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Yoshihiko Sugihara,1 Shigehito Ikushima,1 Mika Miyake,1 Takayoshi Kirisako,2 Yukihiro Yada,3 Daisuke Fujiwara2 1Research Laboratories for Health Science and Food Technologies, 2Central Laboratories for Key Technologies, Research and Development Division, Kirin Company, Limited, Yokohama, 3Doctorate Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Japan Purpose: The present study examined the effect of ingestion of Koji extract containing 14-dehydroergosterol (14-DHE), prepared from Aspergillus kawachii NBRC4308, on improvement of skin conditions among healthy volunteers. Subjects and methods: In a randomized, double-blind, placebo-controlled, parallel-group study, 70 healthy adult women who felt that their skin was dry ingested either a placebo dietary supplement or Koji extract (200 mg/day) supplement containing 0.1% 14-DHE for 12 weeks. Throughout the treatment period and for 4 weeks afterward, objective indicators – including moisture content of the stratum corneum, trans-epidermal water loss (TEWL), and skin wrinkles – were evaluated; in addition, the subjects answered a questionnaire on their skin conditions with ratings on a visual analog scale. Statistical analysis was conducted on the basis of differences from baseline scores. Results: Compared with the placebo group, the Koji extract group showed significantly increased forearm moisture at 4, 8, and 16 weeks (p < 0.05 on unpaired t-test). The questionnaire survey showed a marked improvement in skin conditions, particularly crow’s feet, in the Koji extract group versus the placebo group at 8 weeks (p < 0.05 by unpaired t-test). Furthermore, the Koji extract group showed a trend (p < 0.10) toward improvement in skin moisture (at 4 weeks), dryness around the eyes/mouth (at 4 weeks), and overall skin condition (at 8 weeks) versus the placebo group. Conclusion: Ingestion of Koji extract containing 14-DHE was demonstrated to have positive effects toward improving skin conditions – in particular, on increasing skin moisture in the stratum corneum. Keywords: 14-dehydroergosterol, Aspergillus, Koji, skin, skin moisture, TEWL

Introduction. Antimicrobial resistance (AMR) is a growing global concern. Clonal lineages of CTX-M β-lactamase-producing Escherichia coli (CTXE) and quinolone-resistant E. coli (QREC) were disseminated among the deer population in a famous tourist destination (Nara Park; NP) in Japan. Hypothesis/gap statement. The molecular characteristics of CTXE or QREC isolates, which could pose a threat to public health, have not been elucidated. Aim. This study aimed to characterize the genetic traits of CTXE and QREC isolates derived from NP deer and compare them with lineages prevalent worldwide. Methodology. Sixteen CTXE and three QREC isolates recovered from NP deer faeces between 2018 and 2020 were analysed using whole-genome sequencing (WGS). For endemic lineages, phylogenetic trees were constructed against the isolates registered in the EnteroBase database using the core genome SNP scheme. Results. The most prevalent lineage in NP deer was ST3580. Several pandemic lineages, such as sequence type (ST) 38, ST58 and ST117, were included. The QREC lineages prevalent among deer were designated as extra-intestinal pathogenic E. coli or uropathogenic E. coli (UPEC). Thirteen of the 24 antimicrobial resistance genes (ARGs) were considered high-risk ARG families. Chromosomal integration of bla CTX-M-15 was observed in all plasmid-negative isolates. Phylogenetic analysis suggested relationships between NP isolates and isolates sourced from the environment or poultry. Conclusion. ST3580 has a high potential for clonal dissemination. Furthermore, multiple clinically relevant lineages of CTXE and QREC are endemic in NP deer; however, they could be less virulent than isolates belonging to the same lineages, which could cause severe infectious diseases. Further studies are required to investigate the relationship between chromosomal integration of plasmid-encoded genes and the stable propagation of AMR bacteria in wildlife and the environment., Competing Interests: The authors declare that that there are no conflicts of interest., (Copyright © 2024 The Authors.)

Osimertinib is used as the first-line therapy for patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC). However, early dose reduction is often required due to adverse events (AEs). This study aimed to evaluate the effect of early dose reduction of osimertinib on efficacy and safety. This was a retrospective study including patients with EGFR-mutated NSCLC who were started on osimertinib as the first-line therapy between August 2018 and December 2021. Patients whose doses were reduced to less than 80 mg/day within 6 months of osimertinib initiation or started at 40 mg/day were defined as the dose reduction group. The primary endpoint was progression-free survival (PFS). Factors affecting PFS were explored using the Cox proportional hazards model. A total of 85 patients were included in this study. No significant differences in patient characteristics were observed between the dose reduction (n = 25) and standard dose groups (n = 60). The median PFS in the dose reduction group was significantly prolonged compared with that in the standard dose group (26.0 months vs. 12.0 months, p = 0.03). Multivariable analysis of 84 patients, excluding a patient with unknown brain metastasis, revealed that EGFR exon 21 L858R mutation, malignant pleural effusion or pleural metastasis, liver metastasis, and dose reduction within 6 months were independent factors affecting PFS. Early dose reduction of osimertinib is an effective therapeutic strategy for prolonging PFS in patients with EGFR-mutated NSCLC., (© 2024. The Author(s).)

