Methylation profiling and validation of candidate tDMRs for identification of human blood, saliva, semen and vaginal fluid and its application in forensics.

Masters Degree. University of KwaZulu-Natal, Durban.Identification of body fluids and tissues is an essential step in forensic investigation because it can be used as strong evidence in identifying suspects and victims. Currently in forensic investigations, catalytic, enzymatic and immunological techniques are used to identify body fluids, however, are limited due to lack of sensitivity and specificity. Hence, researchers are always on the lookout for novel methods that can be used to identify and analyse body fluids. Recently, DNA methylation-based markers have proven to be more sensitive and specific than conventional methods for body fluid identification. Genome-wide methylation studies have demonstrated that tissue specific differentially methylated regions (tDMRs) vary in methylation profiles in various cell types and tissues. The differences in methylation profiles of tDMRs can be targeted to be used as biomarkers to differentiate between body fluids and tissues. To date, only a few DNA methylation-based markers have been reported to identify body fluids. To enhance the specificity and robustness of DNA methylation-based identification, novel markers are required. Additionally, methylation-based markers require further interrogation, to evaluate the stability of their methylation profiles under simulated forensics conditions such as UV light, temperature, rain and microbes, which could cause DNA degradation and affect DNA recovery as well as the methylation status of body fluids. In a previous study, based on differential gene expression in blood, saliva, semen and vaginal fluid, gene body CpG islands were selected, in genes Zinc finger protein 282 (ZNF282), Protein tyrosine phosphatase, receptor S (PTPRS) and Hippocalcin like 1 (HPCAL1), that have potential tDMRs to differentiate between, blood, saliva, semen and vaginal fluid. It was proposed that differential gene expression could be possibly due to differences in methylation patterns. The present study was undertaken to establish the methylation status of potential tDMRs in target body fluids by using methylation specific PCR (MSP) and bisulfite sequencing (BS). In both MSP and BS, the methylation status of 3 genes ZNF282, PTPRS and HPCAL1 were analysed in 10 samples of each body fluid. With MSP analysis the ZNF282 and PTPRS1 tDMR displayed semen-specific hypomethylation while HPCAL1 tDMR showed saliva-specific hypomethylation. The PTPRS 2 tDMR did not differentiate between any body fluids due to presence of methylation and unmethylation for all body fluids. With quantitative analysis by BS the ZNF282 tDMR showed statistically significant difference in overall methylation status between semen and all other body fluids as well as at individual CpG sites (p 0.05). The BS study showed that the tDMR for the HPCAL1 gene displayed non-specific amplification therefore was not further analysed. Furthermore, a sensitivity and forensic simulation study was conducted to determine the stability of methylation profiles. To determine the lowest DNA concentration that can be evaluated with MSP, a sensitivity study was conducted using five-fold serial dilution (25, 20, 15, 10, 5, 1 ng) of blood DNA samples. Each DNA dilution was subjected to bisulfite modification, followed by amplification with ZNF282, PTPRS 1, PTPRS 2, and HPCAL1 primers. The results showed that the detection limits were 10 ng for ZNF282 tDMR, 5 ng for PTPRS 1, 15 ng for PTPRS 2, and 5 ng for HPCAL1 tDMR. Thus, it was concluded that a DNA concentration greater than 10 ng would yield successful results with MSP analyses. To evaluate whether environmental conditions has an effect on the stability of methylation profiles of the ZNF282 tDMR, five samples of each body fluid were subjected to five different forensic simulated conditions (dry at room temperature, wet in an exsiccator, outside on the ground, sprayed with alcohol and sprayed with bleach) for 50 days. Following the 50 days, vaginal fluid showed highest DNA recovery under all conditions while semen had least DNA quantity. Under outside on the ground condition, all body fluids except semen showed decrease in methylation level, however, significant decrease in methylation level was observed for saliva. A statistical significant difference was observed for saliva and semen (p < 0.05) in the outside on the ground condition. No differences in methylation level were observed for the ZNF282 tDMR under all conditions for vaginal fluid samples. Thus, ZNF282 tDMR is stable under environmental insults and can be used as reliable semen-specific hypomethylated marker. The analysis of tDMRs represents a unique, efficient and reliable technique that can be used to differentiate between human body fluids. In the future, identification and validation of new tDMRs based markers as well as determining methylation differences in other forensically relevant body fluids will be beneficial for forensics applications.Supervisor on university system as Joshi, Meenu

