Apoptosis repressor with caspase recruitment domain (ARC) inhibits myogenic differentiation

Apoptosis repressor with caspase recruitment domain (ARC), an anti-apoptotic protein, is highly expressed in differentiated heart and skeletal muscle. Apoptosis and differentiation share numerous common pathways; therefore, we examined the impact of ARC on H9c2-myoblast differentiation. We demonstrate that ARC expression levels increase and stabilize upon differentiation. ARC-overexpression in pre-differentiated H9c2-cells suppresses differentiation; indicated by increased myotube formation, nuclear fusion and expression of the differentiation markers myogenin and troponin-T. ARC-overexpression inhibited myoblast differentiation associated caspase-3 activation, suggesting ARC inhibits myogenic differentiation through caspase inhibition. In summary, we show a novel role for ARC in the regulation of muscle differentiation.     

Developing an Extended Genomic Engineering Approach Based on Recombineering to Knock-in Heterologous Genes to <Emphasis Type="Italic">Escherichia coli</Emphasis> Genome

Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase–flippase recognition targets (FLP–FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P _trc and P _araB ), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers [isopropyl β-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.     

Molecular mechanisms,prevalence, and molecular methods for familial combined hyperlipidemia disease: A review

Familial combined hyperlipidemia (FCHL) is the most common genetic dyslipidemia disorder which is accompanied by increasing of triglyceride and cholesterol. This disorder is a complex genetic disease although it also has monogenic forms. The familial form has several criteria for diagnosis that can be distinguished of nonfamilial position. It has been shown that a variety of internal and external risk factors are involved in the pathogenesis of FCHL. Environmental factors and the genetic background also play an important role in the FCHL pathogenesis. Many mechanisms and pathways are involved in lipid metabolism (ie, dysfunctional adipose tissue, hepatic fat and very low-density lipoprotein overproduction, triglyceride-rich lipoproteins, and clearance of low-density lipoprotein particles) that could lead to FCHL. Individuals with a positive family history like those who have a positive family history of cardiovascular diseases are more predispositions for this disorder. To date several methods have been used to identify the genetic background of the FCHL. In the current review, we summarized the prevalence and the molecular mechanisms involved in the FCHL disease. Moreover, we highlighted the used molecular methods for determining the genes involved in the FCHL.     

Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli

The human epidermal growth factor receptor (HER) family plays an important role in cell growth and signaling and alteration of its function has been demonstrated in many different kinds of cancer. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand–HER receptor binding and receptor dimerization. A single chain variable fragment (ScFv) is a valuable alternative to an intact antibody. This report describes the production and purification of an ScFv specific for domain II of the HER2 receptor in Escherichia coli BL21 (DE3) cytoplasm. The majority of expressed of anti-her2his-ScFv protein was produced as inclusion bodies. A Ni-NTA affinity column was used to purify the anti-her2his-ScFv protein. The molecular weight of anti-her2his-ScFv protein was estimated to be approximately 27 kDa, as confirmed by SDS-PAGE and Western blotting assay. The anti-her2his-ScFv showed near 95 % purity and reached a yield of approximately 29 mg/l in flask fermentation. The purified anti-her2his-ScFv showed its biological activity by binding to HER2 receptor on the surface of BT-474 cells. This ScFv may be a potential pharmaceutical candidate for targeting tumour cells overexpressing HER2 receptor.     

Development of Dof (DNA binding with one finger) transcription factor gene-specific primers through data mining as a functional marker and their use for genetic diversity study in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) germplasm

The genetic diversity among Hordeum vulgare L. species were assessed based on PCR amplification pattern derived from 75 set of Dof domain and Dof genes specific primers. Multiple bands showing variability in terms of both number and sizes of bands ranging from 0.1 to 3.0 kbp were observed. Out of a total of 2449 bands, 2328 polymorphic and 121 monomorphic bands were obtained and the percentage of polymorphism ranged from 70.27 to 100%. A very high degree of polymorphism was observed with all the primers except HvDof3, HvDof4, HvDof10, HvDof16, HvDof18, HvDof18, HvDof24, Dof4, Dof11, Dof13, Dof15, Dof16, Dof19, Dof20, Dof21, Dof22, Dof23, Dof28, dof38, sbDof23 and sbDof24 primers. Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on Jaccard’s similarity coefficient matrix. According to results, the genetic resources and diversity in barley germplasm of H. vulgare were rich. The number of polymorphic fragments per primer detected ranged from 11 to 56 bands with an average of 32.65 bands. Average polymorphic information content (PIC) was 0.81 in overall Dof domain and gene specific primers. HvDof 39 showed the highest PIC (0.99) which can be a good candidate primer to verify genetic diversity in H. vulgare. The unweighted pair-group method of the arithmetic average and principal coordinate analysis showed a clear distinction among the genotypes and the genotypes divided into three clusters in the dendrogram results. A model-based structure analysis revealed the presence of three groups. The study showed that genetic variation and population structure are determined among the species of H. vulgare collected from different geographical origins.     

