Iranian Journal of Biotechnology
Volume:14 Issue: 1, Winter 2016

Expansion and differentiation of stem cells relies on the soluble materials as well as the physical conditions of their microenvironment. Several methods have been studied in attempt to enhance the growth and differentiation rates of different adult stem cells extracted from different sources.
The purpose was to improve the three-dimensional (3D) culture condition of the semi-permeable polymeric beads for encapsulation of the human adipose-derived mesenchymal stem cells (hADSCs) by modifying the ratio of the alginate-gelatin composition.
Following isolation and characterization of hADSCs by flow cytometry and their functional differentiation, encapsulation in the alginate and alginate/gelatin compositions were performed. Moreover, the stability, swelling, size frequency, growth kinetics, and cytotoxicity of the beads were measured to meet proper condition in the designed experimental and control culture conditions. Finally, the growth rates of the cells in different experimental groups and control were measured and analyzed statistically.
Viability decreased in 2 and 3 percent alginate once compared to 1% alginate in beads (p£0.05). Moreover swelling of the beads in the alginate/gelatin compositions (50:50 and 70:30) were higher than the pure alginate beads (p£ 0.05). Finally, the cell growth rate in alginate/gelatin (50:50) beads was significantly higher than alginate and alginate/gelatin (70:30) beads (p£0.05).
These findings suggested for the first time that the composite of alginate/gelatin beads with the ratio of 50:50 might provide a suitable culture condition for the encapsulation and in vitro expansion of the hADSCs.

b-Thalassemia is a common autosomal recessive disorder in human caused by a defect in b-globin chain synthesis. The most common mutations causing b-Thalassemia have been found to be splicing mutations. Most of which activate aberrant cryptic splicing/sites without complete disruption of normal splicing. IVSI-110 mutation, a common splicing mutation, leads to a 90% reduction of normal b-globin synthesis and lead to blood transfusion dependency in the homozygote forms. However, modulation of splicing can be achieved by activation or suppression of transacting factors such as SR (Serine, Arginine) amino acids and hnRNPs (Heterogeneous ribonucleoprotein particle) through drugs.
The aim of this study was to investigate the effects of NaBu, isoBu and VPA drugs on restoration of splicing of IVSI-110 b-Thalassemia pre-mRNA in human.
Primary erythroid cells derived from IVSI-110 b-Thalassemia patients were cultured ex vivo and differentiated in the presence of 0.5 and 1 mM of Na-Butyrate (NaBu), 0.5 mM Isobutyramide (isoBu) and 100 mM Valproic acid (VPA). RT- PCR analysis was used to evaluate the effect of the drugs in correction of normal splicing in b-globin mRNAs.
Following treatment with NaBu, isoBu and VPA, the level of normal b-globin mRNA in Primary erythroid cells derived from IVSI-110 b-Thalassemia patients, increased 1.7, 1.5, 1.4 fold, respectively relative to normal b-globin mRNAs. Higher splicing restoration was achieved by NaBu, a histone deacetylase inhibitor, known to upregulate the expression of splicing factors.
The results highlighted the therapeutic potential of splicing modulation for genetic diseases caused by splicing mutations.

Pivotal roles of Nerve growth factor (NGF) in the development and survival of both neuronal and non-neuronal cells indicate its potential for the treatment of neurodegenerative diseases. However, investigation of NGF deficits in different diseases requires the availability of properly folded human b-NGF. In previous studies bacterial expression of hNGF demonstrated the feasibility of its overproduction. However, known limitations in the use of E. coli as an expression host for a protein with three intra-chain disulfide bonds were evident.
Here an optimized system was developed to overexpress the soluble NGF in E. coli.
The gene encoding the b subunit of mature hNGF was optimized based on E. coli codon preference and cloned into pET-32a expression vector providing His- and Trx- tags for detection and increasing the solubility of recombinant protein, respectively. The recombinant DNA was expressed in E. coli Origami (DE3), which enhances the correct formation of disulfide bonds in the cytoplasm of E. coli. Different culture conditions were evaluated to increase soluble expression of the target protein.
The highest soluble expression level was achieved when E. coli Origami (DE3) cells expressing NGF were grown at 30ºC in TB medium with 0.2 mM IPTG induction at OD600nm = 1 for 4 h.
Our results indicated that the recombinant NGF was successfully expressed as a soluble form.