Security in an Omni-Channel environment

Abstract: Omni-Channels ensure that customers have the same shopping experience online as they do in a physical store. Customers should be able to view products online and pay for them accordingly. Customers should also be assured that when they are paying for their items online, all their information is safe and secure. This study examines what organisations use to ensure that their customers private information remains safe at all times. Encryption, one time passwords, audit trails and various other components will be explored. The researcher will also investigate how all these components work together to ensure maximum protection of information at all times. A structured questionnaire was used to measure and rate the overall security features that are used to protect a customer when purchasing online. The results display the preferred features and functions for customers

Fair dismissals : a critical analysis of the 'appropriateness of sanction' in light of recent developments.

Thesis (LL.M.)-University of KwaZulu-Natal, Durban, 2012.No abstract available

Social media and COVID-19 – perceptions and public deceptions regarding colchicine, hydroxychloroquine and ivermectin and resultant lessons for future pandemics

Background: The capacity for social media to influence the consumption of re-purposed medicines to manage COVID-19 despite limited safety and efficacy data at the start of the pandemic is cause for concern. Objective: To ascertain links between social media reports and utilization for three re-purposed medicines (hydroxychloroquine (HCQ), ivermectin and colchicine) to direct future activities. Methods: A combined retrospective analysis of social media posts for these re-purposed medicines was performed in South Africa between January and June 2021 together with utilization and clinical trials data. Utilisation data from IQVIA from three different platforms included private and public markets. Clinical trials data was obtained from various databases. Chloroquine data was analysed in South Africa (HCQ was not available). Results: 77257 posts were collected across key social media platforms during the study period of which 6884 were relevant. Ivermectin had the highest number of posts (55%) followed by HCQ (44%), with limited posts for colchicine (1%). The spike in ivermectin utilisation was closely correlated with social media posts. Similarly, with chloroquine social media interest was enhanced by comments from local politicians. Sentiment analysis showed that the posts regarding the effectiveness of particularly ivermectin and HCQ were positive. Of concern is that the origin of the majority of reporters in social media (85%) was unidentifiable. Conclusion: This is the first study of its kind in South Africa providing evidence that social media is a driver of re-purposed medicine use. Healthcare professionals have a key role to provide evidence-based advice especially with unidentifiable posts

Social media and COVID-19 – perceptions and public deceptions of ivermectin, colchicine and hydroxychloroquine : lessons for future pandemics

The capacity for social media to influence the utilization of re-purposed medicines to manage COVID-19, despite limited availability of safety and efficacy data, is a cause for concern within health care systems. This study sought to ascertain links between social media reports and utili-zation for three re-purposed medicines: hydroxychloroquine (HCQ), ivermectin and colchicine. A combined retrospective analysis of social media posts for these three re-purposed medicines was undertaken, along with utilization and clinical trials data, in South Africa, between January 2020 and June 2021. In total, 77,257 posts were collected across key social media platforms, of which 6884 were relevant. Ivermectin had the highest number of posts (55%) followed by HCQ (44%). The spike in ivermectin use was closely correlated to social media posts. Similarly, regarding chlo-roquine (as HCQ is not available in South Africa), social media interest was enhanced by local politicians. Sentiment analysis revealed that posts regarding the effectiveness of these repurposed medicines were positive. This was different for colchicine, which contributed only a small number of mentions (1%). Of concern is that the majority of reporters in social media (85%) were uniden-tifiable. This study provides evidence of social media as a driver of re-purposed medicines. Healthcare professionals have a key role in providing evidence-based advice especially with unidentifiable post

The impact of provider-initiated (opt-out) HIV testing and counseling of patients with sexually transmitted infection in Cape Town, South Africa: a controlled trial

