A proteomic-based approach to study underlying molecular responses of the small intestine of Wistar rats to genetically modified corn (MON810).

A genetically modified (GM) commercial corn variety, MON810, resistant to European corn borer, has been shown to be non-toxic to mammals in a number of rodent feeding studies carried out in accordance with OECD Guidelines. Insect resistance results from expression of the Cry1Ab gene encoding an insecticidal Bt protein that causes lysis and cell death in susceptible insect larvae by binding to midgut epithelial cells, which is a key determinant of Cry toxin species specificity. Whilst whole animal studies are still recognised as the 'gold standard' for safety assessment, they only provide indirect evidence for changes at the cellular/organ/tissue level. In contrast, omics-based technologies enable mechanistic understanding of toxicological or nutritional events at the cellular/receptor level. To address this important knowledge-gap and to gain insights into the underlying molecular responses in rat to MON810, differential gene expression in the epithelial cells of the small intestine of rats fed formulated diets containing MON810, its near isogenic line, two conventional corn varieties, and a commercial (Purina™) corn-based control diet were investigated using comparative proteomic profiling. Pairwise and five-way comparisons showed that the majority of proteins that were differentially expressed in the small intestine epithelial cells in response to consumption of the different diets in both 7-day and 28-day studies were related to lipid and carbohydrate metabolism and protein biosynthesis. Irrespective of the diet, a limited number of stress-related proteins were shown to be differentially expressed. However these stress-related proteins differed between diets. No adverse clinical or behavioural effects, or biomarkers of adverse health, were observed in rats fed GM corn compared to the other corn diets. These findings suggest that MON810 has negligible effects on the small intestine of rats at the cellular level compared with the well-documented toxicity observed in susceptible insects.

Use of Array Comparative Genomic Hybridization for the Diagnosis of DiGeorge Syndrome in Saudi Arabian Population.

DiGeorge syndrome (DGS) is a genetic disorder known as a clinically variable syndrome with over 180 associated phenotypic features. It is caused by a common human deletion in the 22q11.2 chromosomal region and currently is affecting approximately 1 in 4,000 individuals. Despite the prevalence of inherited diseases mainly due to consanguineous marriages, the current diagnosis of DGS in Saudi Arabia is mainly based on conventional high-resolution chromosome banding (karyotyping) and FISH techniques. However, advanced genome-wide studies for detecting microdeletions or duplications across the whole genome are needed. The aim of this study is to implement and use aCGH technology in clinical diagnosis of the 22q11.2 deletion in Saudi Arabian DGS patients and to confirm its effectiveness compared to conventional FISH and chromosome banding techniques. Thirty suspected DGS patients were assessed for chromosome 22q11.2 deletion using high-resolution G-banding, FISH, and aCGH. The aCGH results were compared with those obtained by the other 2 cytogenetic techniques. G-banding detected the 22q11.2 deletion in only 1 patient in the cohort. Moreover, it detected additional chromosomal aberrations in 3 other patients. Using FISH, allowed for detection of the 22q11.2 deletion in 2 out of 30 patients. Interestingly, the use of aCGH technique showed deletions in the chromosome 22q11.2 region in 8 patients, indicating a 4-fold increase in diagnostic detection capacity compared to FISH. Our results show the effectiveness of aCGH to overcome the limitations of FISH and G-banding in terms of diagnostic yield and allow whole genome screening and detection of a larger number of deletions and/or duplications in Saudi Arabian DGS patients. Except for balanced translocations and inversions, our data demonstrate the suitability of aCGH in the diagnostics of submicroscopic deletion syndromes such as DGS and most chromosomal aberrations or complex abnormalities scattered throughout the human genome. Our results recommend the implementation of aCGH in clinical genomic testing in Saudi Arabia to improve the diagnostic capabilities of health services while maintaining the use of conventional cytogenetic techniques for subsequent validation or for specific and known aberrations whenever required.

