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The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.The comet assay is commonly used to assess DNA damage. This collection of consensus protocols includes adaptations for a wide range of species and sample types, assay formats and detection of different types of DNA lesions.

Homografts are routinely stored by cryopreservation; however, donor cells and remnants contribute to immunogenicity. Although decellularization strategies can address immunogenicity, additional fixation might be required to maintain strength. This study investigated the effect of cryopreservation, decellularization, and decellularization with additional glutaraldhyde fixation on the strength and structure of ovine pulmonary homografts harvested 48 h post-mortem. Cells and cellular remnants were present for the cryopreserved group, while the decellularized groups were acellular. The decellularized group had large interfibrillar spaces in the extracellular matrix with uniform collagen distribution, while the additional fixation led to the collagen network becoming dense and compacted. The collagen of the cryopreserved group was collapsed and appeared disrupted and fractured. There were no significant differences in strength and elasticity between the groups. Compared to cryopreservation, decellularization without fixation can be considered an alternative processing technique to maintain a well-organized collagen matrix and tissue strength of homografts.

Purpose of review Iron deficiency remains highly prevalent in women and young children in low-income and middle-income countries. To prevent the potentially life-long consequences of iron deficiency when occurring during early life, the WHO recommends iron supplementation of pregnant women and young children. However, increasing evidence of limited efficacy and risk of current iron intervention strategies are cause of concern. This review aims to highlight recent advances and challenges of established and novel intervention strategies for the prevention of iron deficiency during the first 1000 days in low-income and middle-income countries. Recent findings Recent meta-analyses and trials challenged the WHO's current recommendation to provide iron-folic acid rather than multiple micronutrient supplements during routine antenatal care. Furthermore, several studies explored optimal windows for iron supplementation, such as prior to conception. Studies are demonstrating that infectious and noninfectious inflammation is compromising the efficacy of iron interventions in vulnerable groups. Therefore, strategies addressing iron deficiency should focus on targeting infection and inflammation while simultaneously providing additional iron. Furthermore, both iron deficiency and iron supplementation may promote an unfavourable gut microbiota. Recent trials in infants indicate that the provision of a prebiotic together with iron may alleviate the adverse effects of iron on the gut microbiome and gut inflammation, and may even enhance iron absorption. Summary Recent studies highlight the need for and potential of novel intervention strategies that increase the efficacy and limit the potential harm of universal iron supplementation.

Reprogramming of cellular metabolism is now considered one of the hallmarks of cancer. Most malignant cells Reprogramming of cellular metabolism is now considered one of the hallmarks of cancer. Most malignant cells present with altered energy metabolism which is associated with elevated reactive oxygen species (ROS) generation. This is also evident for melanoma, the leading cause of skin cancer related deaths. Altered mechanisms affecting mitochondrial bioenergetics pose attractive targets for novel anti-cancer therapies. Antimicrobial peptides have been shown to exhibit selective anti-cancer activities. In this study, the anti-melanoma potential of the antimicrobial peptide, nisin Z, was evaluated in vitro. Nisin Z was shown to induce selective toxicity in melanoma cells compared to non-malignant keratinocytes. Furthermore, nisin Z was shown to negatively affect the energy metabolism (glycolysis and mitochondrial respiration) of melanoma cells, increase reactive oxygen species generation and cause apoptosis. Results also indicate that nisin Z can decrease the invasion and proliferation of melanoma cells demonstrating its potential use against metastasis associated with melanoma. As nisin Z seems to place a considerable extra burden on the energy metabolism of melanoma cells, combination therapies with known anti-melanoma agents may be effective treatment options

Antimicrobial resistance is an imminent threat to the effective prevention and treatment of bacterial infections and alternative antimicrobial strategies are desperately needed. Antimicrobial peptides (AMPs) may be promising alternatives to current antibiotics or act as adjuvants to enhance antibiotic potency. Additionally, the use of biodegradable lipid nanoparticles can enhance the antibacterial activity of antibiotics and antimicrobial peptides. In this study, the interaction of the AMPs, nisin Z and melittin, with conventional antibiotics was investigated on Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli. The effectiveness of nanostructured lipid carriers (NLCs) for the entrapment of nisin Z was also evaluated. Findings revealed that nisin Z exhibited additive interactions with numerous conventional antibiotics. Notable synergism was observed for novobiocin-nisin Z combinations. The addition of the non-antibiotic adjuvant EDTA significantly improved the antimicrobial activity of free nisin Z towards E.coli. NLCs containing nisin Z were effective against Gram-positive species at physiological pH, with an increase in effectiveness in the presence of EDTA. Results indicate that nisin Z may be advantageous as an adjuvant in antimicrobial chemotherapy, while contributing in the battle against antibiotic resistance. NLCs have the potential to enhance the antibacterial activity of nisin Z towards Gram-positive bacterial species associated with skin infections.

