MicroRNA expression profiling in peripheral blood mononuclear cells and serum of type 2 diabetic, pre-diabetic and normo-glycaemic individuals

Thesis (MSc)--Stellenbosch University, 2016.ENGLISH ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNAs that play a fundamental role in cellular function by regulating messenger RNA gene expression. Alterations in miRNA expression are implicated in metabolic dysregulation, with several studies reporting the involvement of miRNAs in the pathophysiology of Type 2 diabetes (T2D). Recently, circulating miRNAs have attracted considerable interest as biomarkers to identify individuals at risk for T2D, thus we hypothesised that circulating miRNA could be used as markers for T2D progression. The aim of this study was to determine whether miRNA expression profiles differ between diabetic, pre-diabetic and normo-glycaemic individuals. Individuals were recruited from local communities and classified as diabetic, pre-diabetic or normo-glycaemic according to World Health Organization criteria, whereafter miRNAs were extracted from peripheral blood mononuclear cells (PBMCs) and serum of age-, gender-, ethnicity- and BMI-matched diabetic (n=4), pre-diabetic (n=4) and normo-glycaemic (n=4) individuals. MiRNAs extracted from PBMCs were sequenced using the Illumina HiSeq 2500 platform, and validated by quantitative real time PCR (qRT-PCR) in PBMCs and serum of these individuals. Moreover, bioinformatics was conducted using various target prediction programs (TargetScan, DIANA and PITA) and the DAVID functional gene annotation tool to assign biological significance to the differentially expressed miRNAs identified by sequencing. Sequencing showed that 267 (pre-diabetics vs. normo-glycaemics), 277 (diabetics vs. normo-glycaemics) and 267 (pre-diabetics vs. diabetics) miRNAs were differentially expressed between groups. Of these, five differentially expressed miRNAs (miR-27b, miR-379, miR-21, miR-98 and miR-143) were selected for validation by qRT-PCR in PBMCs. Only miR-143 and miR-27b were significantly differentially expressed using qRT-PCR, although the results for miR-143 were different compared to the sequencing data. MiR-143 was upregulated in pre-diabetics compared to normo-glycaemic individuals (1.40-fold, p≤0.01), whereas sequencing showed upregulation of miR-143 in diabetics compared to pre-diabetics (1.75-fold, p≤0.05). The differential expression of miR-27b was consistent between qRT-PCR (1.55-fold; p=0.07) and sequencing (1.15-fold; p<0.01), where both methods showed upregulation in pre-diabetics compared to normo-glycaemic individuals. The expression of miR-27b was similarly upregulated in serum of pre-diabetics compared to normo-glycaemic individuals (2.0-fold; p≤0.05). Furthermore, five novel miRNAs identified by sequencing were successfully validated in PBMCs of diabetic, pre-diabetic and normo-glycaemic individual. Sequencing and qRT-PCR showed that miR-27b was upregulated in PBMCs and serum of pre-diabetics compared to normo-glycaemic individuals. Bioinformatics identified peroxisome proliferator-activated receptor gamma (Pparg) as a target for miR-27b. PPARG is an insulin sensitizing agent, thus we speculate that increased miR-27b expression in pre-diabetes suppresses Pparg, thereby inhibiting insulin signaling and subsequently decreasing glucose uptake. The increased insulin and glucose levels observed in the pre-diabetic individuals support this idea, although further work is required to confirm this hypothesis. In conclusion, we showed that miRNA profiles differ during T2D progression, and are able to discriminate between diabetic, pre-diabetic and normo-glycaemic individuals. To our knowledge, this is the first study to report differential expression of miR-27b during T2D, suggesting its potential as a biomarker that could be incorporated into predictive models for the identification of high risk individuals. However, miRNA profiling in a larger sampleAFRIKAANSE OPSOMMING: MikroRNAs (miRNAs ) is klein nie-koderende RNAs wat 'n fundamentele rol in sellulêre funksie speel deur regulering van boodskapperRNA geenuitdrukking. Verskeie studies ïmpliseer veranderings in miRNA ekspresie met metaboliese disregulering en in die patofisiologie van Tipe 2-diabetes (T2D). Onlangs het sirkulerende miRNAs groot belangstelling uitgelok as biomerkers om individue te identifiseer wat „n verhoogde risiko vir T2D het. Ons hipotese stel dus voor dat sirkulerende miRNA gebruik kan word as merkers vir T2D siekteprogressie. Die doel van hierdie studie was om vas te stel of miRNA geenuitdrukkings profiele verskil tussen diabete, prediabete en normoglisemiese individue. Individue wat uit plaaslike gemeenskappe gewerf is, is volgens die Wêreld Gesondheid Organisasie riglyne geklassifiseer as diabete, pre diabete of normoglisemiese individue. Hierna is miRNAs uit die perifere bloed mononukleêreselle (PBMS) en serum van ouderdom, geslag, etniesiteit en liggaamsmassa-indeks vergelykbare diabete (n=4), prediabete (n=4) en normoglisemiese individue (n=4), geïsoleer. Die geenvolgordebepaling van die geïsoleerde miRNAs is bepaal deur „n Illumina HiSeq 2500 platform, en bevestig deur kwantitatiewe “real time PCR” (qRT-PCR). Verder, is bioinformatika uitgevoer met behulp van verskeie teikenvoorspellings programme (TargetScan, Diana en PITA) asook David se funksionele geenannotasie instrument om biologiese betekenis aan die differensieel uitgedrukte miRNAs, te koppel. Geenvolgordebepaling het getoon dat 267 (prediabete vs. normoglisemies), 277 (diabete vs. normoglisemies) and 267 (prediabete vs. diabete) miRNAs differensieel uitgedruk word. Hiervan is vyf differensieel uitgedrukte miRNAs (miR-27b, miR-379, miR-21, miR-98 en miR-143) gekies vir bevestiging deur qRT-PCR in PBMS. MiR-143 en miR-27b differensiasie was deur qRT-PCR bevestig, hoewel die qRT-PCR resultate vir miR-143 verskil het met die geenvolgordebepaling data. Met qRT-PCR is miR-143 opgereguleer in die prediabete teenoor normoglisemiese individue (1,40-voudig, p≤0.01), terwyl met geenvolgordebepaling miR-143 in diabete teenoor prediabete (1,75-voudig, p≤0.05) opgereguleer was. Daar was ooreenstemming in die differensiële uitdrukking van miR-27b tussen die qRT-PCR (1,55-voudig; p=0,07) en geenvolgordebepaling (1,15-voudig; p<0,01), waar albei metodes opregulering gewys het in die prediabete teenoor normoglisemiese individue. In die serum monsters was die uitdrukking van miR-27b soortgelyk opgereguleer in prediabete (2,0-voudig; p≤0.05). Verder is vyf unieke miRNAs geïdentifiseer deur geenvolgordebepaling wat suksesvol bevestig is in PBMS van diabete en prediabete. Bioinformatika het Pparg geïdentifiseer as 'n teiken vir miR-27b. PPARG is 'n insuliensensiterings agent, dus spekuleer ons dat hoër miR-27b ekspresie, in prediabete Pparg onderdruk, wat die insuliensein demp en tot verlaagde glukose opname lei. Die verhoogde insulien en glukose vlakke wat in prediabete voorkom ondersteun hierdie idee, alhoewel verdere werk nodig is om hierdie hipotese te bevestig. Ten slotte, het ons getoon dat miRNA profiele tydens die T2D siekteprogressie verskil, en in staat is om tussen diabete, prediabete en normoglisemiese individue te diskrimineer. Tot ons kennis, dit is die eerste studie wat differensiele uitgedrukking van miR-27b in T2D rapporteer, en die potensiële toepassing as 'n nie-indringende biomerker uitwys. Dit kan moontlik in voorspellende modelle geïnkorporeer kan word vir die identifisering van hoë risiko individue. Maar verdere studies met groter monster getalle en prospektiewe longitudinale studies is nodig om die kliniese toepaslikheid te evalueer

