Study of the electron-doping mechanism in single-walled carbon nanotubes using dimethylbenzimidazole.

Single-walled carbon nanotubes (SWCNTs) exhibit p-type properties in air, necessitating electron doping using n-dopants (e.g., reducing agents) for the development of SWCNT-based electronic devices. Dimethylbenzimidazole (DMBI-H) derivatives serve as effective electron dopants, not only for SWCNTs, but also for various organic semiconducting materials. However, the doping reaction is still a subject of debate. In this study, the electron-doping reactions of ortho-methoxy-substituted DMBI-H for SWCNTs were analyzed in protic and aprotic solvents in the presence and absence of dioxygen (O2). The presence of O2 was found to cause the reduction of O2 on the SWCNT surface in the protic solvent, resulting in the production of DMBI cations and water through proton-coupled electron transfer (PCET) from the n-doped SWCNT and ethanol. This work elucidates the mechanism behind the air-stability of n-type SWCNTs.

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries.

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin-4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti-Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c-erbB-2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call-Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.

Characterization of anti-Müllerian hormone in a case of bovine male pseudohermaphroditism.

The current report aimed to characterize plasma anti-Müllerian hormone (AMH) in bovine male pseudohermaphroditism. The blood AMH concentration in a Japanese Black male pseudohermaphrodite calf was compared with pre- and post-pubertal male and female calves and castrated calves. The concentration in the case was higher than in post-pubertal males, castrated males, and pre- and post-pubertal female calves (p < .05), but similar to that in pre-pubertal male calves. After extraction of the testes, the concentration in the case dropped to a certain extent. The extracted testes expressed AMH, as detected by immunohistochemistry. This study is the first to show the characterization of AMH in a male pseudohermaphrodite calf. AMH levels in peripheral blood might be useful to diagnose male pseudohermaphroditism in cattle.

Recurrence after resection with curative intent for distal cholangiocarcinoma.

BACKGROUND: Few studies have been conducted on patterns of recurrence after resection for distal cholangiocarcinoma (DCC). The aim of this study was to investigate the incidence and pattern of recurrence after resection of DCC, and to evaluate prognostic factors for time to recurrence and recurrence-free survival (RFS). METHODS: Patients who underwent pancreatoduodenectomy with curative intent for DCC between 2001 and 2010 at one of 30 hospitals in Japan were reviewed retrospectively, with special attention to recurrence patterns. The Cox proportional hazards model was used for multivariable analysis. RESULTS: In the study interval, 389 patients underwent pancreatoduodenectomy for DCC with R0/M0 status. Recurrence developed in 213 patients (54·8 per cent). The estimated cumulative probability of recurrence was 54·3 per cent at 5 years. An initial locoregional recurrence occurred in 55 patients (14·1 per cent) and initial distant recurrence in 168 (43·2 per cent), most commonly in the liver. Isolated initial locoregional recurrence occurred in 45 patients (11·6 per cent). Independent prognostic factors for time to recurrence and RFS were perineural invasion (P = 0·001 and P = 0·009 respectively), pancreatic invasion (both P < 0·001) and lymph node metastasis (both P < 0·001). RFS worsened as the number of risk factors increased: the 5-year RFS rate was 70·6 per cent for patients without any risk factors, 50·3 per cent for patients with one factor, 31·8 per cent for those with two factors, and 13·4 per cent when three factors were present. CONCLUSION: More than half of patients with DCC experienced recurrence after R0 resection, usually within 5 years. Perineural invasion, pancreatic invasion and positive nodal involvement are risk factors for recurrence.

Modulating effect of low level-laser therapy on fibrosis in the repair process of the tibialis anterior muscle in rats.