Transmission of antimicrobial-resistant bacteria among humans, animals, and the environment is a growing concern worldwide. The distribution of an international high-risk fluoroquinolone-resistant Escherichia coli clone, ST131, has been documented in clinical settings. However, the transmission of ST131 from humans to surrounding environments remains poorly elucidated. To comprehend the current situation and identify the source of ST131 in nature, we analyzed the genetic features of ST131 isolates from the aquatic environment (lake/river water) and wildlife (fox, raccoon, raccoon dog, and deer) and compared them with the features of isolates from humans in Japan using accessory and core genome single nucleotide polymorphism (SNP) analyses. We identified ST131 isolates belonging to the same phylotype and genome clusters (four of eight clusters were concomitant) with low SNP distance between the human isolates and those from the aquatic environment and wildlife. These findings warn of ST131 transmission between humans and the surrounding environment in Japan., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Wildlife in urban areas have the potential to disseminate antimicrobial-resistant bacteria (ARB) across a wider environment. Using antimicrobial-supplemented agar plates, we isolated extended-spectrum β-lactamase-producing Escherichia coli (EEC) and quinolone-resistant E. coli (QREC) from 144, 23, and 30 deer feces from Nara Park (NP), rural area neighboring NP (RA), and Mt. Odaigahara (MO), respectively. In NP and RA, the prevalence of EEC was 24.3 and 4.3%, respectively; that of QREC was 11.1 and 17.4%, respectively. Neither EEC nor QREC were detected in MO. The pulsotypes of EEC and QREC isolates differed between NP and RA. Our study suggests that deer of the Nara Prefecture are potential carriers of ARB, but long-distance dissemination is unlikely due to limited deer movement.

Background/aim: Osimertinib is a key drug for treating epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC). Genetic differences may be associated to adverse events (AEs) induced by osimertinib. This retrospective observational multicenter study evaluated the association of genotypes, including STAT3 -1697C>G, CYP3A5 6986A>G, and ABCG2 421C>A, with the incidence of osimertinib-induced AEs in patients with EGFR mutation-positive NSCLC., Patients and Methods: A total of 85 patients treated with osimertinib (Institution A: 33 patients, Institution B: 52 patients) were enrolled in the study. Single nucleotide polymorphisms were determined by real-time PCR, and the incidence of AEs was compared for each genotype., Results: Paronychia incidence was 59% for the CC genotype, 19% for the CG genotype, and 19% for the GG genotype at STAT3 -1697C>G. A genotype-related trend was observed (Cochran-Armitage test, p=0.009). Multivariate analysis showed that the CC genotype at STAT3 -1697C>G and female sex were significant independent factors associated with paronychia [odds ratio (OR)=6.41, 95% confidence interval (CI)=1.94-21.20 and OR=3.40, 95%CI=1.03-11.22, respectively]. The incidence of diarrhea was 53% for the CC genotype, 30% for the AC genotype, and 29% for the AA genotype at ABCG2 421C>A, and a genotype-related trend was observed (p=0.048). However, the CC genotype at ABCG2 421C>A was not a significant independent factor associated with diarrhea in multivariate analysis. No significant associations were detected between other polymorphisms and the incidence of AEs., Conclusion: STAT3 -1697C>G may be a novel risk factor for osimertinib-induced paronychia in patients with NSCLC., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Clinical evidence on the increased efficacy of sequential epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy in patients with EGFR-mutated non-small-cell lung cancer (NSCLC) is limited. This study aimed to compare the efficacy of upfront use of first-/second-generation TKI followed by osimertinib with upfront osimertinib therapy for each representative EGFR mutation in Japanese patients with NSCLC. Patients with EGFR-mutated NSCLC were classified into two groups: first-/second-generation TKI followed by osimertinib (sequential TKI group) and upfront osimertinib groups. The total time to treatment failure (TTF) of TKI therapies, progression-free survival (PFS), and overall survival (OS) were retrospectively evaluated. Of the 74 patients included in the analysis, 38 and 34 patients had exon 19 deletion and L858R, respectively, and other two patients had minor mutations. The sequential TKI group had a significantly longer TTF than the upfront osimertinib group in overall patients (33.2 vs. 11.2 months; p = 0.007) and in the subgroup of exon 19 deletion (36.7 vs. 10.0 months; p = 0.004), but not in the subgroup of L858R (22.6 vs. 15.6 months; p = 0.37). The similar tendency was observed in PFS. OS of the sequential TKI group was significantly longer compared with the upfront osimertinib group in overall patients, the subgroup of exon 19 deletion, and the subgroup of L858R. The upfront use of first-/second-generation TKI followed by osimertinib is one of the feasible and effective strategies in Japanese patients with EGFR-mutated NSCLC, especially in patients with exon 19 deletion.

Monitoring the mortality of wildlife provides basic demographic information to support management plan preparation. The utility of mortality records for conservation measures was investigated in the Japanese serow, focusing on temporal trends and spatial distribution. Using the mortality records of Japanese serow from 2006 to 2018 in Gifu prefecture, cause-specific mortality was categorized into five groups (disease, accident, vehicle collision, parapoxvirus infection, and unknown), and the sex ratios were examined. A state space model was used to analyze the time series for the monthly mortalities, and kernel estimation was used for the spatial distribution of the parapoxvirus infection. Land cover type around the detection point was also reported. Disease, accident, and vehicle collision mortality were similar, and 30% of mortality was of anthropogenic origin. The number of mortality records for males was higher, and the larger home range of males could account for this. The state space model showed moderate increases in monthly mortalities over time and a seasonal variation with the highest level in spring and lowest in winter. Land cover analysis demonstrated a temporal increase in the proportion of human settlement areas, suggesting the change of the Japanese serow habitat. The proximity of Japanese serow to human settlements contributed to increase in mortality records. The point pattern analysis indicated spatial clustering for parapoxvirus infection in the area where an epidemic had occurred in the past. Several measures are recommended; however, mortality records can help develop improved conservation plan.

