Generic UPLC-MS/MS and Gyrolab assays with blood microsampling for pharmacokinetic assessments of therapeutic antibodies in mice.

Serial blood sampling from one animal is useful to understand relationship between pharmacokinetics (PK) and pharmacological or toxicological events in individual animals. To assess its feasibility in mice, two therapeutic antibodies were used to evaluate impacts by different blood sampling methods, sampling sites, and assay platforms on PK. Denosumab and Panitumumab were intravenously administered to mice and only 0.05 mL of blood sample per point was collected from jugular vein or tail vein. Blood samples were collected serially from a mouse or collected by traditional composite sampling from each mouse. Plasma concentrations of the two drugs were assayed by a generic ligand binding assay using Gyrolab or by a generic ultra-performance liquid chromatography with tandem mass spectrometry. The two assay platforms showed acceptable accuracy and precision and gave comparable PK parameters of the drugs, suggesting that both assays were successfully applied to the PK assessments. Comparable results in the PK profiles were noted between serial and composite blood samplings and differences in the two sampling sites did not impact PK. These findings suggest that microsampling combined with generic assays is useful to assess PK profiles of therapeutic antibodies in mice.

High heterogeneity and aging state of mineral particles in a slowly-moving dust plume on the southwestern coast of Japan.

Aerosol particles in two size ranges, namely 0.18-1.4 µm (fine) and larger than 1.4 µm (coarse), were collected in the pre-dust, in-dust, and post-dust air during the passage of a slowly-moving dust event at a coastal site in southwestern Japan. We identified the composition and size of individual particles using a scanning electron microscope to investigate the variations during dust passage. The particles could be classified as mineral-seasalt mixtures, non-mixture minerals, sulfur-containing minerals, and seasalt particles, and the number fractions of these type particles in the two size ranges exhibited significant variation across the three periods. In the coarse size range, mixture particles accounted for 17.6 %, 26.8 %, and 37.8 % of the particles in the pre-dust, in-dust, and post-dust air, respectively. Non-mixture particles made up 36.8 %, 29.2 %, and 24.3 % in the same respective periods. In the in-dust air, the average relative ratio of sulfur content in sulfur-containing mineral particles in the coarse range was 5.5 %, whereas in the fine range, it was 17.2 %. The aging state of sea salt components, described by the Cl loss and reflecting the changes in particles due to chemical reactions, exhibited significant differences in the two size ranges. In the fine range, the aging of >90 % particles was predominantly influenced by sulfate formation in the in-dust air. In contrast, nitrate likely played a certain role in both the pre-dust and post-dust air. In the coarse range, the aging was independent of sulfate formation. These results indicate the close dependence of the aging of dust particles on their size and the notable variations of the aged states, underscoring the essentiality to treat dust particles properly according to time and space for a better understanding on their roles in the marine atmosphere.

Number size distribution of bacterial aerosols in terrestrial and marine airflows at a coastal site of Japan.

Size-differentiated concentration of bacterial aerosols is essential for investigating their dissemination via the atmosphere. In this study, the number size distribution of bacterial aerosols was measured at a coastal site in southwestern Japan (32.324°N, 129.993°E) using a size-segregated eight-stage (>11, 7.0-11, 4.7-7.0, 3.3-4.7, 2.1-3.3, 1.1-2.1, 0.65-1.1, and 0.43-0.65µm) sampler. The results showed that the distribution differed according to the source areas: terrestrial air, oceanic air, or a combination of the two. The distribution in the long-distance transported terrestrial air from the Asian continent was monomodal, with a peak of 3.3-4.7 µm. The distribution in local land breeze air was bimodal, with the peaks at 0.43-1.1 and 3.3-4.7 µm. A similar bimodal distribution was encountered when the local island air and long-distance transported terrestrial air mixed. In contrast, the size distribution did not show clear peaks in the air from either nearby or remote marine areas. According to the air mass backward trajectories, the further the distance the air moved in the 72 h before arriving at the site, the lower the concentration of total bacterial aerosols. The estimation of dry deposition fluxes of bacterial cells showed that the deposition was dominated by cells larger than 1.1 µm with a relative contribution from 70.5 % to 93.7 %, except for the local land breeze cases, where the contributions in the size ranges larger and smaller than 1.1 µm were similar. These results show the distinctive number size distributions and removal processes of bacterial aerosols in different types of air. In addition, they indicate that size-dependent characteristics of airborne bacteria should be considered when studying their activities and roles in the atmospheric environment.

