Since the 1960s, researchers have widely used single-case experimental designs (SCEDs) as a standard method in applied behavior analysis, but this research method has rarely attracted the attention ...of scholars in other research fields. Recent years, however, have seen an increased interest in SCEDs in various research fields, including clinical psychology and developmental psychology, partly because of the demand for evidence-based practice. Therefore, this article provides a basic description of SCEDs and explains their fundamental properties as well as the characteristics and problems encountered in typical SCEDs often implemented in applied research. In addition, this study will explain the visual inspection and statistical methods for examining data obtained from SCEDs. It will also describe the standards for evaluating the quality of single-case experimental design studies, especially the criteria to satisfy “Meets WWC SCD Standards without Reservations” proposed by WWC standards version 4.1.
A wide range of aryl boronic 1,1,2,2-tetraethylethylene glycol esters ArB(Epin)s were readily synthesized. Purifying aryl boronic esters by conventional silica gel chromatography is generally ...challenging; however, these introduced derivatives are easily purified on silica gel and isolated in excellent yields. We subjected the purified ArB(Epin) to Suzuki–Miyaura couplings, which provided higher yields of the desired biaryl products than those obtained using the corresponding aryl boronic acids or pinacol esters.
Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of ...patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all
clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor
gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu
, Phe
, Phe
, and His
) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous
gene of a terbinafine-sensitive
(formerly
) strain. All of the generated
transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in
transformants expressing mutant forms of
SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.
Deuterium-labeled compounds are widely utilized in various scientific fields. We summarize the recent advances in the direct deuteration of sugar, saturated fatty acid, and arene derivatives using ...heterogeneous platinum group metal on carbon catalysts by our research group. Hydrogen gas is a key catalyst-activator to facilitate the present H–D exchange reactions. In this review, the direct activation method of catalysts using in situ-generated hydrogen based on the dehydrogenation of alcohols is introduced. The obtained multiple deuterium-labeled products, including bioactive compounds, are expected to contribute to the development of many scientific investigations.
The mechanisms of terbinafine resistance in a set of clinical isolates of
have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of ...TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of
to develop resistance through multidrug efflux transporters. The introduction of a
cDNA library into
allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the
genome, we performed a BLASTp analysis of
,
, and
on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in
Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both
and
were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of
in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in
, which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance.
Trichosporon asahii is a pathogenic fungus that causes severe, deep-seated fungal infections in neutropenic patients. Elucidating the infection mechanisms of T. asahii based on genetic studies ...requires a specific gene-targeting system. Here, we established an efficient gene-targeting system in a highly pathogenic T. asahii strain identified using the silkworm infection model. By comparing the pathogenicity of T. asahii clinical isolates in a silkworm infection model, T. asahii MPU129 was identified as a highly pathogenic strain. Using an Agrobacterium tumefaciens-mediated gene transfer system, we obtained a T. asahii MPU129 mutant lacking the ku70 gene, which encodes the Ku70 protein involved in the non-homologous end-joining repair of DNA double-strand breaks. The ku70 gene-deficient mutant showed higher gene-targeting efficiency than the wild-type strain for constructing a mutant lacking the cnb1 gene, which encodes the beta-subunit of calcineurin. The cnb1 gene-deficient mutant showed reduced pathogenicity against silkworms compared with the parental strain. These results suggest that an efficient gene-targeting system in a highly pathogenic T. asahii strain is a useful tool for elucidating the molecular mechanisms of T. asahii infection.
A protocol for the ruthenium-on-carbon (Ru/C)-catalyzed solvent-free oxidation of alcohols, which proceeds efficiently under solid–solid (liquid)–gas conditions, was developed. Various primary and ...secondary alcohols were transformed to corresponding aldehydes and ketones in moderate to excellent isolated yields by simply stirring in the presence of 10% Ru/C under air or oxygen conditions. The solvent-free oxidation reactions proceeded efficiently regardless of the solid or liquid state of the substrates and reagents and could be applied to gram-scale synthesis without loss of the reaction efficiency. Furthermore, the catalytic activity of Ru/C was maintained after five reuse cycles.