Screening for Gestational Diabetes Mellitus: The Potential of MicroRNAs

Gestational diabetes mellitus (GDM) is associated with short- and long-term complications in both mothers and their offspring. Screening and early diagnosis of GDM is advocated as a strategy to prevent adverse pregnancy outcomes. However, there is currently no test that is amenable to routine screening, particularly in low-and middle-income countries. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression post-transcriptionally. In recent years, miRNAs have been the focus of increasing research due to their important role in regulating biological pathways and their aberrant expression during disease. The discovery of circulating miRNAs in maternal blood, and their altered expression during pregnancy-associated complications have increased interest into their potential as diagnostic biomarkers for GDM. In this review, we summarise studies that have investigated miRNAs in maternal blood thus providing an update of the current status of miRNAs as biomarkers for GDM. We also discuss the challenges of miRNA profiling, and highlight perspectives and recommendations for research

Advances on Dipterology in the 21st century and extinction rates

At least one million extant insect species have been described on Earth, of which 150,000 belong to the megadiverse order Diptera. We here synthesize data from the last 15 years of taxonomic work in Diptera, mapping the world taxonomic productivity in the order. Our data shows an increasing importance of China and Brazil in taxonomic production, along with other traditional centres such as the USA and Europe. We correlate our database with estimates of extinction rates to determine the amount of basic taxonomic research still necessary to have the description of the fly diversity before extinction. Due to the growing recent extinction rates, it is unlikely that we will be able to entirely describe unknown fly diversity before their demise. Even considering that is still a constant and increasing speed of new species description, raising the number of active dipterists in the world is an urgent priority and the only real solution