The emergence and dissemination of resistance to clinically important antimicrobials in wild animals is of great concern. The aim of our study was to reveal the prevalence and intraspecies dissemination of quinolone-resistant Escherichia coli (QREC) in sika deer (Cervus nippon) in Nara Park, a famous tourist spot in Japan. Fecal samples were collected from 59 wild deer in Nara Park between July and October 2018. We isolated QREC using deoxycholate-hydrogen sulfide-lactose agar containing nalidixic acid and subjected it to antimicrobial susceptibility testing. The mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes of the isolates were analyzed and fragment patterns of genomic DNA were compared by pulsed-field gel electrophoresis (PFGE). A total of 105 QREC isolates were obtained from 41 deer (70%). All isolates had mutations within the QRDR. Other than quinolone resistance, QREC isolates also showed resistance to various other antimicrobial agents. The QREC isolates were classified into 15 PFGE clusters, of which seven were observed in multiple deer. Our results suggest clonal transmission of QREC in a high-density deer population. Spread of QREC in deer inhabiting a tourist location could have potential impact on public health., (© Wildlife Disease Association 2021.)

In this retrospective study, we investigated vascular abnormalities in sarcoidosis using fluorescein gonioangiography (FGA) to detect angle neovascularization (ANV), fundus fluorescein angiography (FFA), and pathological specimens from the aspects of microangiopathy. In 57 sarcoidosis patients, clinical data was reviewed by dividing the cases into three groups (Group I: histologically diagnosed; Group II: positive bilateral hilar lymphadenopathy (BHL); Group III: negative BHL). The FFA, FGA, and pathological examination data in the autopsy eyes and trabeculectomy specimens were investigated. FGA and FFA detected ANV (91%) and nodule-associated abnormalities (87%), respectively. No intraocular pressure (IOP) elevation was observed after continuous topical betamethasone, except in the steroid responder group. Maximum IOP had significant correlation with nodules in the angle ( p = 0.02696) and visual field defect ( p = 0.0151). Granulomas adjacent to blood vessels, including the Schlemm's canal, and thickening of the retinal blood vessel wall caused occlusion of those vessels. Photocoagulation was required for retinal tears (14%) and the retinal blood vessel occlusion (7%). Suppression of IOP elevation via continuous topical betamethasone may be important to avoid irreversible outflow-route changes and optic-nerve damage, and the concept of microangiopathy in ocular sarcoidosis may be important for understanding the proper treatment of serious complications.

Retail meats are one of the main routes for spreading antimicrobial-resistant bacteria (ARB) from livestock to humans through the food chain. In African countries, retail meats are often sold at roadside butcheries without chilling or refrigeration. Retail meats in those butcheries are suspected to be contaminated by ARB, but it was not clear. In this study, we tested for the presence of antimicrobial-resistant Escherichia coli from retail meats ( n  = 64) from roadside butcheries in Kampala, Uganda. The meat surfaces were swabbed and inoculated on PetriFilm SEC agar to isolate E. coli . We successfully isolated E. coli from 90.6% of these retail meat samples. We identified the phylogenetic type, antimicrobial susceptibility, and antimicrobial resistance genes prevalence between retail meat isolates ( n  = 89). Phylogenetic type B1 was identified from 70.8% of the retail meat isolates, suggesting that the isolates originated primarily from fecal contamination during meat processing. Tetracycline (TET)-resistant isolates with tetA and/or tetB gene(s) were the most frequently detected (28.1%), followed by ampicillin (AMP) resistance genes with bla TEM (15.7%,) and sulfamethoxazole-trimethoprim (SXT) resistance genes with sul2 (15.7%). No extended-spectrum beta-lactamase-producing isolates were detected. A conjugation assay showed that resistance to AMP, TET, and SXT could be simultaneously transferred to recipients. These findings suggest that antimicrobial-resistant E. coli can easily be transferred from farms to tables from retail meats obtained from roadside butcheries.

Sarcoidosis is a genetically complex systemic inflammatory disease that affects multiple organs. We present a GWAS of a Japanese cohort (700 sarcoidosis cases and 886 controls) with replication in independent samples from Japan (931 cases and 1,042 controls) and the Czech Republic (265 cases and 264 controls). We identified three loci outside the HLA complex, CCL24, STYXL1-SRRM3, and C1orf141-IL23R, which showed genome-wide significant associations (P < 5.0 × 10 -8 ) with sarcoidosis; CCL24 and STYXL1-SRRM3 were novel. The disease-risk alleles in CCL24 and IL23R were associated with reduced CCL24 and IL23R expression, respectively. The disease-risk allele in STYXL1-SRRM3 was associated with elevated POR expression. These results suggest that genetic control of CCL24, POR, and IL23R expression contribute to the pathogenesis of sarcoidosis. We speculate that the CCL24 risk allele might be involved in a polarized Th1 response in sarcoidosis, and that POR and IL23R risk alleles may lead to diminished host defense against sarcoidosis pathogens.

We report the case of a 60-year-old Japanese man with a metastatic brain tumor that caused ataxia. As a consequence of resection of a cerebellar tumor, the tumor was diagnosed as a poorly differentiated adenocarcinoma with choriocarcinomatous features. The patient underwent bronchoscopy, leading to a diagnosis of the same histology as the brain tumor. After the administration of first-line chemotherapy and maintenance therapy due to progressive disease, he was given nivolumab and obtained a partial response; however, 11-months later, computed tomography showed tumor progression. Our experience suggests that nivolumab has strong activity, even in patients with a rare form of lung cancer.

