Optimizing expression of antiviral cyanovirin-N homology gene using response surface methodology and protein structure prediction.

Cyanovirin-N (CVN) is well known as an anti-HIV protein. The efficient production of low cost microbicides for preventing the HIV-infection  has lately become a requirement worldwide. The aim of the present study was to optimize the expression of antiviral Cyanovirin-N homology gene found in the indigenous strain of Nostoc ellipsospourum LZN using Response Surface Methodology (RSM) and Protein Structure Analysis. Optimization of three induction factors (IPTG concentration (0.1, 0.55 and 1mM), temperature for bacterial growth (20, 28.5 and 37°C) and induction time (4, 10 and 16h) was done using RSM and Box-Behnken Design. Total RNA extraction was performed and mRNA levels were quantified in each experimental design by one-step SYBR qPCR. Protein structure was predicted using I-TASSER server. The full-length sequence of LZN-CVN gene is 306 bp in length, due mostly to five mutations. RSM analysis showed that the optimum condition to obtain maximum fold change was a concentration of 0.6mM IPTG, temperature set to 29°C and a 12h long induction time. The extracted protein from periplasmic fraction (8 kDa) was verified via SDS-PAGE. The high percentage of LZN-CVN similarity was demonstrated with PDB (Protein Data Bank) accession code of 2rp3A (CVN domain B mutant) and the ligand binding sites were related to N42, V43, D44, G45, S52, N53 and E56 residues. Different expression systems could assist in the development of anti-HIV proteins in a large scale. The LZN-CVN protein was successfully expressed in the E.coli system. RSM could be applied to a series of mathematical and statistical methods for modeling and analysis of responses which are influenced by various variables of interest.

Lethal and Sublethal Effects of Some Chemical and Biological Insecticides on Tuta absoluta (Lepidoptera: Gelechiidae) Eggs and Neonates.

Tuta absoluta (Meyrick) is one of the serious pests of tomatoes. This study was conducted to evaluate the effects of some chemical and biological insecticides on T. absoluta. The insecticides tested were diazinon, dichlorvos, chlorantraniliprole, deltamethrin, acetamiprid, imidacloprid, spinosad, abamectin, indoxacarb, Bacillus thuringiensis Berliner, Beauveria bassiana (Balsamo) Vuillemin, and Metarhizium anisopliae (Metschnikoff) Sorokin. Sublethal effects for the most effective insecticides tested, namely, abamectin, spinosad, chlorantraniliprole, and indoxacarb were assessed, and life table parameters were calculated. Chlorantraniliprole had the highest lethal effect on T. absoluta followed by spinosad, abamectin, and indoxacarb. On the other hand, imidacloprid was not considerably effective on T. absoluta eggs. Metarhizium anisopliae was 11 and 518 times more effective on the eggs and newly hatched larvae than B. bassiana and B. thuringiensis, respectively. Chlorantraniliprole, spinosad, abamectin, and indoxacarb affected life table parameters of T. absoluta significantly (α = 0.05). Spinosad had the highest sublethal effect on T. absoluta followed by abamectin, chlorantraniliprole, and indoxacarb. The results revealed that chlorantraniliprole, spinosad, abamectin, and indoxacarb had considerable lethal and sublethal effects on T. absoluta, and if they perform similarly in commercial greenhouses and fields, they would be suitable candidates to be considered in IPM programs for this pest.

Novel Chemo-Attractants for Trapping Tomato Leafminer Moth (Lepidoptera: Gelechiidae).

The tomato leafminer moth, Tuta absoluta (Meyrick), is a devastating pest for tomatoes in Iran and throughout the world. This pest reduces tomato yields in farms and greenhouses. It appears some floral odors are significant attractants for tomato leafminer moth. In this study, the effects of three floral compounds, phenylacetaldehyde (PAA), acetic acid (AA), and 3-methyl-1-butanol (MB), were evaluated as trap attractants for tomato leafminer moth. The attractants were tested separately, combined, and blended in binary and tertiary. Lures were tested in delta and water pan traps under field conditions and compared with unbaited traps as controls. Results indicated that water pan traps caught more moths than delta traps. Also treatments with PAA + AA combined and the AA + MB blend were strongly attractive to tomato leafminer moth males compared with other treatments in this study.

Effect of alumina contents on phase stability and mechanical properties of magnesium fluorapatite/alumina composites.