<p>Abstract</p> <p>Background</p> <p>The effectiveness of provider-initiated HIV testing and counseling (PITC) for patients with sexually transmitted infection (STI) in resource-constrained settings are of particular concern for high HIV prevalence countries like South Africa. This study evaluated whether the PITC approach increased HIV testing amongst patients with a new episode of sexually transmitted infection, as compared to standard voluntary counseling and testing (VCT) at the primary care level in South Africa, a high prevalence and low resource setting.</p> <p>Methods</p> <p>The design was a pragmatic cluster-controlled trial with seven intervention and 14 control clinics in Cape Town. Nurses in intervention clinics integrated PITC into standard HIV care with few additional resources, whilst lay counselors continued with the VCT approach in control clinics. Routine data were collected for a six-month period following the intervention in 2007, on new STI patients who were offered and who accepted HIV testing. The main outcome measure was the proportion of new STI patients tested for HIV, with secondary outcomes being the proportions who were offered and who declined the HIV test.</p> <p>Results</p> <p>A significantly higher proportion of new STI patients in the intervention group tested for HIV as compared to the control group with (56.4% intervention versus 42.6% control, p = 0.037). This increase was achieved despite a significantly higher proportion intervention group declining testing when offered (26.7% intervention versus 13.5% control, p = 0.0086). Patients were more likely to be offered HIV testing in intervention clinics, where providers offered the HIV test to 76.8% of new STI patients versus 50.9% in the control group (p = 0.0029). There was significantly less variation in the main outcomes across the intervention clinics, suggesting that the intervention also facilitated more consistent performance.</p> <p>Conclusions</p> <p>PITC was successful in three ways: it increased the proportion of new STI patients tested for HIV; it increased the proportion of new STI patients offered HIV testing; and it delivered more consistent performance across clinics. Recommendations are made for increasing the impact and feasibility of PITC in high HIV prevalence and resource-constrained settings. These include more flexible use of clinical and lay staff, and combining PITC with VCT and other community-based approaches to HIV testing.</p> <p>Trial registration</p> <p>Controlled trial ISRCTN93692532</p

Mitochondrial dysfunction and mitophagy defects in LRRK2-R1441C Parkinson's disease models

Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene have been identified as one of the most common genetic causes of Parkinson’s disease (PD). The LRRK2 PD-associated mutations LRRK2G2019S and LRRK2R1441C, located in the kinase domain and in the ROC-COR domain, respectively, have been demonstrated to impair mitochondrial function. Here, we sought to further our understanding of mitochondrial health and mitophagy by integrating data from LRRK2R1441C rat primary cortical and human induced pluripotent stem cell-derived dopamine (iPSC-DA) neuronal cultures as models of PD. We found that LRRK2R1441C neurons exhibit decreased mitochondrial membrane potential, impaired mitochondrial function and decreased basal mitophagy levels. Mitochondrial morphology was altered in LRRK2R1441C iPSC-DA but not in cortical neuronal cultures or aged striatal tissue, indicating a cell-type-specific phenotype. Additionally, LRRK2R1441C but not LRRK2G2019S neurons demonstrated decreased levels of the mitophagy marker pS65Ub in response to mitochondrial damage, which could disrupt degradation of damaged mitochondria. This impaired mitophagy activation and mitochondrial function were not corrected by the LRRK2 inhibitor MLi-2 in LRRK2R1441C iPSC-DA neuronal cultures. Furthermore, we demonstrate LRRK2 interaction with MIRO1, a protein necessary to stabilize and to anchor mitochondria for transport, occurs at mitochondria, in a genotype-independent manner. Despite this, we found that degradation of MIRO1 was impaired in LRRK2R1441C cultures upon induced mitochondrial damage, suggesting a divergent mechanism from the LRRK2G2019S mutation

Prioritizing Land and Sea Conservation Investments to Protect Coral Reefs

Background: Coral reefs have exceptional biodiversity, support the livelihoods of millions of people, and are threatened by multiple human activities on land (e.g. farming) and in the sea (e.g. overfishing). Most conservation efforts occur at local scales and, when effective, can increase the resilience of coral reefs to global threats such as climate change (e.g. warming water and ocean acidification). Limited resources for conservation require that we efficiently prioritize where and how to best sustain coral reef ecosystems

