Comparing DNA yield from fish scales following different extraction protocols.

Studies on genetic diversity, adaptive potential and fitness of species have become a major tool in conservation biology. These studies require biological material containing a reliable source of DNA which can be extracted and analysed. Recently, non-invasive sampling has become the preferred sampling method of such biological material; particularly when studying endangered species. Elasmoid scales from teleost fish are an example of non-invasive samples from which DNA can successfully be extracted. This study compared different extraction protocols to find an optimal method for extracting DNA from teleost fish scales. This was done with the intent to use the protocol that yielded the highest quantity of DNA on dried, archived scales. The protocols tested in this study included (1) phenol/chloroform with a TNES-urea digestion buffer, (2) phenol/chloroform with an amniocyte digestion buffer and (3) Qiagen DNeasy Blood and Tissue Kit with variations in incubation times and temperatures of each protocol. While the phenol/chloroform with TNES-urea digestion buffer yielded significantly higher concentrations of DNA compared to the other protocols, all protocols followed in this study yielded sufficient quantities of DNA for further downstream applications. Therefore, while there are multiple viable options when selecting a DNA extraction protocol, each research project's individual needs, requirements and resources need to be carefully considered in order to choose the most effective protocol.

Elucidating the Antimycobacterial Mechanism of Action of Decoquinate Derivative RMB041 Using Metabolomics.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), still remains one of the leading causes of death from a single infectious agent worldwide. The high prevalence of this disease is mostly ascribed to the rapid development of drug resistance to the current anti-TB drugs, exacerbated by lack of patient adherence due to drug toxicity. The aforementioned highlights the urgent need for new anti-TB compounds with different antimycobacterial mechanisms of action to those currently being used. An N-alkyl quinolone; decoquinate derivative RMB041, has recently shown promising antimicrobial activity against Mtb, while also exhibiting low cytotoxicity and excellent pharmacokinetic characteristics. Its exact mechanism of action, however, is still unknown. Considering this, we used GCxGC-TOFMS and well described metabolomic approaches to analyze and compare the metabolic alterations of Mtb treated with decoquinate derivative RMB041 by comparison to non-treated Mtb controls. The most significantly altered pathways in Mtb treated with this drug include fatty acid metabolism, amino acid metabolism, glycerol metabolism, and the urea cycle. These changes support previous findings suggesting this drug acts primarily on the cell wall and secondarily on the DNA metabolism of Mtb. Additionally, we identified metabolic changes suggesting inhibition of protein synthesis and a state of dormancy.

Elucidating the Antimycobacterial Mechanism of Action of Ciprofloxacin Using Metabolomics.

In the interest of developing more effective and safer anti-tuberculosis drugs, we used a GCxGC-TOF-MS metabolomics research approach to investigate and compare the metabolic profiles of Mtb in the presence and absence of ciprofloxacin. The metabolites that best describe the differences between the compared groups were identified as markers characterizing the changes induced by ciprofloxacin. Malic acid was ranked as the most significantly altered metabolite marker induced by ciprofloxacin, indicative of an inhibition of the tricarboxylic acid (TCA) and glyoxylate cycle of Mtb. The altered fatty acid, myo-inositol, and triacylglycerol metabolism seen in this group supports previous observations of ciprofloxacin action on the Mtb cell wall. Furthermore, the altered pentose phosphate intermediates, glycerol metabolism markers, glucose accumulation, as well as the reduction in the glucogenic amino acids specifically, indicate a flux toward DNA (as well as cell wall) repair, also supporting previous findings of DNA damage caused by ciprofloxacin. This study further provides insights useful for designing network whole-system strategies for the identification of possible modes of action of various drugs and possibly adaptations by Mtb resulting in resistance.

The effect of Aspalathus linearis (Burm.f.) R.Dahlgren and its compounds on tyrosinase and melanogenesis.

