Avicenna Journal of Medical Biotechnology
Volume:7 Issue: 3, Jul-Sep 2015

The rapid progress of modern biotechnology has presented a number of new and unique ethical and social challenges within the context of human medical science. Research in medical biotechnology has led to increased knowledge of disease, acceleration of the treatment process, improved pharmacotherapy for infectious diseases and hope for the struggle against incurable diseases such as ALS, MS and Alzheimer’s 1. Medical biotechnology promises major advances in human health and therefore, any limitations on the right to freedom of scientific research should be for significant reasons only, and as least restrictive as possible, so as not to impede scientific wisdom and prevent damage to the scientific undertaking. At the same time a duty exists to ensure that research in this area of biotechnology is conducted in ethically acceptable ways. A balance needs to be struck between recognizing the potential benefits which biotechnology research offers to individuals and the community as a whole, and the duty to ensure that research in this area is conducted ethically 2.Researchers in the biotechnology industry face challenges unlike researchers in other sectors. Unlike most other industries, advances and research in the biotechnology industry are often front page news and has to face intense scrutiny by press, academics, government and the public. As biotechnology is a newly emerging field, a further challenge facing the industry is the lack of historical precedence in the sector to provide guidance for the safe and ethical development of the technology. In biotechnology research, the usual ethical principles applicable to health research involving animals and human participants must be observed and such research must be scientifically sound.

Mesenchymal Stem Cells (MSCs) are isolated from different sources like placenta. The placenta and its membranes like Amniotic Membrane (AM) are readily available and easy to work with. There is only limited knowledge on the immunomodulatory properties of human Amniotic Membrane-derived Mesenchymal Stem Cells (hAM-MSCs). The aim of this study was to survey the suppressive activity of hAM-MSCs on T lymphocytes in vitro. Human AMs were obtained after caesarean section births from healthy women. After enzymatic digestion, cells were cultured and hAM-MSCs were obtained. In addition, human T lymphocytes were isolated and co-cultured with hAM-MSCs for 72 hr in the presence or absence of phytohemagglutinin (PHA). Subsequently, proliferation of T cells was analyzed using BrdU and subsequently flow cytometry technique. Besides, the production of IL-4 and IFN-γ was examined by ELISA method. Additionally, the expression of activation markers (CD38, HLA-DR) was studied on T lymphocytes by flow cytometry technique. It was revealed that hAM-MSCs could significantly suppress the proliferation of T lymphocytes (p≤0.01) and significantly decrease the production of IFN-γ by T cells (p<0.05). hAM-MSCs also down regulated the expression of activation markers on the surface of T lymphocytes, CD38 and HLA-DR. The difference was significant between the case and control samples (p<0.05). All the comparisons were carried out between the case (Tcell+PHA+hAM-MSCs) and control (Tcell+PHA) groups. In conclusion, hAM-MSCs could inhibit the (mitogen-activated) T cells even in the absence of blood monocytes. Besides, hAM-MSCs-mediated inhibition of T lymphocytes was combined with down regulation of activation markers.

Microdeletions of the Y chromosome are one of the most frequent genetic causes of spermatogenic failure in infertile men. But their role in gaining weight is unclear. The present study investigated the possible association of these partial microdeletions and obesity. In a case-control study, 180 males were selected. The prevalence of microdeletions was assessed using PCR in AZFc area of Y chromosome and statistical analysis was done using the Fisher exact test and Pearson correlation. In our study, inverse relationship was observed between body mass index and testosterone level (p-value: 0.005). Fisher exact tests showed that there was a significant association between gr/gr mutation and BMI (p-value: 0.044). Our study revealed that Y chromosome microdeletions are more common in obese men. Furthermore, microdeletions such as gr/gr, which were observed in normal men, could cause decreased testosterone level. So, they may contribute to gaining weight.

Human insulin-like growth factor type 1 (hIGF-1) is a protein consisting of 70 amino acids (MW=7.6 kDa) and mainly synthesized by liver. Mecasermin (Trade name INCRELEX) is the synthetic form of the protein which is used as an effective treatment for particular disorders such as short stature, type 1 and 2 diabetes, and wound healing. Current study was aimed to investigate the expression of human insulin-like growth factor type1 in Escherichia coli (E. coli) BL21 (DE3) expression system in order to produce an active recombinant form of the protein. For the purpose of the study, firstly codon optimization was done for hIGF-1 gene, using bioinformatics databases. Then, the gene was synthesized and inserted in pET-24a vector by a cutting strategy included NdeI and BamHI-HF enzymes. In the next step, gene was run in agarose gel and purified. The constructed expression cassette was transformed into E. coli BL21 (DE3) cells through CaCl2 heat shock method. Identification and confirmation of the transformed colonies were performed using screening PCR method. Synthesis of hIGF-1 was induced by IPTG. The expression in induced strains was analyzed by SDS-PAGE and western blotting techniques. Confirmation of cloning and IGF-1 expression cassette was carried out through genetic engineering procedures. Analysis of transformed E. coli strain with SDS-PAGE and western blotting techniques confirmed that gene was expressed in host cells. Molecular weight of the expressed protein was estimated to be 7.6 kDa. hIGF-1 expression cassette for cloning and expression in E. coli was designed and the protein of interest was successfully induced and identified. In addition, E. coli BL21 (DE3) can be used as a suitable host for production of recombinant hIGF-1 and this technology has a potential to be localized.