The aim of the present work was twofold: to prepare biphasic magnesium fluorapatite (MFA) composites with different amounts of alumina using a two-step sintering process, and to evaluate the effects of various amounts of alumina on the mechanical properties, phase stability, and densification of the composite samples. Initially, MFA powders were prepared with different amounts of alumina by mechanical activation and the MFA composite samples were subsequently prepared using the two-step sintering (TSS) method. In order to determine the appropriate temperature of the first step sintering, conventional sintering of MFA/50% alumina was carried out at temperatures in the range of 1000-1300°C. X-ray diffraction and scanning electron microscopy (SEM) techniques were used to characterize the prepared MFA/alumina composites. The results showed fracture toughness and hardness in the MFA/50% alumina composite samples to increase as a result of alumina addition to their maximum values of 5.82±1.05MPam(1/2) and 22.09±3.5GPa, respectively.

Fabrication and evaluation of a single-element Bi0.5Na0.5TiO3-based ultrasonic transducer.

This paper discusses the fabrication and characterization of a single-element ultrasonic transducer with a lead-free piezoelectric active element. A piezoelectric ceramic with composition of 0.88Bi(0.5)Na(0.5)TiO(3)-0.08Bi(0.5)K(0.5)TiO(3)- 0.04Bi(0.5)Li(0.5)TiO(3) was chosen as the active element of the transducer. This composition exhibited a thickness coupling coefficient (kt) of 0.45, a dielectric constant of 440 (at 1 kHz), and a longitudinal piezoelectric coefficient (d(33)) of 84 pC·N(-1). To make the transducer, the ceramic was sandwiched between an epoxy-tungsten backing layer and a silver epoxy matching layer. An epoxy lens was also incorporated into the transducer's design to focus the ultrasound beam. The focused transducer with a center frequency of about 23 MHz demonstrated a -6-dB bandwidth of 55% and an insertion loss of -32 dB; the -20-dB pulsed length was measured to be 150 ns. A phantom made of copper wires (30 µm in diameter) was utilized to investigate the imaging capability of the transducer. The results indicated that the fabricated transducer, with a lateral resolution of 260 µm and a relatively high depolarization temperature, could be considered as a candidate for replacement of lead-based ultrasonic transducers.

Application of novel nanotechnology strategies in plant biotransformation: a contemporary overview.

During the past epoch we have gone through the remarkable progress in plant gene transformation technology. The production of transgenic plants is considered as a valuable tool in plant research and the technology is extensively applied in phytomedicines and agricultural research. Gene transformation in plants is normally carried out by Agrobacterium species, application of some chemicals and physical techniques (electroporation, microprojectile, etc.). Now a days with better efficacy and reproducibility, novel technologies for the direct gene transfer like liposome, positively charged liposome (lipofectin) and nanoparticle based delivery systems are used for genetic transformation of plants. In this review, we have enlightened the novel nanotechnologies like liposome, Carbon nano-tube and nanoparticles with their current status and future prospects in transgenic plant development. Moreover, we have also highlighted the limitations of conventional techniques of gene transfer. Furthermore, we have tried to postulate innovative ideas on the footprints of established nanotechnology and chemical based strategy with improved efficacy, reproducibility and accuracy along with less time consumption.

In vitro propagation of Cichorium intybus L. and quantification of enhanced secondary metabolite (esculin).

In this report, rapid and effective shoot as well as root regeneration system through direct multiplication was successfully developed for Cichorium intybus L. Furthermore, the effect of exogenous growth regulators (TDZ and IAA) at different concentrations on the regulation process of the plant was also studied. Enhanced production of esculin in developed C. intybus L. was evaluated using leaf extract. Only on the expense of 20 days, regeneration was seen and very low dose of TDZ was seen to be more effective. When 0.02 mg/L of TDZ was combined with 1.5mg/L of IAA, nearly 100% of explants produced shoots with the highest number of regenerated shoots (85.37). With further increase in concentration (≥ 0.05 mg/L), the number of shoots per explants get decreased. A lower NAA to IBA ratio (1.0mg/L of IBA and 0.5mg/L of NAA) seemed to be more effective for root generation and considered to be the most effective combination among the tried groups. IBA was more effective in root development than NAA, but both were comparatively effective. On quantitative analysis by RP-HPLC, the 76.23% of Esculin were found in leaf extract of the in vitro developed C. intybus L. This amount was 26.77% higher than normal grown plants.