Investigating Molecular Biomarkers During Gestational Diabetes Mellitus

Introduction: Gestational diabetes mellitus (GDM) is a significant public health concern, due to its association with short- and long-term complications in both mothers and offspring. DNA methylation and single nucleotide polymorphisms (SNPs) offer potential to serve as molecular biomarkers, which may lead to improved detection of GDM with positive effects on health outcomes. Aim: The aim of this study was to investigate whether DNA methylation and SNPs are associated with GDM and may offer potential as molecular biomarkers for GDM in South Africa (SA). Methods: This study followed a two-pronged approach. Firstly, literature searches were conducted to collate and synthesise all published articles reporting on the prevalence of GDM in SA, the screening and diagnostic strategies used, and the current status of DNA methylation and SNPs as biomarkers for GDM. Secondly, we conducted experiments to investigate global (n=201), genome-wide (n=24) and gene-specific DNA methylation (n=286) of the adiponectin gene (ADIPOQ) in whole blood of women with and without GDM, using an Enzyme-Linked Immunosorbent Assay, a methylationEPIC BeadChip Array and pyrosequencing, respectively. In addition, genotype and allele frequencies of ADIPOQ rs266729 and rs17300539, and methylenetetrahydrofolate reductase (MTHFR) rs1801133 were determined, using quantitative real-time PCR (n=449) and DNA sequencing for validation. Results: The literature search showed that the prevalence of GDM in SA has increased over the years. Furthermore, it showed that the lack of uniformity in screening and diagnosis between and within countries hamper the accurate detection of GDM. Lastly, the literature search identified several studies that support the use of DNA methylation and SNPs as potential biomarkers for GDM. Experimentally, we showed no differences in global DNA methylation between GDM and non-GDM groups. Interestingly, global DNA methylation levels were 18% (p=0.012) higher in obese compared to non-obese pregnant women. Genome-wide methylation analysis identified 1046 differentially methylated CpG sites (associated with 939 genes) (Cut-off threshold: M>0.06 and p<0.01). Among the top five CpG sites identified, one CpG mapped to the calmodulin-binding transcription activator 1 (CAMTA1) gene, which has been shown to regulate insulin production and secretion. Two CpG sites (-3410: p=0.048 and -3400: p=0.004) in the ADIPOQ promoter were hypomethylated during GDM in HIV negative, but not in HIV positive women. Lastly, no association between the ADIPOQ and MTHFR polymorphisms and GDM was observed in our population. Conclusion: To our knowledge, this is the first study to investigate the association between DNA methylation or ADIPOQ (rs266729 and rs17300539) and MTHFR (rs1801133) polymorphisms and GDM in SA. Findings suggest that gene-specific, but not global methylation nor SNPs rs266729, rs17300539 and rs1801133, may offer potential as molecular biomarkers of GDM in this population. Future longitudinal studies in larger samples that include both HIV negative and positive pregnant women are warranted to explore the candidacy of DNA methylation as molecular biomarkers for GDM.Thesis (PhD)--University of Pretoria, 2019.National Research Foundation (NRF) of South Africa, Thuthuka Grant (unique grant no. 99391).South African Medical Research Council (SAMRC)Obstetrics and GynaecologyPhDUnrestricte

A simple method to determine soil–water retention curves of compacted active clays

Determining the Soil Water Retention Curve (SWRC) of an active clay constitutes a challenge due to the significant, and sometimes irreversible, volume changes that occur during wetting and drying cycles. A novel yet simple method of experimentally determining the evolution of the SWRCs with moisture cycles is presented based on the results of a rigorous experimental study. Its purpose is to support the modelling of water flux in earthworks exposed to weather cycles that cause deterioration. Firstly, three SWRC branches (the primary drying, a scanning drying, and a scanning wetting branch) are measured and used to fit the proposed generic SWRC semi-empirical model in terms of water ratio, that, in the adsorptive region, is independent of the compaction conditions (void ratio and water content at compaction). Soil Shrink-Swell Curves (SSSCs) in terms of water ratio versus void ratio, that are easy to measure, can be determined for different compaction conditions over several drying and wetting cycles. Finally, the SSSCs are combined with the generic SWRC model to determine the evolution of the SWRCs with moisture cycles for the compaction conditions of interest. This method is demonstrated for two London clays of high and very high plasticity. Samples were compacted in five different conditions, varying in gravimetric water content and dry density, and were cycled six times between 1 and 80 MPa of total suction. The generic SWRC model was fitted to the experimental data. The model was able to estimate the SWRC in terms of degree of saturation over the six drying-wetting cycles without propagation of error. The significance of the research is that SWRC can now be determined over a range of wetting and drying cycles quickly and simply and enable modelling of deterioration of clays fills due to the action of weather to be accurate

