Investigation of the Genetic Diversity of a Rice Core Collection of Japanese Landraces using Whole-Genome Sequencing.

The Rice Core Collection of Japanese Landraces (JRC) consisting of 50 accessions was developed by the genebank at the National Agriculture and Food Research Organization (NARO) in 2008. As a Japanese landrace core collection, the JRC has been used for many research projects, including screening for different phenotypes and allele mining for target genes. To understand the genetic diversity of Japanese Landraces, we performed whole-genome resequencing of these 50 accessions and obtained a total of 2,145,095 single nucleotide polymorphism (SNPs) and 317,832 insertion-deletions (indels) by mapping against the Oryza sativa ssp. japonica Nipponbare genome. A JRC phylogenetic tree based on 1,394 representative SNPs showed that JRC accessions were divided into two major groups and one small group. We used the multiple genome browser, TASUKE+, to examine the haplotypes of flowering genes and detected new mutations in these genes. Finally, we performed genome-wide association studies (GWAS) for agronomical traits using the JRC and another core collection, the World Rice Core Collection (WRC), comprising 69 accessions also provided by the NARO genebank. In leaf blade width, a strong peak close to NAL1, a key gene for the regulation of leaf width, and, in heading date, a peak near HESO1 involved in flowering regulation were observed in GWAS using the JRC. They were also detected in GWAS using the combined JRC + WRC. Thus, JRC and JRC + WRC are suitable populations for GWAS of particular traits.

Accuracy of tibial component placement in unicompartmental knee arthroplasty performed using an accelerometer-based portable navigation system.

PURPOSE: There is a need for new devices to improve the accuracy of implantation in unicompartmental knee arthroplasties (UKAs). The accelerometer-based portable navigation system is expected to improve this accuracy. This study aimed to compare the accuracy of UKAs performed by the portable navigation system with that of the conventional method, and to investigate whether the portable navigation system can complement the surgeon's experience. METHODS: The study comprised of 80 Oxford UKAs. Knees were divided into two groups based on the method of tibial osteotomy: the conventional group (37 UKAs performed by an experienced surgeon using the extra-medullary guide) and the portable navigation group (43 UKAs performed by 2 unaccustomed surgeons using the navigation system). The absolute error from the target angle on the coronal and sagittal plane was measured on whole lower leg X-ray. The incidence of outliers (> 3°) was compared between the groups using Fisher's exact probability test. RESULTS: The incidences of outliers on the coronal plane were 41.0% (15 of 37 knees) in the conventional group and 9.3% (4 of 43 knees) in the portable navigation group (p < 0.0001). The incidences of outliers on the sagittal plane were 13.5% (5 of 37 knees) in the conventional group and 14.0% (6 of 43 knees) in the portable navigation group (p = 0.3772). CONCLUSION: This is the first report on the usefulness of an accelerometer-based portable navigation system in UKA. The use of this system improves the accuracy of implantation of the tibial component beyond the experience of the surgeon. LEVEL OF EVIDENCE: Retrospective comparative study, Level III.

Medium-term clinical results of unicompartmental knee arthroplasty for the treatment for spontaneous osteonecrosis of the knee with four to 15 years of follow-up.

BACKGROUND: Unicompartmental knee arthroplasty (UKA) is an option for the treatment of spontaneous osteonecrosis of the knee (SONK). However, there are limited studies focusing on this area. This study presents medium-term clinical outcome data of UKA for SONK. METHODS: We reviewed 50 SONK knees in 48 patients that were treated by UKA. The mean age, height, and body weight were 73 years, 153 cm, and 57 kg, respectively. The mean follow-up was 8.4 years (range, four to 15 years). Preoperatively, we measured the size and the volume (estimated by width × length × depth) of the necrotic bone mass on T1-weighted magnetic resonance imaging. The clinical results were evaluated serially at follow-up visits radiographically and with the Knee Society Scoring (KSS) and Oxford Knee Scoring (OKS) systems. RESULTS: There were no revisions, re-operations, or major complications. The mean sizes of the necrotic lesions were 17.2 mm (14.7-22.3 mm) in width, 28.2 mm (6.2-34.7 mm) in length, and 11.3 mm (3.2-14.5 mm) in depth. The mean volume was approximately 5.4 cm3 (0.7-10.3 cm3). The mean flexion of the knee, KSS Knee Score, Function Score, and OKS increased from a preoperative 128.7-137.5°, 52.3-91.3, 39.7-90.2, and 21.6-40.2, respectively, at the latest follow-up. At the last follow-up, all patients had good or excellent OKS. CONCLUSIONS: This study demonstrates that UKA is a good option and is reliable for the treatment of SONK irrespective of necrotic bone mass size.

