マンソン住血吸虫のミトコンドリアは予防と治療効果が期待できる創薬標的である

長崎大学学位論文 [学位記番号]博（医歯薬）甲第1386号 [学位授与年月日]令和3年12月1

Mutations in the “a” Determinant Region of Hepatitis B Virus Genotype A among Voluntary Kenyan Blood Donors

Background: Occurrence of mutations within the major antigenic alpha determinant region of hepatitis B surface antigen (HBsAg can alter HBV antigenicity resulting in   failures in diagnosis, vaccine and hepatitis B immunoglobulin therapy. Objective: This study aimed at detection of mutations in the “a” determinant region of HBV surface antigen among voluntary blood donors in Kenya. Design: A cross sectional study involving serology and molecular techniques Settings: This study involved analysis of samples from blood transfusion centers Subjects: A total of 301 blood samples from donor blood were collected for the study. Methods: Sero-status for HBsAg was determined using Enzyme-Linked Immunosorbent Assay (ELISA). A fragment of the S gene including the "a" determinant was amplified by PCR from the HBsAg positive samples and sequenced for mutation analysis. Mutations and phylogenetic analyses were performed using Mega 6 software, Bioedit software and GENETYX® software version 9.1.0. Results: Out of the 301 samples tested 69/301 (22.9%) were Polymerase Chain Reaction (PCR) positive including 2/69(2.9%) were sero-negative for HBsAg. All isolates were genotype A, sub-genotype A1. A total of 29 mutations were observed of which 37.9% were located within the “a” determinant. Mutations T143M and K122R were the most frequent in this study. Escape mutations associated with diagnostic failure, vaccine and immunoglobulin therapy escape were also identified. Conclusions: These findings are important for policies related to vaccine implementation and therapeutic and diagnostic guidelines. Keywords: Escape mutants, genotype, hepatitis B virus, antigenic determinant, surface antigen

Mutations in the “a” Determinant Region of Hepatitis B Virus Genotype A among Voluntary Kenyan Blood Donors

Occurrence of mutations within the major antigenic alpha determinant region of hepatitis B surface antigen (HBsAg can alter HBV antigenicity resulting in   failures in diagnosis, vaccine and hepatitis B immunoglobulin therapy. This study aimed at detection of mutations in the “a” determinant region of HBV surface antigen among voluntary blood donors in Kenya. This was a cross sectional study involving serology and molecular techniques. This study involved analysis of samples from blood transfusion centers. A total of 301 blood samples from donor blood were collected for the study.  Sero-status for HBsAg was determined using Enzyme-Linked Immunosorbent Assay (ELISA). A fragment of the S gene including the "a" determinant was amplified by PCR from the HBsAg positive samples and sequenced for mutation analysis. Mutations and phylogenetic analyses were performed using Mega 6 software, Bioedit software and GENETYX® software version 9.1.0. Out of the 301 samples tested 69/301 (22.9%) were Polymerase Chain Reaction (PCR) positive including 2/69(2.9%) were sero-negative for HBsAg. All isolates were genotype A, sub-genotype A1. A total of 29 mutations were observed of which 37.9% were located within the “a” determinant. Mutations T143M and K122R were the most frequent in this study. Escape mutations associated with diagnostic failure, vaccine and immunoglobulin therapy escape were also identified. These findings are important for policies related to vaccine implementation and therapeutic and diagnostic guidelines. Keywords: Escape mutants, genotype, hepatitis B virus, antigenic determinant, surface antige

Biochemical Studies of Mitochondrial Malate: Quinone Oxidoreductase from Toxoplasma gondii

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis and infects almost one-third of the global human population. A lack of effective drugs and vaccines and the emergence of drug resistant parasites highlight the need for the development of new drugs. The mitochondrial electron transport chain (ETC) is an essential pathway for energy metabolism and the survival of T. gondii. In apicomplexan parasites, malate:quinone oxidoreductase (MQO) is a monotopic membrane protein belonging to the ETC and a key member of the tricarboxylic acid cycle, and has recently been suggested to play a role in the fumarate cycle, which is required for the cytosolic purine salvage pathway. In T. gondii, a putative MQO (TgMQO) is expressed in tachyzoite and bradyzoite stages and is considered to be a potential drug target since its orthologue is not conserved in mammalian hosts. As a first step towards the evaluation of TgMQO as a drug target candidate, in this study, we developed a new expression system for TgMQO in FN102(DE3)TAO, a strain deficient in respiratory cytochromes and dependent on an alternative oxidase. This system allowed, for the first time, the expression and purification of a mitochondrial MQO family enzyme, which was used for steady-state kinetics and substrate specificity analyses. Ferulenol, the only known MQO inhibitor, also inhibited TgMQO at IC50 of 0.822 μM, and displayed different inhibition kinetics compared to Plasmodium falciparum MQO. Furthermore, our analysis indicated the presence of a third binding site for ferulenol that is distinct from the ubiquinone and malate sites