Background: Idiopathic pleuroparenchymal fibroelastosis (IPPFE) is a rare type of interstitial pneumonia characterized by fibroelastosis. Patients with IPPFE as well as idiopathic interstitial pneumonia often have autoimmune diseases, which sometimes coincide with hypothyroidism (HypoT). However, there have been no reports on the association between IPPFE and HypoT. The purpose of this study was to evaluate the correlation between IPPFE and HypoT. We also examined the pathological features of the thyroid glands from autopsied cases., Methods: Thirteen patients diagnosed with IPPFE from among 255 consecutive cases of idiopathic interstitial pneumonia were included in this study; pertinent data were obtained from our hospital's clinical library. We examined the prevalence of HypoT and compared the clinical, radiological, and pathological features between the patients with and those without HypoT. Histological analyses of the lungs and thyroid glands were performed in 4 and 3 cases, respectively., Results: HypoT was identified in 7 of 13 patients (53.8%). Sex, body mass index, survival time, and laboratory test results were not significantly different between patients with and those without HypoT. Radiological and pathological lung findings were similar between both groups of patients. Thyroid gland histology demonstrated perifollicular or interlobular fibrosis without inflammation in all three cases, including a euthyroid case., Conclusions: Although we only analyzed a small number of IPPFE cases, HypoT was prevalent among all of them. Characteristic fibrosis in the thyroid gland was observed even in a euthyroid case. Therefore, patients with IPPFE may potentially have thyroid gland dysfunction through a common pathogenesis in both organs., (Copyright © 2017 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Background: CyberKnife® (CK) is a new, advanced radiotherapy technique. This study aimed to evaluate its efficacy and toxicity in Japanese patients with early-stage primary lung tumor who were medically unfit and inoperable., Methods: This retrospective study investigated patients who received CK treatment for medically inoperable Stage І primary lung tumor at the Japanese Red Cross Medical Center between June 2011 and September 2016. Each patient received a total of 36-48 Gy (median, 43 Gy) administered by CK in 4-5 fractions., Results: Totally, 40 patients (T1a, n = 19; T1b, n = 15; T2a, n = 6) were included. Their median age was 86 (range, 56-95) years. Tracking required the use of fiducial markers in 28 patients and the Xsight Spine Tracking System in 12. The median follow-up was 14.5 (range, 1-51) months. Local recurrence occurred in seven (17.5%) patients. The local progression-free survival rates at 1 and 2 years were 83.9% and 74.0%, respectively. Distant recurrence occurred in regional lymph nodes (n = 5), the lung outside the radiation field (n = 3), and the bone (n = 1). Seven patients died. Overall survival rates at 1 and 2 years were 93.6% and 73.1%, respectively. Radiation pneumonitis was identified in 28 (70%) patients (Grade 1, n = 25; Grade 2, n = 2; Grade 5, n = 1)., Conclusions: CK showed good local control with limited toxicity and could be an alternative treatment modality in medically inoperable patients with Stage І primary lung tumor., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Aims: The lung lesion [immunoglobulin (Ig)G4-L] of IgG4-related disease (IgG4-RD) is a condition that occurs together with IgG4-RD and often mimics the lung lesion [idiopathic multicentric Castleman's disease (iMCD-L)] of idiopathic multicentric Castleman's disease (iMCD). Because no clinical and pathological studies had previously compared features of these diseases, we undertook this comparison with clinical and histological data., Methods and Results: Nine patients had IgG4-L (high levels of serum IgG4 and of IgG4 + cells in lung specimens; typical extrapulmonary manifestations). Fifteen patients had iMCD-L (polyclonal hyperimmunoglobulinaemia, elevated serum interleukin-6 levels and polylymphadenopathy with typical lymphadenopathic lesions). Mean values for age, serum haemoglobin levels and IgG4/IgG ratios were higher in the IgG4-L group and C-reactive protein levels were higher in the iMCD-L group. All IgG4-RD lung lesions showed myxomatous granulation-like fibrosis (active fibrosis), with infiltration of lymphoplasmacytes and scattered eosinophils within the perilymphatic stromal area, such as interlobular septa and pleura with obstructive vasculitis. All 15 lung lesions of iMCD, however, had marked accumulation of polyclonal lymphoplasmacytes in lesions with lymphoid follicles and dense fibrosis, mainly in the alveolar area adjacent to interlobular septa and pleura without obstructive vasculitis., Conclusions: Although both lesions had lymphoplasmacytic infiltration, lung lesions of IgG4-RD were characterized by active fibrosis with eosinophilic infiltration within the perilymphatic stromal area with obstructive vasculitis, whereas lung lesions of iMCD had lymphoplasmacyte proliferating lesions mainly in the alveolar area adjacent to the perilymphatic stromal area. These clinicopathological features may help to differentiate the two diseases., (© 2017 John Wiley & Sons Ltd.)

Aims: To evaluate pulmonary vasculopathy in an autopsy series of patients with combined pulmonary fibrosis and emphysema (CPFE), and compare these findings with those of patients with idiopathic pulmonary fibrosis (IPF) alone and emphysema alone., Methods and Results: We retrospectively analysed the clinical, radiological and pathological features of 26 patients with CPFE, 11 with IPF, and 23 with emphysema. We evaluated pulmonary vascular, venous-venular and arteriolar tissue changes in the fibrotic, emphysematous and relatively unaffected (preserved) areas by using the Heath-Edwards scoring system. We found moderate-to-severe vasculopathy in the CPFE group, but no significant differences in the fibrotic and emphysematous areas among the three groups. However, in the preserved area, the grading was significantly different among the three groups (P < 0.001), and vasculopathy in the CPFE group was the most severe. Although venous-venular and arteriolar changes in almost all fibrotic and emphysematous areas in the three groups showed no significant differences, there were significant differences in venous-venular (P = 0.004) and arteriolar (P < 0.001) changes in the preserved area among the three groups, which were most prevalent in the CPFE group. In the CPFE group, venous-venular changes and vasculopathy by Heath-Edwards grading were highest in the fibrotic area and lowest in the preserved area., Conclusions: These results imply that pulmonary vasculopathy in patients with CPFE could occur in the whole lung tissue. This may explain the tendency for it to lead to the development of pulmonary hypertension in CPFE cases., (© 2016 John Wiley & Sons Ltd.)

