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Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.

Streptomycin (STR) is recommended for the management of multidrug-resistant tuberculosis (MDR-TB). Streptomycin resistance-conferring mutation types and frequency are shown to be influenced by genotypes of circulating strains in a population. This study aimed to characterize the mutations in MDR-TB isolates and examine their relationship with the genotypes in Zambia. A total of 138 MDR-TB isolates stored at the University Teaching Hospital Tuberculosis Reference Laboratory in Zambia were analyzed using spoligotyping and sequencing of STR resistance-associated genes. Streptomycin resistance was observed in 65.9% (91/138) of MDR-TB isolates. Mutations in rpsL, rrs, and gidB accounted for 33%, 12.1%, and 49.5%, respectively. Amino acid substitution K43R in rpsL was strongly associated with the CAS1_Kili genotype (p < 0.0001). The combination of three genes could predict 91.2% of STR resistance. Clustering of isolates based on resistance-conferring mutations and spoligotyping was observed. The clustering of isolates suggests that the increase in STR-resistant MDR-TB in Zambia is largely due to the spread of resistant strains from inadequate treatment. Therefore, rapid detection of STR resistance genetically is recommended before its use in MDR-TB treatment in Zambia.

BackgroundPteropine orthoreovirus (PRV) is an emerging bat-borne zoonotic virus that causes severe respiratory illness in humans. Although PRVs have been identified in fruit bats and humans in Australia and Asia, little is known about the prevalence of PRV infection in Africa. Therefore, this study performed an PRV surveillance in fruit bats in Zambia. MethodsEgyptian fruit bats (Rousettus aegyptiacus, n = 47) and straw-colored fruit bats (Eidolon helvum, n = 33) captured in Zambia in 2017-2018 were screened for PRV infection using RT-PCR and serum neutralization tests. The complete genome sequence of an isolated PRV strain was determined by next generation sequencing and subjected to BLAST and phylogenetic analyses. Replication capacity and pathogenicity of the strain were investigated using Vero E6 cell cultures and BALB/c mice, respectively. ResultsAn PRV strain, tentatively named Nachunsulwe-57, was isolated from one Egyptian fruit bat. Serological assays demonstrated that 98% of sera (69/70) collected from Egyptian fruit bats (n = 37) and straw-colored fruit bats (n = 33) had neutralizing antibodies against PRV. Genetic analyses revealed that all 10 genome segments of Nachunsulwe-57 were closely related to a bat-derived Kasama strain found in Uganda. Nachunsulwe-57 showed less efficiency in viral growth and lower pathogenicity in mice than another PRV strain, Miyazaki-Bali/2007, isolated from a patient. ConclusionsA high proportion of Egyptian fruit bats and straw-colored fruit bats were found to be seropositive to PRV in Zambia. Importantly, a new PRV strain (Nachunsulwe-57) was isolated from an Egyptian fruit bat in Zambia, which had relatively weak pathogenicity in mice. Taken together, our findings provide new epidemiological insights about PRV infection in bats and indicate the first isolation of an PRV strain that may have low pathogenicity to humans. Author summaryPteropine orthoreovirus (PRV) is a causative agent of acute respiratory illness in humans i

Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.

The Higashi-Shikagoe limestone quarry is an open-pit mine situated in Hokkaido Prefecture, Japan, that has experienced four slope failure incidents since 1996. The rock slope behaviour has been monitored since the first failure event by measuring the rock slope surface displacement using an automated polar system. Recent measurements have revealed a gradual decrease of the distance between the beam generator and mirrors over time; however, the displacements and decrease rate differs between the centre and left- and right-hand sides of the quarry. This implies that the deformation characteristics of the rock slope and factors influencing the slope deformation differ at the centre and left- and right-hand sides of the quarry. In this study, the two-dimensional finite element method was used to identify the causes of slope deformation by investigating the effects of limestone excavation at the foot of the rock slope, the deterioration of a similar to 70m-thick clay layer at the rock slope foot wall, and shear failure owing to rainfall infiltration. The numerical results show that slope deformation on the left-hand side and centre of the quarry are induced by clay deterioration, whereas the right-hand side of the quarry is deformed owing to floor excavation and/or shear sliding. The rock slope is presently stable because the magnitude of the rate of displacement decrease is small and no acceleration is observed.

