Methylome analysis using MeDIP-seq with low DNA concentrations

DNA methylation is an epigenetic mark that has a crucial role in many biological processes. To understand the functional consequences of DNA methylation on phenotypic plasticity, a genome-wide analysis should be embraced. This in turn requires a technique that balances accuracy, genome coverage, resolution and cost, yet is low in DNA input in order to minimize the drain on precious samples. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) fulfils these criteria, combining MeDIP with massively parallel DNA sequencing. Here we report an improved protocol using 100-fold less genomic DNA than that commonly used. We show comparable results for specificity (>97%) and enrichment (>100-fold) over a wide range of DNA concentrations (5,000-50 ng) and demonstrate the utility of the protocol for the generation of methylomes from rare bone marrow cells using 160-300 ng of starting DNA. The protocol described here, i.e., DNA extraction to generation of MeDIP-seq library, can be completed within 3-5 d.     

Mutational analysis of the subunit C (Vma5p) of the yeast vacuolar H+-ATPase.

Subunit C is a V(1) sector subunit found in all vacuolar H(+)-ATPases (V-ATPases) that may be part of the peripheral stalk connecting the peripheral V(1) sector with the membrane-bound V(0) sector of the enzyme (Wilkens, S., Vasilyeva, E., and Forgac, M. (1999) J. Biol. Chem. 274, 31804--31810). To elucidate subunit C function, we performed random and site-directed mutagenesis of the yeast VMA5 gene. Site-directed mutations in the most highly conserved region of Vma5p, residues 305--325, decreased catalytic activity of the V-ATPase by up to 48% without affecting assembly. A truncation mutant (K360stop) identified by random mutagenesis suggested a small region near the C terminus of the protein (amino acids 382--388) might be important for subunit stability. Site-directed mutagenesis revealed that three aromatic amino acids in this region (Tyr-382, Phe-385, and Tyr-388) in addition to four other conserved aromatic amino acids (Phe-260, Tyr-262, Phe-296, Phe-300) are essential for stable assembly of V(1) with V(0), although alanine substitutions at these positions support some activity in vivo. Surprisingly, three mutations (F260A, Y262A, and F385A) greatly decrease the stability of the V-ATPase in vitro but increase its k(cat) for ATP hydrolysis and proton transport by at least 3-fold. The peripheral stalk of V-ATPases must balance the stability essential for productive catalysis with the dynamic instability involved in regulation; these three mutations may perturb that balance.     

Racial identity,aesthetic surgery and Yorùbá African Values

The question of racial identity in the process and outcome of aesthetic surgery is gaining increasing attention in bioethical discourse. This paper attempts an ethical examination of the racial identity issues involved in aesthetic surgery. Dominant moral values in Western culture are explored in the evaluation of aesthetic surgery. The paper argues that African values are yet to receive the universal attention they arguably deserve especially in the rethinking of values underlying aesthetic surgery as racial transformation. Through a consideration of some moral‐aesthetic values in the Yorùbá‐African culture, this paper further re‐evaluates the ethics of aesthetic surgery. The paper contends against the propagation of aesthetic surgery as a new form of bolstering racial divides and identity in the evolving cosmopolitan age. The position defended in the paper is that some values from Yorùbá‐African culture are useful in the consideration of the ethics of aesthetic surgery and more importantly, in avoiding the racial identity bias embedded in aesthetic surgery. The paper concludes that if due consideration is perhaps given to some African moral‐aesthetic values in the global aesthetic surgery industry, some of the evolving moral and racial complexities would be better mediated.     

African Bioethics vs. Healthcare Ethics in Africa: A Critique of Godfrey Tangwa

It is nearly two decades now since the publication of Godfrey Tangwa's article, ‘Bioethics: African Perspective’, without a critical review. His article is important because sequel to its publication in Bioethics, the idea of ‘African bioethics’ started gaining some attention in the international bioethics literature. This paper breaks this relative silence by critically examining Tangwa's claim on the existence of African bioethics. Employing conceptual and critical methods, this paper argues that Tangwa's account of African bioethics has some conceptual, methodic and substantive difficulties, which altogether do not justify the idea of African bioethics, at least for now. Contra Tangwa, this article establishes that while African bioethics remains a future possibility, it is more cogent that current efforts in the name of ‘African bioethics’ be primarily re‐intensified towards ‘Healthcare ethics in Africa’.     

Cytotoxic Activity of Herbal Medicines as Assessed in Vitro: A Review

Since time immemorial, human beings have sought natural medications for treatment of various diseases. Weighty evidence demonstrates the use of chemical methodologies for sensitive evaluation of cytotoxic potentials of herbal agents. However, due to the ubiquitous use of cytotoxicity methods, there is a need for providing updated guidance for the design and development of in vitro assessment. The aim of this review is to provide practical guidance on common cell-based assays for suitable assessment of cytotoxicity potential of herbal medicines and discussing their advantages and disadvantages Relevant articles in authentic databases, including PubMed, Web of Science, Science Direct, Scopus, Google Scholar and SID, from 1950 to 2022 were collected according to selection criteria of in vitro cytotoxicity assays and protocols. In addition, the link between cytotoxicity assay selection and different factors such as the drug solvent, concentration and exposure duration were discussed.     

Mass administration of medicines in changing contexts: Acceptability,adaptability and community directed approaches in Kaduna and Ogun States,Nigeria

Nigeria has the highest burden of NTDs in sub-Saharan Africa. Commitments to reach the control and elimination of many Neglected Tropical Diseases (NTDs), particularly those amenable to preventive chemotherapy (onchocerciasis, schistosomiasis, soil transmitted helminths, lymphatic filariasis and trachoma) by 2020 are detailed in the London declaration. Strategies to reach targets build on existing approaches, one of which is the use of community directed intervention (CDI) methods to deliver the mass administration of medicines (MAM). However, treatment using this approach has been inconsistent and there are questions about the acceptability and adaptability of these interventions during periods of programmatic, social, and political change. This paper explores the current strengths and weaknesses of CDI approaches in MAM delivery. We consider the acceptability and adaptability of existing MAM approaches to ensure equity in access to essential treatments. Using qualitative methods, we explore implementer perspectives of MAM delivery. We purposively selected programme implementers to ensure good programmatic knowledge and representation from the different levels of health governance in Nigeria. Data collection took place across two States (Kaduna and Ogun). Our results indicate that CDI approaches have underpinned many historic successes in NTD programme acceptance in Nigeria, specifically in Kaduna and Ogun State. However, our results also show that in some contexts, factors that underpin the success of CDI have become disrupted presenting new challenges for programme implementers. Capturing the tacit knowledge of health implementers at varying levels of the health system, we present the current and changing context of MAM delivery in Kaduna and Ogun States and consolidate a platform of evidence to guide future programme delivery and research studies. We situate our findings within the broader NTD literature, specifically, in identifying how our findings align to existing reviews focused on factors that shape individual acceptance of MAM.     

Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency

In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (M_r 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs⁻¹, with a K_m value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs⁻¹ and 0.0766 µMs⁻¹, with a K_m value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII k_cat/K_m ratios were 0.12 µM⁻¹s⁻¹and 0.13 µM⁻¹s⁻¹ and 0.216 µM⁻¹s⁻¹ and 0.094 µM⁻¹s⁻¹ for for cis- and trans-dienelactone, respectively. The k_cat/K_m ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though K_m value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.     

The rapid purification of a phosphotransferase from wheat shoots

A phosphotransferase from wheat shoots which specifically phosphorylates the 5'-position of nucleosides has been purified by a simple procedure involving chromatography on Matrex Gel Blue A.