A biochemical assessment of Ochratoxin A stress responses in vitro, with resveratrol as a possible therapeutic intervention.

Doctor of Philosophy Medical Biochemistry. University of KwaZulu-Natal. Durban, 2017.Abstract available in PDF file

The phytoalexin, resveratrol ameliorates ochratoxin A genotoxicity in human embryonic kidney (HEK293) cells.

M. Med. Sc. University of KwaZulu-Natal, Durban 2014.Background: Ochratoxin A (OTA) is a mycotoxin produced by fungal species of Aspergillus and Penicillium. OTA is nephrotoxic and carcinogenic in several animal models; it frequently contaminates human and animal food products. Chronic exposure is associated with progressive renal fibrosis in humans (Balkan endemic nephropathy). Resveratrol is a phytoalexin that possesses both anti-cancer and antioxidant properties. We investigated the mechanism of cellular oxidative stress induced by OTA in the human embryonic kidney (HEK293) cell line. Methods: An IC50 value of 1.5μM was determined from a dose-dependent cell viability curve using the methylthiazol tetrazolium (MTT) assay on HEK293 cells treated with a range of OTA concentrations (0.25μM–50μM) for 24hrs. Glutathione levels were quantified by luminometry and gene expression of Nrf2, OGG1, CAT, SOD and GPx was determined by qPCR. Protein expression of Nrf2 and phosphorylated SIRT1 (pSIRT1) was assessed by western blot, DNA damage was determined using the comet assay, and flow cytometry was employed for intracellular ROS detection. Results: Resveratrol decreased mRNA expression of OGG1 (p<0.05) and OTA significantly increased OGG1 expression (p<0.05). The comet assay proved that while OTA induced DNA damage, resveratrol protected the DNA against strand breaks. Both resveratrol and OTA significantly increased antioxidant defence gene expression (Nrf2, CAT, GPx and SOD) (p<0.05). OTA decreased intracellular ROS, while resveratrol-treated cells exhibited the lowest percentage of intracellular ROS. Luminometry analysis showed the OTA+Resveratrol co-treatment to have a synergistic effect on the concentration of GSH and GSSG. Western blot analysis of protein showed that resveratrol significantly increased the levels of pSIRT1 while concomitantly decreasing the protein levels of Nrf2 (p<0.05) and OTA significantly decreased pSIRT1 protein levels

The phytoalexin resveratrol ameliorates ochratoxin a toxicity in human embryonic kidney (HEK293) cells

Ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by Aspergillus and Penicillium fungi. It contaminates human and animal food products, and chronic exposure is associated with renal fibrosis in humans (Balkan endemic nephropathy). Resveratrol, a phytoalexin, possesses anti-cancer and antioxidant properties. We investigated the mechanism of cellular oxidative stress induced by OTA, and the effect of resveratrol in human embryonic kidney (HEK293) cells over 24 and 48 h. Cells were exposed to OTA [IC50¼1.5 mM (24 h) and 9.4 mM (48 h) determined using MTT assay] and 25mM resveratrol. Glutathione was quantified by luminometry and gene expression of Nrf2 and OGG1 was determined by qPCR. Protein expression of Nrf2, LonP1, SIRT3, and pSIRT1 was assessed by Western blot, DNA damage (comet assay), and intracellular reactive oxygen species (flow cytometry). At 24 h, resveratrol increased mRNA expression of the DNA repair enzyme, OGG1 (P<0.05), whereas OTA and OTAþresveratrol significantly decreased OGG1 expression (P<0.05). OGG1 expression increased during 48-h exposure to resveratrol and OTAþresveratrol (P<0.05). Comet tail lengths doubled in 48-h OTA-treated cells, whereas at both time periods, OTAþresveratrol yielded shorter comet tails (P<0.0001). During 24- and 48-h exposure, OTA, resveratrol, and OTAþresveratrol significantly decreased mRNA expression of Nrf2 (P<0.05). Luminometry analysis of GSH revealed an increase by OTAþresveratrol for 24 and 48 h (P<0.05 and P<0.001, respectively). Western blot analysis showed decreased Nrf2 protein expression during 24-h exposure, but increased Nrf2 expression during 48 h. LonP1 protein expression increased during 24-h exposure to OTA (P<0.05) and OTAþresveratrol (P<0.0011) and during 48-h exposure to resveratrol (P<0.0005).http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-46442016-12-31hb201

Methylenetetrahydrofolate (MTHFR), the One-Carbon Cycle, and Cardiovascular Risks