Mammal responses to global changes in human activity vary by trophic group and landscape

Wildlife must adapt to human presence to survive in the Anthropocene, so it is critical to understand species responses to humans in different contexts. We used camera trapping as a lens to view mammal responses to changes in human activity during the COVID-19 pandemic. Across 163 species sampled in 102 projects around the world, changes in the amount and timing of animal activity varied widely. Under higher human activity, mammals were less active in undeveloped areas but unexpectedly more active in developed areas while exhibiting greater nocturnality. Carnivores were most sensitive, showing the strongest decreases in activity and greatest increases in nocturnality. Wildlife managers must consider how habituation and uneven sensitivity across species may cause fundamental differences in human–wildlife interactions along gradients of human influence.Peer reviewe

Dolutegravir twice-daily dosing in children with HIV-associated tuberculosis: a pharmacokinetic and safety study within the open-label, multicentre, randomised, non-inferiority ODYSSEY trial

Background: Children with HIV-associated tuberculosis (TB) have few antiretroviral therapy (ART) options. We aimed to evaluate the safety and pharmacokinetics of dolutegravir twice-daily dosing in children receiving rifampicin for HIV-associated TB. Methods: We nested a two-period, fixed-order pharmacokinetic substudy within the open-label, multicentre, randomised, controlled, non-inferiority ODYSSEY trial at research centres in South Africa, Uganda, and Zimbabwe. Children (aged 4 weeks to <18 years) with HIV-associated TB who were receiving rifampicin and twice-daily dolutegravir were eligible for inclusion. We did a 12-h pharmacokinetic profile on rifampicin and twice-daily dolutegravir and a 24-h profile on once-daily dolutegravir. Geometric mean ratios for trough plasma concentration (Ctrough), area under the plasma concentration time curve from 0 h to 24 h after dosing (AUC0–24 h), and maximum plasma concentration (Cmax) were used to compare dolutegravir concentrations between substudy days. We assessed rifampicin Cmax on the first substudy day. All children within ODYSSEY with HIV-associated TB who received rifampicin and twice-daily dolutegravir were included in the safety analysis. We described adverse events reported from starting twice-daily dolutegravir to 30 days after returning to once-daily dolutegravir. This trial is registered with ClinicalTrials.gov (NCT02259127), EudraCT (2014–002632-14), and the ISRCTN registry (ISRCTN91737921). Findings: Between Sept 20, 2016, and June 28, 2021, 37 children with HIV-associated TB (median age 11·9 years [range 0·4–17·6], 19 [51%] were female and 18 [49%] were male, 36 [97%] in Africa and one [3%] in Thailand) received rifampicin with twice-daily dolutegravir and were included in the safety analysis. 20 (54%) of 37 children enrolled in the pharmacokinetic substudy, 14 of whom contributed at least one evaluable pharmacokinetic curve for dolutegravir, including 12 who had within-participant comparisons. Geometric mean ratios for rifampicin and twice-daily dolutegravir versus once-daily dolutegravir were 1·51 (90% CI 1·08–2·11) for Ctrough, 1·23 (0·99–1·53) for AUC0–24 h, and 0·94 (0·76–1·16) for Cmax. Individual dolutegravir Ctrough concentrations were higher than the 90% effective concentration (ie, 0·32 mg/L) in all children receiving rifampicin and twice-daily dolutegravir. Of 18 children with evaluable rifampicin concentrations, 15 (83%) had a Cmax of less than the optimal target concentration of 8 mg/L. Rifampicin geometric mean Cmax was 5·1 mg/L (coefficient of variation 71%). During a median follow-up of 31 weeks (IQR 30–40), 15 grade 3 or higher adverse events occurred among 11 (30%) of 37 children, ten serious adverse events occurred among eight (22%) children, including two deaths (one tuberculosis-related death, one death due to traumatic injury); no adverse events, including deaths, were considered related to dolutegravir. Interpretation: Twice-daily dolutegravir was shown to be safe and sufficient to overcome the rifampicin enzyme-inducing effect in children, and could provide a practical ART option for children with HIV-associated TB