Pigmentation, a process controlled by melanogenesis, plays a vital role in protecting the skin against harmful ultraviolet rays. The level of protection is compromised in case of hypopigmentation. This study aimed to evaluate an Aspalathus linearis extract, fractions and phytoconstituents, for their efficacy on melanogenesis stimulation. Fifteen compounds were kinetically assessed against tyrosinase; the rate-limiting enzyme of melanogenesis. Aspalathin and catechin significantly (p value < 0.001) increased the enzymatic rate, showing 50% stimulatory effects at 119.70 ± 2.06 µg/mL and 143.30 ± 2.74 µg/mL, respectively, by acting as subversive substrates. Five compounds inhibited the enzyme's activity, of which four exhibited competitive inhibition. To investigate the molecular interactions between the compounds and the active site, molecular docking was done, using tyrosinase (PBD: 2Y9X) and tyrosinase related protein 1 (PBD: 5M8P). All the compounds docked successfully with acceptable docking scores. Further quantitative structure-activity relationship analysis identified potential functional groups, linked to the specific activity. The crude extract, its fractions, and compounds exhibited low antiproliferative activity with 50% cell viability at concentrations higher than 100 µg/mL. Finally, both aspalathin and catechin exhibited a significant increase (4.5%) in melanin production at 119.82 µg/mL and 76.92 µg/mL, respectively. This is the first report of A. linearis' compounds on skin re-pigmentation.

Anti-SARS-CoV Natural Products With the Potential to Inhibit SARS-CoV-2 (COVID-19).

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), known to cause the disease COVID-19, was declared a pandemic in early 2020. The objective of this review was to collate information regarding the potential of plants and natural products to inhibit coronavirus and targets associated with infection in humans and to highlight known drugs, which may have potential activity against SARS-CoV-2. Due to the similarity in the RNA genome, main proteases, and primary host receptor between SARS-CoV and SARS-CoV-2, a review was conducted on plants and secondary metabolites, which have shown activity against SARS-CoV. Numerous scientific reports on the potential of plants and secondary metabolites against SARS-CoV infection were found, providing important information on their possible activity against SARS-CoV-2. Based on current literature, 83 compounds have been identified with the potential to inhibit COVID-19. The most prominent selectivity was found for the alkaloid, lycorine, the lignan, savinin, and the abietane terpenoid, 8-beta-hydroxyabieta-9(11),13-dien-12-one with selectivity index values greater than 945, 667, and 510, respectively. Plants and their secondary metabolites, with activity against targets associated with the SARS-CoV infection, could provide valuable leads for the development into drugs for the novel SARS-CoV-2. The prospects of using computational methods to screen secondary metabolites against SARS-CoV targets are briefly discussed, and the drawbacks have been highlighted. Finally, we discuss plants traditionally used in Southern Africa for symptoms associated with respiratory viral infections and influenza, such as coughs, fever, and colds. However, only a few of these plants have been screened against SARS-CoV. Natural products hold a prominent role in discovering novel therapeutics to mitigate the current COVID-19 pandemic; however, further investigations regarding in vitro, in vivo, pre-clinical, and clinical phases are still required.

Selection on age of female reproduction in the marula fruit fly, Ceratitis cosyra (Walker) (Diptera: Tephritidae), decreases total antioxidant capacity and lipid peroxidation.

The oxidative damage caused to cells by Reactive Oxygen Species (ROS) is one of several factors implicated in causing ageing. Oxidative damage may also be a proximate cost of reproductive effort that mediates the trade-off often observed between reproduction and survival. However, how the balance between oxidative damage and antioxidant protection affects life-history strategies is not fully understood. To improve our understanding, we selected on female reproductive age in the marula fruit fly, Ceratitis cosyra, and quantified the impact of selection on female and male mortality risk, female fecundity, male sperm transfer, calling and mating. Against expectations, upward-selected lines lived shorter lives and experienced some reductions in reproductive performance. Selection affected oxidative damage to lipids and total antioxidant protection, but not in the direction predicted; longer lives were associated with elevated oxidative damage, arguing against the idea that accumulated oxidative damage reduces lifespan. Greater reproductive effort was also associated with elevated oxidative damage, suggesting that oxidative damage may be a cost of reproduction, although one that did not affect survival. Our results add to a body of data showing that the relationship between lifespan, reproduction and oxidative damage is more complex than predicted by existing theories.