The 5-10-methylenetetrahydrofolate reductase (MTHFR) enzyme is vital for cellular homeostasis due to its key functions in the one-carbon cycle, which include methionine and folate metabolism and protein, DNA, and RNA synthesis. The enzyme is responsible for maintaining methionine and homocysteine (Hcy) balance to prevent cellular dysfunction. Polymorphisms in the MTHFR gene, especially C677T, have been associated with various diseases, including cardiovascular diseases (CVDs), cancer, inflammatory conditions, diabetes, and vascular disorders. The C677T MTHFR polymorphism is thought to be the most common cause of elevated Hcy levels, which is considered an independent risk factor for CVD. This polymorphism results in an amino acid change from alanine to valine, which prevents optimal functioning of the enzyme at temperatures above 37 &deg;C. Many studies have been conducted to determine whether there is an association between the C677T polymorphism and increased risk for CVD. There is much evidence in favour of this association, while several studies have concluded that the polymorphism cannot be used to predict CVD development or progression. This review discusses current research regarding the C677T polymorphism and its relationship with CVD, inflammation, diabetes, and epigenetic regulation and compares the evidence provided for and against the association with CVD

Diagnosis and Treatment of MODY: An Updated Mini Review

Maturity-Onset Diabetes of the Young (MODY) is the most common form of monogenic diabetes resulting from a single gene mutation. It is characterized by mild hyperglycemia, autosomal dominant inheritance, early onset of diabetes (&lt;25 years), insulin resistance, and preservation of endogenous insulin secretion. Currently, 14 MODY subtypes have been identified, with differences in incidence, clinical features, diabetes severity and related complications, and treatment response. This type of diabetes is mostly misdiagnosed as either type 1 or type 2 diabetes mellitus because it is difficult to differentiate between these forms of diabetes due to clinical similarities, the high cost of genetic testing, and lack of awareness. As a result, thousands of patients are not receiving appropriate treatment. Accurate diagnosis would allow for more effective therapeutic management and treatment strategies that are distinct from those used for type 1 and type 2 diabetes. This review serves to explore MODY subtypes, diagnosis, and treatment, and increase awareness of MODY incidence

Incidence of HNF1A and GCK MODY Variants in a South African Population

Background and Aim: Maturity-onset diabetes of the young (MODY) is the result of single gene variants. To date, fourteen different MODY subtypes have been described. Variants in genes coding for glucokinase (GCK, MODY2) and hepatic nuclear factor 1 alpha (HNF1A, MODY3) are most frequently encountered. MODY patients are often misdiagnosed with type 1 or type 2 diabetes, resulting in incorrect treatment protocols. At the time of reporting, no data are available on MODY prevalence in populations from Africa. Our study aimed to investigate and report on the incidence of MODY-related variants, specifically HNF1A variants, in a population from the Western Cape. Methods: Study participants were recruited (1643 in total, 407 males, 1236 females) and underwent anthropometric tests. Thereafter, blood was collected, and real-time PCR was used to screen for specific variants in HNF1A and GCK genes. Results: Ninety-seven individuals (5.9%) were identified with a specific HNF1A gene polymorphism (rs1169288) and twelve (0.9%) with a GCK polymorphism (rs4607517). Conclusion: In total, 6.6% of the study population expressed MODY variants. To our knowledge, we are the first to report on MODY incidence in Africa. This research provides the basis for MODY incidence studies in South Africa, as well as data on non-Caucasian populations

The Relationship between the Oral Microbiota and Metabolic Syndrome

The oral microbiota plays a crucial role in both systemic inflammation and metabolic syndrome (MetS), which is characterised by low-grade inflammation. Studies have analysed the gut microbiota using stool specimens from subjects with MetS; however, the etiological role of the oral microbiota in the development of MetS is still uncertain. We investigated the oral microbiota of 128 subgingival plaque samples from a South African cohort with and without MetS. After a comprehensive analysis of the oral microbiota, we observed a significant increase in Gram-positive aerobic and anaerobic microbiota in those with MetS. We observed an abundance of Actinomyces, Corynebacterium, and Fusobacterium genera in the MetS group, which differed significantly from previous studies, which found Granulicatella to be enriched in MetS. To further assess the impact of the metabolic parameters (FBG, Waist C, HDL, TGs, and BP) on the oral microbiota, we calculated the odds ratio (ORs) for significant oral microbiota identified between the MetS groups. We found that different species were associated with at least four MetS risk factors. This study has shown that the oral microbiota is disrupted in MetS and may promote inflammation providing a gateway to other systemic diseases, including diabetes and cardiovascular diseases