The most frequently occurring cancers are those of the skin, with melanoma being the leading cause of death due to skin cancer. Breakthroughs in chemotherapy have been achieved in certain cases, though only marginal advances have been made in treatment of metastatic melanoma. Strategies aimed at inducing redox dysregulation by use of reactive oxygen species (ROS) inducers present a promising approach to cancer chemotherapy. Here we use a rational combination of an oxidant drug combined with a redox or pro-oxidant drug to optimize the cytotoxic effect. Thus we demonstrate for the first time enhanced activity of the amino-artemisinin artemisone and novel prenylated piperazine derivatives derived from dihydroartemisinin as the oxidant component, and elesclomol-Cu(II) as the redox component, against human malignant melanoma cells A375 in vitro. The combinations caused a dose dependent decrease in cell numbers and increase in apoptosis. The results indicate that oxidant-redox drug combinations have considerable potential and warrant further investigation.

Antibiotic resistance is an imminent threat to the effective treatment of bacterial infections, and alternative antibiotic strategies are urgently required. The golden epoch of antibiotics is coming to an end, and the development of new therapeutic agents to combat bacterial infections should be prioritized. This article will review the potential of antimicrobial peptides (AMPs) to combat the threat of antimicrobial resistance. The modern-day antimicrobial resistance dilemma is briefly discussed followed by a review of the potential of AMPs to be used alone or in combination with current antibiotics in order to enhance antibacterial properties of antibiotics while also potentially combatting resistance. This article reiterates that many AMPs exhibit direct microbial killing activity and also play an integral role in the innate immune system. These properties make AMPs attractive alternative antimicrobial agents. Furthermore, AMPs are promising candidates to be used as adjuvants in combination with current antibiotics in order to combat antibiotic resistance. Combinations of AMPs and antibiotics are less likely to develop resistance or transmit cross-resistance. The further identification and therapeutic development of AMPs and antibiotic-AMP combinations are strongly recommended.

PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2018 The rise in antibiotic resistance and the lack of the development of new antibiotics poses a considerable threat to human health. This is especially of concern in individuals who are immune-compromised (due to immunosuppressive diseases or chemotherapy). There is a desperate need for intervention aimed at strengthening our current arsenal of antibiotics or developing new antibiotics to prevent high death rates due to infections with antibiotic resistant superbugs. In spite of the higher susceptibility to bacterial infections in cancer patients undergoing chemotherapy, resistance to conventional chemotherapy agents also poses a threat to the successful treatment of cancer. Antimicrobial peptides (AMPs) are multifunctional and several peptides have both antibacterial and anticancer activities, as well as displaying immune-modulatory properties. Therefore, AMPs may be considered alternatives to antibiotics/chemotherapy agents or as adjuvants to conventional antibiotics/chemotherapy agents. In this thesis, the antibacterial as well as anticancer activities of the Generally Regarded as Safe (GRAS) status AMP, nisin Z, were evaluated. The antibacterial activity was assessed with regard to the interaction of nisin Z with conventional antibiotics on Staphylococcus aureus, S. epidermidis and Escherichia coli. Additionally, the use of biodegradable lipid nanoparticles has been shown to enhance the antibacterial activity of antibiotics and AMPs. Therefore, the effectiveness of nanostructured lipid carriers (NLCs) for the entrapment of nisin Z was also assessed. The anticancer activity of nisin Z was evaluated against cultured melanoma cells. Reprogramming of cellular metabolism is now considered one of the hallmarks of cancer. Most malignant cells present with altered energy metabolism which is associated with elevated reactive oxygen species (ROS) generation. This is also evident for melanoma, the leading cause of skin cancer related deaths. Altered mechanisms affecting mitochondrial bioenergetics pose attractive targets for novel anticancer therapies. In this study, the anti-melanoma potential of nisin Z was evaluated in vitro. The underlying anticancer mechanism of nisin Z with regard to the ability of this AMP to induce ROS production, apoptosis, disrupt the energy metabolism (glycolysis and mitochondrial respiration) and inhibit cell proliferation and invasion of melanoma cells was investigated. Likewise, the ability of nisin Z to enhance the cytotoxicity and selectivity of conventional chemotherapeutic agents was also investigated. Finally, synergistic interactions between nisin Z and conventional chemotherapeutic agents were examined. Results indicated that nisin Z exhibited additive interactions with numerous conventional antibiotics. Notable synergism was observed for novobiocin-nisin Z combinations. The addition of the non-antibiotics adjuvant ethylenediaminetetraacetic acid (EDTA) significantly improved the antimicrobial activity of free nisin Z towards E.coli. NLCs containing nisin Z were effective against Gram positive species at physiological pH, with an increase in effectiveness in the presence of EDTA. Results indicate that nisin Z may be advantageous as an adjuvant in antimicrobial chemotherapy, while contributing in the battle against antibiotic resistance. NLCs have the potential to enhance the antibacterial activity of nisin Z towards Gram-positive bacterial species associated with skin infections. The minimum inhibitory concentrations (MICs) and half maximal inhibitory concentrations (IC50) were used as a measure of the toxicity and selectivity of nisin Z to bacterial and mammalian cells, respectively. Based on the results from this study, nisin Z displays selective toxicity to bacterial and cancer cells, compared to non-malignant cells. Furthermore, nisin Z was shown to negatively affect the energy metabolism (glycolysis and mitochondrial respiration) of melanoma cells, increase ROS production and cause apoptosis. Results also indicate that nisin Z can decrease the invasion and proliferation of melanoma cells demonstrating its potential use against metastasis associated with melanoma. In the current study it was found that combinations of nisin Z with 5-fluoruracil, hydroxy urea and etoposide were able to enhance the cytotoxicity of these conventional chemotherapeutic agents to melanoma cells. The etoposide-nisin Z combination also displayed a synergistic interaction. In conclusion nisin Z with its GRAS status, in addition to displaying direct antibacterial and anticancer properties, shows great potential to be used as an adjuvant with conventional antibiotics and chemotherapy agents National Research Foundation (NRF) North-West University (NWU) Doctoral