Dynamic flexibility of double-stranded RNA activated PKR in solution

PKR, an interferon-induced double-stranded RNA activated serine-threonine kinase, is a component of signal transduction pathways mediating cell growth control and responses to stress and viral infection. Analysis of separate PKR functional domains by NMR and X-ray crystallography has revealed details of PKR RNA binding domains and kinase domain, respectively. Here, we report the structural characteristics, calculated from biochemical and neutron scattering data, of a native PKR fraction with a high level of autophosphorylation and constitutive kinase activity. The experiments reveal association of the protein monomer into dimers and tetramers, in the absence of double-stranded RNA or other activators. Low-resolution structures of the association states were obtained from the large angle neutron scattering data and reveal the relative orientation of all protein domains in the activated kinase dimer. Low-resolution structures were also obtained for a PKR tetramer-monoclonal antibody complex. Taken together, this information leads to a new model for the structure of the functioning unit of the enzyme, highlights the flexibility of PKR and sheds light on the mechanism of PKR activation. The results of this study emphasize the usefulness of low-resolution structural studies in solution on large flexible multiple domain proteins.     

High sensitivity enzyme-linked immunosorbent assay (ELISA) method for measuring protein carbonyl in samples with low amounts of protein

The oxidative modification of proteins has been shown to play a major role in a number of pathological processes. One such modification is the addition of the carbonyl groups to the amino acid residue in proteins. For the measurement of the carbonyl groups in low concentration protein samples, we have modified the ELISA (enzyme-linked immunosorbent assay) method that was developed by Buss et al. [Buss, I. H; Chan, T. P.; Sluis, K. B.; Domigan, N. M.; Winterbourn, C. C. Protein carbonyl measurement by a sensitive ELISA method. Free Radic. Biol. Med.23:361-366; 1997 ]. In the modified method, protein samples diluted in phosphate-buffered saline were adsorbed to wells of an ELISA plate and then reacted with dinitrophenylhydrazine (DNPH). The protein-conjugated DNPH was probed by a commercial anti-DNPH antibody, and then a second antibody conjugated with horseradish peroxidase was added for quantification. The method was calibrated using oxidized albumin, and required only 5 mug protein. This obviated the need to concentrate protein in experimental and clinical samples with low amounts of protein. In addition the effect of TCA on carbonyl measurement is eliminated. The standard curve was linear in the range of 0-3.36 nmol carbonyls/mg protein, which is the range within which clinical samples fell. The results correlated well with the colorimetric carbonyl assay. The method was used to analyze the amount of protein carbonyl in aqueous humor and diluted plasma samples.     

Role of CT scan in theranostic and management of traumatic spinal cord injury

Traumatic spinal cord injury (TSCI) is a condition with suffering of neural structures from acute trauma with short-term or permanent sensory and motor problems. This study was conducted with the aim of determining the prevalence of TSCI in Tehran with emphasis on demographic characteristics of patients and to evaluate the effect of computed tomography (CT) in determining fracture type and severity grade of injury among TSCI patients. In a cross-sectional study, all TSCI and spinal fracture patients (N = 520) who referred to the main trauma center in Tehran, Iran, in 2013 and 2014 were selected. Radiography and CT scan were prepared and reported blindedly by two radiologists. Majority of the patients was 21–30 years male, married and their most common occupation was car driver. A significant difference was observed between gender and etiology (P = 0.001). The main etiology was traffic accident followed by falling from height. While the most common location of injury for males was thoracic vertebrae followed by lumbar vertebrae; for females it was lumbar followed by thoracic. Majority of patients had ASIA (American Spinal Injury Association) impairment scale of E (normal), followed by B (sensory incomplete). Most of the cases were hospitalized less than one week. Age of the patient and duration of hospitalization had a significant association (P = 0.015). The results showed that in traumatic spinal cord events, traffic accident and falling from height are the main etiologies; hence, authorities in Iranian health system could consider preventive policies to decline the load and TSCI effects in hospitals and population.     

Upregulation of microRNA-146a was not accompanied by downregulation of pro-inflammatory markers in diabetic kidney

The present study was designed to evaluate whether microRNA-146a and its adapter proteins (TRAF6 and IRAK1) are involved in the pathogenesis of diabetes-induced kidney damage. Male Sprague–Dawley rats were divided into control and diabetic groups (n = 6 in each). Diabetes was induced by injection of streptozotocin (55 mg/kg; i.p.) in 12 h fasted rats. Diabetic kidney damage was diagnosed by renal hypertrophy, thickened glomerular basement membrane, widened filtration slits, mesangial expansion, as well as by elevated levels of blood urea and creatinine in diabetic rats 2 months after induction of diabetes. While the expression of NF-κB mRNA and miR-146a were increased in diabetic kidney compared to the sham controls (p < 0.01 for both comparisons), the mRNA levels of IRAK1 and TRAF6 did not statistically reduce. The NF-κB activity and the concentrations of TNF-α, IL-6 and IL-1β in the kidney of diabetic rats were higher than the kidney of controls (p < 0.05 for TNF-α and NF-κB; p < 0.01 for IL-6 and IL-1β). Our results indicate that the upregulation of miR-146a was not accompanied by downregulation of inflammatory mediators in diabetic kidney. It is possible that a defect in the miR-146a-mediated negative loop provides a situation for sustained activation of NF-κB and its targets to promote cells toward abnormalities.