Decrease of bioaerosols in westerlies from Chinese coast to the northwestern Pacific: Case data comparisons.

The dissemination of bioaerosols in the westerly wind from the Asian continent to the northwestern Pacific constantly links the land and marine ecosystems. Several observation campaigns targeting bioaerosols were conducted in the coastal city Qingdao of China (QD), at a coast site of Kumamoto in southwestern Japan (KM), and in the northwestern Pacific (NP) between 2014 and 2016. We compared the concentration of bioaerosols in the range of 1.1-7.0 µm obtained in those campaigns to investigate their variation in the westerly wind. The substantial influence of westerlies on bioaerosol concentration was confirmed in the three areas. In the case of non-dust air, the arrival of the continental air led to a 29 % decrease of bioaerosols at KM while a 57 % increase at NP, indicating that the concentration in non-dust air was lower than the local level in the island air while higher than that in the remote marine air. In case of dust occurrence, bioaerosols in the air decreased with the distance from the Asian continent at KM and NP consecutively, and the arrival of the air caused a 2-fold increase at KM and a 1.7-fold increase at NP. The relative concentration increase rate of bioaerosols (IRRC), defined as the ratio of the increment of bioaerosols caused by long-distance transported air to the local level in each area, decreased rapidly after the air left the continent in the dust cases, which is similar to the decrease of the dry deposition flux of dust reported in the literature. This result indicates that the reduction of bioaerosols in the dusty air was likely dominated by the removal of bioaerosols attached to dust particles.

Asian dust exposure triggers acute myocardial infarction.

AIMS: To elucidate whether Asian dust is associated with the incidence of acute myocardial infarction (AMI) and to clarify whether patients who are highly sensitive to Asian dust will develop AMI. METHODS AND RESULTS: Twenty-one participating institutions located throughout Kumamoto Prefecture and capable of performing coronary intervention were included in the study. Data for ground-level observations of Asian dust events were measured at the Kumamoto Local Meteorological Observatory. Data collected between 1 April 2010 and 31 March 2015 were analysed, and 3713 consecutive AMI patients were included. A time-stratified case-crossover design was applied to examine the association between Asian dust exposure and AMI. The occurrence of Asian dust events at 1 day before the onset of AMI was associated with the incidence of AMI [odds ratio (OR), 1.46; 95% confidence interval (CI), 1.09-1.95] and especially, non-ST-segment elevation myocardial infarction was significant (OR 2.03; 95% CI, 1.30-3.15). A significant association between AMI and Asian dust was observed in patients with age ≥75 years, male sex, hypertension, diabetes mellitus, never-smoking status, and chronic kidney disease (CKD). However, Asian dust events had a great impact on AMI onset in patients with CKD (P < 0.01). A scoring system accounting for several AMI risk factors was developed. The occurrence of Asian dust events was found to be significantly associated with AMI incidence among patients with a risk score of 5-6 (OR 2.45; 95% CI: 1.14-5.27). CONCLUSION: Asian dust events may lead to AMI and have a great impact on its onset in patients with CKD.

Notch signaling may be involved in the abnormal differentiation of epidermal keratinocytes in psoriasis.