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and the treatment of choice is ofloxacin (OFX). Specific amino acid substitutions in DNA gyrase of M. leprae have been reported leading to resistance against the drug. In our previous study, WQ-3810, a fluoroquinolone with a new R1 group (6-amino-3,5-difluoropyridin-2-yl) was shown to have a strong inhibitory activity on OFX-resistant DNA gyrases of M. leprae, and the structural characteristics of its R1 group was predicted to enhance the inhibitory activity. Methodology/Principal Finding: To further understand the contribution of the R1 group, WQ-3334 with the same R1 group as WQ-3810, WQ-4064, and WQ-4065, but with slightly modified R1 group, were assessed on their activities against recombinant DNA gyrase of M. leprae. An in silico study was conducted to understand the molecular interactions between DNA gyrase and WQ compounds. WQ-3334 and WQ-3810 were shown to have greater inhibitory activity against M. leprae DNA gyrase than others. Furthermore, analysis using quinolone-resistant M. leprae DNA gyrases showed that WQ-3334 had greater inhibitory activity than WQ-3810. The R8 group was shown to be a factor for the linkage of the R1 groups with GyrB by an in silico study. Conclusions/Significance: The inhibitory effect of WQ compounds that have a new R1 group against M. leprae DNA gyrase can be enhanced by improving the binding affinity with different R8 group molecules. The information obtained by this work could be applied to design new fluoroquinolones effective for quinolone-resistant M. leprae and other bacterial pathogens.

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and the treatment of choice is ofloxacin (OFX). Specific amino acid substitutions in DNA gyrase of M. leprae have been reported leading to resistance against the drug. In our previous study, WQ-3810, a fluoroquinolone with a new R1 group (6-amino-3,5-difluoropyridin-2-yl) was shown to have a strong inhibitory activity on OFX-resistant DNA gyrases of M. leprae, and the structural characteristics of its R1 group was predicted to enhance the inhibitory activity. Methodology/Principal Finding: To further understand the contribution of the R1 group, WQ-3334 with the same R1 group as WQ-3810, WQ-4064, and WQ-4065, but with slightly modified R1 group, were assessed on their activities against recombinant DNA gyrase of M. leprae. An in silico study was conducted to understand the molecular interactions between DNA gyrase and WQ compounds. WQ-3334 and WQ-3810 were shown to have greater inhibitory activity against M. leprae DNA gyrase than others. Furthermore, analysis using quinolone-resistant M. leprae DNA gyrases showed that WQ-3334 had greater inhibitory activity than WQ-3810. The R8 group was shown to be a factor for the linkage of the R1 groups with GyrB by an in silico study. Conclusions/Significance: The inhibitory effect of WQ compounds that have a new R1 group against M. leprae DNA gyrase can be enhanced by improving the binding affinity with different R8 group molecules. The information obtained by this work could be applied to design new fluoroquinolones effective for quinolone-resistant M. leprae and other bacterial pathogens.

Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and the treatment of choice is ofloxacin (OFX). Specific amino acid substitutions in DNA gyrase of M. leprae have been reported leading to resistance against the drug. In our previous study, WQ-3810, a fluoroquinolone with a new R1 group (6-amino-3,5-difluoropyridin-2-yl) was shown to have a strong inhibitory activity on OFX-resistant DNA gyrases of M. leprae, and the structural characteristics of its R1 group was predicted to enhance the inhibitory activity. Methodology/Principal Finding: To further understand the contribution of the R1 group, WQ-3334 with the same R1 group as WQ-3810, WQ-4064, and WQ-4065, but with slightly modified R1 group, were assessed on their activities against recombinant DNA gyrase of M. leprae. An in silico study was conducted to understand the molecular interactions between DNA gyrase and WQ compounds. WQ-3334 and WQ-3810 were shown to have greater inhibitory activity against M. leprae DNA gyrase than others. Furthermore, analysis using quinolone-resistant M. leprae DNA gyrases showed that WQ-3334 had greater inhibitory activity than WQ-3810. The R8 group was shown to be a factor for the linkage of the R1 groups with GyrB by an in silico study. Conclusions/Significance: The inhibitory effect of WQ compounds that have a new R1 group against M. leprae DNA gyrase can be enhanced by improving the binding affinity with different R8 group molecules. The information obtained by this work could be applied to design new fluoroquinolones effective for quinolone-resistant M. leprae and other bacterial pathogens.