Here we describe the development of tightly regulated expression switches in yeast, by engineering distant homologues of Escherichia coli TetR, including the transcriptional regulator PhlF from Pseudomonas and others. Previous studies demonstrated that the PhlF protein bound its operator sequence (phlO) in the absence of 2,4-diacetylphloroglucinol (DAPG) but dissociated from phlO in the presence of DAPG. Thus, we developed a DAPG-Off system in which expression of a gene preceded by the phlO-embedded promoter was activated by a fusion of PhlF to a multimerized viral activator protein (VP16) domain in a DAPG-free environment but repressed when DAPG was added to growth medium. In addition, we constructed a DAPG-On system with the opposite behavior of the DAPG-Off system; i.e., DAPG triggers the expression of a reporter gene. Exposure of DAPG to yeast cells did not cause any serious deleterious effect on yeast physiology in terms of growth. Efforts to engineer additional Tet repressor homologues were partially successful and a known mammalian switch, the p-cumate switch based on CymR from Pseudomonas, was found to function in yeast. Orthogonality between the TetR (doxycycline), CamR (d-camphor), PhlF (DAPG), and CymR (p-cumate)-based Off switches was demonstrated by evaluating all 4 ligands against suitably engineered yeast strains. This study expands the toolbox of "On" and "Off" switches for yeast biotechnology.

As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiae -based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.

Some patients with idiopathic pulmonary fibrosis (IPF) do not have honeycombing on high-resolution computed tomography (HRCT) at their initial evaluation. The clinical course and sequential changes in HRCT findings in these patients are not fully understood. We reviewed the cases of 43 patients with IPF without honeycombing on initial HRCT from institutions throughout Japan. All patients were diagnosed with IPF based on a surgical lung biopsy. Multidisciplinary discussions were held five times between 2011 and 2014, to exclude alternative etiologies. We evaluated the sequential changes in HRCT findings in 30 patients with IPF. We classified these 30 patients into three groups based on their HRCT patterns and clarified the clinical characteristics and prognosis among the groups. The patterns of all 30 patients on initial HRCT corresponded to a possible usual interstitial pneumonia (UIP) pattern which was described in the 2011 International Statement. On long-term follow-up (71.0±38.7 standard deviation [SD] months), honeycombing was seen in 16 patients (53%, the HoneyCo group); traction bronchiectasis or cysts without honeycombing was observed in 12 patients (40%, the NoHoneyCo group), and two patients showed no interval change (7%, the NoChange group) on HRCT. The mean survival periods of the HoneyCo and NoHoneyCo groups were 67.1 and 61.2 months, respectively (p = 0.76). There are some patients with IPF whose conditions chronically progress without honeycombing on HRCT. The appearance of honeycombing on HRCT during the follow-up might not be related to prognosis., Competing Interests: The authors have declared that no competing interests exist.

The edible, nitrate assimilating, yeast Candida utilis is a commercial food additive, and it is a potentially useful host for heterologous protein expression. A number of ATP-binding cassette (ABC) transporters are multidrug efflux pumps that can cause multidrug resistance in opportunistic pathogens. In order to develop optimal novel antimicrobial agents it is imperative to understand the structure, function and expression of these transporters. With the ultimate aim of developing an alternative yeast host for the heterologous expression of eukaryotic membrane transporters, and to identify ABC transporters potentially associated with C. utilis multidrug resistance, we classified the entire repertoire of 30 C. utilis ABC proteins. We named the open reading frame most similar to the archetype multidrug efflux pump gene C. albicans CDR1 as CuCDR1 Overexpression of CuCDR1 in Saccharomyces cerevisiae ADΔ caused multidrug resistance similar to that of cells overexpressing CaCDR1 Unlike CaCdr1p, however, the C-terminally green fluorescent protein (GFP) tagged CuCdr1p-GFP was functionally impaired and did not properly localize to the plasma membrane. CuCdr1p function could be recovered however by adding a 15 amino acid linker -GAGGSAGGSGGAGAG- between CuCdr1p and the C-terminal GFP tag., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

TAFRO syndrome is a systemic inflammatory disorder characterized by thrombocytopenia, anasarca including pleural effusion and ascites, fever, renal insufficiency, and organomegaly including hepatosplenomegaly and lymphadenopathy. Its onset may be acute or sub-acute, but its etiology is undetermined. Although several clinical and pathological characteristics of TAFRO syndrome resemble those of multicentric Castleman disease (MCD), other specific features can differentiate between them. Some TAFRO syndrome patients have been successfully treated with glucocorticoids and/or immunosuppressants, including cyclosporin A, tocilizumab and rituximab, whereas others are refractory to treatment, and eventually succumb to the disease. Early and reliable diagnoses and early treatments with appropriate agents are essential to enhancing patient survival. The present article reports the 2015 updated diagnostic criteria, disease severity classification and treatment strategy for TAFRO syndrome, as formulated by Japanese research teams. These criteria and classification have been applied and retrospectively validated on clinicopathologic data of 28 patients with this and similar conditions (e.g. MCD with serositis and thrombocytopenia).