Detection of an interaction between prion protein and neuregulin I-ß1 by fluorescence resonance energy transfer analysis.

Cellular prion protein (PrP) copurifies with neuregulin type I-ß1 (NRG I-ß1), but no interaction has been detected by a general immunoprecipitation study. We speculate that PrP interacts with NRG I-ß1. Here, the interaction of PrP with NRG I-ß1 was detected by measuring fluorescence resonance energy transfer (FRET) between enhanced blue (EBFP) and enhanced green (EGFP) fluorescent protein-fusion proteins. Full-length PrP interacted with EGFP in addition to NRG I-ß1. From this result, we deduced that PrP interacts with EGFP through its unstructured N-terminal domain. We therefore detected FRET between PrP deleting the N-terminal domain and NRG I-ß1. In contrast, the C-terminal domain of PrP interacted with NRG I-ß1 and the proteins dissociated completely in the presence of sodium chloride. This interaction occurs at the nanomolar level, which is important for the reaction to be functional in organisms. We concluded that PrP interacted with NRG I-ß1 through its C-terminal domain.

Role of calcium-binding sites in calcium-dependent membrane association of annexin A4.

Annexin A4 (Anx4) is a cytosolic calcium-binding protein with four repeat domains, each containing one calcium-binding site (CBS). The protein interacts with the phospholipid membrane through the CBS-coordinated calcium ion, although the role of each CBS in the calcium-dependent association is unclear. To determine the role of each CBS, 15 CBS-abolished variants were produced in various combinations by substitution of a calcium-liganding residue on each CBS by Ala. Various mutant combinations produced different influences on calcium-dependent membrane-binding behavior and on the sodium-dependent dissociation of membrane-bound Anx4. Our data suggest the interaction of Anx4 with the lipid membrane consists of strong and weak interactions. CBSs I and IV mediate formation of strong interactions, while CBSs II and III are important for weak interactions. We also suggest Anx4 binds the lipid membrane through CBSs I and IV in the cytoplasmic fluids.

Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity.

To cause food-borne botulism, botulinum neurotoxin (BoNT) in the gastrointestinal lumen must traverse the intestinal epithelial barrier. However, the mechanism by which BoNT crosses the intestinal epithelial barrier remains unclear. BoNTs are produced along with one or more non-toxic components, with which they form progenitor toxin complexes (PTCs). Here we show that serotype A1 L-PTC, which has high oral toxicity and makes the predominant contribution to causing illness, breaches the intestinal epithelial barrier from microfold (M) cells via an interaction between haemagglutinin (HA), one of the non-toxic components, and glycoprotein 2 (GP2). HA strongly binds to GP2 expressed on M cells, which do not have thick mucus layers. Susceptibility to orally administered L-PTC is dramatically reduced in M-cell-depleted mice and GP2-deficient (Gp2(-/-)) mice. Our finding provides the basis for the development of novel antitoxin therapeutics and delivery systems for oral biologics.

A novel mucosal vaccine targeting Peyer's patch M cells induces protective antigen-specific IgA responses.

Mucosal vaccines can induce mucosal immunity, including antigen-specific secretory IgA production, to protect from invasion by pathogens and to neutralize toxins at mucosal surfaces. We established an effective antigen-delivering fusion protein, anti-GP2-SA, as a mucosal vaccine. The anti-GP2-SA consists of streptavidin (SA) fused to the antigen-binding fragment region from a mAb against glycoprotein 2 (GP2), an antigen-uptake receptor specifically expressed on M cells. Anti-GP2-SA targets antigen-sampling M cells in the follicle-associated epithelium covering Peyer's patches. Immunofluorescence showed that anti-GP2-SA specifically bound to M cells. Orally administered biotinylated ovalbumin peptide (bOVA) conjugated with anti-GP2-SA more efficiently induced OVA-specific fecal IgA secretion compared with bOVA alone or bOVA conjugated with SA. Furthermore, mice immunized by oral administration of the biotinylated Salmonella enterica serovar Typhimurium (S. Typhimurium) lysate conjugated with anti-GP2-SA were significantly better protected from subsequent infection by virulent S. Typhimurium than mice treated with the bacterial lysate alone or conjugated with SA. These results suggest that anti-GP2-SA-based M-cell-targeting vaccines are a novel strategy for inducing efficient mucosal immunity.