Streptomycin (STR) is recommended for the management of multidrug-resistant tuberculosis (MDR-TB). Streptomycin resistance-conferring mutation types and frequency are shown to be influenced by genotypes of circulating strains in a population. This study aimed to characterize the mutations in MDR-TB isolates and examine their relationship with the genotypes in Zambia. A total of 138 MDR-TB isolates stored at the University Teaching Hospital Tuberculosis Reference Laboratory in Zambia were analyzed using spoligotyping and sequencing of STR resistance-associated genes. Streptomycin resistance was observed in 65.9% (91/138) of MDR-TB isolates. Mutations in rpsL, rrs, and gidB accounted for 33%, 12.1%, and 49.5%, respectively. Amino acid substitution K43R in rpsL was strongly associated with the CAS1_Kili genotype (p < 0.0001). The combination of three genes could predict 91.2% of STR resistance. Clustering of isolates based on resistance-conferring mutations and spoligotyping was observed. The clustering of isolates suggests that the increase in STR-resistant MDR-TB in Zambia is largely due to the spread of resistant strains from inadequate treatment. Therefore, rapid detection of STR resistance genetically is recommended before its use in MDR-TB treatment in Zambia.

BackgroundPteropine orthoreovirus (PRV) is an emerging bat-borne zoonotic virus that causes severe respiratory illness in humans. Although PRVs have been identified in fruit bats and humans in Australia and Asia, little is known about the prevalence of PRV infection in Africa. Therefore, this study performed an PRV surveillance in fruit bats in Zambia. MethodsEgyptian fruit bats (Rousettus aegyptiacus, n = 47) and straw-colored fruit bats (Eidolon helvum, n = 33) captured in Zambia in 2017-2018 were screened for PRV infection using RT-PCR and serum neutralization tests. The complete genome sequence of an isolated PRV strain was determined by next generation sequencing and subjected to BLAST and phylogenetic analyses. Replication capacity and pathogenicity of the strain were investigated using Vero E6 cell cultures and BALB/c mice, respectively. ResultsAn PRV strain, tentatively named Nachunsulwe-57, was isolated from one Egyptian fruit bat. Serological assays demonstrated that 98% of sera (69/70) collected from Egyptian fruit bats (n = 37) and straw-colored fruit bats (n = 33) had neutralizing antibodies against PRV. Genetic analyses revealed that all 10 genome segments of Nachunsulwe-57 were closely related to a bat-derived Kasama strain found in Uganda. Nachunsulwe-57 showed less efficiency in viral growth and lower pathogenicity in mice than another PRV strain, Miyazaki-Bali/2007, isolated from a patient. ConclusionsA high proportion of Egyptian fruit bats and straw-colored fruit bats were found to be seropositive to PRV in Zambia. Importantly, a new PRV strain (Nachunsulwe-57) was isolated from an Egyptian fruit bat in Zambia, which had relatively weak pathogenicity in mice. Taken together, our findings provide new epidemiological insights about PRV infection in bats and indicate the first isolation of an PRV strain that may have low pathogenicity to humans. Author summaryPteropine orthoreovirus (PRV) is a causative agent of acute respiratory illness in humans i

The Higashi-Shikagoe limestone quarry is an open-pit mine situated in Hokkaido Prefecture, Japan, that has experienced four slope failure incidents since 1996. The rock slope behaviour has been monitored since the first failure event by measuring the rock slope surface displacement using an automated polar system. Recent measurements have revealed a gradual decrease of the distance between the beam generator and mirrors over time; however, the displacements and decrease rate differs between the centre and left- and right-hand sides of the quarry. This implies that the deformation characteristics of the rock slope and factors influencing the slope deformation differ at the centre and left- and right-hand sides of the quarry. In this study, the two-dimensional finite element method was used to identify the causes of slope deformation by investigating the effects of limestone excavation at the foot of the rock slope, the deterioration of a similar to 70m-thick clay layer at the rock slope foot wall, and shear failure owing to rainfall infiltration. The numerical results show that slope deformation on the left-hand side and centre of the quarry are induced by clay deterioration, whereas the right-hand side of the quarry is deformed owing to floor excavation and/or shear sliding. The rock slope is presently stable because the magnitude of the rate of displacement decrease is small and no acceleration is observed.

Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (Mtb), caused by amino acid substitutions in DNA gyrase, has been increasingly reported worldwide. WQ-3810 is a newly developed FQ that is highly active against FQ-resistant pathogens; however, its activity against Mtb has not been evaluated. Herein we examined the efficacy of WQ-3810 against Mtb through the use of recombinant Mtb DNA gyrases. In addition, in vitro anti-mycobacterial activity of WQ-3810 was evaluated against recombinant Mtb var. bovis Bacille Calmette-Guerin strains in which gyrase-coding genes were replaced with Mtb variants containing resistance-conferring mutations. WQ-3810 showed a higher inhibitory activity than levofloxacin against most recombinant DNA gyrases with FQ-resistance mutations. Furthermore, WQ-3810 showed inhibition even against a DNA gyrase variant harboring a G88C mutation which is thought to confer the highest resistance against FQs in clinical Mtb isolates. In contrast, the FQ susceptibility test showed that WQ-3810 had relatively weak mycobactericidal activity compared with moxifloxacin. However, the combination of WQ-3810 and ethambutol showed the greatest degree of synergistic activity against recombinant strains. Since FQs and ethambutol have been used in multi-drug therapy for tuberculosis, WQ-3810 might represent a new, potent anti-tuberculosis drug that can be effective even against FQ-resistant Mtb strains.

Background: Mycobacterium leprae causes leprosy and ofloxacin is used to control this bacterium. However, specific amino acid substitutions in DNA gyrases of M. leprae interferes with the effect of ofloxacin. Methodology/principal findings: Here we tested the inhibitory effect of WQ-3810 on DNA gyrases in M. leprae, using recombinant gyrases. We theorized that WQ-3810 and DNA gyrases interacted, which was tested in silico. Compared with control drugs like ofloxacin, WQ-3810 showed a better inhibitory effect on ofloxacin-resistant DNA gyrases. The in-silico study showed that, unlike control drugs, a specific linkage between a R1 group in WQ-3810 and aspartic acid at position 464 in the subunit B of DNA gyrases existed, which would enhance the inhibitory effect of WQ-3810. This linkage was confirmed in a further experiment, using recombinant DNA gyrases with amino acid substitutions in subunits B instead. Conclusions/significance: The inhibitory effect of WQ-3810 was likely enhanced by the specific linkage between a R1 group residue in its structure and DNA gyrases. Using interactions like the one found in the present work may help design new fluoroquinolones that contribute to halt the emergence of antibiotic-resistant pathogens. (c) 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Background: Mycobacterium leprae causes leprosy and ofloxacin is used to control this bacterium. However, specific amino acid substitutions in DNA gyrases of M. leprae interferes with the effect of ofloxacin. Methodology/principal findings: Here we tested the inhibitory effect of WQ-3810 on DNA gyrases in M. leprae, using recombinant gyrases. We theorized that WQ-3810 and DNA gyrases interacted, which was tested in silico. Compared with control drugs like ofloxacin, WQ-3810 showed a better inhibitory effect on ofloxacin-resistant DNA gyrases. The in-silico study showed that, unlike control drugs, a specific linkage between a R1 group in WQ-3810 and aspartic acid at position 464 in the subunit B of DNA gyrases existed, which would enhance the inhibitory effect of WQ-3810. This linkage was confirmed in a further experiment, using recombinant DNA gyrases with amino acid substitutions in subunits B instead. Conclusions/significance: The inhibitory effect of WQ-3810 was likely enhanced by the specific linkage between a R1 group residue in its structure and DNA gyrases. Using interactions like the one found in the present work may help design new fluoroquinolones that contribute to halt the emergence of antibiotic-resistant pathogens. (c) 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (Mtb), caused by amino acid substitutions in DNA gyrase, has been increasingly reported worldwide. WQ-3810 is a newly developed FQ that is highly active against FQ-resistant pathogens; however, its activity against Mtb has not been evaluated. Herein we examined the efficacy of WQ-3810 against Mtb through the use of recombinant Mtb DNA gyrases. In addition, in vitro anti-mycobacterial activity of WQ-3810 was evaluated against recombinant Mtb var. bovis Bacille Calmette-Guerin strains in which gyrase-coding genes were replaced with Mtb variants containing resistance-conferring mutations. WQ-3810 showed a higher inhibitory activity than levofloxacin against most recombinant DNA gyrases with FQ-resistance mutations. Furthermore, WQ-3810 showed inhibition even against a DNA gyrase variant harboring a G88C mutation which is thought to confer the highest resistance against FQs in clinical Mtb isolates. In contrast, the FQ susceptibility test showed that WQ-3810 had relatively weak mycobactericidal activity compared with moxifloxacin. However, the combination of WQ-3810 and ethambutol showed the greatest degree of synergistic activity against recombinant strains. Since FQs and ethambutol have been used in multi-drug therapy for tuberculosis, WQ-3810 might represent a new, potent anti-tuberculosis drug that can be effective even against FQ-resistant Mtb strains.

