Development of an Expression Vector Engineering Strategy Based on tDNA Insulator Element for the Stable Expression of Vascular Endothelial Growth Factor Receptor-Fc Fusion Protein.

During the past decades, tremendous advances have occurred in manufacturing recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. Nevertheless, the production of stable high-producing cell lines has remained a major obstacle in the development process of the CHO cell line. It has been shown that genomic regulatory elements can promote cell line development efficiency by improving transgenes' productivity and stability. Such elements include insulators, ubiquitous chromatin opening elements, scaffold/matrix attachment regions, and antirepressors. In addition, tDNA elements are shown to act as insulators in mammalian cells. This study examines the effect of the tDNA insulator on stable expression of a vascular endothelial growth factor receptor-Fc fusion protein.

Demethylation of CDKN2A in systemic lupus erythematosus and rheumatoid arthritis: a blood biomarker for diagnosis and assessment of disease activity.

INTRODUCTION/OBJECTIVES: Considering the phenotypic and serological heterogeneity of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), significant challenges may intervene with the precise diagnosis. In this regard, numerous studies have shown that changes in DNA methylation levels can be used to distinguish between healthy individuals and those with SLE and RA, as well as to predict disease activity and prognosis. METHODS: In the current study, we evaluated quantitative methylation level of CDKN2A promoter in peripheral blood mononuclear cells (PBMCs) of SLE and RA patients, and healthy controls by methylation-quantification of endonuclease-resistant DNA (MethyQESD), a bisulfite conversion-independent method. RESULTS: Our findings revealed an excessive hypomethylation of CDKN2A in SLE and RA patients compared to healthy individuals (P < 0.001). Besides, by determining efficient cutoff value, the specificity of CDKN2A for correct diagnosis of healthy subjects was measured to be 77.30% and the sensitivity for SLE and RA diagnosis were 81.33%, and 72%, respectively. Furthermore, CDKN2A methylation level was shown to be positively associated with C3 and C4 levels and negatively associated with anti­dsDNA concentration (P < 0.001). Moreover, a statistically significant difference in the DNA methylation levels of CDKN2A promoter was identified between SLE cases with age of ≤ 18 and patients with > 18 years of age (P = 0.025). CONCLUSION: Our findings demonstrated that CDKN2A methylation levels in PBMCs of SLE and RA patients could be used as a promising diagnostic biomarker. The significant association between hypomethylation of CDKN2A promoter and disease activity factors in SLE patients, is suggesting that CDKN2A hypomethylation could be used as an alternative biomarker for assessment of disease activity. Key Points • Several studies have reported increased expression of CDKN2A in SLE and RA suggesting that it may be involved in the pathogenesis of these disorders. • CDKN2A hypomethylation has been implicated in different autoimmune diseases. • Our findings demonstrated that CDKN2A methylation levels in PBMCs of SLE and RA patients could be used as a promising diagnostic and prognostic biomarker.

Promoter methylation of matrix metallopeptidase 9 in peripheral blood mononuclear cells: A novel biomarker in a promising source for noninvasive colorectal cancer diagnosis.

OBJECTIVES: Colorectal cancer (CRC) has been described as a "silent disease," which can be readily treated in most patients when discovered in its early stages. Considering the limitations of the current conventional tests for the diagnosis of CRC, researchers strive to find noninvasive and more valid biomarkers for the early detection of CRC. It has been shown that tumor-specific methylation patterns can also be identified in peripheral blood mononuclear cells (PBMCs) and are reliable sources of methylation analysis for CRC screening. MATERIALS AND METHODS: We carried out a quantitative methylation analysis on matrix metallopeptidase 9 (MMP9) promoter using methylation quantification endonuclease-resistant DNA (MethyQESD) method. A total of 70 patients with CRC and 70 normal controls were enrolled in this study for methylation analysis in the PBMCs. RESULTS: Our findings discovered a considerable hypermethylation of MMP9 promoter in CRC patients compared with healthy controls (mean: 47.30% and 20.31%, respectively; P > 0.001). The sensitivity and specificity of the MMP9 gene for the diagnosis of CRC were 88% and 78%, respectively. In addition, on the basis of area under the curve values, the diagnostic power of the MMP9 gene was 0.976 (P < 0.001). Moreover, our analysis established that MMP9 methylation was significantly different between the different stages of CRC (P: 0.034). CONCLUSIONS: Our results showed that MMP9 promoter methylation in PBMCs can be used as an outstanding biomarker for CRC diagnosis. Besides, we confirmed that PBMCs are reliable sources of methylation analysis for CRC screening and MethyQESD is an accurate and fast method for quantitative methylation analyses.

Analysis of SDC2 gene promoter methylation in whole blood for noninvasive early detection of colorectal cancer.