Analysis of the spatio-temporal distribution of Eurygaster integriceps (Hemiptera: Scutelleridae) by using spatial analysis by distance indices and geostatistics.

Eurygaster integriceps Puton (Hemiptera: Scutelleridae) is the most serious insect pest of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) in Iran. In this study, spatio-temporal distribution of this pest was determined in wheat by using spatial analysis by distance indices (SADIE) and geostatistics. Global positioning and geographic information systems were used for spatial sampling and mapping the distribution of this insect. The study was conducted for three growing seasons in Gharamalek, an agricultural region to the west of Tabriz, Iran. Weekly sampling began when E. integriceps adults migrated to wheat fields from overwintering sites and ended when the new generation adults appeared at the end of season. The adults were sampled using 1- by 1-m quadrat and distance-walk methods. A sweep net was used for sampling the nymphs, and five 180° sweeps were considered as the sampling unit. The results of spatial analyses by using geostatistics and SADIE indicated that E. integriceps adults were clumped after migration to fields and had significant spatial dependency. The second- and third-instar nymphs showed aggregated spatial structure in the middle of growing season. At the end of the season, population distribution changed toward random or regular patterns; and fourth and fifth instars had weaker spatial structure compared with younger nymphs. In Iran, management measures for E. integriceps in wheat fields are mainly applied against overwintering adults, as well as second and third instars. Because of the aggregated distribution of these life stages, site-specific spraying of chemicals is feasible in managing E. integriceps.

Direct detection and discrimination of double-stranded oligonucleotide corresponding to hepatitis C virus genotype 3a using an electrochemical DNA biosensor based on peptide nucleic acid and double-stranded DNA hybridization.

Development of an electrochemical DNA biosensor for the direct detection and discrimination of double-stranded oligonucleotide (dsDNA) corresponding to hepatitis C virus genotype 3a, without its denaturation, using a gold electrode is described. The electrochemical DNA sensor relies on the modification of the gold electrode with 6-mercapto-1-hexanol and a self-assembled monolayer of 14-mer peptide nucleic acid probe, related to the hepatitis C virus genotype 3a core/E1 region. The increase of differential pulse voltammetric responses of methylene blue, upon hybridization of the self-assembled probe with the target ds-DNA to form a triplex is the principle behind the detection and discrimination. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the developed DNA sensor responds selectively to the ds-DNA target. Diagnostic performance of the biosensor is described and the detection limit was found to be 1.8 x 10(-12) M in phosphate buffer solution, pH 7.0. The relative standard deviation of measurements of 100 pM of target ds-DNA performed with three independent probe-modified electrodes was 3.1%, indicating a remarkable reproducibility of the detection method.

Electrochemical detection of short sequences of hepatitis C 3a virus using a peptide nucleic acid-assembled gold electrode.

Development of an electrochemical DNA biosensor, using a gold electrode modified with a self-assembled monolayer composed of a peptide nucleic acid (PNA) probe and 6-mercapto-1-hexanol, is described. The sensor relies on covalent attachment of the 14-mer PNA probe related to the hepatitis C virus genotype 3a (pHCV3a) core/E1 region on the electrode. Covalently self-assembled PNA could selectively hybridize with a complementary sequence in solution to form double-stranded PNA-DNA on the surface. The increase of peak current of methylene blue (MB), upon hybridization of the self-assembled probe with the target DNA in the solution, was observed and used to detect the target DNA sequence. Some hybridization experiments with noncomplementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. Diagnostic performance of the biosensor is described and the detection limit was found to be 5.7 x 10(-11)M with a relative standard deviation of 1.4% in phosphate buffer solution, pH 7.0. This sensor exhibits high reproducibility and could be used for detection of the target DNA for seven times after the regeneration process.

Resistance status of the Colorado potato beetle, Leptinotarsa decemlineata, to endosulfan in East Azarbaijan and Ardabil provinces of Iran.

Three Colorado potato beetle, Leptinotarsa decemlineata, populations, from Ardabil, Bostanabaad and Ajabshir, were collected from potato fields in East Azarbaijan and Ardabil provinces in Iran and assayed for resistance to endosulfan. Possible resistance mechanisms were investigated using synergism studies and biochemical assays. Laboratory tests showed that the Bostanabaad strain was 220 and 109 times resistant compared with the susceptible strain in 2003 and 2004, respectively. The resistance ratios for the Ajabshir and Ardabil strains were 19 and 18, respectively. Since considerably more resistance was observed in the Bostanabaad strain compared with the other two, further investigation of the origin of resistance was done on this strain. Two insecticide synergists, piperonyl butoxide and S,S,S-tributylphosphorotrithioate, reduced resistance 2.3 and 3.5 times, respectively. These small degrees of synergism suggest that metabolism is not the source of the considerable difference in susceptibility between the two strains. This was supported by the results obtained from the biochemical assays that showed that glutathione S-transferase activity in the Bostanabaad strain did not significantly differ from the susceptible strain. These results suggest that target site insensitivity may be involved.