Wharton’s Jelly-Mesenchymal Stem Cells (WJ-MSCs) are pluripotent cells with differentiation capability into most cell lineages. The aim of the current work was to examine the role of Retinoic Acid (RA) in differentiation process of these cells into hepatocyte-like cells and determine the morphological and functional patterns. Human WJ-MSCs were extracted, cultured and expanded; after approximately 95% of confluence, the cells were treated with hepatogenic media containing RA. The cells were subsequently analyzed for morphological changes, glycogen storage, albumin production, and specific gene expression. WJ-MSCs expressed high levels of CD90 (93.6%) and CD105 (90.7%), but low levels of CD34 (0.3%) and CD45 (0.8%). Albumin production had significant difference in the two groups (p≤0.05). The data showed specific characteristics in favor of considering the differentiated cells as hepatocyte-like cells such as obtaining morphologic, functional, and αFP and HNF1-α expression patterns which in turn were higher in cells exposed to RA. Based on the data of present study, RA is an effective molecule in inducing differentiation of WJ-MSCs into hepatocyte-like cells; therefore, it may be considered as a promising factor for targeting therapy of liver disorders.

Coenzyme Q10 (CoQ10) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ10-producing Escherichia coli (E. coli). Two CoQ10-producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or PBAD promoters. Over-expression of ispA under the control of PBAD promoter led to a relative increase in CoQ10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 µg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. The reduction in CoQ10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ10.

The human ADIPOQ gene encodes adiponectin protein hormone, which is involved in regulating glucose levels as well as fatty acid breakdown. It is exclusively produced by adipose tissue and abundantly present in the circulation, with concentration of around 0.01% of total serum proteins, with important effect on metabolism. Most deleterious nonsynonymous single nucleotide polymorphisms in the coding region of the ADIPOQ gene were investigated using SNP databases, and detected nonsynonymous variants were analyzed in silico from the standpoint of relevant protein function and stability by using SIFT, PolyPhen-2, PROVEAN and MUpro, I-Mutant2.0 tools, respectively. A total of 58 nonsynonymous SNPs consisting of 55 missense variations, 3 nonsense variations were found in the ADIPOQ gene. Next, 14 of the 55 missense variants were predicted to be damaging or deleterious by three different software programs (PolyPhen-2, SIFT, and PROVEAN), and 38 of them were predicted to be less stable (I-Mutant 2.0 and MUpro software). Totally, 10 variants out of 55 missense variants were predicted to be both deleterious and reduce protein stability. Additionally, 3 nonsense variants were predicted to produce a truncated ADIPOQ protein. RMSD and total energy were calculated for 4 nsSNPs out of 10 nsSNPs which were both deleterious and showed a decrease in protein stability. rs144526209 has high root-mean-square deviation (RMSD) and lower total energy value compared to the native modeled structure. It was concluded that this nsSNP, potentially functional and polymorphic in the ADIPOQ gene, might be associated with diabetes, obesity, and inflammation.

Interleukin-16 (IL-16) is an important regulator of T cell activation and was reported to act as a chemoattractant agent. There are evidences that IL16 can control the neuroinflammatory processes in Alzheimer’s Disease (AD). This study was performed to investigate the role or association of IL16 polymorphisms, rs11556218 and rs4778889 with the risk of late-onset Alzheimer’s disease (LOAD) in Iranian population. Totally, 148 AD patients and 137 nondemented and age-matched subjects were recruited in this study. Genotyping of rs11556218 T/G and rs4778889 T/C polymorphisms was performed by PCR-RFLP method using the NdeI and AhdI restriction enzymes, respectively. Statistical analysis of rs11556218 genotypes showed a protective effect against AD in the heterozygote genotype (p=0.001, OR=0.16) as well as rs4778889 (p=0.001, OR=0.23). Frequency of rs11556218 allele T was higher in controls than patients (p=0.001, OR=0.32). However, there was no significant difference in the frequencies of rs4778889 alleles between the AD patients and controls. Our results indicate that the rs11556218 and rs4778889 polymorphisms have a protective role in the development of sporadic AD in Iranian population.