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1.  Pharmacist-managed dose adjustment feedback using therapeutic drug monitoring of vancomycin was useful for patients with methicillin-resistant Staphylococcus aureus infections: a single institution experience 
Vancomycin (VCM) requires dose adjustment based on therapeutic drug monitoring. At Aomori Prefectural Central Hospital, physicians carried out VCM therapeutic drug monitoring based on their experience, because pharmacists did not participate in the dose adjustment. We evaluated the impact of an Antimicrobial Stewardship Program (ASP) on attaining target VCM trough concentrations and pharmacokinetics (PK)/pharmacodynamics (PD) parameters in patients with methicillin-resistant Staphylococcus aureus (MRSA) infections.
Materials and methods
The ASP was introduced in April 2012. We implemented a prospective audit of prescribed VCM dosages and provided feedback based on measured VCM trough concentrations. In a retrospective pre- and postcomparison study from April 2007 to December 2011 (preimplementation) and from April 2012 to December 2014 (postimplementation), 79 patients were treated for MRSA infection with VCM, and trough concentrations were monitored (pre, n=28; post, n=51). In 65 patients (pre, n=15; post, n=50), 24-hour area under the concentration–time curve (AUC 0–24 h)/minimum inhibitory concentration (MIC) ratios were calculated.
Pharmacist feedback, which included recommendations for changing dose or using alternative anti-MRSA antibiotics, was highly accepted during postimplementation (88%, 29/33). The number of patients with serum VCM concentrations within the therapeutic range (10–20 μg/mL) was significantly higher during postimplementation (84%, 43/51) than during preimplementation (39%, 11/28) (P<0.01). The percentage of patients who attained target PK/PD parameters (AUC 0–24 h/MIC >400) was significantly higher during postimplementation (84%, 42/50) than during preimplementation (53%, 8/15; P=0.013). There were no significant differences in nephrotoxicity or mortality rate.
Our ASP increased the percentage of patients that attained optimal VCM trough concentrations and PK/PD parameters, which contributed to the appropriate use of VCM in patients with MRSA infections.
PMCID: PMC5072573  PMID: 27789965
antimicrobial stewardship; prospective audit and feedback; therapeutic drug monitoring; vancomycin
2.  Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea 
PLoS ONE  2015;10(10):e0141586.
The basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.
PMCID: PMC4624876  PMID: 26510163
3.  Grouping of multicopper oxidases in Lentinula edodes by sequence similarities and expression patterns 
AMB Express  2015;5:63.
The edible white rot fungus Lentinula edodes possesses a variety of lignin degrading enzymes such as manganese peroxidases and laccases. Laccases belong to the multicopper oxidases, which have a wide range of catalytic activities including polyphenol degradation and synthesis, lignin degradation, and melanin formation. The exact number of laccases in L. edodes is unknown, as are their complete properties and biological functions. We analyzed the draft genome sequence of L. edodes D703PP-9 and identified 13 multicopper oxidase-encoding genes; 11 laccases in sensu stricto, of which three are new, and two ferroxidases. lcc8, a laccase previously reported in L. edodes, was not identified in D703PP-9 genome. Phylogenetic analysis showed that the 13 multicopper oxidases can be classified into laccase sensu stricto subfamily 1, laccase sensu stricto subfamily 2 and ferroxidases. From sequence similarities and expression patterns, laccase sensu stricto subfamily 1 can be divided into two subgroups. Laccase sensu stricto subfamily 1 group A members are mainly secreted from mycelia, while laccase sensu stricto subfamily 1 group B members are expressed mainly in fruiting bodies during growth or after harvesting but are lowly expressed in mycelia. Laccase sensu stricto subfamily 2 members are mainly expressed in mycelia, and two ferroxidases are mainly expressed in the fruiting body during growth or after harvesting, and are expressed at very low levels in mycelium. Our data suggests that L. edodes laccases in same group share expression patterns and would have common biological functions.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-015-0151-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4573974  PMID: 26384343
Fruiting body; Laccase; Lentinula edodes; Lignin degradation; Multicopper oxidase
4.  Retrospective analysis of mortality and Candida isolates of 75 patients with candidemia: a single hospital experience 