Objectives: Considering the limitations of the current approaches to colorectal cancer (CRC) screening, scientists strived to find noninvasive and more powerful biomarkers for the early diagnosis of CRC. Nowadays, there are different sources of biomarkers for CRC diagnosis. Blood-based samples including circulating cell-free tumor DNA (ctDNA) and DNA extracted from leukocytes in peripheral blood might be promising sources of noninvasive cancer biomarkers such as cancer-specific methylation patterns. In this study, we aimed to evaluate the noninvasive early diagnosis of CRC via quantitative promotor methylation analysis of SDC2 gene in whole blood. Materials and Methods: Sixty-five CRC patients and 65 healthy participants were enrolled to assess promoter methylation of SDC2 gene in whole blood using the methylation quantification endonuclease-resistant DNA (MethyQESD) technique. Results: Our findings demonstrated drastic hypermethylation of SDC2 in blood samples from CRC subjects (37.91%) compared with non-malignant individuals (17.02%) (P < 0.001). The sensitivity for detection of CRC by methylation of SDC2 was 81.54%, with a specificity of 69.23%. The ROC curve analysis demonstrated that the AUC was 0.847 (P < 0.001), indicating that the status of SDC2 promoter methylation in whole blood is an excellent biomarker of CRC diagnosis. Furthermore, our results showed that methylation level in CRC patients significantly increased in higher tumor stages, demonstrating that an increased percentage of methylation is correlated with tumor progression (P < 0.001). Conclusion: SDC2 promoter methylation status in blood samples is a valuable cancer biomarker and holds high power and accuracy in distinguishing CRC patients from healthy subjects in the early stages of the disease.

Expression of ERBB gene family in females with breast cancer and its correlation with clinicopathological characteristics of the disease.

BACKGROUND: Breast cancer (BC) is the most prevalent and fatal cancer in women. Given that there are very few studies investigating the overexpression of four members of ERBB genes, we decided to investigate the correlation between these four genes with clinicopathological characteristics in breast cancer cases. METHODS: Tumoural tissue of 50 patients with sporadic invasive ductal BC was recruited. Also, control samples were provided from adjacent non-cancerous tissues (ANCTs) of the same patients. The expression of four ERBB genes was evaluated by real-time PCR and its correlation with clinicopathological characteristics was assessed. RESULTS: Only ERBB2 (HER2) was overexpressed in tumoural tissue compared with ANCTs. Our data showed a significant relationship between ERBB1 overexpression with triple-negative tumors, ER, and PR negativity (P < 0.05). Also, ERBB2 overexpression indicated a significant correlation with several pathological characteristics such as age < 50, tumor size larger than 2 cm, early and advanced stages, negative involved lymph nodes, luminal B, triple-negative, ERBB2-enrich, estrogen receptor (ER) and progesterone receptor (PR) negative tumors, Ki-67 mutation more than 15%, and finally HER2/neu immunohistochemistry (IHC) positive and intermediate (P < 0.05). Moreover, this study demonstrated that ERBB4 overexpression had a significant correlation with tumor size smaller than 2 cm, grade I and II tumors (early-stage tumors), luminal A, ER and PR positive tumors, HER-2/neu IHC intermediate, and tumors that had a Ki-67 mutation lower than 15% (P < 0.05). Besides, our analysis showed a significant correlation between the expression of ERBB1 with ERBB2 and ERBB3 with ERBB4 (P < 0.05). CONCLUSIONS: Our findings showed a significant relationship between unfavorable clinicopathological characteristics with ERBB1 and ERBB2 overexpression, but overexpression of ERBB4 was correlated with favorable outcomes.

A novel splice site mutation in the SDCCAG8 gene in an Iranian family with Bardet-Biedl syndrome.

PURPOSE: Bardet-Biedl syndrome (BBS: OMIM 209,900) is a rare ciliopathic human genetic disorder that affects many parts of the body systems. BBS is a genetically heterogeneous disorder with a wide spectrum of clinical manifestations which makes its diagnosis and management more challenging. RetNet reports 18 genes that cause BBS and each of genes has had several known mutations. Genetic studies suggesting that serologically defined colon cancer antigen 8 (SDCCAG8) gene mutations are a major cause of BBS. MATERIALS AND METHODS: In this section, we investigated the consanguineous Iranian family members with BBS. Whole-exome sequencing and Sanger sequencing, were performed to screen and confirm the suspicious pathogenic mutations. The identified mutation was investigated using bioinformatics tools to predict the effect of the mutation on protein structure. RESULTS: Sequential analysis identified a novel splice site mutation c.1221 + 2 T > A in the SDCCAG8 gene in BBS patients. Structure-based approaches have predicted significant structural alterations in SDCCAG8 protein. CONCLUSIONS: This study was conducted to show the aberrant alternative splicing as one of the single splicing mutations identified can cause BBS by affecting the function of SDCCAG8 protein.