Metformin improves the angiogenic potential of human CD34⁺ cells co-incident with downregulating CXCL10 and TIMP1 gene expression and increasing VEGFA under hyperglycemia and hypoxia within a therapeutic window for myocardial infarction.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with diabetes mellitus (DM). To identify the most effective treatment for CVD, it is paramount to understand the mechanism behind cardioprotective therapies. Although metformin has been shown to reduce CVD in Type-2 DM clinical trials, the underlying mechanism remains unexplored. CD34(+) cell-based therapies offer a new treatment approach to CVD. The aim of this study was to investigate the effect of metformin on the angiogenic properties of CD34(+) cells under conditions mimicking acute myocardial infarction in diabetes. METHODS: CD34(+) cells were cultured in 5.5 or 16.5 mmol/L glucose ± 0.01 mmol/L metformin and then additionally ± 4 % hypoxia. The paracrine function of CD34(+) cell-derived conditioned medium was assessed by measuring pro-inflammatory cytokines, vascular endothelial growth factor A (VEGFA), and using an in vitro tube formation assay for angiogenesis. Also, mRNA of CD34(+) cells was assayed by microarray and genes of interest were validated by qRT-PCR. RESULTS: Metformin increased in vitro angiogenesis under hyperglycemia-hypoxia and augmented the expression of VEGFA. It also reduced the angiogenic-inhibitors, chemokine (C-X-C motif) ligand 10 (CXCL10) and tissue inhibitor of metalloproteinase 1 (TIMP1) mRNAs, which were upregulated under hyperglycemia-hypoxia. In addition metformin, increased expression of STEAP family member 4 (STEAP4) under euglycemia, indicating an anti-inflammatory effect. CONCLUSIONS: Metformin has a dual effect by simultaneously increasing VEGFA and reducing CXCL10 and TIMP1 in CD34(+) cells in a model of the diabetic state combined with hypoxia. Therefore, these angiogenic inhibitors are promising therapeutic targets for CVD in diabetic patients. Moreover, our data are commensurate with a vascular protective effect of metformin and add to the understanding of underlying mechanisms.

Reference genes for expression studies in hypoxia and hyperglycemia models in human umbilical vein endothelial cells.

Human umbilical vein endothelial cell (HUVEC)-based gene expression studies performed under hypoxia and/or hyperglycemia show huge potential for modeling endothelial cell response in cardiovascular disease and diabetes. However, such studies require reference genes that are stable across the whole range of experimental conditions. These reference genes have not been comprehensively defined to date. We applied human genome-wide microarrays and quantitative real-time PCR (qRT-PCR) on RNA obtained from primary HUVEC cultures that were incubated for 24 hr either in euglycemic or in hyperglycemic conditions and then subjected to short-term CoCl2-induced hypoxia for 1, 3, or 12 hr. Using whole-transcript arrays, we selected 10 commonly used reference genes with no significant expression variation across eight different conditions. These genes were ranked using NormFinder software according to their stability values. Consequently, five genes were selected for validation by qRT-PCR. These were ribosomal protein large P0 (RPLP0), transferrin receptor (TFRC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-glucuronidase (GUSB), and ß-actin (ACTB). All five genes displayed stable expression under hyperglycemia. However, only RPLP0 and TFRC genes were stable under hypoxia up to 12 hr. Under hyperglycemia combined with hypoxia up to 12 hr, the expression of RPLP0, TFRC, GUSB, and ACTB genes remained unchanged. Our findings strongly confirm that RPLP0 and TFRC are the most suitable reference genes for HUVEC gene expression experiments subjected to hypoxia and/or hyperglycemia for the given experimental conditions. We provide further evidence that even commonly known references genes require experimental validation for all conditions involved.

Molecular diagnosis of fragile X syndrome using methylation sensitive techniques in a cohort of patients with intellectual disability.