It was previously shown that the activity of a serine protease from a moderately halotolerant Bacillus aquimaris VITP4 strain is active in a wide range of pH and temperatures and could be modulated by the presence of the divalent metal ions.
In the present study, a quantitative analysis was done in order to explore the parameters that are contributing to the protease activity.
Changes in the secondary structure of the enzyme was determined by circular dichroism analysis. The conditions for the optimal activity was investigated by Response Surface Methodology. Stability of the enzyme was determined by thermal inactivation experiments.
The initial one-factor-at-a-time experiments have indicated that the activity of the enzyme could be enhanced not only by the presence of low concentrations of NaCl but also by divalent metal ions, such as Ca2, Mn2 and Cu2. A clear dependence of the activity to the secondary structure of the enzyme could be established using circular dichroism spectroscopy. In the next level of optimization, four factors; viz. pH, temperature, concentration of Ca2, and Mn2 were used to optimize the conditions required for the maximal activity of the enzyme by Response Surface Methodology, and the data could be explained using quadratic model. Under optimal condition of 43°C, pH 8.0, 8.2 mM Ca2, and 4.3 mM Mn2 a 1.5 times enhancement in the enzyme activity could be achieved. The storage stability of the enzyme under these selected conditions has indicated a non-linear relation between the conditions for the enzymatic activity as well as stability. However, the condition for the maximal stability (267±18 min) has corresponded to that of the optimal conditions for the maximal activity.
This study, for the first time, has explored the possibility of using statistical methods for identifying the optimal conditions for alkaline protease activity isolated from the halotolerant Bacillus aquimaris VITP4.

Enzyme engineering by immobilization techniques has proven to be well compatible with the other chemical or biological approaches aiming to improve enzyme’s functions and stability. Zeolites are porous alumino-silicates with a wide range of porosity and particle size along with the other remarkable properties such as high surface area, high stability against a wide range temperatures, pHs, as well as organic solvents.
Nano-zeolites are a class of advanced materials that have special properties that has made them ideal candidate for a wide range of applications.
In this study, a nano-zeolite which has been synthesized and characterized in our previous work, was used to immobilize a-amylase and activated with glutaraldehyde as a bi-functional agent to improve enzyme properties.
Studies have shown an increased stability of the immobilized enzyme compared to the free enzyme against a range of temperature change and pHs as well. Also the stability of the immobilized enzyme was increased with respect to storage. The calculated binding efficiency shows that the immobilized a-amylase conserved 58.44 % of its native activity.
Using nano pore zeolite for covalent attachment of the a-amylase resulted in an increased resistance of this enzyme against denaturation. The immobilized enzyme demonstrated higher stability compared to the free enzyme at higher temperatures and pH variations. Immobilization also caused an increase in the enzyme stability during storage.

Bacterial resistance to the commonly used antibacterial agents is an increasing challenge in the medicine, and a major problem for the health care systems; the control of their spread is a constant challenge for the hospitals.
In this study, we have investigated the antimicrobial activity of the Zinc Oxide nanoparticles against clinical sample; Yersinia intermedia bacteria.
Nanoparticle susceptibility constants and death kinetic were used to evaluate the antimicrobial characteristics of the Zinc Oxide (ZnO) against the bacteria. Antimicrobial tests were performed with 108 cfu.mL-1 at baseline. At first, Minimum Inhibitory Concentration (MIC) of ZnO was determined and then nanoparticle suspension at one and two times of the MIC was used for death kinetic and susceptibility constant assay at 0 to 360 min treatment time.
ZnO nanoparticles with size ranging from 10 to 30 nm showed the highest susceptibility reaction against Y. intermedia (Z=39.06 mL.mg-1). The process of Y. intermedia death in ZnO suspension was assumed to follow the first-order kinetics and the survival ratio of bacteria decreased with the increasing treatment time. An increased concentration of the nanoparticle was seen to enhance the bactericidal action of the nanoparticle. Then we performed the best ratio of the nanoparticles on semi-sensitive and resistance antibiotic for the bacteria. However, based on experimental results, synergy of ZnO nanoparticles and Oxacilin was determined and Y. intermedia showed a higher sensitivity compared to the ZnO nanoparticles alone.
The results of the present study illustrates that ZnO has a strong antimicrobial effect and could potentially be employed to aid the bacterial control. It could also improve- antibacterial effects in combination with the antibiotics.

