Production of Recombinant Streptavidin and Optimization of Refolding Conditions for Recovery of Biological Activity.

BACKGROUND: Streptavidin is a protein produced by Streptomyces avidinii with strong biotin-binding ability. The non-covalent, yet strong bond between these two molecules has made it a preferable option in biological detection systems. Due to its extensive use, considerable attention is focused on streptavidin production by recombinant methods. METHODS: In this study, streptavidin was expressed in Escherichia coli (E. coli) BL21 (DE3) pLysS cells and purified by affinity chromatography. Various dialysis methods were employed to enable the protein to refold to its natural form and create a strong bond with biotin. RESULTS: Streptavidin was efficiently expressed in E. coli. Streptavidin attained its natural form during the dialysis phase and the refolded protein bound biotin. The addition of proline or arginine to the dialysis buffer resulted in a refolded streptavidin with greater affinity for biotin than refolding in dialysis buffer with no added amino acids. CONCLUSION: Dialysis of recombinant streptavidin in the presence of arginine or proline resulted in proper refolding of the protein. The recombinant dialyzed streptavidin bound biotin with affinity as great as that of a commercial streptavidin.

Association between chemerin rs17173608 and rs4721 gene polymorphisms and gestational diabetes mellitus in Iranian pregnant women.

Gestational diabetes mellitus (GDM) is defined as hyperglycemia detected during pregnancy and its risk is increased with obesity. Chemerin, an adipokine, has been proposed as potential mediators of insulin resistance in GDM. This case-control study was designed to assess the relation between chemerin SNPs rs4721 (or rs10278590) and rs17173608 and the development of GDM. One hundred thirty GDM pregnant women with GDM and 160 healthy pregnant women were enrolled in this study. The diagnosis of GDM was based on the International Association of Diabetes and Pregnancy Study Group (IADPSG) criteria. Chemerin rs4721 polymorphism gene was amplified through PCR, and SNP was detected using restriction enzyme AluI. Genotyping for chemerin rs17173608 polymorphism was performed by using tetra-amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR). Blood glucose level was measured by an enzymatic method. Our finding showed that the genotypes frequency of chemerin rs4721 polymorphism was significantly different between GDM and non-GDM groups (χ2 = 7.44, P = 0.02). The genotype of rs4721 was significantly associated with GDM in co-dominant and dominant genotypes (GG vs GT, OR = 2.3, 95%CI = 1.24-4.24, P = 0.008, and GG vs GT + TT, OR = 2.21, 95%CI = 1.23-3.99, P = 0.008, respectively). No significant difference was observed in allele frequency between case and control groups (P = 0.62). Moreover, the genotypes and allele frequencies of chemerin rs17173608 polymorphism did not show significant differences between GDM and non-GDM (P > 0.05). We concluded that the genotype of rs4721 was found to contribute significant risk to GDM while genotype of rs17173608 could not predict the risk of GDM.

Detection of KPC Carbapenemase in Pseudomonas aeruginosa Isolated From Clinical Samples Using Modified Hodge Test and Boronic Acid Phenotypic Methods and Their Comparison With the Polymerase Chain Reaction.

BACKGROUND: Bacterial resistance to antibiotics has become a major source of concern for public health. Pseudomonas aeruginosa strains are important opportunistic pathogens. These bacteria have a high resistance to a wide range of existing antimicrobials and antibiotics. OBJECTIVES: The present study was performed to evaluate the frequency of KPC in P. aeruginosa isolated from clinical samples of educational hospitals of Arak University of Medical Sciences, using the mentioned phenotypic and genotypic methods. MATERIALS AND METHODS: One hundred and eight non-duplicate clinical isolates of P. aeruginosa were collected from hospitals of Arak University of Medical Sciences, Arak, Iran. Antibacterial susceptibility was determined by the disk diffusion method. KPC production was confirmed by the Modified Hodge Test (MHT), which is a phenotypic test, and combined-disk test with boronic acid and the Polymerase Chain Reaction (PCR). RESULTS: In the present study, 13 isolates (12%) of P. aeruginosa were positive for KPC, using PCR. Comparison of the two phenotypic methods used in this study showed that boronic acid is more sensitive than MHT in identification of KPC-producing strains (84.6% vs. 77%). CONCLUSIONS: Utilization of reliable methods for identifying carbapenemase-producing strains and determining their antibiotic resistance pattern could have a very important role in treatment of infections caused by these strains. A substantial amount of P. aeruginosa isolated from clinical samples of hospitals in Arak (Iran) produce KPC carbapenemase. Due to their low specificity, MHT and boronic acid phenotypic methods could not completely identify KPC-producing P. aeruginosa. However, the sensitivity of boronic acid phenotypic method in detection of KPC was higher than MHT.

Association between vitamin D receptor ApaI and TaqI gene polymorphisms and gestational diabetes mellitus in an Iranian pregnant women population.

It has been proposed that variants of the nuclear vitamin D receptor (VDR) gene are associated with a susceptibility to type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus (GDM). Our study was aimed to evaluate a possible association between the VDR ApaI (rs7975232) and TaqI (rs731236) gene polymorphisms and susceptibility to GDM in an Iranian pregnant women population. This case-control study was performed on a population of pregnant Iranian women, including 157 GDM and 157 non-GDM subjects.VDR ApaI and TaqI polymorphisms were assessed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).Our finding showed that the genotypes frequency of VDR ApaI polymorphism was significantly different between GDM and non-GDM groups (χ(2)=8.5, P=0.014). The CC genotype increased the risk of GDM as compared to the AA genotype (AA vs.CC, OR=2.996, 95% CI=1.278-7.022, P=0.012). The genotype and allele frequencies of VDR TaqI polymorphism were significantly different between GDM and non-GDM subjects (χ(2)=7.27, P=0.026, χ(2)=4.08, P=0.043 respectively). A significant protection was shown against GDM in VDR TaqI genotypes and allele (TT vs.TC, OR=0.523, 95% CI=0.23-0.84, P=0.007, TT vs. TC+CC, OR=0.546, 95% CI=0.35-0.86, P=0.009, T vs. C, OR=0.711, 95% CI=0.511-0.99, P=0.043). In conclusion, our findings show a significant association between VDR ApaI and TaqI gene polymorphisms and the GDM at the investigated loci.

