concentrations were studied from 0 to 28g/L. Eventually, 15g/L has shown the best concentration for glucose in term of growth rate (2.63h-1) and carbon balance (99.76% recovery). Comparison for types of carbohydrate was also conducted. 15g/L of xylose (monosaccharide) and starch (complex carbohydrate) was tested.  In term of growth rate and productivity, glucose showed the best carbohydrates. Results for this study showed that glucose and xylose produced more than 80% of acetic acid and less than 20% of butyric acid. Meanwhile, 63.1% of acetic acid and 36.9% of butyric acid were produced from starch. 

Lactic Acid-Chitosan Films’ Properties and Their in vivo Wound Healing Activity

Chitosan is a derivative of chitin, a compound usually isolated from the shells of some crustaceans such as crab, lobster and shrimp. It has biocompatible, biodegradable, and antimicrobial properties. To use these properties of chitosan in biomedical fields, chitosan films (1%, 2%, 3% and 4%) were prepared by using l% lactic acid as solvent. The effects of chitosan films on tensile strength, elongation at break, degree of swelling, thickness, morphology, allergic and irritation reactions and antibacterial property were evaluated. Staphylococcus aureus and Escherichia coli were used as tested microorganisms. In vivo wound healing activities of chitosan films were investigated using mice model. As results, Chitosan films have similar appearance and good swelling properties and 4% chitosan film showed the better swelling activity and the greatest elongation ratio than the other chitosan films. They also showed their good activity of wound healing in mice model. Moreover, the results showed that the films did not produce any unwilling symptoms (allergy or irritation). In conclusion, it is evident that the chitosan film has the potentiality to use as wound healing biofilms in the biomedical fields.

Optimization of Lipase Production Using Bacillus subtilis by Response Surface Methodology

A total of 6 isolates of Bacillus subtilis were isolated from oil mill waste collected in Namakkal district, Tamilnadu, India. The isolated bacteria were screened using lipase screening medium containing Tween 80. BS-3 isolate exhibited a greater clear zone than the others, indicating higher lipase activity. Therefore, this isolate was selected for media optimization studies. Ten process variables were screened using Plackett–Burman design and were further optimized by central composite design of response surface methodology for lipase production in submerged fermentation. Maximum lipase production of 16.627 U/min/ml were predicted in medium containing yeast extract (9.3636g), CaCl2 (0.8986g) and incubation periods (1.813 days). A mean value of 16.98 ± 0.2286 U/min/ml of lipase was acquired from real experiments.

Production and Extraction of Quercetin and (+)-Catechin from Phyllanthus niruri Callus Culture

Quercetin and (+)-catechin are metabolites present in Phyllanthus niruri plant, have potential in medicinal uses as anticancer and antioxidant agents. Studies on production of quercetin and (+)-catechin from P. niruri callus culture via in vitro technique were carried out and the results were compared to the intact plant. P. niruri explants were cultured on Murashige and Skoog (MS) solidified media supplemented with several phytohormone combinations for one month. The metabolites were extracted from P. niruri callus and intact plant by using carbon dioxide supercritical fluid extraction (SFE) with ethanol as modifier and solvent extraction techniques. The extracts were analyzed by means of HPLC method. Results showed that P. niruri callus culture was successfully established. The highest content of quercetin (1.72%) was found from P. niruri callus grown in media supplemented with 0.8mg/L kinetin and 0.2mg/L 2,4-dicholophenoxyacetic acid (2,4-D), which was 1.2 fold higher than intact plant. Meanwhile, the highest amounts of (+)-catechin (0.63%) was found from P. niruri callus grown in media with addition of 0.2mg/L 1-naphthalene acetic acid (NAA) and 0.8mg/L 2,4-D. The SFE condition in this study showed better extraction efficiency when higher contents of selected metabolites were found in all SFE extracts compared to the common solvent extracts.

Partial Purification of Cytotoxic Peptides against Gastric Cancer Cells from Protein Hydrolysate of Euphorbia hirta Linn.