Localization of each keratin isoform differs among epidermal layers. Proliferating basal cells synthesize keratin 14 (K14) and suprabasal cells express keratin 10 (K10) in normal skin. Notch signaling is essential for keratinocyte differentiation. Notch1 is expressed in all epidermal layers, Notch2 in the basal cell layer and Notch3 in basal cell and spinous cell layers in normal epidermis. It has been poorly elucidated how localization and expression levels of Notch molecules are related to epidermal molecular markers K10 and K14 in psoriatic skin with abnormal differentiation of epidermal tissue. This study aimed to investigate the relationship between abnormal differentiation of epidermal cells in psoriatic skin and expression of Notch molecules. We investigated keratins (K14 and K10) and Notches (1, 2, 3 and 4) using immunohistochemistry in psoriatic skin (n=30) and normal skin (n=10). In normal skin, K14 and K10 were discretely observed in the basal cell layer and suprabasal layer, respectively. In psoriatic skin, K14 was expressed in the pan epidermal layer while it and K10 were co-expressed in some middle suprabasal layer cells. Notch1, 2, 3, and 4 localized in all epidermal layers in normal skin. In psoriatic skin, Notch1, 2, and 4 mainly localized in suprabasilar layers and Notch3 is lacalized in pan epidermal, suprabasilar, and basilar layers. Protein and mRNA of Notch1, 2, and 3 isoforms decreased in psoriatic epidermis compared with normal epidermis. These data suggest that decrements in these Notch molecules might cause aberrant expression of K10 and K14 leading to anomalous differentiation of the epidermis in psoriatic lesions.

Lipid droplets of arbuscular mycorrhizal fungi emerge in concert with arbuscule collapse.

Plants share photosynthetically fixed carbon with arbuscular mycorrhizal (AM) fungi to maintain their growth and nutrition. AM fungi are oleogenic fungi that contain numerous lipid droplets in their syncytial mycelia during most of their life cycle. These lipid droplets are probably used for supporting growth of extraradical mycelia and propagation; however, when and where the lipid droplets are produced remains unclear. To address these issues, we investigated the correlation between intracellular colonization stages and the appearance of fungal lipid droplets in roots by a combination of vital staining of fungal structures, selective staining of lipids and live imaging. We discovered that a surge of lipid droplets coincided with the collapse of arbuscular branches, indicating that arbuscule collapse and the emergence of lipid droplets may be associated processes. This phenomenon was observed in the model AM fungus Rhizophagus irregularis and the ancestral member of AM fungi Paraglomus occultum. Because the collapsing arbuscules were metabolically inactive, the emerged lipid droplets are probably derived from preformed lipids but not de novo synthesized. Our observations highlight a novel mode of lipid release by AM fungi.

Complete genome sequence of Enterococcus mundtii QU 25, an efficient L-(+)-lactic acid-producing bacterium.

Enterococcus mundtii QU 25, a non-dairy bacterial strain of ovine faecal origin, can ferment both cellobiose and xylose to produce l-lactic acid. The use of this strain is highly desirable for economical l-lactate production from renewable biomass substrates. Genome sequence determination is necessary for the genetic improvement of this strain. We report the complete genome sequence of strain QU 25, primarily determined using Pacific Biosciences sequencing technology. The E. mundtii QU 25 genome comprises a 3 022 186-bp single circular chromosome (GC content, 38.6%) and five circular plasmids: pQY182, pQY082, pQY039, pQY024, and pQY003. In all, 2900 protein-coding sequences, 63 tRNA genes, and 6 rRNA operons were predicted in the QU 25 chromosome. Plasmid pQY024 harbours genes for mundticin production. We found that strain QU 25 produces a bacteriocin, suggesting that mundticin-encoded genes on plasmid pQY024 were functional. For lactic acid fermentation, two gene clusters were identified-one involved in the initial metabolism of xylose and uptake of pentose and the second containing genes for the pentose phosphate pathway and uptake of related sugars. This is the first complete genome sequence of an E. mundtii strain. The data provide insights into lactate production in this bacterium and its evolution among enterococci.

Isolation and phenotypic characterization of Lotus japonicus mutants specifically defective in arbuscular mycorrhizal formation.