Oxford phase 3 unicompartmental knee arthroplasty in Japan--clinical results in greater than one thousand cases over ten years.

There are few reports of the Oxford unicompartmental knee arthroplasty (UKA) survival rate in Asia. This study describes outcomes of 1279 Oxford UKAs for Japanese patients. The mean follow-up was 5.2 years. We divided patients into two groups based on preoperative indications (extended indications group and strict indications group). The Oxford knee score improved from 22.3 to 40.8 (P=0.041). The 10-year survival rate using revision was 95%. A total of 25 UKAs (2.0%) required revision. The most common reason was subsidence of tibial component. The 5-year cumulative survival rate of the strict indications group was significantly higher than that of the extended indications group (99.1% vs. 93.8%, P<0.001). When we followed inclusion criteria strictly, good clinical results were achieved in Asia.

Crystal structure of Sus scrofa quinolinate phosphoribosyltransferase in complex with nicotinate mononucleotide.

We have determined the crystal structure of porcine quinolinate phosphoribosyltransferase (QAPRTase) in complex with nicotinate mononucleotide (NAMN), which is the first crystal structure of a mammalian QAPRTase with its reaction product. The structure was determined from protein obtained from the porcine kidney. Because the full protein sequence of porcine QAPRTase was not available in either protein or nucleotide databases, cDNA was synthesized using reverse transcriptase-polymerase chain reaction to determine the porcine QAPRTase amino acid sequence. The crystal structure revealed that porcine QAPRTases have a hexameric structure that is similar to other eukaryotic QAPRTases, such as the human and yeast enzymes. However, the interaction between NAMN and porcine QAPRTase was different from the interaction found in prokaryotic enzymes, such as those of Helicobacter pylori and Mycobacterium tuberculosis. The crystal structure of porcine QAPRTase in complex with NAMN provides a structural framework for understanding the unique properties of the mammalian QAPRTase active site and designing new antibiotics that are selective for the QAPRTases of pathogenic bacteria, such as H. pylori and M. tuberculosis.

Crystallization and preliminary X-ray crystallographic analysis of quinolinate phosphoribosyltransferase from porcine kidney in complex with nicotinate mononucleotide.

Quinolinate phosphoribosyltransferase (QAPRTase) is a key enzyme in NAD biosynthesis; it catalyzes the formation of nicotinate mononucleotide (NAMN) from quinolinate and 5-phosphoribosyl-1-pyrophosphate. In order to elucidate the mechanism of NAMN biosynthesis, crystals of Sus scrofa QAPRTase (Ss-QAPRTase) purified from porcine kidney in complex with NAMN were obtained and diffraction data were collected and processed to 2.1 Šresolution. The Ss-QAPRTase-NAMN cocrystals belonged to space group P321, with unit-cell parameters a=119.1, b=119.1, c=93.7 Å, γ=120.0°. The Matthews coefficient and the solvent content were estimated as 3.10 Å3 Da(-1) and 60.3%, respectively, assuming the presence of two molecules in the asymmetric unit.

Establishment of true niacin deficiency in quinolinic acid phosphoribosyltransferase knockout mice.