BACKGROUND: Fragile X syndrome, the most common form of inherited intellectual disability, is caused by expansion of CGG trinucleotide repeat at the 5' untranslated region of the FMR1 gene at Xq27. In affected individuals, the CGG repeat expansion leads to hypermethylation and the gene is transcriptionally inactive. Our aim was to identify fragile X syndrome among children with intellectual disability in Saudi Arabia. PATIENTS AND METHODS: The study included 63 patients (53 males, 10 females) presented with intellectual disability, 29 normal subjects, and 23 other family members. DNA samples from six patients previously diagnosed with fragile X syndrome by Southern blot technique were used as positive controls. The method was based on bisulfite treatment of DNA followed by two different techniques. The first technique applied polymerase chain reaction amplification using one set of primers specific for amplifying methylated CpG dinucleotide region; another set designed to amplify the unmethylated CGG repeats. The second technique used the methylation-specific melting curve analysis for detection of methylation status of the FMR1 promoter region. RESULTS: Molecular testing using methylation sensitive polymerase chain reaction had shown amplified products in all normal subjects using unmethylated but not methylated primers indicating normal alleles, whereas amplified products were obtained using methylated polymerase chain reaction primers in fragile X syndrome-positive samples and in 9 of 53 males, indicating affected individuals. Molecular testing using melting curve analysis has shown a single low melting peak in all normal males and in (44/53) patients indicating unmethylated FMR1 gene, whereas high melting peak indicating methylated gene was observed in the fragile X syndrome-positive samples and in 9 of 53 patients. We found 100% concordance between results of both techniques and the results of Southern blot analysis. Three samples have shown both methylated and unmethylated alleles, indicating possible mosaicism. No female patients or carriers could be detected by both techniques. CONCLUSION: The technique can be applied for the rapid screening for fragile X syndrome among patients with intellectual disability. The impact of mosaicism on clinical severity needs further investigation.

Detection of FLT3 oncogene mutations in acute myeloid leukemia using conformation sensitive gel electrophoresis.

FLT3 (fms-related tyrosine kinase 3) is a receptor tyrosine kinase class III that is expressed on by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. FLT3 is also expressed on leukemia blasts in most cases of acute myeloid leukemia (AML). In order to determine the frequency of FLT3 oncogene mutations, we analyzed genomic DNA of adult de novo acute myeloid leukemia (AML). Polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) were used for FLT3 exons 11, 14, and 15, followed by direct DNA sequencing. Two different types of functionally important FLT 3 mutations have been identified. Those mutations were unique to patients with inv(16), t(15:17) or t(8;21) and comprised fifteen cases with internal tandem duplication (ITD) mutation in the juxtamembrane domain and eleven cases with point mutation (exon 20, Asp835Tyr). The high frequency of the flt3 proto-oncogene mutations in acute myeloid leukemia AML suggests a key role for the receptor function. The association of FLT3 mutations with chromosomal abnormalities invites speculation as to the link between these two changes in the pathogenesis of acute myeloid leukemiaAML. Furthermore, CSGE method has shown to be a rapid and sensitive screening method for detection of nucleotide alteration in FLT3 gene. Finally, this study reports, for the first time in Saudi Arabia, mutations in the human FLT3 gene in acute myeloid leukemia AML patients.

Urinary enzymes and microalbuminuria as indicators of renal involvement in patients with diabetes mellitus in saudi arabia.

There is a high prevalence of diabetes mellitus in the population of Saudi Arabia with the majority having non-insulin dependent diabetes mellitus (NIDDM). Random mid-day urine samples were obtained from 100 male [37 insulin dependants DM (IDDM) and 63 NIDDM] and 100 female (51 IDDM and 49 NIDDM) diabetic patients. Eighty-four patients were hypertensive (46 males and 28 females). One hundred and fifty-five subjects, not under medication and without clinical evidence of renal disease, hypertension, or diabetes mellitus were used as controls. Two urinary enzymes, N-acetyl-beta-D-glucosaminidase (NAG) and alanine amino-peptidase (AAP) were measured in the urine, together with total protein and creatinine concentration. Microalbuminuria, glucose and pH were measured using test strips. Increased levels of both NAG and AAP were found in the diabetic subjects. Increased excretion of both these enzymes as well as microalbuminuria was found in the hypertensive groups. The high levels of urinary enzymes found, suggest that renal complications were prevalent in the groups studied. Because of the high incidence of diabetes in Saudi Arabia, a screening program should be established which would include urinary biomarkers for the early detection of renal damage.