Several symbiotic mutants of legume plants defective in nodulation have also been shown to be mutants related to arbuscular mycorrhizal (AM) symbiosis. The origin of the AM symbiosis can be traced back to the early land plants. It has therefore been postulated that the older system of AM symbiosis was partially incorporated into the newer system of legume-rhizobium symbiosis. To unravel the genetic basis of the establishment of AM symbiosis, we screened about 34,000 plants derived from ethyl methanesulfonate (EMS)-mutagenized Lotus japonicus seeds by microscopic observation. As a result, three lines (ME778, ME966 and ME2329) were isolated as AM-specific mutants that exhibit clear AM-defective phenotypes but form normal effective root nodules with rhizobial infection. In the ME2329 mutant, AM fungi spread their hyphae into the intercellular space of the cortex and formed trunk hyphae in the cortical cells, but the development of fine branches in the arbuscules was arrested. The ME2329 mutant carried a nonsense mutation in the STR-homolog gene, implying that the line may be an str mutant in L. japonicus. On the ME778 and ME966 mutant roots, the entry of AM fungal hyphae was blocked between two adjacent epidermal cells. Occasionally, hyphal colonization accompanied by arbuscules was observed in the two mutants. The genes responsible for the ME778 and ME966 mutants were independently located on chromosome 2. These results suggest that the ME778 and ME966 lines are symbiotic mutants involved in the early stage of AM formation in L. japonicus.

Genomic features of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid.

Lactococcus lactis IO-1 (JCM7638) produces L-lactic acid predominantly when grown at high xylose concentrations, and its utilization is highly desired in the green plastics industry. Therefore it is worthwhile studying its genomic traits. In this study, we focused on (i) genes of possible horizontal transfer derivation (prophages, the nisin-sucrose transposon, and several restriction-modification systems), and (ii) genes for the synthetic pathways of amino acids and vitamins in the IO-1 genome. In view of the results of this analysis, we consider their meanings in strain IO-1.

Different circadian expression of major matrix-related genes in various types of cartilage: modulation by light-dark conditions.

We screened circadian-regulated genes in rat cartilage by using a DNA microarray analysis. In rib growth-plate cartilage, numerous genes showed statistically significant circadian mRNA expression under both 12:12 h light-dark and constant darkness conditions. Type II collagen and aggrecan genes--along with several genes essential for post-translational modifications of collagen and aggrecan, including prolyl 4-hydroxylase 1, lysyl oxidase, lysyl oxidase-like 2 and 3'-phosphoadenosine 5'-phosphosulphate synthase 2--showed the same circadian phase. In addition, the mRNA level of SOX9, a master transcription factor for the synthesis of type II collagen and aggrecan, has a similar phase of circadian rhythms. The circadian expression of the matrix-related genes may be critical in the development and the growth of various cartilages, because similar circadian expression of the matrix-related genes was observed in hip joint cartilage. However, the circadian phase of the major matrix-related genes in the rib permanent cartilage was almost the converse of that in the rib growth-plate cartilage under light-dark conditions. We also found that half of the oscillating genes had conserved clock-regulatory elements, indicating contribution of the elements to the clock outputs. These findings suggest that the synthesis of the cartilage matrix macromolecules is controlled by cell-autonomous clocks depending upon the in vivo location of cartilage.

Long-term efficacy of psoriasis vulgaris treatments: analysis of treatment with topical corticosteroid and/or vitamin D3 analog, oral cyclosporin, etretinate and phototherapy over a 35-year period, 1975-2010.

Various therapies have been tried for psoriasis. In Japan, biologics began to be used for psoriasis treatment in January 2010. Their clinical efficacy is well known, but biologics cannot be used in all psoriasis patients for reasons such as side-effects and cost. It is necessary to evaluate the effect of long-term psoriasis treatment, but there have been no reports evaluating long-term treatment. Therefore, the outcomes of patients who had been treated at the Tokai University Hospital for more than 5 years, before biological agents were released, were examined. Three categories, classified by initial severity, changes in severity by method of treatment and background characteristics, were investigated. In conclusion, cases of long-term treatment with a combination of topical corticosteroid and topical vitamin D3 analog or oral cyclosporin were found to be effective therapies. Patients with a history of diabetes mellitus or cardiovascular disease of psoriasis were likely to be treatment resistant.

