Critical appraisal of two Box-Cox formulae for their utility in determining reference intervals by realistic simulation and extensive real-world data analyses.

BACKGROUND: The reference interval (RI) is defined as the central 95 % range of reference values (RVs) from healthy individuals. The ideal method for determining RIs is to transform RV distribution into Gaussian and estimate its 95 % range parametrically. One-parameter Box-Cox formula (1pBC) is widely used for correcting skewness (Sk) or kurtosis (Kt) in data distribution. However, 1pBC is not popular for computing RIs due to its unreliability in Gaussian transformation. While its two-parameter version (2pBC) is not used due to a challenge in fitting power (λ) and shift (α) parameters simultaneously. In this study, technical issues in fitting both formulae are assessed, and an alternative algorithm for successful use of 2pBC is proposed. METHODS: For fitting 1pBC, optimal λ was determined by stepwise linear search. For 2pBC, optimal [λ, α] combination was pursued in two ways: by grid search of λ and α (2pBCgrid) or by using the grid search but keeping α-range close to the reference distribution (2pBCopt). Their accuracy and precision in determining RIs were compared by generating power-normal distributions simulating RVs of 23 major chemistry analytes. Additionally, their practical utilities were compared by analyzing 776 real-world datasets comprising test results of 25 analytes that were obtained from the global multicenter RV study of IFCC. For comparison, the performance of nonparametric method was evaluated in both settings. RESULTS: For analytes with not-much-skewed distributions, unbiased estimation of RIs was accomplished by all methods. Nevertheless, when reference distributions are located far from zero, λ estimated by1pBC and 2pBCgrid fluctuated widely, which was attributable to virtually flat goodness-of-fit profile for [λ, α]. For highly skewed distributions, 1pBC caused bias in estimating RI and λ due to remotely peaked goodness-of-fit profile. Real-world data analyses revealed that 2pBCopt and 1pBC achieved Gaussian transformation (|Sk|<0.1 and |Kt|<0.3) in 82.4 % and 66.9 % among 776 datasets, respectively. Fitting bias signified by Kt<-0.4 was more common to 1pBC. The practical utility of 2pBCopt was unbiased prediction of analyte-specific distribution-shape (λ). Whereas nonparametric method gave highly variable RIs for both simulated and real-world datasets. CONCLUSIONS: 2pBCopt is suitable for calculating RIs and grasping distribution-shape from the estimated λ.

Induction of dwarf and early flowering phenotypes in Tricyrtis sp. by ectopic expression of LEAFY from Arabidopsis thaliana.

LEAFY (LFY), which encodes a plant-specific transcription factor, plays an important role in the transition from vegetative to reproductive development. Ectopic expression of LFY has been reported to induce dwarfism and early flowering in some model plants. In order to examine the possibility of using LFY for molecular breeding of ornamental plants, we produced and characterized transgenic plants ectopically expressing LFY from Arabidopsis thaliana (AtLFY) in the liliaceous ornamental plant Tricyrtis sp. Nine independent transgenic plants have been obtained, and all of them exhibited dwarf phenotypes compared with the vector control. These transgenic plants could be classified into three types according to the degree of dwarfism: one showed an extreamly dwarf phenotype with smaller leaves (Type I); two showed moderately dwarf phenotypes (Type II); and six showed slightly dwarf phenotypes (Type III). All of Type I, Type II and Type III transgenic plants produced flower buds 1-3 weeks earlier than the vector control. Vector control and Type III transgenic plants produced 1-4 apical flower buds, whereas Type I and Type II transgenic plants produced only a single apical flower bud. Type I and Type II transgenic plants often produced non-fully-opened flowers. Quantitative real-time reverse transcription-polymerase chain reaction analysis showed that the AtLFY expression level generally correlated with the degree of dwarfism. These results indicate that morphological alterations observed in the transgenic plants was induced by ectopic expression of AtLFY. Lower levels of ectopic expression of LFY may be valuable for producing dwarf and early flowering ornamental plants.

Establishment of large canine hepatocyte spheroids by mixing vascular endothelial cells and canine adipose-derived mesenchymal stem cells.

INTRODUCTION: Differentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs). METHODS: Spheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes (ALB, AFP, α1-AT, CDH1, CYP2E1, CYP3A12, and TAT) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes. RESULTS: The ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P < 0.001). Histological evaluation revealed that the three-mix spheroids formed large canine hepatocyte spheroids (LcHS), and hepatocytes were distributed in the center of the spheroids. Quantitative gene expression analysis of LcHS showed that liver-related genes expression were maintained the same levels with that of a culture of cHep alone from days 4-7. CONCLUSION: These results revealed that the three-mix culture method using cHep, HUVECs, and cASCs was capable of promoting LcHS without impairing liver function in cHep, suggesting that LcHS could be used for the application of high-volume culture techniques in dogs.