The treatment of muscle injuries is a common practice at rehabilitation centers. Low-level laser therapy (LLLT) has demonstrated positive effects regarding the modulation of the inflammatory response, the enhancement of the tissue repair process and the prevention of fibrosis. The aim of the present study was to evaluate the effects of LLLT on morphological aspects of muscle tissue, collagen remodeling and activity of matrix metalloproteinase 2 (MMP-2) in rat skeletal muscle following acute injury. Wistar rats were divided into five groups: (1) control group (n = 10), (2) sham group (n = 10), (3) LLLT group (n = 30), (4) non-treated injury group (n = 30) and (5) injury + LLLT group (n = 30). Cryoinjury was performed on the belly of the tibialis anterior (TA) muscle. LLLT was performed daily with an AlGaAs laser (780 nm; beam spot of 0.04 cm(2), output power of 40 mW, power density of 1 W/cm(2), energy density of 10 J/cm(2) and 10-s exposure time). Animals were euthanized at 1, 3 and 7 days. The TA muscles were removed and weighed. Morphological aspects were evaluated using H & E staining. The amount and distribution of collagen fibers were evaluated by picrosirius staining. Characterization and activity of MMP-2 were evaluated by zymography and Western blot techniques, respectively. The results revealed that LLLT induced a reduction in inflammatory infiltrate and myonecrosis after 1 day, an increase in the number of blood vessels after 3 and 7 days as well as an increase in the number of immature muscle fibers and MMP-2 gelatinase activity after 7 days. In conclusion, LLLT has a positive effect on the inflammatory process, MMP2 activity and collagen organization and distribution in the repair process of rat skeletal muscle.

Carbenoxolone alters the morphology of adipose tissues and downregulates genes involved in adipogenesis, glucose transport and lipid metabolism in high-fat diet-fed mice.

Glucocorticoid (GC) excess promotes adipose tissue accumulation, and 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) plays an important role in the local amplification of GC. Therefore, in this study, we investigated the effects of carbenoxolone (CBX), an 11ß-HSD1 inhibitor, on morphological changes in visceral fat, and the expression of genes involved in adipogenesis and lipid metabolism in high-fat (HF) diet-fed mice. Mice were fed a HF diet from 5 weeks of age. At 10 weeks of age, the mice received an intraperitoneal injection of CBX or vehicle every day for 2 weeks. CBX decreased body weight and visceral fat mass, and improved insulin sensitivity in HF-fed mice. This was accompanied by reduced adipocyte size and a decrease in large-sized adipocytes in visceral fat. The expression of adipogenesis (PPARγ and C/EBPα), glucose transport (GLUT4) and lipid metabolism (LPL, ATGL, and HSL)-related genes were suppressed in CBX mice. CBX treatment induced beneficial morphological changes in visceral fat and decreased the expression of adipogenesis, glucose transport and lipid metabolism-related genes. These findings reveal a potential mechanism underling the effects of CBX on reduced fat accumulation and improved insulin sensitivity.

Altered gene expressions of ghrelin, PYY, and CCK in the gastrointestinal tract of the hyperphagic intrauterine growth restriction rat offspring.

Intrauterine growth restriction (IUGR) is associated with a substantially greater incidence of metabolic syndrome in adulthood. Animal studies have shown that IUGR offspring are hyperphagic during the early postnatal period and therefore exhibit obesity. The molecular mechanisms underlying food intake regulation in the gastrointestinal tract have not been clarified in IUGR. In the present study, we utilized a rat model of IUGR by restricting the food intake of the mother (50% of the normal intake, ad libitum; FR group) from day 7 of gestation until delivery. Pups from undernourished mothers were fostered by control mothers. We examined the food intake and assessed the gene expressions of ghrelin, peptide YY (PYY), and cholecystokinin (CCK) in the alimentary tract of male newborns (postnatal day1) and adult offspring (age, 7 months). Compared to the offspring whose mothers received the standard diet ad libitum (CON offspring), FR offspring were hyperphagic from the weaning time until the end of the experiment, and resulted in a heavier final weight. Both newborn and adult FR offspring had higher ghrelin gene expression in the stomach and higher ghrelin plasma levels than did the controls. Although the gastrointestinal gene expressions and plasma levels of the anorexic peptides, PYY and CCK, were elevated in the FR newborns, they decreased in the FR adults. Our findings suggest that the altered gene expressions of orexigenic and anorexigenic gut peptides in the gastrointestinal tract in the maternal undernutrition-induced IUGR offspring provide a potential mechanism to explain hyperphagia and obesity seen in these offspring.

Analysis of sex difference in the tubular reabsorption of lithium in rats.