Psoriasis affects patient's quality of life more seriously in female than in male in Japan.

OBJECTIVE: The aim of this study was to assess the quality of life (QOL) of patients with psoriasis in Japan using Dermatology Life Quality Index (DLQI). Furthermore, we had evaluated the correlation between DLQI and clinical severity of psoriasis. METHODS: The Japanese version of DLQI was used to assess the QOL of patients. Psoriasis area and severity index (PASI) and Itch visual analogue scale (VAS) were used to assess clinical severity of psoriasis. RESULTS: The subjects were 102 Japanese patients with mild to severe psoriasis (77 males, 25 females, mean age 55.2 ± 14.2). There were no statistically significant differences in age, PASI, and itch VAS between male and female. The mean DLQI scores in total were 3.6 ± 3.2 in male and 7.2 ± 1.2 in female. The mean total DLQI scores in female were higher than that in male (p = 0.0016). Significant correlation was observed between DLQI scores and PASI score (p < 0.001) or itch VAS score (p < 0.001). CONCLUSIONS: The mean total DLQI scores in female were significantly higher than that in male. Also, we confirmed the correlation between DLQI and clinical severity of psoriasis. These findings suggest that QOL assessment plays a greater role in females than in the males, when assessing the severity of psoriasis.

Real-time monitoring in three-dimensional hepatocytes reveals that insulin acts as a synchronizer for liver clock.

Resetting the peripheral clock and understanding the integration between the circadian rhythm and metabolic pathways are fundamental questions. To test whether insulin acts as a synchronizer for the hepatic clock by cell-autonomous mechanisms, the phase-resetting capabilities of insulin were investigated in cultured hepatic cells. We provide evidence that three-dimensional (3D) cell culture conditions that preserve the differentiated state of primary hepatocytes sustained the robustness of the molecular clock, while this robustness rapidly dampened under classical monolayer cell culture conditions. Herein, we established a 3D cell culture system coupled with a real-time luciferase reporter, and demonstrated that insulin directly regulates the phase entrainment of hepatocyte circadian oscillators. We found that insulin-deficient diabetic rats had a pronounced phase advance in their hepatic clock. Subsequently, a single administration of insulin induced phase-dependent bi-directional phase shifts in diabetic rat livers. Our results clearly demonstrate that insulin is a liver clock synchronizer.

A case of chromomycosis treated by surgical therapy combined with preceded oral administration of terbinafine to reduce the size of the lesion.

Chromomycosis is a chronic fungal disease of the skin and subcutaneous tissues caused by a group of dematiaceous black fungi. Small lesions can be removed with excision, but other cases are difficult to treat. We report a case of chromomycosis caused by Fonsecaea pedrosoi (F. pedrosoi). The case involved a 74-year-old man, who had noted a lesion on the back of the right thigh, that was gradually enlarging and reaching up to 30 cm in diameter, in 20-years. From microscopic examination, sclerotic cells were seen. We diagnosed this case as chromomycosis caused by F. pedrosoi on mycological examination. The patient was initially treated with oral terbinafine (250 mg/day) as the lesion was very large. After the 18 months treatment, the size of the lesion reduced to 1 cm, then the remaining lesion was excised.

Natural versus anthropogenic inhalable aerosol chemistry of transboundary East Asian atmospheric outflows into western Japan.