The mortality rate for candidemia is approximately 30%–60%. However, prognostic factors in patients with candidemia have not yet been elucidated in detail. The aim of the present study was to analyze prognostic factors for candidemia using the mortality rate and Candida isolates of patients with candidemia. Seventy-five patients with candidemia were analyzed between January 2007 and December 2013. The main outcome of this study was the 30-day mortality rate after the diagnosis of candidemia. The acute physiology and chronic health evaluation II score (APACHE II score) was measured in 34 patients (45.3%). Odds ratios (ORs) for death due to candidemia were analyzed using a multivariate stepwise logistic regression analysis. Twenty (26.6%) patients died within 30 days of being diagnosed with candidemia. Non-survivors had a significantly higher APACHE II score (n=7, mean; 18.9±4.5) than that of survivors (n=27, mean; 14.0±5.0). Advanced age (OR =1.1, 95% confidence interval =1.01–1.23, P=0.04) was a significant risk factor for a high mortality rate, whereas removal of a central venous catheter (OR =0.03, 95% confidence interval =0.002–0.3, P=0.01) was associated with a lower mortality rate. Seventy-six Candida spp. were isolated from blood cultures: Candida albicans 28 (36.8%), Candida parapsilosis 23 (30.2%), Candida guilliermondii 16 (21.0%), Candida glabrata four (5.2%), Candida tropicalis two (2.6%), and Candida spp. three (3.9%) that could not be identified. C. parapsilosis was the most frequently isolated species in younger patients (<65 years), whereas C. albicans was the most frequently isolated in elderly patients (≥65 years). Physicians who treat candidemia need to consider removing the central venous catheter and pay attention to the general condition of patients, particularly that of elderly patients.
PMCID: PMC4501221  PMID: 26185460
aging; Candida; candidemia; central venous catheter; Japan
5.  Effect of Electrical Stimulation on Fruit Body Formation in Cultivating Mushrooms 
Microorganisms  2014;2(1):58-72.
The effect of high-voltage electrical stimulation on fruit body formation in cultivating mushrooms was evaluated using a compact pulsed power generator designed and based on an inductive energy storage system. An output voltage from 50 to 130 kV with a 100 ns pulse width was used as the electrical stimulation to determine the optimum amplitude. The pulsed high voltage was applied to a sawdust-based substrate of Lyophyllum decastes and natural logs hosting Lentinula edodes, Pholiota nameko, and Naematoloma sublateritium. The experimental results showed that the fruit body formation of mushrooms increased 1.3–2.0 times in terms of the total weight. The accumulated yield of Lentinula edodes for four cultivation seasons was improved from 160 to 320 g by applying voltages of 50 or 100 kV. However, the yield was decreased from 320 to 240 g upon increasing the applied voltage from 100 to 130 kV. The yield of the other types of mushrooms showed tendencies similar to those of Lentinula edodes when voltage was applied. An optimal voltage was confirmed for efficient fruit body induction. The hypha activity was evaluated by the amount of hydrophobin release, which was mainly observed before the fruit body formation. The hydrophobin release decreased for three hours after stimulation. However, the hydrophobin release from the vegetative hyphae increased 2.3 times one day after the stimulation.
PMCID: PMC5029503  PMID: 27694776
pulsed power; electrical stimulation; Lentinula edodes; mushroom
6.  Short-term training of upper gastrointestinal endoscopy for resident doctors in Sotogahama Central Hospital in Aomori, Japan 
It is essential for young physicians in municipal hospitals to be familiar with the technique of upper gastrointestinal (GI) endoscopy. Endoscopy is an exciting subspecialty in primary care medicine. Endoscopic procedures are primarily performed by general physicians in Japan. However, a standardized strategy for teaching diagnostic GI endoscopy is still lacking, and there is not sufficient time for young physicians to effectively learn the upper GI endoscopy technique. To elucidate how young physicians can be trained in the skills of GI endoscopy in a short time period, we initiated a 12-week training course. Two young physicians performed upper GI endoscopies for outpatients and inpatients 2 or 3 days a week from April 2010 to March 2012. The total number of cases undergoing GI endoscopy during the training course in each year was 117 and 111, respectively. The young physicians were trained in this technique by the attending physician. The short-term training course included four phases. During these phases, the young physicians learned how to insert the endoscope through the nasal cavity or oral cavity into the esophageal inlet, how to pass the endoscope from the esophageal inlet into the duodenum, how to take pictures with the endoscope, and how to stain the gastric and duodenal mucosa and take mucosal biopsy samples. The young physicians experienced 20–30 cases in each phase. In week five, they performed endoscope insertion into the duodenum along the folds of the greater curvature of the stomach. They viewed the entire stomach and took pictures until week ten of the course. The pictures taken in week ten were of a better quality for examining the disease lesions than those taken in week six. In the last 2 weeks of the training course, the young physicians stained the gastric and duodenal mucosa and took mucosal biopsy samples. The short-term training course of 100–120 cases in 12 weeks was effective for teaching young physicians how to perform GI endoscopies independently.
