Differentially expressed miR-152, a potential biomarker for in-stent restenosis (ISR) in peripheral blood mononuclear cells (PBMCs) of coronary artery disease (CAD) patients.

BACKGROUND AND AIMS: In-stent restenosis (ISR) remains the most daunting challenge of current treatments of coronary artery disease (CAD). MicroRNAs (miRNAs) are prominent regulators of key pathological processes leading to restenosis and used as diagnostic tools in different studies. miR-152 and miR-148a are implicated to contribute in the putative intracellular mechanisms of ISR. The aim of present study is to investigate the potential early-stage diagnostic role of miR-152 and miR-148a expression levels for ISR in peripheral blood mononuclear cells (PBMCs) of patients who underwent stent implantation. METHODS AND RESULTS: The miRNAs that are supposed to be involved in the ISR were nominated by bioinformatics approach mainly using miRWalk3. Then by quantitative real-time PCR, we determined the relative expression of miR-152 and miR-148a of PBMCs from ISR patients with their age/sex-matched controls. RESULTS: The presence of ISR significantly coincided with a decrease in the relative expression of miR-152. The area under the curve (AUC) for miR-152 receiver operating characteristic (ROC) curve was 0.717 (95% CI; 0.60-0.83) with a sensitivity of 70% and a specificity of 67%, suggesting that the miRNA expression level might be employed to identify patients at risk of ISR. CONCLUSIONS: To the best of our knowledge, this is the first work to show that the miR-152 expression level can possibly be applied to predict CAD patients at risk of ISR. The results suggest that the expression levels of miR-152 in PBMCs may serve as a biomarker for ISR. Our finding suggests the importance of miRNA levels in PBMCs as a novel biological tool to detect diseases in their early clinical stages.

Type 1 diabetes genetic risk score discriminates between monogenic and Type 1 diabetes in children diagnosed at the age of <5 years in the Iranian population.

AIM: To examine the extent to which discriminatory testing using antibodies and Type 1 diabetes genetic risk score, validated in European populations, is applicable in a non-European population. METHODS: We recruited 127 unrelated children with diabetes diagnosed between 9 months and 5 years from two centres in Iran. All children underwent targeted next-generation sequencing of 35 monogenic diabetes genes. We measured three islet autoantibodies (islet antigen 2, glutamic acid decarboxylase and zinc transporter 8) and generated a Type 1 diabetes genetic risk score in all children. RESULTS: We identified six children with monogenic diabetes, including four novel mutations: homozygous mutations in WFS1 (n=3), SLC19A2 and SLC29A3, and a heterozygous mutation in GCK. All clinical features were similar in children with monogenic diabetes (n=6) and in the rest of the cohort (n=121). The Type 1 diabetes genetic risk score discriminated children with monogenic from Type 1 diabetes [area under the receiver-operating characteristic curve 0.90 (95% CI 0.83-0.97)]. All children with monogenic diabetes were autoantibody-negative. In children with no mutation, 59 were positive to glutamic acid decarboxylase, 39 to islet antigen 2 and 31 to zinc transporter 8. Measuring zinc transporter 8 increased the number of autoantibody-positive individuals by eight. CONCLUSIONS: The present study provides the first evidence that Type 1 diabetes genetic risk score can be used to distinguish monogenic from Type 1 diabetes in an Iranian population with a large number of consanguineous unions. This test can be used to identify children with a higher probability of having monogenic diabetes who could then undergo genetic testing. Identification of these individuals would reduce the cost of treatment and improve the management of their clinical course.

Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode.

AIMS: The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase® cultivation mode and a redox mutant strain in the E. coli expression system. METHODS AND RESULTS: A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis® . CONCLUSIONS: This study demonstrated that the combination of SUMO fusion technology, EnBase® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. SIGNIFICANCE AND THE IMPACT OF THE STUDY: The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments.

A fed-batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric-truncated form of tissue plasminogen activator.

AIMS: A novel chimeric-truncated form of tissue-type plasminogen activator (t-PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed-batch processes. METHODS AND RESULTS: The expression cassette for the novel t-PA was prepared in pET-28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase(®) Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t-PA Assay Kit. The fed-batch fermentation mode, coupled with a Ni-NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46.66 IU mg(-1) ) compared to traditional batch mode (35.8 IU mg(-1) ). CONCLUSIONS: Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed-batch cultivation methods showed the potential to replace miss-folded formats of protein with proper folded, soluble form with improved potency. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post-translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.

[Image quality of CT angiography of coronary arteries depending on the degree of coronary calcification using a dual source CT scanner]. / Bildqualität von CT-Angiografien der Koronararterien in Abhängigkeit vom Ausmass der Koronarverkalkungen bei Einsatz eines Dual-Source-CTs.

PURPOSE: To evaluate the influence of severe calcification on image quality of CT angiography (CTA) of coronary arteries acquired with dual source CT. METHOD: 171 dual source CTAs in spiral mode were evaluated. Depending on the Agatston score (CS), patients where divided into four subgroups (I: CS 0 - 200, II: CS 201 - 600, III: CS 601 - 1000, IV: CS > 1000). Image quality was evaluated by two independent, blinded readers using a three-stage scale and the image quality of each subgroup was also shown with the help of an image quality index (BQI). In addition, a comparison with coronary catheter angiography was undertaken in a subgroup. RESULTS: Dual source CTA in spiral mode showed good image quality even in patients with Agatston scores of 601 - 1000 and > 1000. The four classes showed no significant difference in image quality. (BQI = 1,164 [I], BQI = 1,212 [II], BQI = 1,281 [III], BQI = 1,111 [IV]; p = 0,3145). The detailed analysis showed particularly good quality in patients with a heart rate < 75 bpm. The smaller subgroup of patients with CS > 200 and heart rates > 75 bpm showed a statistically significant reduction in image quality (BQI = 1,625; p = 0.0016). There were no gender differences in the image quality (p = 0.9881) although the calcium scores differed significantly in men (CS = 313 [+/- 449]) and women (CS = 145 [+/- 304]). The comparison with coronary catheter angiography showed a good correlation (92%). CONCLUSION: CTA of coronary arteries performed with dual source CT in spiral mode provides good image quality even in patients with higher calcification scores. The reduction of heart rates > 75 bpm improves the image quality significantly in patients with higher calcification scores.