Circulating miR-30a-5p and miR-182-5p in prediabetes and screen-detected diabetes mellitus

CITATION: Weale C. J. et al. 2020. Circulating miR-30a-5p and miR-182-5p in Prediabetes and Screen-Detected Diabetes Mellitus. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 13:5037-5047. doi:10.2147/DMSO.S286081The original publication is available at https://www.dovepress.com/diabetes-metabolic-syndrome-and-obesity-targets-and-therapy-journalBackground: microRNAs (miRNAs) have been touted as potential diagnostic and prognostic biomarkers for various diseases. The aim of the present study was to evaluate the diagnostic value of miR-30a-5p and miR-182-5p for prediabetes and screen-detected type 2 diabetes mellitus (T2DM). Methods: The study included 1270 participants (207 prediabetes, 94 screen-detected diabetes and 969 normotolerant) from the Vascular and Metabolic Health (VMH) study. Whole blood levels of miR-30a-5p and miR-182-5p were quantitated by RT-qPCR. Multivariable logistic regressions were used to relate miRNAs with prediabetes or T2DM and receiver operating characteristic (ROC) curves were used to evaluate the ability of each miRNA to diagnose these conditions. Results: Both miRNAs were significantly highly expressed in individuals with prediabetes or T2DM (both ≥3.2-fold, and p<0.001). We also observed significant under-expression in T2DM relative to prediabetes for miR-182-5p (0.49-fold, p=0.001). Age, sex and BMI-adjusted partial correlation coefficient analysis revealed a significant correlation between the two miRNAs across glucose tolerance statuses (r≥0.932, p<0.001). In normotolerant individuals, both miRNAs showed a negative correlation with waist circumference and positive correlation with HDL-cholesterol whilst in T2DM they correlated positively with hip circumference, 2-hour insulin, HDL- and LDL-cholesterol. Multivariable logistic regressions revealed both miRNAs to be consistently and continuously associated with prediabetes or T2DM (OR≥1.18, 95% 95% CI: 1.10-1.28, p<0.001), while only miR-182-5p associated with a reduced prevalence of T2DM relative to prediabetes (OR: 0.89, 95% CI: 0.83-0.96, p=0.003). In ROC analyses, miR-182-5p almost outperformed HbA1c in diagnosing prediabetes; area under the curve 0.74 vs 0.69. Conclusion: Our findings demonstrate that miR-30a-5p and miR-182-5p are associated with dysglycaemia and could potentially predict prediabetes, particularly miR-182-5p.https://www.dovepress.com/circulating-mir-30a-5p-and-mir-182-5p-in-prediabetes-and-screen-detect-peer-reviewed-fulltext-article-DMSOPublishers versio

MicroRNAs-1299, -126-3p and -30e-3p as Potential Diagnostic Biomarkers for Prediabetes

This cross-sectional study investigated the association of miR-1299, -126-3p and -30e-3p with and their diagnostic capability for dysglycaemia in 1273 (men, n = 345) South Africans, aged &gt;20 years. Glycaemic status was assessed by oral glucose tolerance test (OGTT). Whole blood microRNA (miRNA) expressions were assessed using TaqMan-based reverse transcription quantitative-PCR (RT-qPCR). Receiver operating characteristic (ROC) curves assessed the ability of each miRNA to discriminate dysglycaemia, while multivariable logistic regression analyses linked expression with dysglycaemia. In all, 207 (16.2%) and 94 (7.4%) participants had prediabetes and type 2 diabetes mellitus (T2DM), respectively. All three miRNAs were significantly highly expressed in individuals with prediabetes compared to normotolerant patients, p &lt; 0.001. miR-30e-3p and miR-126-3p were also significantly more expressed in T2DM versus normotolerant patients, p &lt; 0.001. In multivariable logistic regressions, the three miRNAs were consistently and continuously associated with prediabetes, while only miR-126-3p was associated with T2DM. The ROC analysis indicated all three miRNAs had a significant overall predictive ability to diagnose prediabetes, diabetes and the combination of both (dysglycaemia), with the area under the receiver operating characteristic curve (AUC) being significantly higher for miR-126-3p in prediabetes. For prediabetes diagnosis, miR-126-3p (AUC = 0.760) outperformed HbA1c (AUC = 0.695), p = 0.042. These results suggest that miR-1299, -126-3p and -30e-3p are associated with prediabetes, and measuring miR-126-3p could potentially contribute to diabetes risk screening strategies