Protein hydrolysates prepared from a number of medicinal plants are promising sources of various bioactive peptides. In this work, proteins from dried whole plant of Euphorbia hirta Linn. were extracted and digested with pepsin for 12h. The hydrolysates of lesser than 3 KDa were fractionated by a cut-off membrane. The peptide hydrolysate was then purified by an anion-exchange chromatography on DEAE-Sephacel™ column and reverse-phase chromatography on Sep-pak C18 column, respectively. The cytotoxic effect of each peptide fraction against a gastric carcinoma cell line (KATO-III, ATCC No. HTB103) was investigated using colorimetric MTT viability assay. A human liver cell line (Chang Liver, CLS No. 300139) was used as a control normal cell line. Two purified peptide peaks, peak l and peak ll at 100µg peptides mL-1 affected cell viability of the gastric cancer cell lines to 63.85±4.94 and 66.92±6.46%, respectively. Our result showed for the first time that the peptide fractions derived from protein hydrolysate of Euphorbia hirta Linn. have anti-gastric cancer activity, which offers a potential novel and natural anti-gastric cancer remedy.

Identification of Single Nucleotide Polymorphism in 5'-UTR of CYP11B1 Gene in Pakistani Sahiwal Cattle

A major goal in animal genetics is to understand the role of common genetic variants in diseases susceptibility and production traits. Sahiwal cattle can be considered as a global animal genetic resource due to its relatively high milk producing ability, resistance against tropical diseases and heat tolerant. CYP11B1 gene provides instructions for making a mitochondrial enzyme called steroid 11-beta-hydroxylase. It catalyzes the 11deoxy-cortisol to cortisol and 11deoxycorticosterone to corticosterone in cattle. The bovine CYP11B1 gene is positioned on BTA14q12 comprises of eight introns and nine exons and protein is associated with mitochondrial epithelium. The present study was aimed to identify the single-nucleotide polymorphisms in CYP11B1 gene in Sahiwal cattle breed of Pakistan. Four polymorphic sites were identified in exon one of CYP11B1 gene through sequencing approach. Significant finding was the incidence of the C→T polymorphism in 5'-UTR, causing amino acid substitution from alanine to valine (A30V) in Sahiwal cattle breed. That Ala/Val polymorphism may serve as a powerful genetic tool for the development of DNA markers that can be used for the particular traits for different local cattle breeds.

An Integrated Predictor for Cis-Regulatory Modules

Various cis-regulatory module (CRM) predictors have been proposed in the last decade. Several well-established CRM predictors adopted different categories of prediction strategies, including window clustering, probabilistic modeling and phylogenetic footprinting. Appropriate integration of them has a potential to achieve high quality CRM prediction. This study analyzed four existing CRM predictors (ClusterBuster, MSCAN, CisModule and MultiModule) to seek a predictor combination that delivers a higher accuracy than individual CRM predictors. 465 CRMs across 140 Drosophila melanogaster genes from the RED fly database were used to evaluate the integrated CRM predictor proposed in this study. The results show that four predictor combinations achieved superior performance than the best individual CRM predictor.

The Potency of Sandfish (Holothuria scraba) as a Source of Natural Aphrodisiacs

Sandfish is one of marine biota that has a biomedicine (bioactive compound) potency. People in Gorontalo Province, Indonesia, have been sandfish as an aphrodisiac for men as it is believed that sandfish has a steroid hormone potency. This research aims at studying using the steroid hormone potency from every fraction of sandfish (meat and innards) and its activity of male reproduction (rooster) as an aphrodisiac. Steroid extraction was done using Touchstone and Kasparow method, and then it was utilized to study the effectiveness of bioassay of rooster. This research had five treatments and was done in complete randomized design. Based on Lieberman-Burchard and bioassay test, the author found that sandfish extract contains steroid hormone. Sandfish extract was able to enrich testosterone and cholesterol concentration in blood serum; fastening secondary reproduction characteristics of the rooster, and increasing growth as well as improving rooster’s comb. Therefore, sandfish steroid is potential to be used as an aphrodisiac for men.