Efficient full spin-orbit torque switching in a single layer of a perpendicularly magnetized single-crystalline ferromagnet.

Spin-orbit torque (SOT), which is induced by an in-plane electric current via large spin-orbit coupling, enables an innovative method of manipulating the magnetization of ferromagnets by means of current injection. In conventional SOT bilayer systems, the magnetization switching efficiency strongly depends on the interface quality and the strength of the intrinsic spin Hall Effect. Here, we demonstrate highly efficient full SOT switching achieved by applying a current in a single layer of perpendicularly magnetized ferromagnetic semiconductor GaMnAs with an extremely small current density of ∼3.4 × 105 A cm-2, which is two orders of magnitude smaller than that needed in typical metal bilayer systems. This low required current density is attributed to the intrinsic bulk inversion asymmetry of GaMnAs as well as its high-quality single crystallinity and large spin polarization. Our findings will contribute to advancements in the electrical control of magnetism and its practical application in semiconductor devices.

Portal encasement: Significant CT findings to diagnose local recurrence after pancreaticoduodenectomy for pancreatic cancer.

BACKGROUND/OBJECTIVES: To demonstrate the utility of portal encasement as a criterion for early diagnosis of local recurrence (LR) after pancreaticoduodenectomy (PD) for pancreatic ductal adenocarcinoma (PDAC). METHODS: A total of 61 patients who underwent PD for PDAC were included in this retrospective study. Portal stenosis was evaluated by sequential postoperative computed tomography (CT) scans and correlated with disease recurrence. In addition to the conventional LR diagnostic criterion of a growing soft tissue mass, LR was evaluated using portal encasement as an additional diagnostic criterion. Portal encasement was defined as progressive stenosis of the portal system accompanied by a soft tissue mass, notwithstanding the enlargement of the mass. RESULTS: Benign portal stenosis was found on the first postoperative CT imaging in 16 patients. However, stenosis resolved a median of 81 days later in all but one patient whose stenosis was due to portal reconstruction during PD. Portal encasement could be distinguished from benign portal stenosis based on the timing of emergence of the portal stenosis. Portal encasement developed in 13 of the 19 patients with LR, including 6 patients in whom the finding of portal encasement led to the diagnosis of LR a median of 147 days earlier with our diagnostic criterion compared with the conventional diagnostic criteria. CONCLUSIONS: Portal encasement should be considered as a promising diagnostic criterion for earlier diagnosis of LR after PD for PDAC.

Genetic Factors of Low-responsiveness to Hepatitis B Virus Vaccine Confirms the Importance of Human Leukocyte Antigen Class II Types in a Japanese Young Adult Population.

We investigated the genetic mechanisms underlying the association between human leukocyte antigen (HLA) types and the immune response to hepatitis B virus (HBV) vaccination in 84 healthy Japanese adults, and found that the HLA-DRB1＊04 and HLA-DQB1＊03 frequencies were higher in the low responders (<10 mIU/ml; n=9, 10.7%) compared to the responders (≥10 mIU/ml, n=75, 89.3%). The combination of DRB1＊04 and DQB1＊03 was associated with a low response to vaccination. The DRB1＊04 and DQB1＊03 haplotypes' frequencies were significantly higher in the low responders compared to responders. Novel candidate HLA types may be important in Japanese individuals.

Influence of single nucleotide polymorphisms of cytokine genes on anti-HBs antibody production after hepatitis B vaccination in a Japanese young adult population.

Hepatitis B (HB) vaccination is one of the most efficient tools to prevent the transmission of the virus. Considerable variability exists in HB vaccine responses, with 5-10% of healthy Japanese adults demonstrating no response following a standard vaccination. Recently, polymorphisms of immune-regulatory genes, such as cytokine genes, have been reported to influence the immune response to HB vaccine. The aim of this study was to investigate the underlying mechanisms of the genetic association between several cytokine gene polymorphisms and the immune response to HB vaccination in a Japanese population. One hundred and twenty three vaccinated young adults were classified according to the level of antibody-titer (anti-HBs). Single nucleotide polymorphism typing for IFN-γ (+874, 3'-UTR), IL-10 (-591, -819, -1082), and TNF-α (-308, -857), was accomplished using the PCR-RFLP or SSP-PCR method. The TNF-α (-857) CC type and the IL-10 (-1082) AG type were present more frequently in the low titer group than in the high titer group. The TNF-α (-857) CC type was found to be significantly associated with low response of serum anti-HBs. The anti-HBs antibody was not readily produced in the IL-10 (-1082) AG and TNF-α (-857) CC haplotype. Conversely, the antibody was readily produced in the IL-10 (-1082) AA and TNF-α (-857) CC haplotype, and the IL-10 (-1082) AA and TNF-α (-857) CT haplotype, suggesting a high likelihood of the IL-10 (-1082) AG type to be included in the low anti-HBs group, and high anti-HBs antibody production in those with the TNF-α (-857) CT type. These SNPs may produce ethnically-specific differences in the immune response to HB vaccine in the Japanese population. J. Med. Invest. 63: 256-261, August, 2016.