Lithium is used in the treatment of bipolar disorder. We previously demonstrated that two types of transporters mediate the tubular reabsorption of lithium in rats, and suggested that sodium-dependent phosphate transporters play a role in lithium reabsorption with high affinity. In the present study, we examined sex differences in lithium reabsorption in rats. When lithium chloride was infused at 60 µg/min, creatinine clearance and the renal clearance of lithium were lower, and the plasma concentration of lithium was higher in female rats. These values reflected the higher fractional reabsorption of lithium in female rats. In rats infused with lithium chloride at 6 µg/min, the pharmacokinetic parameters of lithium examined were all similar in both sexes. The fractional reabsorption of lithium was decreased by foscarnet, a representative inhibitor of sodium-dependent phosphate transporters, in male and female rats when lithium chloride was infused at the low rate. Among the candidate transporters mediating lithium reabsorption examined herein, the mRNA expression of only PiT2, a sodium-dependent phosphate transporter, exhibited sexual dimorphism. The present results demonstrated sex differences in the tubular reabsorption of lithium with low affinity in rats.

Inappropriate use of ozone generators and their sales status: questionnaire survey of healthcare providers and investigation of online sales.

Ozone generators have attracted attention as a result of the spread of severe acute respiratory syndrome coronavirus-2. In a questionnaire survey targeting healthcare facilities, 20 (91%) used ozone generators in patient areas, and five (23%) used them in indoor spaces occupied by people. A search for ozone generators on the Amazon Japan website revealed that 76% of products lacked information on ozone emission rate, coverage area and/or use time. These results suggest that ozone generators may be used inappropriately in hospitals and clinics, and have been sold to the general public without adequate information for assessing their safety and efficacy.

Tumor microenvironment in triple-negative breast cancer: the correlation of tumor-associated macrophages and tumor-infiltrating lymphocytes.

PURPOSE: Immune cells such as cytotoxic T cells, helper T cells, B cells or tumor-associated macrophages (TAMs) contribute to the anti-tumor response or pro-tumorigenic effect in triple negative breast cancer (TNBC). The interrelation of TAMs, T and B tumor-infiltrating lymphocytes (TILs) in TNBC has not been fully elucidated. METHODS: We evaluated the association of tumor-associated macrophages, T and B TILs in TNBC. RESULTS: TNBCs with a high CD68+, CD163+ TAMs and low CD4+, CD8+, CD20+ TILs had a significantly shorter relapse-free survival (RFS) and overall survival (OS) than those with low CD68+, CD163+ TAMs and high CD4+, CD8+, CD20+ TILs. TNBCs with high CD68+ TAMs/low CD8+ TILs showed a significantly shorter RFS and OS and a significantly poorer prognosis than those with high CD68+ TAMs/high CD8+ TILs, low CD68+ TAMs/high CD8+ TILs, and low CD68+/low CD8+. TNBCs with high CD163+ TAMs/low CD8+, low CD20 + TILs showed a significantly shorter RFS and OS and a significantly poorer prognosis than those with high CD163+ TAMs/high CD8+ TILs and high CD163+ TAMs /high CD20+ TILs. CONCLUSIONS: Our study suggests that TAMs further create an optimal tumor microenvironment (TME) for growth and invasion of cancer cells when evasion of immunoreactions due to T and B TILs occurs. In TNBCs, all these events combine to affect prognosis. The process of TME is highly complex in TNBCs and for an improved understanding, larger validation studies are necessary to confirm these findings.

Stereoselective binding of chiral ligands to single nucleotide polymorphisms of the human organic cation transporter-1 determined using cellular membrane affinity chromatography.

Membranes from stably transfected cell lines that express two point mutations of the human organic cation transporter-1 (hOCT1), R488M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form two cellular membrane affinity chromatography (CMAC) columns, CMAC(hOCT1(G465R)) and CMAC(hOCT1(R488M)). Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [(3)H] MMP(+) (1-methyl-4-phenylpyridinium) as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol, and isoproterenol. The chromatographic data obtained were used to refine a previously developed pharmacophore for hOCT1.

Effects of maternal high-fat diet on serum lipid concentration and expression of peroxisomal proliferator-activated receptors in the early life of rat offspring.