Toxic Effect of Sodium Nitrate on Germinating Seeds of Vigna radiata

Sodium nitrate has been used industrially in a number of work fields ranging from agriculture to food industry. Sodium nitrate and nitrite are associated with a higher risk of cancer in human beings. In present study, the effect of sodium nitrate on germinating seeds was studied. Two different sets of ungerminated Vigna radiata seeds were taken. In one set Vigna radiata seeds were soaked in distilled water for 4 hours and they were allowed to germinate in distilled water (Control) and 0.1 to 1% and 10% concentrations of sodium nitrate (NaNo3). In soaked seed set, on 2nd day radical developed in control and 0.1 to 1% concentrations of sodium nitrate. Seeds size was enlarged in 1% and 10% concentrations of sodium nitrate. On 3rd day in 0.1% sodium nitrate length of the radicle was 7.5cm with one leaf let and control sample showed 9cm with one leaflet. On 5th day in 0.1% sodium nitrate length of the radicle was 10 cm with one leaf let and control sample showed 11.5cm with one leaflet. No radicle developed in 1 and 10% NaNo3 concentrations. On 10th day all plants including control were dead. More number of mitotic cells was observed in apical root meristems of control germinating seeds and less mitotic cells were observed in 0.1% NaNo3 germinating seeds. But cells were elongated in 0.9%NaNo3 concentration and particles are deposited in the cells and no mitotic cells were observed. In other sets, dry seeds were allowed to germinate in Distilled water (control) and in 0.1 to 1% and 10% concentrations of sodium nitrate. In dry seed set, on 2nd day radicle developed from control set. In 0.1 to 1% concentrations of sodium nitration seed enlarged in size but but not allowed germination. But in 10% NaNo3 seeds coat colour was changed from dark green to brown. On 3rd day the radicle was developed in 0.1% concentration of NaNo3. No growth of radicle was observed in 0.3 to 10% concentrations of NaNo3 but plumule was observed in control plant. Seed coat color was changed from dark green to brown in color in 1% and 10% NaNo3. On 5th day in control seeds the radicle growth was 11cm and 0.1% NaNo3 concentration was 1.3 cm. On 10th day all plants including control were dead. More number of mitotic cells was observed in apical root meristems of control germinating seeds and less mitotic cells were observed in 0.1% NaNo3 germinating seeds. At higher concentrations of NaNo3 allowed seed germination in soaked seeds but produced radicle decay. In comparison to it, in dry seed set, germination of seeds observed only in 0.1% NaNo3 concentration. The inhibitory effect of NaNo3 on seed germination is due to reduction of water imbibition and mitotic activity.

Solvent Effect on Antioxidant Activity and Total Phenolic Content of Betula alba and Convolvulus arvensi

The potential of using herbal Betula alba (BA) and Convolvulus arvensis (CA) as a natural antioxidant for food applications were investigated. Each plant extract was prepared by using pure ethanol, different concentration of ethanol aqueous solutions, including 50% and 75%, 50% methanol aqueous and water. Total phenolic content (TPC) was determined using Folin–Ciocalteau method and antioxidant activity were analyzed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, trolox equivalent antioxidant capacity (TEAC), Oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) respectively. Ethanol extract of CA exhibited the highest TPC and antioxidant activity; however BA showed varies of antioxidant activity value in each assay. The BA and CA exhibit the potential sources of natural antioxidant for food commodities.

The Expression of a Novel Gene Encoding an Ankyrin-Repeat Protein, DRA1, is Regulated by Drought-Responsive Alternative Splicing

Drought stress is a critical environmental factor that adversely affects crop productivity and quality. Because of their immobile nature, plants have evolved mechanisms to sense and respond to drought stress. We identified a novel locus of Arabidopsis, designated DRA1 (drought responsive ankyrin1), whose disruption leads to increased drought-stress tolerance. DRA1 encodes a transmembrane protein with an ankyrin-repeat motif that has been implicated in diverse cellular processes such as signal transduction. RT-PCR analysis revealed that there were at least two splicing variants of DRA1 transcripts in wild-type plants. In response to drought stress, the levels of DRA1 transcripts retaining second and third introns were increased, whereas these introns were removed under unstressed conditions. These results suggest that DRA1 protein may negatively regulate plant drought tolerance and that the expression of DRA1is regulated in response to drought stress by alternative splicing.