Pyrazinamide forms a core part of treatment for all types of tuberculosis (TB) in Zambia. Due to challenges associated with pyrazinamide testing, little information is available to indicate the frequency of resistance to this drug in Zambia. To determine the frequency of pyrazinamide (PZA) resistance and its correlation with mutation in pncA in Mycobacterium tuberculosis isolated from patients in Lusaka, Zambia, BACTEC MGIT M960 was used for phenotypic PZA susceptibility testing while sequencing was used to determine resistance-conferring mutations in the pncA. Of the 131 isolates analyzed, 32 were phenotypically resistant to PZA. Among multidrug-resistant (MDR) M. tuberculosis isolates, the frequency of PZA resistance was 21 of 35 (58.3%). And 27 of 32 PZA resistant isolates had mutations in the pncA that seem to confer resistance. With BACTEC MGIT 960 as the reference standard, gene sequencing showed 84.4% sensitivity and 100% specificity. Nine new mutations were identified and the single nucleotide substitution T104G and C195T were the most frequent mutations. However, they were observed in both susceptible and resistant strains and indicating that they are non-resistance conferring mutations. This study has demonstrated that PZA susceptibility testing is necessary especially in patients suffering from MDR-TB as approximately half of the patients have PZA resistant TB. Similar studies will have to be carried out in other provinces to get an accurate estimate of PZA resistance in Zambia. Mutations in pncA were the major mechanism of PZA resistance with no involvement of rpsA and panD genes. However, the presence of mutations among phenotypically PZA susceptible M. tuberculosis isolates makes it challenging to independently use genotyping method for the determination of PZA resistance.

Pyrazinamide forms a core part of treatment for all types of tuberculosis (TB) in Zambia. Due to challenges associated with pyrazinamide testing, little information is available to indicate the frequency of resistance to this drug in Zambia. To determine the frequency of pyrazinamide (PZA) resistance and its correlation with mutation in pncA in Mycobacterium tuberculosis isolated from patients in Lusaka, Zambia, BACTEC MGIT M960 was used for phenotypic PZA susceptibility testing while sequencing was used to determine resistance-conferring mutations in the pncA. Of the 131 isolates analyzed, 32 were phenotypically resistant to PZA. Among multidrug-resistant (MDR) M. tuberculosis isolates, the frequency of PZA resistance was 21 of 35 (58.3%). And 27 of 32 PZA resistant isolates had mutations in the pncA that seem to confer resistance. With BACTEC MGIT 960 as the reference standard, gene sequencing showed 84.4% sensitivity and 100% specificity. Nine new mutations were identified and the single nucleotide substitution T104G and C195T were the most frequent mutations. However, they were observed in both susceptible and resistant strains and indicating that they are non-resistance conferring mutations. This study has demonstrated that PZA susceptibility testing is necessary especially in patients suffering from MDR-TB as approximately half of the patients have PZA resistant TB. Similar studies will have to be carried out in other provinces to get an accurate estimate of PZA resistance in Zambia. Mutations in pncA were the major mechanism of PZA resistance with no involvement of rpsA and panD genes. However, the presence of mutations among phenotypically PZA susceptible M. tuberculosis isolates makes it challenging to independently use genotyping method for the determination of PZA resistance.

Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.

Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.