Peroxisomal proliferator-activated receptors (PPARs) play an important role in the regulation of lipid metabolism. The aim of this study was to investigate the effects of a maternal high-fat (HF) diet on serum lipid concentration and PPAR gene expression in liver and adipose tissue in the early life of the rat offspring. Female Sprague-Dawley rats were fed either an HF or control (CON) diet 6 weeks before mating and throughout gestation and lactation. Blood and tissue samplings of male offspring were carried out at birth or weaning. Birth weights were similar and serum triglyceride (TG) and nonesterified fatty acid (NEFA) levels showed no significant difference between HF and CON newborns, despite greatly increased hepatic PPARα mRNA expression in the HF newborns (p<0.05). Both HF newborns and weanlings revealed significantly decreased hepatic PPARγ expression compared with controls (p<0.0001). Hepatic PPARα expression in the HF weanlings was reduced markedly compared with CON weanlings (p<0.0001) and showed a negative correlation with serum TG levels (r=-0.743, p<0.05). However, epididymal expression of PPARγ in the HF weanlings was upregulated significantly compared with controls (p<0.05) and demonstrated a positive correlation with epididymal fat mass (r=0.733, p<0.05). These were accompanied by obesity as well as a rise in serum TG by 79% (p<0.05) and NEFA concentration by 36% (p<0.05) in these HF weanlings. Our findings suggest that maternal HF diet leads to alterations in PPAR gene expression in the weanling offspring, which is associated with the disturbed lipid homeostasis.

When should we intervene to control the 2009 influenza A(H1N1) pandemic?

We simulated the early phase of the 2009 influenza A(H1N1) pandemic and assessed the effectiveness of public health interventions in Japan. We show that the detection rate of border quarantine was low and the timing of the intervention was the most important factor involved in the control of the pandemic, with the maximum reduction in daily cases obtained after interventions started on day 6 or 11. Early interventions were not always effective.

Disseminated Pneumocystosis in a Toy Poodle.

A 1-year and 7-months-old neutered male toy poodle was presented with persistent respiratory distress, gradual weight loss and melaena. Thoracic radiography showed an unstructured interstitial lung pattern. Histopathological examination of tissues collected at necropsy examination revealed disseminated infection by Pneumocystis carinii. The organisms were detected in the lungs, lymph nodes, liver, heart, kidneys, spleen, gastrointestinal tract and pancreas. In the lungs, the organisms were present in the alveolar space and interstitial tissue, and calcified foci containing P. carinii were observed. The presence of the organism in non-thoracic lymph nodes provided evidence of lymphogenous spread. A definitive diagnosis of disseminated pneumocystosis was achieved through the use of Grocott methenamine silver staining, immunohistochemistry (IHC) and polymerase chain reaction for P. carinii. Depletion of cells expressing immunoglobulin (Ig)A and IgG was confirmed by IHC of lymphoid tissue, suggesting possible underlying immunodeficiency.

Light-dependent translocation of a phytochrome B-GFP fusion protein to the nucleus in transgenic Arabidopsis.

Phytochrome is a ubiquitous photoreceptor of plants and is encoded by a small multigene family. We have shown recently that a functional nuclear localization signal may reside within the COOH-terminal region of a major member of the family, phytochrome B (phyB) (Sakamoto, K., and A. Nagatani. 1996. Plant J. 10:859-868). In the present study, a fusion protein consisting of full-length phyB and the green fluorescent protein (GFP) was overexpressed in the phyB mutant of Arabidopsis to examine subcellular localization of phyB in intact tissues. The resulting transgenic lines exhibited pleiotropic phenotypes reported previously for phyB overexpressing plants, suggesting that the fusion protein is biologically active. Immunoblot analysis with anti-phyB and anti-GFP monoclonal antibodies confirmed that the fusion protein accumulated to high levels in these lines. Fluorescence microscopy of the seedlings revealed that the phyB-GFP fusion protein was localized to the nucleus in light grown tissues. Interestingly, the fusion protein formed speckles in the nucleus. Analysis of confocal optical sections confirmed that the speckles were distributed within the nucleus. In contrast, phyB-GFP fluorescence was observed throughout the cell in dark-grown seedlings. Therefore, phyB translocates to specific sites within the nucleus upon photoreceptor activation.

Immunohistochemical Profile of Ameloblastic Carcinoma Arising from an Amyloid-Producing Odontogenic Tumour in a Miniature Dachshund.