Trousseau's syndrome (cancer-associated thrombosis) is the second leading cause of death in cancer patients, after death from cancer itself. The risk of a venous thromboembolism is 4- to 7-fold higher in patients with cancer than in those without cancer. The causes of this impaired coagulation are associated with general patient-related risk factors, and other factors that are specific to the particular cancer or treatment. It is important to assess the risk of thrombotic events in cancer patients and administer effective prophylaxis and treatment. Effective prophylaxis and treatment of venous thromboembolism reduces morbidity and mortality, and improves patients' quality of life. Low molecular weight heparin is the first-line treatment for venous thromboembolism, as an effective and safe means for prophylaxis and treatment, according to guidelines released by international scientific societies. Oral anticoagulation therapy with warfarin is preferable to no therapy. However, warfarin has low efficacy and is associated with high rates of recurrence. If low molecular weight heparin is unavailable, some guidelines recommend the use of vitamin K antagonists that have a target international normalized ratio in the range of 2-3, as acceptable alternatives. Novel oral anticoagulants that directly inhibit factor Xa or thrombin are promising for the prophylaxis of high-risk cancer patients and in the long-term treatment of venous thromboembolism. However, to date, there is insufficient evidence to support the use of these new anticoagulants., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A 56-year-old woman, a never-smoker, had postoperative recurrence of anaplastic lymphoma kinase rearranged lung cancer. She achieved a partial response to treatment with an anaplastic lymphoma kinase tyrosine kinase inhibitor, crizotinib. After the tumor regrowth, crizotinib was switched to alectinib; once again a partial response was observed. At the second recurrence, transbronchial needle aspiration of the right paratracheal node was performed, which revealed cytological findings of small-cell carcinoma. While treatment with cisplatin-irinotecan chemotherapy made reduction of some tumor shadows, including the biopsied mediastinal lymph nodes, new, small, nodular shadows, highly suggestive of pulmonary metastases, were detected in both lung fields. This case may show proof of the transformation to small-cell lung cancer as a mechanism of resistance to anaplastic lymphoma kinase tyrosine kinase inhibitors in anaplastic lymphoma kinase rearranged tumor. However, this transformation may also be only one part of the resistance mechanism of the heterogeneous tumor., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Chronic pulmonary aspergillosis (CPA) is a refractory disorder that needs long-term antifungal treatment and occasionally results in fatal respiratory failure and hemoptysis. However, the pathological features of the disorder have not been thoroughly delineated. Thirty cases were therefore analyzed clinically and histologically to elucidate the pathophysiology of CPA. The subjects comprised 14 individuals who underwent surgical removal and 16 patients who died. No subject exhibited a severely immunocompromised state. The main symptoms included cough, hemosputum, and dyspnea. Chest computed tomography (CT) findings revealed a cavity, fungus ball, and consolidation and/or ground glass opacity (GGO); 27 serial CT scans showed enlarged consolidation and/or GGO (70%), dilatation of the cavity (26%), and extension to the opposite lung (22%). Histopathological findings revealed a cavity with ulceration, bronchitis, and various degrees of organizing pneumonia (OP) that were correlated with the area of consolidation and GGO on the CT scan. The essential pathophysiology of CPA can be understood as an active state of ulceration of the cavity and/or erosive bronchitis caused by contact with the fungus ball, which may play a significant role in the development of OP. Consequently, OP is thought to reflect respiratory failure that relates to the prognosis of CPA.

We herein describe a case of sarcoid myositis with anti-Ku antibody positivity. Pathological findings of the muscle were compatible with sarcoidosis, but could not be completely distinguished from myositis diseases that arise from other causes. According to a physical examination, pathological findings, the detection of anti-Ku antibody and the human leukocyte antigen (HLA)-DPB1 allele, we strongly suspected that the patient developed both sarcoidosis and polymyositis. Sarcoidosis is often complicated by autoimmune diseases. This case suggests the possibility that sarcoidosis and other autoimmune diseases may have common causal genetic factors.

Background: Although Mycobacterium gordonae isolation from respiratory samples is usually regarded as contamination, M. gordonae can cause definite pulmonary disease. The establishment of a standard diagnostic criteria of pulmonary disease that is caused by this low virulence mycobacterium is obviously necessary., Methods: We performed clinical research on over 200 cases in which M. gordonae was isolated over an 8-year period, focusing on the M. gordonae subtype. Sequence analysis of rpoB was performed to identify the genotypes., Results: A total of 287 respiratory samples (209 cases) were positive for M. gordonae. Twenty-seven cases (12.9%) had a positive culture more than twice and 11 of these cases (5.3%) had more than three positive cultures. Ultimately, three cases (1.4%) were newly diagnosed as M. gordonae pulmonary disease using our own diagnostic criteria. In all of the identified M. gordonae cases, the cultures tested positive with a Mycobacteria Growth Indicator Tube test at 24 days; however, in patients with definitive pulmonary disease, the cultures were positive at 9 days. A subtype analysis revealed that all of the definitive disease cases had subtype C., Conclusion: The time taken to detect a positive culture and subtype of the isolates could be used as the diagnostic criteria for definite M. gordonae pulmonary disease., (© The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Here we describe the engineering of a distant homolog of the Tet repressor, CamR, isolated from Pseudomonas putida, that is regulated by camphor, a very inexpensive small molecule (at micromolar concentrations) for use in Saccharomyces cerevisiae. The repressor was engineered by expression from a constitutive yeast promoter, fusion to a viral activator protein cassette, and codon optimization. A suitable promoter responsive to the CamR fusion protein was engineered by embedding a P. putida operator binding sequence within an upstream activating sequence (UAS)-less CYC1 promoter from S. cerevisiae. The switch, named the Camphor-Off switch, activates expression of a reporter gene in camphor-free media and represses it with micromolar concentrations of camphor., (Copyright © 2015 Ikushima et al.)