The eastward transport of aerosols exported from mainland Asia strongly influences air quality in the Japanese archipelago. The bulk of the inhalable particulate matter (PM(10)) in these intrusions comprises either natural, desert-derived minerals (mostly supermicron silicates) or anthropogenic pollutants (mostly submicron sulphates), in various states of mixing. We analyse PM(10) collected in Kumamoto, SW Japan, during three contrasting types of aerosol intrusions, the first being dominated by desert PM which became increasingly mixed with anthropogenic components as time progressed, the second being a relatively minor event mixing fine, distal desert PM with anthropogenic materials, and the third being dominated by anthropogenic pollutants. Whereas the chemistry of the natural mineral component is characterised by "crustal" elements (Si, Al, Fe, Mg, K, Li, P, Sc, V, Rb, Sr, Zr, Th, lanthanoids), the anthropogenic component is rich in secondary inorganic compounds and more toxic metallic elements (NH(4)(+), SO(4)(2-), As, Pb, Cd, Cu, Zn, Sn, Bi, Sb, and Ge). Some desert-dust (Kosa) intrusions are more calcareous than others, implicating geologically different source areas, and contain enhanced levels of NO(3)(-), probably as supermicron Ca(NO(3))(2) particles produced by chemical reaction between NOx pollutants (mostly from industry and traffic) and carbonate during atmospheric transport. The overall trace element chemistry of aerosol intrusions into Kumamoto shows low V/Rb, low NO(3)(-)/SO(4)(2-), enhanced As levels, and unfractionated La/Ce values, which are all consistent with anthropogenic sources including coal emissions rather than those derived from the refining and combustion of oil fractionates. Geographically dispersed, residual sulphatic plumes of this nature mix with local traffic (revealed by OC and EC concentrations) and industrial emissions and dissipate only slowly, due to the dominance of submicron accumulation mode PM which is atmospherically persistent, and raise questions over the chronic health effects of breathing finely respirable sulphatic aerosol containing enhanced amounts of toxic metals.

Complete genome sequence of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid.

We report the complete genome sequence of Lactococcus lactis IO-1 (= JCM7638). It is a nondairy lactic acid bacterium, produces nisin Z, ferments xylose, and produces predominantly L-lactic acid at high xylose concentrations. From ortholog analysis with other five L. lactis strains, IO-1 was identified as L. lactis subsp. lactis.

Cellular DBP and E4BP4 proteins are critical for determining the period length of the circadian oscillator.

The phenotypes of mice carrying clock gene mutations have been critical to understanding the mammalian clock function. However, behavior does not necessarily reflect cell-autonomous clock phenotypes, because of the hierarchical dominance of the central clock. We performed cell-based siRNA knockdown and cDNA overexpression and monitored rhythm using bioluminescent reporters of clock genes. We found that knockdown of DBP, D-box positive regulator, in our model led to a short-period phenotype, whereas overexpressing of DBP produced a long-period rhythm when compared to controls. Furthermore, knockdown and overexpressing of E4BP4, D-box negative regulator, led to an opposite effect of DBP. Our experiments demonstrated that D-box regulators play a crucial role in determining the period length of Per1 and Per2 promoter-driven circadian rhythms in Rat-1 fibroblasts.

High-grade endometrial stromal sarcoma with smooth muscle and skeletal muscle differentiation: report of a case with cytomorphologic and immunocytologic analysis.

We report a case of high-grade endometrial stromal sarcoma with cytological and immunocytochemical findings. Cytologically, major tumor cells showed round-to-short spindle shapes with round- to oval-shaped nuclei and moderately abundant delicate cytoplasm. Tumor cells with tapered shapes and eccentric nuclei were also observed. A few spindle cells having enlarged cigar-shaped nuclei with conspicuous nucleoli and delicate wispy cytoplasm, which resembled leiomyosarcoma, were intermingled. One rhabdomyoblast cell with both α-sarcomeric muscle actin and myoglobin was also observed. Most of the tumor cells, including the leiomyosarcomatous spindle cells, were positive for CD10, and negative for desmin and h-caldesmon. Accordingly, when relatively monotonous round-to-short spindle tumor cells and taper-shaped tumor cells are observed in the female genital tract, high-grade endometrial stromal sarcoma should be considered in the differential diagnosis. Immunocytochemistry contributed to the correct diagnosis. This case was high-grade endometrial stromal sarcoma with smooth muscle and skeletal muscle differentiation.