Laboratory evaluation of five chitin synthesis inhibitors against the colorado potato beetle, Leptinotarsa decemlineata.

Results of laboratory experiments are reported that tested the effects of five chitin synthesis inhibitors, diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron. on second instars of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Crysomelidae), originally collected from potato fields of Bostanabaad, a town 66 km southeast of Tabriz, Iran. In bioassays, the larvae were fed potato leaves dipped in aqueous solutions containing chitin synthesis inhibitors. The mortalities and abnormalities of the treated larvae were recorded 72 hours after treatments. LC(50) values were 58.6, 69.6, 27.3, 0.79 and 81.4 mg ai/ L for diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron, respectively. Compared with phosalone, which is one of the common insecticides used for controlling this pest in Iran, lufenuron and hexaflumuron seem to be much more potent, and if they perform equally well in the field, they would be suitable candidates to be considered as reduced risk insecticides in management programs for L. decemlineata due to much wider margin of safety for mammals and considerably fewer undesirable environmental side effects.

Immobilization and voltammetric detection of human interleukine-2 gene on the pencil graphite electrode.

The immobilization and differential pulse anodic voltammetry (DPAV) of a 20-mer oligonucleotide related to the human interleukine-2 (hIL-2) using renewable pencil graphite electrode (PGE) is described. The influences of electrochemical pretreatment of PGE on the ability of the electrode in hIL-2 adsorption, and conditions of hiIL-2 immobilization on PGE including immobilization potential and time, sodium chloride concentration as well as stirring of the solution were studied and optimum conditions were suggested. Accordingly, the electrochemical pretreatment of the polished PGE by electrostatic procedure at 1.80V for 5min in 0.50M acetate buffer solution of pH 4.8 is proposed as the optimum pre-treatment procedure. Similarly, the obtained optimum conditions for immobilization of hIL-2 on the activated PGE was an immobilization duration of 5min at applied potential of 0.50V. Trace levels of hIL-2 was readily detected following only 5min immobilization period with detection limit of 6nM.

Asthma and other allergic diseases in 13-14-year-old schoolchildren in Urmia, Iran. [corrected].

We determined the prevalence and risk factors of asthma, allergic rhinitis and atopic eczema in 3000 13-14-year-old schoolchildren in Urmia, Islamic Republic of Iran. We used the International Study of Asthma and Allergies in Childhood (ISAAC) written and video questionnaires. With the written questionnaire, the prevalence of current symptoms (within the past 12 months) was: wheeze 14.5%, allergic rhinitis 23.6% and eczema 10.1%. Self-reported asthma ever was only 2.1%. With the video questionnaire, the prevalence of wheeze was lower; 7.4% for wheeze at rest ever and 4.6% during the past 12 months. Boys had a significantly higher prevalence for most items examined except for eczema.

Asthma and other allergic diseases in 13-14-year-old schoolchildren in Urmia: an ISAAC study

We determined the prevalence and risk factors of asthma, allergic rhinitis and atopic eczema in 3000 13-14-year-old schoolchildren in Urmia, Islamic Republic of Iran. We used the International Study of Asthma and Allergies in Childhood [ISAAC] written and video questionnaires. With the written questionnaire, the prevalence of current symptoms [within the past 12 months] was: wheeze 14.5%, allergic rhinitis 23.6% and eczema 10.1%. Self-reported asthma ever was only 2.1%. With the video questionnaire, the prevalence of wheeze was lower; 7.4% for wheeze at rest ever and 4.6% during the past 12 months. Boys had a significantly higher prevalence for most items examined except for eczema

Developing an electrochemical deoxyribonucleic acid (DNA) biosensor on the basis of human interleukine-2 gene using an electroactive label.