Relative Suitability Evaluation of Two Methods of Particle-Size Analysis for Selected Soils of Sudan Savanna of Nigeria

The two widely used methods base on the sedimentation principle (Bouyoucos hydrometer and International pipette) for particle-size analysis were comparatively evaluated on soils collected from various locations in Sudan savanna of Nigeria particularly from Sokoto and Zamfara States. The hydrometer method under-estimated the silt and over-estimated the clay content. Also, the hydrometer reading proved difficult and tended to submerge when floated for clay reading in the suspension of very sandy soils (900g kg-1 sand). Furthermore, the results from the two methods were validated by subjecting the data to USDA soil textural triangle to determine their textural class names. The outcome was that 91.67 % of the experimental soils retained the same textural class names irrespective of the method. Thus, Bouyoucos hydrometer method may conveniently find a place in routine work in view of its simplicity, rapidity, and strong correlation with the pipette method.

Preparation of POMA Nanofibers by Electrospinning and Its Applications in Tissue Engineering

In this manuscript, we produced neat electrospun poly(o-methoxyaniline) (POMA) fibers and utilized it for applying the growth of neural stem cells. The transparency and morphology of as-prepared POMA fibers was characterized by UV-visible spectroscopy and scanning electron microscopy, respectively. It was found to have no adverse effects on the long-term proliferation of the neural stem cells (NSCs), retained the ability to self-renew, and exhibit multipotentiality. Results of immunofluorescence staining studies confirmed that POMA electrospun fibers could provide a great environment for NSCs and enhance its differentiation.

Statistical Screening of Medium Components on Ethanol Production from Cashew Apple Juice using Saccharomyces diasticus

In the present study, effect of critical medium components (a total of fifteen components) on ethanol production from waste cashew apple juice (CAJ) using yeast Saccharomyces diasticus was studied. A statistical response surface methodology (RSM) based Plackett-Burman Design (PBD) was used for the design of experiments. The design contains a total of 32 experimental trails. The effect of medium components on ethanol was studied at two different levels such as low concentration level (-) and high concentration levels (+). The dependent variables selected in this study were ethanol concentration (g/L) and cellmass concentration (g/L). Data obtained from RSM on ethanol production were subjected to analysis of variance (ANOVA). In general, initial substrate concentration significantly influenced the microbial growth and product formation. Of the medium components evaluated, CAJ concentration, yeast extract, (NH4)2SO4, and malt extract showed significant effect on ethanol fermentation. A second-order polynomial model was used to predict the experimental data and the model fitted the data with a high correlation coefficient (R2 > 0.98). Maximum ethanol (15.3 g/L) and biomass (6.4 g/L) concentrations were obtained at the optimum medium composition and at optimum condition (temperature-30°C; initial pH-6.8) after 72 h fermentation using S.diasticus.

Cloning and Functional Characterization of Promoter Elements of the D Hordein Gene from the Barley (Hordeum vulgare L.) by Bioinformatic Tools

The low level of foreign genes expression in transgenic plants is a key factor that limits plant genetic engineering. Because of the critical regulatory activity of the promoters on gene transcription, they are studied extensively to improve the efficiency of the plant transgenic system. The strong constitutive promoters, such as CaMV 35S promoter and Ubiqutin 1 maize are usually used in plant biotechnology research. However the expression level of the foreign genes in all tissues is often undesirable. But using a strong seed-specific promoter to limit gene expression in the seed solves such problems. The purpose of this study is to isolate one of the seed specific promoters of Hordeum vulgare. So one of the common varieties of Hordeum vulgare in Iran was selected and their genomes extracted then the D-Hordein promoter amplified using the specific designed primers. Then the amplified fragment of the insert cloned in an appropriate vector and then transformed to E. coli. At last for the final admission of accuracy the cloned fragments sent for sequencing. Sequencing analysis showed that the cloned fragment DHPcontained motifs; like TATA box, CAAT-box, CCGTCC-box, AMYBOX1 and E-box etc., which constituted the seed-specific promoter activity. The results were compared with sequences existing in data banks. D-Hordein promoters of Alger has 99% similarity at 100 % coverage. The results also showed that D-Hordein promoter of barley and HMW promoter of wheat are too similar.