Deformity of Buttonhole Entry Site Causes Higher Frequency of Vascular Access-Related Infection.

BACKGROUND: Vascular access-related infection is more frequent in patients using the buttonhole method for cannulation of the arteriovenous access for hemodialysis. Deformity of buttonhole entry sites is frequently observed among patients on the buttonhole method for extended periods of time. With deformed buttonhole entry sites, moreover, scabs are often incompletely removed at the time of buttonhole cannulation. METHOD: In 166 patients using the buttonhole method at Hino Clinic in Osaka, Japan as of June 30, 2014, the shapes of buttonhole entry sites were categorized into the following 3 types: flat, depressive deformity, and bulging deformity. A multivariate logistic regression method was used to analyze associations between various data including shapes of buttonhole entry sites and occurrence of access-related infection. We also examined microscopic features of the buttonhole entry site tissue that was removed from a patient who died after 3 years of buttonhole cannulation. RESULTS: For the flat buttonhole entry sites, frequency of access-related infection was 0.12 events/1,000 arteriovenous fistulas as compared to 0.47 events/1,000 arteriovenous fistulas for the entry sites with bulging deformity. Such infection did not occur for the entry sites with depressive deformity. The multivariate logistic regression analysis revealed a significant association between an entry site with bulging deformity and occurrence of access-related infection (odds ratio = 5.369, p = 0.0085). Furthermore, the microscopic section showed granulations beneath the skin at the buttonhole entry site and around the buttonhole tract. CONCLUSION: A significant association was shown between an entry site with bulging deformity and occurrence of access-related infection. The microscopic features of the buttonhole entry site of the patient on the buttonhole method for 3 years suggest that the entity of bulging deformity at the entry site is hypertrophic granulation.

Multiscale analysis of changes in an anisotropic collagen gel structure by culturing osteoblasts.

Mimicking the complicated anisotropic structures of a native tissue is extremely important in tissue engineering. In a previous study, we developed an anisotropic collagen gel scaffold (ACGS) having a hierarchical structure and a properties gradient. In this study, our objective was to see how cells remodel the scaffolds through the cells-ACGS interaction. For this purpose, we cultured osteoblastic cells on ACGS, which we regarded as a model system for the cells-extracellular matrix (cell-ECM) interaction. Changes in the ACGS-cell composites structure by cell-ECM interactions was investigated from a macroscopic level to a microscopic level. Osteoblastic cells were also cultured on an isotropic collagen gel (ICGS) as a control. During the cultivation, mechanical stimuli were applied to collagen-cell composites for adequate matrix remodeling. Confocal laser scanning microscope (CLSM) was used to observe macroscopic changes in the ACGS-cell composite structure by osteoblastic cells. Small-angle X-ray scattering (SAXS) measurements were performed to characterize microscopic structural changes in the composites. Macroscopic observations using CLSM revealed that osteoblastic cells remained only in the diluted phase in ACGS and they collected collagen fibrils or formed a toroidal structure, depending on the depth from the ACGS surface in the tubular diluted phase. The cells were uniformly distributed in ICGS. SAXS analysis suggests that collagen fibrils were remodeled by osteoblastic cells, and this remodeling process would be affected by the structure difference between ACGS and ICGS. These results suggest that we directly regulate cell-ECM interaction by the unique anisotropic and hierarchical structure of ACGS. The cell-gel composite presented in this study would promise an efficient scaffold material in tissue engineering.

[A case of neuroendocrine small cell carcinoma of the common bile duct].

A 75-year-old woman admitted for jaundice was found to have a tumor occupying the lumen of the middle bile duct on diagnostic imaging. Treatment with endoscopic retrograde bile drainage (ERBD) improved the jaundice. Bile cytology was defined as class III. We diagnosed the condition as middle bile duct cancer with a replaced right hepatic artery, and therefore, curative resection was possible. Pancreatoduodenectomy was performed and the replaced right hepatic artery was resected. Histological examination using hematoxylin and eosin( HE) staining and immunological staining with chromogranin A, synaptophysin, and CD56 revealed small cell carcinoma. Small cell carcinoma of the bile duct is a highly malignant disease. Fortunately, it is rare and only 22 cases have been reported in the Japanese literature.