A 13-year-old female miniature dachshund was presented with a centrally-located sublingual mass in the rostral mandibular region. The focally ulcerated growth completely covered the left (305) and right (405) premolar teeth and partially covered the right canine teeth (404). A punch biopsy sample revealed neoplastic proliferation of odontogenic epithelium arranged in irregular cords with frequent comedo-like necrosis. Following the initial diagnosis of ameloblastic carcinoma, a bilateral rostral hemimandibulectomy was performed. Although the detailed examination of the resected mass was consistent with the initial diagnosis, it also contained birefringent congophilic, amelogenin-labelled amyloid deposits similar to an amyloid-producing odontogenic tumour (APOT) in 30-40% of the mass, in continuity with the ameloblastic carcinoma. All neoplastic cells had diffuse moderate expression of cytokeratin (CK) AE1/AE3 and CK5, diffuse mild expression of CK14 and multifocal moderate expression of CK19. Because the APOT-like growth in the mass was histologically benign, the tumour was diagnosed as an ameloblastic carcinoma arising from an APOT.

Bovine Endocarditis Associated with Mycoplasma bovis.

Mycoplasma bovis is a microorganism associated with pneumonia, mastitis, arthritis and otitis media of cattle; however, there are no reports of this organism causing bovine endocarditis. Five adult cattle with endocarditis characterized by caseated lesions (diameter 5-12 cm) of the endocardial surface of the left atrium, but without lesions in heart valves or affecting the right side of the heart, were identified in slaughterhouses in Japan. M. bovis was successfully isolated from the lesions and M. bovis antigen was detected immunohistochemically within the lesions. The results suggest that the lesions may have been associated with M. bovis alone. To our knowledge, this is the first demonstration of bovine endocarditis associated with M. bovis.

CAUSES OF DEATH AND DETECTION OF ANTIBODIES AGAINST JAPANESE ENCEPHALITIS VIRUS IN MISAKI FERAL HORSES (EQUUS CABALLUS) IN SOUTHERN JAPAN, 2015-17.

We performed postmortem examinations on seven Misaki feral horses (Equus caballus) and evaluated Misaki feral horses, Japanese wild boars (Sus scrofa leucomystax), domestic pigs (Sus scrofa), and wild Japanese macaques (Macaca fuscata fuscata) from 2015 to 2017 in Cape Toi, Kushima, Miyazaki Prefecture, southern Japan, for antibodies against Japanese encephalitis virus (JEV). Strongylus vulgaris infection with severe arterial lesions and hemomelasma ilei was present in all necropsied horses. We frequently found intestinal ulcers, perihepatitis filamentosa, and poor body condition. We recorded degenerative arthropathy in metacarpophalangeal joints in two cases and a fracture of the rib with diaphragmatic rupture in one case. A total of 73% (177/242) of horses were seropositive for JEV as tested by hemagglutination inhibition (HI). The HI data also revealed that 74% (59/80) of the wild boars, 67% (60/90) of the pigs, and 29% (22/75) of the wild monkeys were seropositive for JEV. Our findings showed that Strongylus spp. are still a risk to horses in this region, and that environmental factors such as topographic location of the pasture and steep slope may have caused of degenerative arthropathy and bone fracture. Our results showed that JEV is endemic in Japan. The wild boars and pigs were presumed to act as strong amplifiers and sources of infection, with subsequent risk to humans.

Mitochondria frozen with trehalose retain a number of biological functions and preserve outer membrane integrity.

In apoptosis, Bcl-2-family proteins regulate the barrier function of the mitochondrial outer membrane (MOM), controlling the release of proapoptotic proteins from the intermembrane space into the cytoplasm. This process can be studied in vitro with freshly isolated mouse liver mitochondria. Unfortunately, mitochondria frozen/thawed in standard sucrose-mannitol buffers become leaky and useless for apoptosis research. However, here we show that mitochondria frozen in buffer containing the sugar, trehalose, maintained MOM integrity and responsiveness to Bcl-2-family proteins, much like fresh mitochondria. Trehalose also preserved ultrastructure, as well as biological functions such as ATP synthesis, calcium-induced swelling, transmembrane potential, and the import and processing of protein precursors. However, bioenergetic function was somewhat reduced. Thus, trehalose-frozen mitochondria retained most of the biological features of mitochondria including MOM integrity. Although not ideal for studies involving bioenergetics, this method will facilitate research on apoptosis and other mitochondrial functions that rely on an intact MOM.