The number of multi-drug-resistant tuberculosis (MDR-TB) cases is rising worldwide. As a countermeasure against this situation, the implementation of rapid molecular tests to identify MDR-TB would be effective. To develop such tests, information on the frequency and distribution of mutations associating with phenotypic drug resistance in Mycobacterium tuberculosis is required in each country. During 2010, the common mutations in the rpoB, katG and inhA of 178 phenotypically MDR M. tuberculosis isolates collected by the National Tuberculosis Control Program (NTP) in Myanmar were investigated by DNA sequencing. Mutations affecting the 81-bp rifampicin (RIF) resistance-determining region (RRDR) of the rpoB were identified in 127 of 178 isolates (71.3%). Two of the most frequently affected codons were 531 and 526, with percentages of 48.3% and 14.0% respectively. For isoniazid (INH) resistance, 114 of 178 MDR-TB isolates (64.0%) had mutations in the katG in which a mutation-conferring amino acid substitution at codon 315 from Ser to Thr was the most common. Mutations in the inhA regulatory region were also detected in 20 (11.2%) isolates, with the majority at position -15. Distinct mutation rate and pattern from surrounding countries might suggest that MDR-TB has developed and spread domestically in Myanmar. (C) 2016, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Background: Fluoroquinolones are a class of antibacterial agents used for leprosy treatment. Some new fluoroquinolones have been attracting interest due to their remarkable potency that is reportedly better than that of ofloxacin, the fluoroquinolone currently recommended for treatment of leprosy. For example, DC-159a, a recently developed 8-methoxy fluoroquinolone, has been found to be highly potent against various bacterial species. Nonetheless, the efficacy of DC-159a against Mycobacterium leprae is yet to be examined. Methodology/Principal Findings: To gather data that can support highly effective fluoroquinolones as candidates for new remedies for leprosy treatment, we conducted in vitro assays to assess and compare the inhibitory activities of DC-159a and two fluoroquinolones that are already known to be more effective against M. leprae than ofloxacin. The fluoroquinolone-inhibited DNA supercoiling assay using recombinant DNA gyrases of wild type and ofloxacin-resistant M. leprae revealed that inhibitory activities of DC-159a and sitafloxacin were at most 9.8-and 11.9-fold higher than moxifloxacin. Also the fluoroquinolone-mediated cleavage assay showed that potencies of those drugs were at most 13.5-and 9.8-fold higher than moxifloxacin. In addition, these two drugs retained their inhibitory activities even against DNA gyrases of ofloxacin-resistant M. leprae. Conclusions/Significance: The results indicated that DC-159a and sitafloxacin are more effective against wild type and mutant M. leprae DNA gyrases than moxifloxacin, suggesting that these antibacterial drugs can be good candidates that may supersede current fluoroquinolone remedies. DC-159a in particular is very promising because it is classified in a subgroup of fluoroquinolones that is known to be less likely to cause adverse effects. Our results implied that DC-159a is well worth further investigation to ascertain its in vivo effectiveness and clinical safety for humans.

Amino acid substitutions providing quinolone resistance to Campyloabcter jejuni have been found in the quinolone resistance-determining region of protein DNA gyrase subunit A (GyrA), with the highest frequency at position 86 followed by position 90. In this study, wild-type and mutant recombinant DNA gyrase subunits were expressed in Escherichia coli and purified using Ni-NTA agarose column chromatography. Soluble 97 kDa GyrA and 87 kDa DNA gyrase subunit B were shown to reconstitute ATP-dependent DNA supercoiling activity. A quinolone-inhibited supercoiling assay demonstrated the roles of Thr86Ile, Thr86Ala, Thr86Lys, Asp90Asn, and Asp90Tyr amino acid substitutions in reducing sensitivity to quinolones. The marked effect of Thr86Ile on all examined quinolones suggested the advantage of this substitution in concordance with recurring isolation of quinolone-resistant C. jejuni. An analysis of the structure-activity relationship showed the importance of the substituent at position 8 in quinolones to overcome the effect of Thr86Ile. Sitafloxacin (SIT), which has a fluorinate cyclopropyl ring at R-1 and a chloride substituent at R-8, a characteristic not found in other quinolones, showed the highest inhibitory activity against all mutant C. jejuni gyrases including ciprofloxacin-resistant mutants. The results suggest SIT as a promising drug for the treatment of campylobacteriosis caused by CIP-resistant C. jejuni. Copyright (C) 2016 John Wiley & Sons, Ltd.