Predictive Binding Affinity of Plant-Derived Natural Products Towards the Protein Kinase G Enzyme of Mycobacterium tuberculosis (MtPknG).

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a growing public health concern worldwide, especially with the emerging challenge of drug resistance to the current drugs. Efforts to discover and develop novel, more effective, and safer anti-TB drugs are urgently needed. Products from natural sources, such as medicinal plants, have played an important role in traditional medicine and continue to provide some inspiring templates for the design of new drugs. Protein kinase G, produced by M. tuberculosis (MtPKnG), is a serine/threonine kinase, that has been reported to prevent phagosome-lysosome fusion and help prolong M. tuberculosis survival within the host's macrophages. Here, we used an in silico, target-based approach (docking) to predict the interactions between MtPknG and 84 chemical constituents from two medicinal plants (Pelargonium reniforme and Pelargonium sidoides) that have a well-documented historical use as natural remedies for TB. Docking scores for ligands towards the target protein were calculated using AutoDock Vina as the predicted binding free energies. Ten flavonoids present in the aerial parts of P. reniforme and/or P. sidoides showed docking scores ranging from -11.1 to -13.2 kcal/mol. Upon calculation of all ligand efficiency indices, we observed that the (-G/MW) ligand efficiency index for flavonoids (4), (5) and (7) was similar to the one obtained for the AX20017 control. When taking all compounds into account, we observed that the best (-G/MW) efficiency index was obtained for coumaric acid, coumaraldehyde, p-hydroxyphenyl acetic acid and p-hydroxybenzyl alcohol. We found that methyl gallate and myricetin had ligand efficiency indices superior and equal to the AX20017 control efficiency, respectively. It remains to be seen if any of the compounds screened in this study exert an effect in M. tuberculosis-infected macrophages.

Sideritis Perfoliata (Subsp. Perfoliata) Nutritive Value and Its Potential Medicinal Properties.

Sideritis perfoliata L. subsp. perfoliata is an endemic species of the Eastern Mediterranean region with several uses in traditional medicine. The present study aims to explore the unknown properties of S. perfoliata investigating the nutritional content as well as the antioxidant, anticancer, antituberculosis, antiwrinkle, anti-acne, hyper/hypo-pigmentation and antibacterial activities. Mineral content, nutritional value, the composition and antioxidant properties of the essential oil, the antityrosinase, the antibacterial activity and anti-elastase potential of the extract, were evaluated. The antiproliferative activity of S. perfoliata against cervical cancer (HeLa), human melanoma (UCT-Mel-1), human hepatocellular carcinoma (HepG2) and human epidermoid carcinoma (A431) was investigated. Cytotoxic effects on normal human keratinocyte (HaCat) and kidney epithelial (Vero) cell lines were also determined. Sideritis perfoliata exhibited high nutritional value of proteins and minerals (K, P, Mg, Fe, Zn, Cu). The most abundant components of the essential oil were found to be α-pinene, ß-phelladrene, valeranone, ß-pinene and sabinene. The ethanolic extract of S. perfoliata displayed moderate antioxidant potential and antibacterial activity against Prevotella intermedia. Noteworthy elastase and moderate anticancer potential against the human liver cancer cell line (HepG2) was observed with IC50 values of 57.18 ± 3.22 µg/mL and 64.27 ± 2.04 µg/mL respectively. The noteworthy in vitro activity of S. perfoliata could be due to the presence of flavonoids and phenols in the leaves, having high nutritional value. Sideritis perfoliata could potentially be useful to reduce the appearance of wrinkles and for the treatment of liver cancer. The moderate antibacterial, antioxidant and elastase activity of the plant can be linked to the traditional use of S. perfoliata for the treatment of wounds and inflammation.

Finding stories in noise: Mitochondrial portraits from RAD data.