In the Krebs cycle, succinate is oxidized to fumarate by succinate dehydrogenase (SDH), followed by the conversion of fumarate to malate by fumarate hydratase (FH). In cells with defective SDH and FH, the Krebs cycle is congested, respiration impaired and fumarate and succinate accumulates. Several studies have indicated that the accumulation of these substrates are associated with cytotoxicity and oncogenesis. High levels of succinate and fumarate induce hypoxia inducible factor (HIF1A) hydroxylases, leading to the activation of oncogenic HIF pathways. However, the role of HIF as primary inducer of oncogenic change has been questioned, as other non-enzymatic mechanisms have been shown to interfere with cellular metabolism, cell signalling as well as disrupting protein function. Owing to the essential roles that SDH and FH play in cellular energy metabolism, and their associated tumor suppressor capacity, it is vital to understand the biochemical effects resulting from the accumulation of their associated metabolites. Therefore, in this study, we investigated the effect of high concentrations of succinate and fumarate exposure on cell viability, genome integrity and global DNA methylation using a human hepatocellular carcinoma (HepG2) cell culture model. It was found that relatively high concentrations of succinate and fumarate cause a loss of cell viability, which seems to be orchestrated through an apoptotic pathway. Cells exposed to high levels of succinate also presented with elevated caspase 3 and/or caspase 7 levels. In addition, elevated levels of fumarate lead to extensive DNA fragmentation, which may contribute pathophysiologically by inducing chromosomal instability, while succinate demonstrated lower genotoxicity. Furthermore, both succinate and fumarate altered the global DNA methylation patterns via significant DNA hypermethylation. Since numerous studies have reported correlations between aberrant DNA methylation and oncogenesis, hypermethylation may contribute to the oncogenesis observed in cells exposed to high concentrations of these metabolites.

Recently, research into the development of new antimicrobial agents has been driven by the increase in resistance to traditional antibiotics and Emerging Infectious Diseases. Antimicrobial peptides (AMPs) are promising candidates as alternatives to current antibiotics in the treatment and prevention of microbial infections. AMPs are produced by all known living species, displaying direct antimicrobial killing activity and playing an important role in innate immunity. To date, more than 2000 AMPs have been discovered and many of these exhibit broad-spectrum antibacterial, antiviral and anti-parasitic activity. Neglected tropical diseases (NTDs) are caused by a variety of pathogens and are particularly wide-spread in low-income and developing regions of the world. Alternative, cost effective treatments are desperately needed to effectively battle these medically diverse diseases. AMPs have been shown to be effective against a variety of NTDs, including African trypanosomes, leishmaniosis and Chagas disease, trachoma and leprosy. In this review, the potential of selected AMPs to successfully treat a variety of NTD infections will be critically evaluated National Research Foundation (NRF) of South Africa