A specific polymerase chain reaction method to identify Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a multidrug-resistant nosocomial pathogen that is difficult to identify unequivocally using current methods. Accordingly, because the presence of this microorganism in a patient may directly determine the antimicrobial treatment, conventional polymerase chain reaction (PCR) and real-time PCR assays targeting 23S rRNA were developed for the specific identification of S. maltophilia. The PCR protocol showed high specificity when tested against other species of Stenotrophomonas, non-fermentative Gram-negative bacilli and 100 clinical isolates of S. maltophilia previously identified using the Vitek system.Pontificia Universidade Catolica do Rio Grande do Su Faculdade de Biociencias Departamento de Biologia Celular e MolecularUniversidade Federal de São Paulo (UNIFESP) Departamento de BiofisicaUNIFESP, Depto. de BiofisicaSciEL

Changes in subcutaneous adipose tissue microRNA expression in response to exercise training in African women with obesity

The mechanisms that underlie exercise-induced adaptations in adipose tissue have not been elucidated, yet, accumulating studies suggest an important role for microRNAs (miRNAs). This study aimed to investigate miRNA expression in gluteal subcutaneous adipose tissue (GSAT) in response to a 12-week exercise intervention in South African women with obesity, and to assess depot-specific differences in miRNA expression in GSAT and abdominal subcutaneous adipose tissue (ASAT). In addition, the association between exercise-induced changes in miRNA expression and metabolic risk was evaluated. Women underwent 12-weeks of supervised aerobic and resistance training (n = 19) or maintained their regular physical activity during this period (n = 12). Exercise-induced miRNAs were identified in GSAT using Illumina sequencing, followed by analysis of differentially expressed miRNAs in GSAT and ASAT using quantitative real-time PCR. Associations between the changes (pre- and postexercise training) in miRNA expression and metabolic parameters were evaluated using Spearman’s correlation tests

Intimate partner violence : a risk factor for gestational diabetes

CITATION: Pheiffer, C., Dias, S., & Adam, S. 2020. Intimate Partner Violence: A Risk Factor for Gestational Diabetes. International journal of environmental research and public health, 17(21). doi:10.3390/ijerph17217843The original publication is available at https://www.mdpi.com/journal/ijerphThe early detection and management of gestational diabetes mellitus (GDM) is an important public health goal. GDM, which is defined as a glucose intolerance that develops during pregnancy, affects about 14% of pregnancies globally, and without effective treatment, it is associated with adverse short- and long-term maternal and neonatal outcomes. Risk-factor screening is an acceptable and affordable strategy to enable risk stratification and intervention. However, common biological risk factors such as overweight or obesity, excessive gestational weight gain, and family history of diabetes often have poor predictive ability, failing to identify a large proportion of women at risk of developing GDM. Accumulating evidence implicate psychosocial factors in contributing to GDM risk. As such, intimate partner violence (IPV), through its contributing effects on maternal stress and depression, presents a plausible risk factor for GDM. Experiencing IPV during pregnancy may dysregulate the hypothalamus-pituitary-adrenal (HPA) axis, leading to increased cortisol secretion and insulin resistance. These effects may exacerbate the insulin-resistant environment characteristic of pregnancy, thus increasing GDM risk. This review explores the relationship between IPV and GDM. We highlight studies that have linked IPV with GDM and propose a biological mechanism that connects IPV and GDM. Recommendations for IPV screening strategies to prevent GDM are discussed.https://www.mdpi.com/1660-4601/17/21/7843Publishers versio

Differences in Rate of Perceived Exertion and Workload Intensity in Males and Females during Submaximal Arm and Leg Ergometry

International Journal of Exercise Science 15(4): 1222-1235, 2022. Purpose: Arm ergometry (AE) is necessitated for individuals unable to perform leg ergometry (LE) exercise. This study explored gender differences in RPE and workload (WL) during AE and LE at submaximal target heart rates (THR). Methods: 35 healthy college-aged individuals were randomly allocated to begin exercise on either AE or LE. Participants exercised on both modes with increasing WL to achieve submaximal THRs of 110, 120, 130, 140 and 150 beats per minute (bpm). Factorial ANOVAs tested for differences in RPE and WL. Results: No significant differences were found in RPE between genders, as well as between arm and leg exercise (p \u3e 0.001). For WL, a significant main effect was found for mode with LE greater than AE (p \u3c 0.001), and gender, with males greater than females (p \u3c 0.001). A significant interaction effect was also found for HR and mode, with a greater increase in WL during LE compared to AE in both genders (p = 0.001). Conclusions: Exercise specialists typically prescribe exercise based on a chosen THR. The results of this study provide meaningful data on mean RPE and WL responses that a given THR elicits for ergometry. The finding of no differences in RPE between AE and LE informs the clinician that at any given submaximal THR, similar RPE scores can be expected during AE and LE. Further research is warranted to investigate differences in wider populations