Recently, applications of albumin nanoparticles as drug delivery carriers have increased. Most toxicology studies have shown that surface chemistry and size of nanoparticles play an important role in biocompatibility and toxicity.
The effect of desolvating agents with different chemical properties on the size of synthesized HSA NPs was investigated.
Acetone, ethanol, methanol, and acetonitrile were used to synthesize HSA NPs with controllable size by desolvation method. Scanning electron microscopy (SEM), dynamic light scattering (DLS), and circular dichroism (CD) were employed to characterize produced particles. Finally, the toxicity of HSA NPs synthesized under different conditions was evaluated on PC-12 cells.
The sizes of synthesized particles differed according to the different solvents used. The sizes were 275.3 nm, 155.3 nm, 100.11 nm, and 66.2 nm for acetonitrile, ethanol, acetone, and methanol, respectively. CD showed that larger NPs had more changes in the secondary structures. Finally, the toxicity monitored on the cultured PC-12 cells showed no significant toxic effect through treating with these NPs at different concentrations (0-500 mg.mL-1).
The size of HSA NPs has a strong dependency on the desolvating agent. The mechanism in which the desolvating agent affects the size of HSA NPs is complex. Various factors such as dielectric constant, polarity, functional groups, and hydrogen bonding of the solvents have the potential to affect the size and structure of HSA NPs. CD analysis suggested that the solvent denaturing capability had a critical effect on the HSA particle size. The stronger denaturing capability of the solvent resulted in the larger HSA particle size.

Rosa centifoliais commercially propagated by asexual means but in vitro propagation ensure the production of disease free and healthy plants and browning of explants creates hurdle in their multiplication.
The aim was to reduce oxidative browning of shoots of R. centifolia in MS medium during in vitro propagation.
Axillary buds of R. centifolia were sterilized with 70% ethyl alcohol for 4 min and 5% sodium hypochlorite for 2 min followed by three washing with sterilized double distilled water. In order to control oxidative browning, Ascorbic acid (100 mg.L-1), citric acid (100 mg.L-1) and activated charcoal (3 g.L-1) were used while to control withering of shoots, different concentrations (3.0 mg.L-1, 6.0 mg.L-1, 9.0 mg.L-1) of either glutamine, asparagine and proline were put into trial. Different concentrations of Benzyl aminopurine (BAP) and naphthalene acetic acid (NAA) were used for in vitro shoot and root formation.
Minimum browning percentage (20%) was achieved in the presence of activated charcoal (3.0 g.L-1) and pretreatment of explants with running tap water. Asparagin (9.0 mg.L-1) produced maximum shooting (93%), minimum withering (6.67%), and it took longer period (27 days) for shoots to wither. BAP (3.0 mg.L-1) NAA (0.5 mg.L-1) was produced the highest number of shoots (1.63), in a shortest periods (9 days). For root production, NAA (1.5 mg.L-1) BAP (0.5 mg.L-1) reduced the time to 11 days with maximum number of roots (4.33) and root length (4.20 cm).
The supplement of activated charcoal (3.0 g.L-1), a sparagin (9.0 mg.L-1) and combination of BAP and NAA in the MS medium is effective for in vitro propagation of R. centifolia.

Worldwidely cultivated, melon is commercially an important fruit crop, as it is in Iran.
Establishment of an efficient in vitro plant regeneration system plays a pivotal role in the plant transformation, hence, the importance of regeneration protocol for Iranian melon (Cucumis melo L. var. Gorgab) has encouraged us to work on in vitro melon regeneration.
Material and The effect of selective media, including various concentrations of the 6-benzyladenine (BA), cefotaxime, as well as indole-3-acetic acid (IAA) on regeneration of the cotyledonary petioles derived from a 6-day-old in vitro grown seedlings were assessed.
The highest frequency of regeneration rate was recorded at 1.5 mg.L-1 of the BA plus 250 mg.L-1 cefotaxime in addition to 1 mg.L-1 BA plus 1000 mg.L-1 cefotaxime. The highest percentage of the shoot formation (100%) was recorded at 1 mg.L-1 BA plus 1000 mg.L-1 cefotaxime, while, it was relatively lower (75%) on than the medium containing 1.5 mg.L-1 BA in combination with 250 mg.L-1 cefotaxime. The highest root induction was observed in the medium containing 500 mg.L-1 cefotaxime 0.1 mg.L-1 IAA. A significant positive influence on roots and leaves formation, as well as their number, in addition to regeneration of shoots was observed as well.
This is the first work reporting an appropriate regeneration procedure for the melon, an Iranian native crop.