MicroRNA binding site polymorphism in inflammatory genes associated with colorectal cancer: literature review and bioinformatics analysis.

Inflammation, among environmental risk factors, is one of the most important contributors to colorectal cancer (CRC) development. In this way, studies revealed that the incidence of CRC in inflammatory bowel disease patients is up to 60% higher than the general population. MicroRNAs (miRNAs), small noncoding RNA molecules, have attracted excessive attention due to their fundamental role in various aspects of cellular biology, such as inflammation by binding to the 3'-untranslated regions (3'-UTR) of pro and anti-inflammatory genes. Based on multiple previous studies, SNPs at 3'-UTR can affect miRNA recognition elements by changing the thermodynamic features and secondary structure. This effect can be categorized, based on the number of changes, into four groups, including break, decrease, create, and enhance. In this paper, we will focus on functional variants in miRNA binding sites in inflammatory genes, which can modulate the risk of CRC by both investigating previous studies, regarding miRSNPs in inflammatory genes associated with CRC and recruiting in silico prediction algorithms to report putative miRSNPs in 176 inflammatory genes. In our analysis, we achieved 110 miRSNPs in 3'-UTR of 67 genes that seem good targets for future researches.

Application of Multiplex Ligation-Dependent Probe Amplification in Determining the Copy Number Alterations of HER Gene Family Members in Invasive Ductal Breast Carcinoma.

BACKGROUND: The aim of this study was to assess the usability of multiplex ligation-dependent probe amplification (MLPA) for copy number determination of HER gene family members (ERBB1-4) in invasive breast carcinoma and to explore the association of ERBB1-4 gene copy numbers with clinicopathological characteristics of breast cancer (BC) patients. METHODS: Clinical and immunohistochemical characteristics were assessed in 104 BC patients and the molecular subtype was determined for each tumor sample. Furthermore, HER-2/neu status was assessed by immunohistochemistry (IHC) and equivocal results were confirmed by Fluorescent in situ hybridization (FISH). The copy numbers of ERBB1-4 genes were determined by MLPA. RESULTS: Twenty-five percent of all patients showed ERBB2 gene-amplification by MLPA, whereas 14.4% of cases showed ERBB-2/neu overproduction at the protein level (IHC). Moreover, only 2.9% and 1.9% of patients showed amplification in ERBB1 and ERBB4, respectively. No copy number changes were observed in ERBB3. Our results indicated a significant association between ERBB2 copy number gain and histological grade (p value= 0.01), stage (p value= 0.02), and tumor subtypes (p value= <0.001). In addition, we found MLPA more accurate in assessing HER2 status with 15.4% and 9.6% gene amplification detection in early stages (1, 2A and 2B) and advanced tumor stages (3A, 3B, and 4), respectively, compared to IHC (early stages= 13.5% and advanced stages= 4.7%). CONCLUSION: According to our findings, MLPA is a fast, precise and low-cost technique to detect ERBB2 amplification, especially in advanced tumor stages. However, due to infrequent amplification found in ERBB1 and ERBB4 as well as the lack of amplification in ERBB3, their importance in the prognostic evaluation of BC patients remains controversial.

Association Study of IL-4 -590 C/T and DDX39B -22 G/C Polymorphisms with the Risk of Late-Onset Alzheimer's Disease in Iranian Population.

Interleukin-4 (IL-4), an important anti-inflammatory cytokine, is elucidated to regulate amyloid ß-induced production of the inflammatory cytokines such as IL-1 and IL-6. It is assumed that IL-4 may involve in the inflammation pathology of surrounding senile plaques in Alzheimer's disease (AD) patients. DEAD (Asp-Glu-Ala-Asp) box polypeptide 39B (DDX39B), appears to be involved in regulation of the inflammatory cytokines which are in correlation with AD pathology. This study was conducted to investigate the two single nucleotide polymorphisms (SNPs), IL-4 -590 C/T and DDX39B -22 G/C, association with the risk of late-onset AD (LOAD) in Iranian population. In the present study, therefore, a cohort of 153 LOAD cases and 153 age-matched unrelated, non-dementia control subjects were analyzed for the two polymorphisms by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). Our results successfully demonstrate a protective association between the IL-4 -590 T allele, IL-4 -590 C/T heterozygous genotype (P= 0.01, OR= 0.53 and P= 0.041; OR= 0.56, respectively) and LOAD in Iranian population. A resemblance significant association was detected in female population when subjects were stratified by sex: the IL-4 -590 T allele (P= 0.02, OR= 0, 40) and the heterozygous genotype (P= 0.009, OR= 0.29). However, no significant association was observed between the DDX39B -22 G/C polymorphism in the cases and controls. Furthermore, it is clarified that the protective effect of IL-4 -590 is independent from APOE protective genotypes. Accordingly, the IL-4 -590 T allele may be applied as a protective marker in the development of LOAD in Iranian population.