Mitochondria as a Potential Target for the Development of Prophylactic and Therapeutic Drugs against Schistosoma mansoni Infection

The emergence of parasites resistant to praziquantel, the only therapeutic agent, and its ineffectiveness as a prophylactic agent (inactive against the migratory/juvenile Schistosoma mansoni), make the development of new antischistosomal drugs urgent. The parasite’s mitochondrion is an attractive target for drug development, because this organelle is essential for survival throughout the parasite’s life cycle. We investigated the effects of 116 compounds against Schistosoma mansoni cercaria motility that have been reported to affect mitochondrion-related processes in other organisms. Next, eight compounds plus two controls (mefloquine and praziquantel) were selected and assayed against the motility of schistosomula (in vitro) and adults (ex vivo). Prophylactic and therapeutic assays were performed using infected mouse models. Inhibition of oxygen consumption rate (OCR) was assayed using Seahorse XFe24 analyzer. All selected compounds showed excellent prophylactic activity, reducing the worm burden in the lungs to less than 15% of that obtained in the vehicle control. Notably, ascofuranone showed the highest activity, with a 98% reduction of the worm burden, suggesting the potential for the development of ascofuranone as a prophylactic agent. The worm burden of infected mice with S. mansoni at the adult stage was reduced by more than 50% in mice treated with mefloquine, nitazoxanide, amiodarone, ascofuranone, pyrvinium pamoate, or plumbagin. Moreover, adult mitochondrial OCR was severely inhibited by ascofuranone, atovaquone, and nitazoxanide, while pyrvinium pamoate inhibited both mitochondrial and nonmitochondrial OCRs. These results demonstrate that the mitochondria of S. mansoni are a feasible target for drug development.長崎大学学位論文 学位記番号:博(医歯薬)甲第1386号 学位授与年月日:令和3年12月1日Author: Keith Kiplangat Talaam, Daniel Ken Inaoka, Takeshi Hatta, Daigo Tsubokawa, Naotoshi Tsuji, Minoru Wada, Hiroyuki Saimoto, Kiyoshi Kita, Shinjiro HamanoCitation: Antimicrobial Agents and Chemotherapy, 65(10), art. no. e00418-21; 2021Nagasaki University (長崎大学)課程博

Anti-bacterial efficacy of alcoholic hand rubs in the Kenyan market, 2015

Abstract Background Hand hygiene is known to be effective in preventing hospital and community-acquired infections. The increasing number of hand sanitizer brands in Kenyan hospitals and consumer outlets is of concern. Thus the main aim of this study was to evaluate the anti-bacterial efficacy and organoleptic properties of these hand sanitizers in Kenya. Methods This was an experimental, laboratory-based study of 14 different brands of hand sanitizers (coded HS1-14) available in various retail outlets and hospitals in Kenya. Efficacy was evaluated using standard non-pathogenic Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) as per the European Standard (EN). The logarithmic reduction factors (RF) were assessed at baseline and after treatment, and log reduction then calculated. Ten and 25 healthy volunteers participated in the efficacy and organoleptic studies respectively. Results Four (28.6%) hand sanitizers (HS12, HS9, HS13 and HS14) showed a 5.9 reduction factor on all the three bacteria strains. Seven (50%) hand sanitizers had efficacies of <3 against all the three bacteria strains used. Efficacy on E. Coli was higher compared to the other pathogens. Three hand sanitizers were efficacious on one of the pathogens and not the other. In terms of organoleptic properties, gel-based formulations were rated far higher than the liquid based formulations brands. Conclusion Fifty percent (50%) of the selected hand sanitizers in the Kenyan market have efficacy that falls below the World Health Organization (WHO) and DIN EN 1500:2013. Of the 14 hand sanitizers found in the Kenyan market, only four showed efficacies that were comparable to the WHO-formulation. There is a need to evaluate how many of these products with <3 efficacy that have been incorporated into the health system for hand hygiene and the country\u2019s policy on regulations on their usage