Mitochondrial DNA (mtDNA) has formed the backbone of phylogeographic research for many years; however, recent trends focus on genome-wide analyses. One method proposed for calibrating inferences from noisy next-generation data, such as RAD sequencing, is to compare these results with analyses of mitochondrial sequences. Most researchers using this approach appear to be unaware that many single nucleotide polymorphisms (SNPs) identified from genome-wide sequence data are themselves mitochondrial, or assume that these are too few to bias analyses. Here, we demonstrate two methods for mining mitochondrial markers using RAD sequence data from three South African species of yellowfish, Labeobarbus. First, we use a rigorous SNP discovery pipeline using the program stacks, to identify variant sites in mtDNA, which we then combine into haplotypes. Second, we directly map sequence reads against a mitochondrial genome reference. This method allowed us to reconstruct up to 98% of the Labeobarbus mitogenome. We validated these mitogenome reconstructions through blast database searches and by comparison with cytochrome b gene sequences obtained through Sanger sequencing. Finally, we investigate the organismal consequences of these data including ancient genetic exchange and a recent translocation among populations of L. natalensis, as well as interspecific hybridization between L. aeneus and L. kimberleyensis.

Exploring the phylogeography of a hexaploid freshwater fish by RAD sequencing.

The KwaZulu-Natal yellowfish (Labeobarbus natalensis) is an abundant cyprinid, endemic to KwaZulu-Natal Province, South Africa. In this study, we developed a single-nucleotide polymorphism (SNP) dataset from double-digest restriction site-associated DNA (ddRAD) sequencing of samples across the distribution. We addressed several hidden challenges, primarily focusing on proper filtering of RAD data and selecting optimal parameters for data processing in polyploid lineages. We used the resulting high-quality SNP dataset to investigate the population genetic structure of L. natalensis. A small number of mitochondrial markers present in these data had disproportionate influence on the recovered genetic structure. The presence of singleton SNPs also confounded genetic structure. We found a well-supported division into northern and southern lineages, with further subdivision into five populations, one of which reflects north-south admixture. Approximate Bayesian Computation scenario testing supported a scenario where an ancestral population diverged into northern and southern lineages, which then diverged to yield the current five populations. All river systems showed similar levels of genetic diversity, which appears unrelated to drainage system size. Nucleotide diversity was highest in the smallest river system, the Mbokodweni, which, together with adjacent small coastal systems, should be considered as a key catchment for conservation.

Diallyl Polysulfides from Allium sativum as Immunomodulators, Hepatoprotectors, and Antimycobacterial Agents.

Mycobacterium tuberculosis remains one of the world's deadliest killers, with an annual death rate of ∼1.5 million. The medicinal effects of garlic have been well documented, and natural products have been shown to have antimycobacterial activity. The current study evaluated the efficacy of six Allium sativum L. polysulfide mixtures as antimycobacterial agents together with their cytotoxic, immunomodulatory, and hepatoprotective activities. The microtitre PrestoBlue assay was used to determine the minimum inhibitory concentrations (MIC). Cytotoxicity was evaluated by using peripheral blood mononuclear cells (PBMC). Excreted cytokine levels were determined by utilizing an enzyme-linked immunosorbent assay (ELISA), by exposing isolated PBMCs to varying concentrations of polysulfide mixtures. Human C3A liver cells were utilized in the hepatoprotective study, to assess the protective effect against the toxicity induced by acetaminophen. Samples with higher amounts of diallyl trisulfide (Sample G4) showed the highest antimycobacterial activity, exhibiting an MIC of 2.5 µg/mL against M. tuberculosis H37Rv. Five samples showed moderate toxicity in PBMC, with G1 showing no toxicity. The selective index of G4 was the highest, with a selectivity index close to one. Two samples, G3 and G6 containing higher amounts of diallyl tetrasulfide and lower amounts of diallyl trisulfide, showed >50% hepatoprotection. This is comparable to a hepatoprotective agent, Silymarin, which showed a hepatoprotective effect of 30% at the tested concentration. Diallyl tetrasulfide showed significant antimycobacterial activity. A combination of higher diallyl tetrasulfide and lower diallyl trisulfide was indicative of hepatoprotective activity.

In vitro and In vivo antimycobacterial, hepatoprotective and immunomodulatory activity of Euclea natalensis and its mode of action.