Development of an electrochemical DNA biosensor based on a human interleukine-2 (IL-2) gene probe, using a pencil graphite electrode (PGE) as transducer and methylene blue (MB) as electroactive label is described. The sensor relies on the immobilization of a 20-mer single stranded oligonucleotide probe (hIL-2) related to the IL-2 gene on the electrode. The hybridization between the probe and its complementary sequence (chIL-2) as the target was studied by square wave voltammetry (SWV) of MB accumulated on the PGE. In this approach the extent of hybridization is evaluated on the basis of the difference between SWV signals of MB accumulated on the probe-PGE and MB accumulated on the probe-target-PGE. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. Some experimental variables affecting the performance of the biosensor including: polishing of PGE, its electrochemical activation conditions (i.e., activation potential and activation time) and probe immobilization conditions on the electrodes (i.e., immobilization potential and time) were investigated and the optimum values of 1.80 V and 300 s for PGE activation, and -0.5 V and 400s for the probe immobilization on the electrode were suggested.

Mechanism of extraction of beta-carotene from microalga Dunaliellea salina in two-phase bioreactors.

We show that it is possible to extract beta-carotene selectively from Dunaliella salina in two-phase bioreactors. The cells continue to produce beta-carotene and the extracted part is substituted by newly produced molecules. This process is called "milking." We performed several experiments to understand the exact mechanism of the extraction process. The results show that direct contact between the cells and the biocompatible organic solvent was not a requirement for the extraction but it accelerated the extraction. Electron microscopy photographs showed an undulated shape of the cell membrane and a space between the cell and the chloroplast membranes in the cells growing in the presence of dodecane (a biocompatible solvent). Extra-chloroplast beta-carotene globules located in the space between the cell and the chloroplast membranes were observed in these cells as well. It was shown that dodecane was taken up by the cells. The concentration of dodecane in the cells was about 13 pg.cll(-1). It can be concluded that dodecane uptake by the cells is responsible for the morphological changes in the cells and leads to more activity in the cell membrane. The results suggest two possible modes of extraction. One of the mechanisms is transport of the globules from the chloroplast to the space between the cell and the chloroplast membranes and subsequently from there to the outside by exocytosis. Another possible mode for the extraction could be release of beta-carotene from the globules as a result of alterations in the membrane in response to the uptake of dodecane. beta-Carotene molecules diffuse from the chloroplast to the space between the cell and the chloroplast membranes and from there to the medium either by diffusion or by exocytosis after accumulation in the vesicles.

Modulation of thyroidal radioiodine uptake by theophylline.

Diagnostic and therapeutic use of radioiodine in the management of thyroid disorders depends on the ability of thyroid cells to concentrate radioiodine, a process regulated by thyrotropin and dependent on the intracellular increase in cAMP. We tested the ability of theophylline, a drug known to increase intracellular cAMP via inhibition of phosphodiesterase, to modulate the thyroidal radioiodine uptake in FRTL-5 cells, in mice and in humans. In FRTL-5 cells, theophylline increased the uptake of radioactive iodine and intracellular cAMP only at low concentrations (1 microM). In mice, theophylline increased slightly the radioiodine uptake, although this increase varied from 1.5- to 6.6-fold. In humans, theophylline decreased slightly the radioiodine uptake, a decrease that became more pronounced with time after radioiodine administration. These studies suggest that theophylline modulates the radioiodine uptake in a dose-dependent fashion, although the modulation is mild and probably not applicable to the clinical setting.

Photocatalytic degradation of an organophosphorus pesticide phosalone in aqueous suspensions of titanium dioxide.

The present work deals with photocatalytic degradation of an organophosphorus pesticide, phosalone, in water in the presence of TiO2 particles under UV light illumination (1000 W). The influence of the basic photocatalytic parameters such as pH of the solution, amount of TiO2, irradiation time, stirring rate, and distance from UV source, on the photodegradation efficiency of phosalone was investigated. The degradation rate of phosalone was not high when the photolysis was carried out in the absence of TiO2 and it was negligible in the absence of UV light. The half-life (DT50) of a 20 ppm aqueous solution of phosalone was 15 min in optimized conditions. The plot of lnC (phosalone) vs. time was linear, suggesting first order reaction (K=0.0532 min(-1)). The half-life time of photomineralization in the concentration range of 7.5-20 ppm was 13.02 min. The efficiency of the method was also determined by measuring the reduction of Chemical Oxygen Demand (COD). During the mineralization under optimized conditions, COD decreased by more than 45% at irradiation time of 15 min. The photodegradation of phosalone was enhanced by addition of proper amount of hydrogen peroxide (150 ppm).