We have adapted the Golden Gate DNA assembly method to the assembly of transcription units (TUs) for the yeast Saccharomyces cerevisiae, in a method we call yeast Golden Gate (yGG). yGG allows for the easy assembly of TUs consisting of promoters (PRO), coding sequences (CDS), and terminators (TER). Carefully designed overhangs exposed by digestion with a type IIS restriction enzyme enable virtually seamless assembly of TUs that, in principle, contain all of the information necessary to express a gene of interest in yeast. We also describe a versatile set of yGG acceptor vectors to be used for TU assembly. These vectors can be used for low or high copy expression of assembled TUs or integration into carefully selected innocuous genomic loci. yGG provides synthetic biologists and yeast geneticists with an efficient new means by which to engineer S. cerevisiae.

Lactococcus lactis subsp. lactis JCM 5805(T) is a dairy lactic acid bacterium that induces plasmacytoid dendritic cell (pDC) activation. Here, we report the 2.55-Mb draft genome and annotation of Lactococcus lactis JCM 5805(T). This genome information will provide further insights into the mechanisms underlying the immunomodulatory function of this strain., (Copyright © 2015 Fujii et al.)

Background: Clinical evaluation to differentiate the characteristic features of pulmonary fibrosis and emphysema is often difficult in patients with combined pulmonary fibrosis and emphysema (CPFE), but diagnosis of pulmonary fibrosis is important for evaluating treatment options and the risk of acute exacerbation of interstitial pneumonia of such patients. As far as we know, it is the first report describing a correlation among clinical, radiological, and whole-lung pathological features in an autopsy cases of CPFE patients., Methods: Experts retrospectively reviewed the clinical charts and examined chest computed tomography (CT) images and pathological findings of an autopsy series of 22 CPFE patients, and compared these with findings from 8 idiopathic pulmonary fibrosis (IPF) patients and 17 emphysema-alone patients., Results: All patients had a history of heavy smoking. Forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC%) was significantly lower in the emphysema-alone group than the CPFE and IPF-alone groups. The percent predicted diffusing capacity of the lung for carbon monoxide (DLCO%) was significantly lower in the CPFE group than the IPF- and emphysema-alone groups. Usual interstitial pneumonia (UIP) pattern was observed radiologically in 15 (68.2%) CPFE and 8 (100%) IPF-alone patients and was pathologically observed in all patients from both groups. Pathologically thick-cystic lesions involving one or more acini with dense wall fibrosis and occasional fibroblastic foci surrounded by honeycombing and normal alveoli were confirmed by post-mortem observation as thick-walled cystic lesions (TWCLs). Emphysematous destruction and enlargement of membranous and respiratory bronchioles with fibrosis were observed in the TWCLs. The cystic lesions were always larger than the cysts of honeycombing. The prevalence of both radiological and pathological TWCLs was 72.7% among CPFE patients, but no such lesions were observed in patients with IPF or emphysema alone (p=0.001). The extent of emphysema in CPFE patients with TWCLs was greater than that in patients without such lesions. Honeycombing with emphysema was also observed in 11 CPFE patients., Conclusions: TWCLs were only observed in the CPFE patients. They were classified as lesions with coexistent fibrosing interstitial pneumonia and emphysema, and should be considered an important pathological and radiological feature of CPFE.

Background: In 1999, a Japanese epidemiological survey of chronic hypersensitivity pneumonitis (HP) showed that summer-type HP was the most prevalent variant of the disease. The number of reported cases of chronic HP has recently been increasing, and the clinical features of the disease seem to have changed. We conducted another nationwide epidemiological survey of chronic HP in Japan to determine better estimates of the frequency and clinical features of the disease., Methods: A questionnaire was sent to qualified hospitals throughout Japan, and data on cases of chronic HP diagnosed between 2000 and 2009 were collected., Results: In total, 222 cases of chronic HP from 22 hospitals were studied. Disease subtypes included bird-related HP (n=134), summer-type HP (n=33), home-related HP (n=25), farmer's lung (n=4), isocyanate-induced HP (n=3), and other types (n=23). The median proportion of lymphocytes in bronchoalveolar lavage fluid was high (24.5%). The primary findings of computed tomography of the chest were ground-glass attenuation and interlobular septal thickening. Centrilobular fibrosis was the major pathological finding on examination of surgical lung biopsy specimens from 93 patients. The median survival time was 83 months., Conclusions: The proportion of bird-related HP was higher than that in the previous epidemiological survey, and the proportions of isocyanate-induced HP and farmer's lung were lower. A crucial step in diagnosing chronic HP is to thoroughly explore the possibility of antigen exposure., (© 2013 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

A genetically-engineered strain of the yeast Candida utilis harboring genes encoding (1) an acetoacetyl-CoA transferase from Clostridium acetobutylicum ATCC 824, (2) an acetoacetate decarboxylase, and (3) a primary-secondary alcohol dehydrogenase derived from Clostridium beijerinckii NRRL B593 produced up to 0.21 g/L of isopropanol. Because the engineered strain accumulated acetate, isopropanol titer was improved to 1.2 g/L under neutralized fermentation conditions. Optimization of isopropanol production was attempted by the overexpression and disruption of several endogenous genes. Simultaneous overexpression of two genes encoding acetyl-CoA synthetase and acetyl-CoA acetyltransferase increased isopropanol titer to 9.5 g/L. Moreover, in fed-batch cultivation, the resultant recombinant strain produced 27.2 g/L of isopropanol from glucose with a yield of 41.5 % (mol/mol). This is the first demonstration of the production of isopropanol by genetically engineered yeast.