PMCID: PMC3746972  PMID: 23976870
endoscopy; gastroenterology; general medicine; medical education; young physicians
7.  Genetic engineering of yellow betalain pigments beyond the species barrier 
Scientific Reports  2013;3:1970.
Betalains are one of the major plant pigment groups found in some higher plants and higher fungi. They are not produced naturally in any plant species outside of the order Caryophyllales, nor are they produced by anthocyanin-accumulating Caryophyllales. Here, we attempted to reconstruct the betalain biosynthetic pathway as a self-contained system in an anthocyanin-producing plant species. The combined expressions of a tyrosinase gene from shiitake mushroom and a DOPA 4,5-dioxygenase gene from the four-o'clock plant resulted in successful betalain production in cultured cells of tobacco BY2 and Arabidopsis T87. Transgenic tobacco BY2 cells were bright yellow because of the accumulation of betaxanthins. LC-TOF-MS analyses showed that proline-betaxanthin (Pro-Bx) accumulated as the major betaxanthin in these transgenic BY2 cells. Transgenic Arabidopsis T87 cells also produced betaxanthins, but produced lower levels than transgenic BY2 cells. These results illustrate the success of a novel genetic engineering strategy for betalain biosynthesis.
PMCID: PMC3679504  PMID: 23760173
8.  Endo-β-1,3-Glucanase GLU1, from the Fruiting Body of Lentinula edodes, Belongs to a New Glycoside Hydrolase Family ▿ † 
Applied and Environmental Microbiology  2011;77(23):8350-8354.
The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).
PMCID: PMC3233070  PMID: 21965406
9.  Characterization of β-N-acetylhexosaminidase (LeHex20A), a member of glycoside hydrolase family 20, from Lentinula edodes (shiitake mushroom) 
AMB Express  2012;2:29.
We purified and cloned a β-N-acetylhexosaminidase, LeHex20A, with a molecular mass of 79 kDa from the fruiting body of Lentinula edodes (shiitake mushroom). The gene lehex20a gene had 1,659 nucleotides, encoding 553 amino acid residues. Sequence analysis indicated that LeHex20A belongs to glycoside hydrolase (GH) family 20, and homologues of lehex20a are broadly represented in the genomes of basidiomycetes. Purified LeHex20A hydrolyzed the terminal monosaccharide residues of β-N-acetylgalactosaminides and β-N-acetylglucosaminides, indicating that LeHex20A is a β-N-acetylhexosaminidase classified into EC The maximum LeHex20A activity was observed at pH 4.0 and 50°C. The kinetic constants were estimated using chitooligosaccharides with degree of polymerization 2-6. GH20 β-N-acetylhexosaminidases generally prefer chitobiose among natural substrates. However, LeHex20A had the highest catalytic efficiency (kcat/Km) for chitotetraose, and the Km values for GlcNAc6 were 3.9-fold lower than for chitobiose. Furthermore, the enzyme partially hydrolyzed amorphous chitin polymers. These results indicate that LeHex20A can produce N-acetylglucosamine from long-chain chitomaterials.
PMCID: PMC3430601  PMID: 22656067
β-N-acetylglucosaminide; Chitin; Fungal cell wall; Glycoside hydrolase family 20; Basidiomycete

Results 1-9 (9)