ETHNOPHARMACOLOGICAL RELEVANCE: The Natal gwarri or Natal ebony (Euclea natalensis A.DC.) is a deciduous tree found widespread throughout southern Africa, especially in Kwazulu-Natal and the southern cost. It has been widely used by indigenous communities such as the Zulus, Tsongas and Vendas for symptoms related to tuberculosis (TB). The decoctions made from the plant parts are administered for chest diseases to treat complications such as chest pains, bronchitis, pleurisy and asthma. TB is prevalent in immune-compromised patients and it is evident that TB-drugs cause hepatotoxicity. The objective of the present study was therefore to evaluate the antimycobacterial activity of the ethanolic extract of E. natalensis against TB and its hepatoprotective and immunomodulatory activities. MATERIALS AND METHODS: The antimycobacterial, antioxidant, hepatoprotective, immunomodulatory activity and cytotoxicity of the ethanolic extract of the shoots of E. natalensis were determined in vitro. The mechanism of action of the antituberculosis activity was determined by investigating the inhibitory effect on mycothiol disulfide reductase enzyme. Furthermore, the acute, sub-acute toxicity (50-2000mg/kg) and antimycobacterial effect (300mg/kg) of E. natalensis shoot extract were investigated in Balb/c mice. Hepatoprotective activity of the extract (50-150mg/kg) was evaluated on isoniazid and rifampicin (50mg/kg; i.p.) induced hepatic damage in a rat model. RESULTS: The minimum inhibitory concentration of the extract was found to be 125µg/ml against Mycobacterium tuberculosis. The extracts 50% inhibitory concentration (IC50) against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical was found to be 22.55µg/ml. The plant showed a hepatoprotective effect (50% at 12.5µg/ml) and the ability to increase T-helper 1 cell cytokines; Interleukin 12, Interleukin 2 and Interferon α by up to 12 fold and the ability to decrease the T-helper 2 cell cytokine Interleukin 10 4 fold when compared to baseline cytokine production. No cellular toxicity was observed in primary peripheral blood mononuclear cells (PBMC's) and two secondary cell lines; U937 monocytes and Chang liver cells (a derivative of the HepG2 cell line). During mechanistic studies, the extract showed a 50% inhibition of mycothiol reductase activity at 38.62µg/ml. During the acute and sub-acute studies, E. natalensis exhibited no toxic effect and the 50% lethal dose (LD50) was established to be above 2000mg/kg. The extract was able to reduce the mycobacterial load (1.5-fold reduction) in infected mice. Isoniazid and rifampicin caused significant hepatic damage in rats, and the extract was able to reduce the toxicity by 15% and 40% at 50 and 150mg/kg respectively. CONCLUSION: The present study supports the traditional usage of the plant against tuberculosis symptoms. The study showed the ability of E. natalensis shoot extract to inhibit mycobacterial growth, stimulate an appropriate immune response and have a hepatic protective effect. Due to the extract's significant results for hepatoprotective, immunomodulatory effects and antimycobacterial activity, it may prove to be effective to serve as an adjuvant for TB-patients.

Viability Reagent, PrestoBlue, in Comparison with Other Available Reagents, Utilized in Cytotoxicity and Antimicrobial Assays.

This study compared different commercially available viability reagents. The growth indicator reagents include p-iodonitrotetrazolium violet (INT), PrestoBlue, and Alamar Blue which were used for antimicrobial analysis against Streptococcus mutans, Prevotella intermedia, Propionibacterium acnes, and Mycobacterium tuberculosis. PrestoBlue and Alamar Blue are resazurin based reagents that resulted in a quick and easily distinguishable colour change that allowed for visual readings. INT and Sodium 3'-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate (XTT) are tetrazolium based reagents which are converted to a formazan dye in the presence of metabolically active mitochondria enzyme. For cell viability analysis, reagents XTT and PrestoBlue were compared. PrestoBlue was able to clearly indicate the minimum inhibitory concentration (MIC) of various positive drug controls on various microbial strains. PrestoBlue was also a good indicator of the 50% inhibitory concentration (IC50) of positive drug controls on various cell lines.