We previously reported the construction of a recombinant Candida utilis strain expressing mXYL1, XYL2 and XYL3, which encode mutated Candida shehatae xylose reductase K275R/N277D, C. shehatae xylitol dehydrogenase and Pichia stipitis xylulokinase to produce ethanol from xylose. However, its productivity was low. In this study, to breed a strain with higher productivity of ethanol from xylose, we used a cocktail multicopy integration method to attain optimized gene dosage of the three enzymes. Gene expression cassettes of the xylose-metabolizing enzymes were simultaneously integrated into C. utilis chromosomes in one step. Measurement of integrated gene copy number and xylose fermentability in all of the resulting integrant strains revealed that the copy number ratio of XYL2/mXYL1 in strains with higher ethanol yield was higher than that in strains with lower ethanol yield, whereas the copy number ratio of mXYL1/XYL3 was lower in strains with higher ethanol yield. The resultant strain CIS35, which was found to be the best producer of ethanol from xylose produced 29.2 g/L of ethanol, yielding 0.402 g ethanol/g xylose. This result provides that C. utilis may be a good candidate as a host for ethanol production from xylose., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

An understanding of the diagnostic performance of interferon-gamma release assays (in terms of parameters such as specificity, positive predictive value, and likelihood ratio) is important in the diagnosis of active tuberculosis in elderly Japanese patients because the high proportion of a prior history of tuberculosis among these patients can lead to misleading results. To elucidate the diagnostic performance of such assays, we examined the results of the QuantiFERON-TB Gold In-Tube test (QFT-GIT) in 65 patients in a younger group, and 52 patients in an elderly group who were suspected of having active tuberculosis and who had received the QFT-GIT. The median ages of the younger patients and elderly patients were 54 and 78 years, respectively. Among patients with active tuberculosis, the number of those with positive results on the QFT-GIT was similar in the two age groups [15 out of 17 (88.2 %) in the younger patients compared with 7 out of 8 (87.5 %) in the elderly patients]. However, in the patients with other diseases the number of those with positive QFT-GIT results was significantly different in the younger and elderly groups, being 6.3 and 27.3 %, respectively (P = 0.01). Although the sensitivity, negative predictive value, and negative likelihood ratio were similar in the two groups, the specificity, positive predictive value, and positive likelihood ratio were significantly lower in the elderly patients, being 72.7% (95 % confidence interval [CI], 57.2-85.0) compared with 93.8% (95 % CI, 82.8-98.7), 36.8% (95 % CI, 16.3-61.6) compared with 83.8% (95 % CI, 58.6-96.4), and 3.21 (95 % CI, 1.85-5.56) compared with 14.12 (95 % CI, 4.66-42.81), respectively. In the elderly patients with positive results on the test, a comparison of clinical data between those with active tuberculosis and those with other diseases demonstrated that the only clinical parameter showing a significant difference between these two groups was the radiological finding of small nodules in the patients with active tuberculosis (P < 0.01). The QFT-GIT may be less accurate in elderly patients, and radiological findings can be helpful in the clinical evaluation of patients with positive results on the test.

We have reported that a recombinant Candida utilis strain expressing a Candida shehatae xylose reductase K275R/N277D, a C. shehatae xylitol dehydrogenase, and xylulokinase from Pichia stipitis produced ethanol from xylose, but its productivity was low. In the present study, metabolomic (CE-TOF MS) and transcriptomic (microarray) analyses were performed to characterize xylose metabolism by engineered C. utilis and to identify key genetic changes contributing to efficient xylose utilization. The metabolomic analysis revealed that the xylose-fermenting strain accumulated more pentose phosphate pathway intermediates, more NADH, and more glycolytic intermediates upstream of glyceraldehyde 3-phosphate than the wild-type. Transcriptomic analysis of the strain grown on xylose indicated a significant increase in expression of the genes encoding tricarboxylic acid cycle enzymes, respiratory enzymes, and enzymes involved in ethanol oxidation. To decrease the NADH/NAD(+) ratio and increase the ethanol yield of the fermentation of xylose, ADH1 encoding NADH-dependent alcohol dehydrogenase was overexpressed. The resulting strain exhibited a 17% increase in ethanol production and a 22% decrease in xylitol accumulation relative to control.

Aims: To evaluate the histological characteristics differentiating chronic hypersensitivity pneumonitis (chronic HP) with a usual interstitial pneumonia (UIP)-like pattern from idiopathic pulmonary fibrosis (IPF)/UIP., Methods and Results: Surgical lung biopsy specimens from 22 patients with chronic HP diagnosed as having a UIP-like pattern upon histological examination and 13 patients with IPF/UIP were examined and the incidences of bronchiolitis, perilobular fibrosis, centrilobular fibrosis, bridging fibrosis, organizing pneumonia, fibroblastic foci, honeycombing, granulomas, giant cells, lymphocytic alveolitis and lymphoid follicles were compared. Bronchiolitis, centrilobular fibrosis, bridging fibrosis, organizing pneumonia, granulomas, giant cells and lymphocytic alveolitis were significantly more frequent among patients with chronic HP than among patients with IPF (all P<0.01)., Conclusions: Centrilobular fibrosis, bridging fibrosis and organizing pneumonia, in addition to bronchiolitis, granulomas and giant cells, are characteristic features of chronic HP with a UIP-like pattern. These features are therefore important in differentiating chronic HP from IPF/UIP, as management strategies differ for the two disorders., (© 2012 Blackwell Publishing Limited.)