Abstract
Malassezia restricta is the most predominant fungus in the microbiome of human skin. This microorganism can cause or exacerbate Malassezia-associated skin dermatitis, seborrheic dermatitis, ...atopic dermatitis, and pityriasis versicolor. The virulence factors of M. restricta have not been analyzed because a gene recombination system has not been developed. In this study, we established an Agrobacterium tumefaciens-mediated gene transfer (ATMT) system, optimized for generating gene-deficient mutants of M. restricta. A mutant of FKB1 gene, which encodes the FKBP12 protein that binds to the calcineurin inhibitor tacrolimus, was generated using the ATMT system. Subsequently, the FKB1 gene was reintroduced into the FKB1 gene-deficient mutant for obtaining a gene-complemented strain. The wild-type strain of M. restricta was sensitive to tacrolimus, whereas the FKB1 gene-deficient mutant was resistant to tacrolimus; the phenotypic drug susceptibility in the mutant was restored by reintroducing the FKB1 gene. Contrastingly, the FKB1 gene-deficient mutant was not resistant to cyclosporine A, which also inhibits calcineurin by binding to cyclophilin A. The gene recombination system for M. restricta will facilitate in elucidating the molecular mechanisms causing Malassezia-associated dermatitis.
Activated carbon (AC) is a porous solid with a larger surface area and lower cost than chromatography resins. AC has been used in the field of biopharmaceutical manufacturing for plasma-derived ...products and recombinant monoclonal antibodies (mAb). In our previous study, AC was employed in the purification process of therapeutic mAb as a replacement for Protein A affinity chromatography (PrA). In addition, we designed an all flow-through purification process using AC. In these investigations, greater effective clearance of high-molecular-weight species (HMW), low-molecular-weight species (LMW), host cell proteins (HCP), and DNA was observed compared to that of the conventional Protein A platform purification process. However, it was revealed that mAb recovery from the AC step was lower than that from the PrA step. In this work, to improve mAb recovery from the AC step while maintaining the effective removal of impurities, a pretreatment procedure conducted prior to the AC treatment was investigated. We found that both an ultrafiltration/dilution and reduction in the conductivity of the filtered cell culture supernatant after acid precipitation could improve both the impurity clearance and mAb recovery from the AC treatment. From the obtained results, we designed a two-step purification process in which AC treatment is followed by either cation exchange column chromatography or anion exchange column chromatography, and we compared this against the Protein A platform purification process. Excellent impurity clearance was achieved, even in the one-column process. Furthermore, we designed an innovative column-free flow-through purification process based on acid precipitation, clarification, ultrafiltration/dilution, and the implementation of an AC filter membrane and an anion exchange chromatography membrane. With this process in the pilot-scale, HCP level can be reduced to below 10 ng/mg, and HMW and LMW can be removed to below 1% while improving mAb recovery. From these results, it is strongly expected that AC is a promising candidate for the next generation of mAb purification processes to improve the economy and efficiency of the process.
•Monoclonal antibody purification using activated carbon was presented.•All flow-through and column-free process using an activated carbon membrane filter was designed.•Effective antibody recovery and impurities clearance were obtained.
•We examined soil bacterial communities across a forested headwater catchment.•Soil bacterial communities showed rapid turnover along slopes.•Slope-induced soil properties explained bacterial ...community structures.
Despite a rapidly growing body of literature on the spatial variability of soil microbial communities at landscape to regional scales, the community structures of forest soil bacteria at a small catchment scale and their relationships with environmental conditions have been poorly explored. Here, we analyzed soil bacterial communities in the O- and surface mineral horizons at 53 sampling points within a forested headwater catchment in central Japan. Non-metric multidimensional scaling based on the Simpson dissimilarity index was used to analyze compositional changes of soil bacterial communities within the catchment. We evaluated the importance of topographic features, soil chemical properties, and forest type in shaping bacterial communities in each soil horizon using structural equation modeling (SEM). Furthermore, we performed hierarchical clustering and indicator analyses to identify characteristic soil bacterial communities in the catchment. The observed bacterial communities exhibited rapid turnover along slopes. According to the SEM results, the indirect effect of normalized height, a continuous measure of slope position, explains the bacterial community structure in each soil horizon through the effects of pH in the O-horizon and pH and water content in the mineral horizon. Cluster analysis revealed three major spatial units of soil bacterial communities within the catchment in each soil horizon: the lower, middle, and upper slopes. The observed indicators of soil bacterial taxa and metabolic functions differed markedly across slope positions, e.g., ammonifiers were typical of the upper and middle slopes in the mineral horizon, whereas denitrifiers were specific to the lower slopes of the O-horizon. This indicates that the bacterial community functions in this catchment are also constructed to reflect variations in soil pH or moisture along the slopes. This study highlights that the community structure of surface soil bacteria in the forested headwater catchment can be explained by soil chemical properties closely associated with slope positions.
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