Evaluation of In Vivo Transfection Efficiency of Eudragit Coated Nanoparticles of Chitosan-DNA: A pH-sensitive System Prepared for Oral DNA Delivery.

BACKGROUND: Success of any gene therapy protocol relies mostly on using an efficient carrier to direct nucleic acid to the place of action. The system should also have transfection ability at release site. Different routes are available for delivering genetic materials to the target organs, amongst them; oral delivery is particularly attractive for certain reasons. However, serious obstacles, like acidic environment of stomach and presence of protease and nuclease enzymes in gastrointestinal (GI) tract, make oral route a highly challenging option. OBJECTIVES: The present study suggests preparation of gene nanoparticles (NPs) of chitosan within a layer of Eudragit L100 for oral delivery of nucleic acid. The nanoparticles have some features both in size and polymer properties that can be penetrating enough to transfect epithelial layer cells of intestine and protect the entrapped materials against stomach harsh condition. MATERIALS AND METHODS: In this experimental study, conducted in Iran, particles were prepared by coacervation technique followed by encapsulation of nanoparticle within a coat of Eudragit L100 using solvent evaporation technique. Formulation behavior was monitored both in vitro and in vivo. Stability of particle construction and release profile of DNA were examined at pH of ± 0.8 environ pKa of Eudragit. Size and zeta potential of particles were measured. To demonstrate transfection efficiency of the constructed carrier, reverse transcription polymerase chain reaction (RT-PCR) was carried out using human insulin specific primers on total RNA extracted from upper part of small intestine of 48-hour post-transfected rats (sampled by simple random selection, n = 3). RESULTS: The mean size and zeta potential of particles were 300 ± 4 nm and 14 ± 0.5 mV, respectively. Encapsulation of this system was 89.6 ± 1.2%. DNA release from batches was less than 12% at pH = 5.2 and more than 60% at pH = 6.8 with significant difference of P < 0.05. RT-PCR product confirmed the presence of insulin transcript of 437 bp in upper intestinal extracts of the transfected rats. No band of DNA was seen after RT-PCR of placebo form of nanoparticles received group. CONCLUSIONS: Eudragit coated nanoparticle of chitosan is an efficient choice for oral delivery of DNA to upper part of GI tract.

A mutation in HOXA2 is responsible for autosomal-recessive microtia in an Iranian family.

Microtia, a congenital deformity manifesting as an abnormally shaped or absent external ear, occurs in one out of 8,000-10,000 births. We ascertained a consanguineous Iranian family segregating with autosomal-recessive bilateral microtia, mixed symmetrical severe to profound hearing impairment, and partial cleft palate. Genome-wide linkage analysis localized the responsible gene to chromosome 7p14.3-p15.3 with a maximum multi-point LOD score of 4.17. In this region, homeobox genes from the HOXA cluster were the most interesting candidates. Subsequent DNA sequence analysis of the HOXA1 and HOXA2 homeobox genes from the candidate region identified an interesting HOXA2 homeodomain variant: a change in a highly conserved amino acid (p.Q186K). The variant was not found in 231 Iranian and 109 Belgian control samples. The critical contribution of HoxA2 for auditory-system development has already been shown in mouse models. We built a homology model to predict the effect of this mutation on the structure and DNA-binding activity of the homeodomain by using the program Modeler 8v2. In the model of the mutant homeodomain, the position of the mutant lysine side chain is consistently farther away from a nearby phosphate group; this altered position results in the loss of a hydrogen bond and affects the DNA-binding activity.

A novel TECTA mutation confirms the recognizable phenotype among autosomal recessive hearing impairment families.

Mutations in the TECTA gene result in sensorineural non-syndromic hearing impairment. TECTA-related deafness can be inherited autosomal dominantly (designated as DFNA8/12) or autosomal recessively (as DFNB21). The alpha-tectorin protein, which is encoded by the TECTA gene, is one of the major components of the tectorial membrane in the inner ear. Six mutations in the TECTA gene have already been reported in families segregating autosomal recessive non-syndromic hearing impairment. In this study, seventy-five Iranian families segregating autosomal recessive non-syndromic hearing impairment were analyzed for homozygosity at the DFNB21 locus by genotyping two short tandem repeat markers closely linked to the TECTA gene. Allelic segregation consistent with possible linkage to the DFNB21 locus was found in 1/75 families studied. By sequencing all 23 coding exons of TECTA, a 16bp deletion (c.6203-6218del16) in exon 21, leading to a frameshift, segregating with the hearing loss was found. All 3 affected individuals of this family have moderate-to-severe hearing loss across all frequencies, which is more pronounced in the mid frequencies. This new mutation, as well as the six previously reported mutations in the TECTA gene, is inactivating. All of these mutations lead to an easily recognized audiometric profile of moderate to severe hearing impairment as presented by the family in this study too. The TECTA autosomal recessive non-syndromic deafness phenotype differs from the typical profound deafness phenotype that is seen in most families segregating autosomal recessive non-syndromic deafness. On the basis of the recognizable phenotype, we recommend mutation screening of TECTA in families with this hearing phenotype.