Pyridine nucleotide coenzymes are involved in >500 enzyme reactions and are biosynthesized from the amino acid L-tryptophan (L-Trp) as well as the vitamin niacin. Hence, "true" niacin-deficient animals cannot be "created" using nutritional techniques. We wanted to establish a truly niacin-deficient model animal using a protocol that did not involve manipulating dietary L-Trp. We generated mice that are missing the quinolinic acid (QA) phosphoribosyltransferase (QPRT) gene. QPRT activity was not detected in qprt(-/-)mice. The qprt(+/+), qprt(+/-), or qprt(-/-) mice (8 wk old) were fed a complete diet containing 30 mg nicotinic acid (NiA) and 2.3 g L-Trp/kg diet or an NiA-free diet containing 2.3 g L-Trp/kg diet for 23 d. When qprt(-/-)mice were fed a complete diet, food intake and body weight gain did not differ from those of the qprt(+/+) and qprt(+/-) mice. On the contrary, in the qprt(-/-) mice fed the NiA-free diet, food intake and body weight were reduced to 60% (P < 0.01) and 70% (P < 0.05) of the corresponding values for the qprt(-/-) mice fed the complete diet at d 23, respectively. The nutritional levels of niacin, such as blood and liver NAD concentrations, were also lower in the qprt(-/-) mice than in the qprt(+/+) and the qprt(+/-) mice. Urinary excretion of QA was greater in the qprt(-/-) mice than in the qprt(+/+) and qprt(+/-) mice (P < 0.01). These data suggest that we generated truly niacin-deficient mice.

Production of a recombinant full-length prion protein in a soluble form without refolding or detergents.

Recombinant prion protein has been produced in insoluble form and refolded following solubilization with denaturants. It is, however, preferable to use a soluble recombinant protein prepared without artificial solubilization. In this study, a soluble recombinant prion protein was produced in Escherichia coli cells by coexpression of neuregulin I-ß1 and purified to high purity.

Crystallization and preliminary X-ray crystallographic analysis of human quinolinate phosphoribosyltransferase.

Quinolinate phosphoribosyltransferase (QPRTase) is a key NAD-biosynthetic enzyme which catalyzes the transfer of quinolinic acid to 5-phosphoribosyl-1-pyrophosphate, yielding nicotinic acid mononucleotide. Homo sapiens QPRTase (Hs-QPRTase) appeared as a hexamer during purification and the protein was crystallized. Diffraction data were collected and processed at 2.8 Šresolution. Native Hs-QPRTase crystals belonged to space group P2(1), with unit-cell parameters a=76.2, b=137.1, c=92.7 Å, ß=103.8°. Assuming the presence of six molecules in the asymmetric unit, the calculated Matthews coefficient is 2.46 Å3 Da(-1), which corresponds to a solvent content of 49.9%.

Eosin-shadow method: a selective enhancement of light-microscopic visualization of pancreatic zymogen granules on hematoxylin-eosin sections.

We developed a novel method for enhancing light-microscopic visualization of pancreatic zymogen granules in a selective manner on hematoxylin and eosin-stained sections. By using an absorption filter that transmits light with wavelength from 510 to 550 nm, corresponding to the narrow absorption spectrum of eosin, only eosinophilic tissue and cellular components were remarkably highlighted as distinct shadows against lighter background consisting of basophilic components. Using a pair of mirror sections of the pancreas, immunocytochemistry with anti-amylase antibody confirmed that the shadows observed through the filter represented zymogen granules. Immersion in formalin for 36 h at room temperature was the optimal fixation condition. Here we designate the procedure as the "eosin-shadow method" and propose that this technique is convenient and useful to help investigators identify zymogen granules more easily in routine pathological examination and histological studies.

Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.

The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.

Crystal structures of sodium-bound annexin A4.

Annexin A4 (Anx4) possesses four repeat domains with one Ca(2+)-binding site (CBS) in each domain. In this study, we resolved two crystal structures of the Na(+)-bound form at high resolution (1.58 and 1.35 A). This is the first report that Anx4 binds the Na(+) ion in CBSs. Electron density maps, valence screening, and atomic absorbance spectrometry confirmed that Anx4 bound the Na(+) ion. One structure (1.58 A) bound the Na(+) ion in CBS I, whereas another structure (1.35 A) bound the Na(+) ion in CBS II and CBS III. We compared the two Na(+)-bound forms by superimposing their C(alpha) traces. The C(alpha) atoms of CBS III largely moved by coordination of the Na(+) ion. In the C(alpha) atoms of CBS I, however, little change resulted from Na(+)-coordination. Only the side chain of Glu71 was moved by Na(+)-coordination in CBS I. These results indicate that Anx4 also binds not only Ca(2+) but also Na(+) ion in the CBS.

Comparison between highly cross-linked and conventional polyethylene in total knee arthroplasty.