Amino acid substitutions providing quinolone resistance to Campyloabcter jejuni have been found in the quinolone resistance-determining region of protein DNA gyrase subunit A (GyrA), with the highest frequency at position 86 followed by position 90. In this study, wild-type and mutant recombinant DNA gyrase subunits were expressed in Escherichia coli and purified using Ni-NTA agarose column chromatography. Soluble 97 kDa GyrA and 87 kDa DNA gyrase subunit B were shown to reconstitute ATP-dependent DNA supercoiling activity. A quinolone-inhibited supercoiling assay demonstrated the roles of Thr86Ile, Thr86Ala, Thr86Lys, Asp90Asn, and Asp90Tyr amino acid substitutions in reducing sensitivity to quinolones. The marked effect of Thr86Ile on all examined quinolones suggested the advantage of this substitution in concordance with recurring isolation of quinolone-resistant C. jejuni. An analysis of the structure-activity relationship showed the importance of the substituent at position 8 in quinolones to overcome the effect of Thr86Ile. Sitafloxacin (SIT), which has a fluorinate cyclopropyl ring at R-1 and a chloride substituent at R-8, a characteristic not found in other quinolones, showed the highest inhibitory activity against all mutant C. jejuni gyrases including ciprofloxacin-resistant mutants. The results suggest SIT as a promising drug for the treatment of campylobacteriosis caused by CIP-resistant C. jejuni. Copyright (C) 2016 John Wiley & Sons, Ltd.

The number of multi-drug-resistant tuberculosis (MDR-TB) cases is rising worldwide. As a countermeasure against this situation, the implementation of rapid molecular tests to identify MDR-TB would be effective. To develop such tests, information on the frequency and distribution of mutations associating with phenotypic drug resistance in Mycobacterium tuberculosis is required in each country. During 2010, the common mutations in the rpoB, katG and inhA of 178 phenotypically MDR M. tuberculosis isolates collected by the National Tuberculosis Control Program (NTP) in Myanmar were investigated by DNA sequencing. Mutations affecting the 81-bp rifampicin (RIF) resistance-determining region (RRDR) of the rpoB were identified in 127 of 178 isolates (71.3%). Two of the most frequently affected codons were 531 and 526, with percentages of 48.3% and 14.0% respectively. For isoniazid (INH) resistance, 114 of 178 MDR-TB isolates (64.0%) had mutations in the katG in which a mutation-conferring amino acid substitution at codon 315 from Ser to Thr was the most common. Mutations in the inhA regulatory region were also detected in 20 (11.2%) isolates, with the majority at position -15. Distinct mutation rate and pattern from surrounding countries might suggest that MDR-TB has developed and spread domestically in Myanmar. (C) 2016, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Background: Fluoroquinolones are a class of antibacterial agents used for leprosy treatment. Some new fluoroquinolones have been attracting interest due to their remarkable potency that is reportedly better than that of ofloxacin, the fluoroquinolone currently recommended for treatment of leprosy. For example, DC-159a, a recently developed 8-methoxy fluoroquinolone, has been found to be highly potent against various bacterial species. Nonetheless, the efficacy of DC-159a against Mycobacterium leprae is yet to be examined. Methodology/Principal Findings: To gather data that can support highly effective fluoroquinolones as candidates for new remedies for leprosy treatment, we conducted in vitro assays to assess and compare the inhibitory activities of DC-159a and two fluoroquinolones that are already known to be more effective against M. leprae than ofloxacin. The fluoroquinolone-inhibited DNA supercoiling assay using recombinant DNA gyrases of wild type and ofloxacin-resistant M. leprae revealed that inhibitory activities of DC-159a and sitafloxacin were at most 9.8-and 11.9-fold higher than moxifloxacin. Also the fluoroquinolone-mediated cleavage assay showed that potencies of those drugs were at most 13.5-and 9.8-fold higher than moxifloxacin. In addition, these two drugs retained their inhibitory activities even against DNA gyrases of ofloxacin-resistant M. leprae. Conclusions/Significance: The results indicated that DC-159a and sitafloxacin are more effective against wild type and mutant M. leprae DNA gyrases than moxifloxacin, suggesting that these antibacterial drugs can be good candidates that may supersede current fluoroquinolone remedies. DC-159a in particular is very promising because it is classified in a subgroup of fluoroquinolones that is known to be less likely to cause adverse effects. Our results implied that DC-159a is well worth further investigation to ascertain its in vivo effectiveness and clinical safety for humans.

The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.

The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.

Trans. of Daiichi Tsusho K. K. (Daiichi Trading Co.) (China), p1-32, Jan 60.