Studies on the formation mechanism and the structure of the anisotropic collagen gel prepared by dialysis-induced anisotropic gelation.

We have found that dialysis of 5 mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 151 mM [corrected] at 25 °C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that the collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.

Chemotherapeutic targeting of etoposide to various tissues on the basis of polyamine level.

The effects of etoposide on body weight, organ weights and the concentrations of putrescine, spermidine and spermine in 14 different tissues were examined in rats that had been given the drug for five consecutive days. Etoposide reduces all polyamines, which are associated with tumor cell growth, in the thymus and reduces polyamines of two kinds in the spleen, heart, small intestine, skeletal muscle and lung but it increases putrescine in the prostate and spleen, and spermine in the large intestine.

Bacterial endophthalmitis after small-incision cataract surgery. effect of incision placement and intraocular lens type.

PURPOSE: To determine whether endophthalmitis after small-incision cataract surgery is affected by the incision site (superior sclerocorneal versus temporal cornea) or the foldable intraocular lens (IOL) material (silicone versus acrylic). SETTING: Multicenter study. METHODS: Patients who had small-incision cataract surgery at Toyama Medical and Pharmaceutical University Hospital and affiliated hospitals from March 1998 to March 2001 were examined prospectively. The patients were randomized into 3 groups. In Group A, an acrylic IOL (MA60BM, Alcon) was implanted through a temporal corneal incision. In Group B, an acrylic IOL (MA60BM) was implanted via a superior sclerocorneal incision. In Group C, a silicone poly(methyl methacrylate) IOL (SI-40NB, Allergan) was implanted via a superior sclerocorneal incision. Each patient was followed for more than 6 months. RESULTS: Initially, 7622 patients (12 317 eyes) agreed to participate in the study. The final study included 3831 eyes in Group A, 3901 eyes in Group B, and 3863 eyes in Group C. Postoperative endophthalmitis was diagnosed clinically in 11 eyes (0.29%) in Group A, 2 (0.05%) in Group B, and 2 (0.05%) in Group C. Endophthalmitis proven by culture was found in 9 eyes in Group A, 2 in Group B, and 2 in Group C. The incidence of endophthalmitis in Group A was higher than in the other groups. The incidence of endophthalmitis in Group B was similar to that in Group C. The relative risk of postoperative endophthalmitis proven by culture in Groups B and C was 4.6 times (P =.037) lower than in Group A. CONCLUSION: The findings suggest that a temporal corneal incision may lead to an increased risk of postoperative endophthalmitis and that the IOL material does not affect the incidence of this complication.

Chemotherapeutic targeting of etoposide to regions of the brain on the basis of polyamine level.

The effects of etoposide on body weight, regional weights, and the concentrations of putrescine, spermidine and spermine in the cerebellum, hippocampus, corpus striatum, cortex, the combined regions of the thalamus and hypothalamus and the diencephalon of the brain were examined in rats. Etoposide seems to be a better choice for management of cortical and hippocampal tumors because it reduces polyamines, which are associated with tumor cell growth, but not for the management of tumors of the diencephalon and corpus striatum because it increases polyamines.

Determination of absolute configurations of light-atom molecules by means of direct detection of Bijvoet differences.

The absolute configurations of two light-atom molecules were determined sufficiently well by direct detection of Bijvoet differences. The compounds examined were (I) beta-cytidine C9H13N3O5 and (II) (S)-3-[(R)-4,4,4-Trifluoro-3-[4-methoxyphenyl]butanoyl]-4-(phenylmethyl)oxazolidin-2-one, C21H20NO4F3. Both compounds crystallize in orthorhombic system with the space group P2(1)2(1)2(1) and Z = 4. The crystal structures were carefully refined by the technique of conventional structure analysis. All possible reflections were measured on a laboratory diffractometer with Cu Kalpha radiation. The multiple-diffraction effect was often observed especially as remarkable intensity enhancement in weak reflections. After such unreliable reflections were eliminated by comparisons among the equivalent mates, data were averaged to a set of Bijvoet pairs. Afterwards, additional measurements by the psi-scan technique were tested. Since psi-scan data showed a slight systematic error probably owing to some shape-effect, an artificial absorption correction DIFABS was adopted to remove the error. Small but significant intensity differences could be detected for many Bijvoet pairs, and the absolute configurations were correctly determined without ambiguity in all cases. The R and wR values for separate refinements of enantiomorphs supported the results with slight differences. Flack parameters indicated no contradictions as well.