The use of highly cross-linked polyethylene in total knee prostheses is still controversial. The aim of the present study was to compare radiographic and clinical results of using conventional and highly cross-linked polyethylene in cruciate retaining total knee prostheses of completely the same design. Two hundred and two consecutive total knee arthroplasties (NexGen CR, Zimmer) were performed using the same procedure. The first consecutive 113 knees had conventional polyethylene insert and following consecutive 89 knees had highly cross-linked polyethylene insert (Prolong, Zimmer). Differences in the age, gender, and diagnosis between two groups were not statistically significant. Preoperative range of motion (ROM) of the knee, and Knee Society Score (KSS) was better in highly cross-linked polyethylene group. Clinical and radiographic results were evaluated at two years after operation. The difference of ROM and KSS between groups was not statistically significant. There was no revision surgery. No knee exhibited osteolysis, aseptic loosening, or polyethylene failure. There was no early catastrophic clinical failure due to use of the new material.

Range of motion of standard and high-flexion cruciate retaining total knee prostheses.

Recently, use of high-flexion design was introduced in cruciate-retaining (CR) total knee prostheses. The purpose of this study was to prospectively compare the ranges of motion (ROMs) of 89 knees with standard and 87 knees with high-flexion CR total knee prostheses. Differences in age, gender, diagnosis, preoperative ROM of the knee, and Knee Society Score between the 2 groups were not statistically significant. At 12-month follow-up, average ROM was 112.0 degrees +/- 12.6 degrees for standard, and 115.3 degrees +/- 13.4 degrees for high-flexion CR prosthesis (P = .101). To our knowledge, this is the first report on the ROM with the high-flexion CR total knee prosthesis. Using the technique of anterior referencing for femoral component sizing and using a fixed 7 degrees slope for the tibial component, we found no significant differences between groups with regard to ROM, clinical, or radiographic parameters.

Production of a soluble recombinant prion protein fused to blue fluorescent protein without refolding or detergents in Escherichia coli cells.

The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E. coli) cells to 15 degrees C. Soluble PrP-EBFP was purified on cation exchange and heparin-affinity columns to yield high purity protein. This is the first report of the preparation of soluble recombinant PrP without refolding following solubilization using denaturants or disruption using detergents. To confirm the integrity of PrP-EBFP, anisotropy was estimated under physiological conditions in the presence of heparin, which interacts with PrP. The dissociation constant was determined to be 0.88+/-0.07 microM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules.

Phthalate esters enhance quinolinate production by inhibiting alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), a key enzyme of the tryptophan pathway.

Tryptophan is metabolized to alpha-amino-beta-carboxymuconate-epsilon-semialdehyde (ACMS) via 3-hydroxyanthranilate (3-HA). ACMS decarboxylase (ACMSD) directs ACMS to acetyl CoA; otherwise ACMS is non-enzymatically converted to quinolinate (QA), leading to the formation of NAD and its degradation products. Thus, ACMSD is a critical enzyme for tryptophan metabolism. Phthalate esters have been suspected of being environmental endocrine disrupters. Because of the structural similarity of phthalate esters with tryptophan metabolites, we examined the effects of phthalate esters on tryptophan metabolism. Phthalate esters containing diets were orally given to rats and the urinary excreted tryptophan metabolites were quantified. Of the phthalate esters with different side chains tested, di(2-ethylhexyl)phthalate (DEHP) and its metabolite, mono(2-ethylhexyl)phthalate (MEHP), most strongly enhanced the production of QA and degradation products of nicotinamide, while 3-HA was unchanged. This pattern of metabolic change led us to assume that these esters lowered ACMSD protein or its activity. Although DEHP could not be tested because of its low solubility, MEHP reversibly inhibited ACMSD from rat liver and mouse kidney, and also the recombinant human enzyme. Correlation between inhibition of ACMSD by phthalate esters with different side chains and urinary excretion of QA supports the notion that phthalate esters perturb tryptophan metabolism by inhibiting ACMSD. Quinolinate is a potential endogenous toxin and has been implicated in the pathogenesis of various disorders. Although toxicity of phthalate esters through accumulation of QA remains to be investigated, they may be detrimental by acting as metabolic disrupters when intake of a tryptophan-rich diet and exposure to phthalate esters occur coincidentally.