Integrated genome-wide differentiation and association analyses identify causal genes underlying breeding-selected grain quality traits in japonica rice.

Improving grain quality is a primary objective in contemporary rice breeding. Japanese modern rice breeding has developed two different types of rice, eating and sake-brewing rice, with different grain characteristics, indicating the selection of variant gene alleles during the breeding process. Given the critical importance of promptly and efficiently identifying genes selected in past breeding for future molecular breeding, we conducted genome scans for divergence, genome-wide association studies, and map-based cloning. Consequently, we successfully identified two genes, OsMnS and OsWOX9D, both contributing to rice grain traits. OsMnS encodes a mannan synthase that increases the white core frequency in the endosperm, a desirable trait for sake brewing but decreases the grain appearance quality. OsWOX9D encodes a grass-specific homeobox-containing transcription factor, which enhances grain width for better sake brewing. Furthermore, haplotype analysis revealed that their defective alleles were selected in East Asia, but not Europe, during modern improvement. In addition, our analyses indicate that a reduction in grain mannan content during African rice domestication may also be caused a defective OsMnS allele due to breeding selection. This study not only reveals the delicate balance between grain appearance quality and nutrition in rice but also provides a new strategy for isolating causal genes underlying complex traits, based on the concept of "breeding-assisted genomics" in plants.

Characterization of Cooked Nonglutinous Rice Cultivars Based on Flavor Volatiles and Their Change during Storage.

Characterizing fleshly cooked rice cultivars according to the volatile aroma compounds helps consumers select a favorite and is useful for the development of new cultivars that will have a pleasant aroma. In the present study, six Japanese nonglutinous cultivars, which were freshly harvested in 2021, were characterized based on their flavor volatiles after being freshly cooked. In order to extract the volatile compounds just after cooking, the vaporized compounds were extracted for 5 min using a solid-phase microextraction (SPME) fiber and were measured via gas chromatography/mass spectrometry (GC/MS). Multiple comparison tests statistically detected four volatile aroma compounds: 2-pentylfuran, nonanal, 4-vinylphenol, and indole. From among the six rice cultivars tested, the proportions of the latter two compounds showed significant differences, and in principal component analysis of cooked rice, these two best characterized freshly harvested and freshly cooked Japanese nonglutinous rice cultivars; indole was indicative of Nipponbare, and 4-vinylphenol was indicative of Koshihikari and Ichihomare. In the present study, changes in the volatile aroma compounds of the freshly cooked rice cultivars were found to slightly differentiate according to storage times: 2-pentylfuran tended to increase, nonanal first increased and then decreased, and 4-vinylphenol and indole either remained almost unchanged or were only slightly decreased during storage. Therefore, establishing the differences in rice cultivar types revealed that the characteristics of the flavor volatiles of freshly cooked rice after long-term storage significantly depend on how the rice cultivar is stored.

A novel mutant with triple the dietary fiber content in white rice.

Dietary fiber has high functional value in relation to gut flora. We searched for a high-lysine mutant of the most popular rice cultivar in Japan, 'Koshihikari', as a route to a higher dietary fiber content like a success case in new barley cultivar, 'Beau-fiber'. We found several promising high-lysine mutants with high dietary fiber content. One of these, 'WFE5', has three times the dietary fiber content in white rice. Two rounds of backcrossing to Koshihikari produced a near-isogenic line with a high fiber content. The line's agronomic traits were close to those of Koshihikari except for yield and eating quality. As these two traits are critical, we discuss how to improve them.

Binasal hemianopia caused by bilateral optic perineuritis due to sarcoidosis.

•We describe the first case of binasal hemianopia due to bilateral optic perineuritis.•Bilateral optic perineuritis should be considered as a causative disease of binasal hemianopia.•Early diagnosis of optic perineuritis is crucial to avoid irreversible visual impairment.

Using SPME-GC/REMPI-TOFMS to Measure the Volatile Odor-Active Compounds in Freshly Cooked Rice.

The volatile odor-active compounds of cooked rice were evaluated using a method that combined solid-phase microextraction (SPME) with gas chromatography-resonance-enhanced multiphoton ionization time-of-flight mass spectrometry (GC/REMPI-TOFMS). An SPME fiber was held at the upper levels of the cooked rice and given an extraction time of 5 min. By using a nanosecond ultraviolet (266 nm) pulsed laser for ionization, two compounds, 4-vinylphenol and indole, which are considered to be important for the characteristic flavor of cooked rice, could be detected from all types of cultivars measured in the present study-nonglutinous rice, glutinous rice, and aromatic rice. In the case of fresh nonglutinous rice, the amounts of introduction for 4-vinylphenol and indole to GC were ca. 70 and 20 pg, respectively. While both peak areas decreased with increases in the time needed to maintain warmth, the decreasing behaviors differed slightly with a noteworthy rapid decrease for indole. For nonglutinous rice, the peak areas for 4-vinylphenol were almost the same, whether it was fresh (measured within 1 month from harvest) or aged (measured 6-12 months after harvest), but those of indole significantly decreased following storage. We also found differences among cultivars: the peak area for 4-vinylphenol in nonglutinous rice was somewhat strong; the peak area for indole was intensely strong in glutinous rice; however, the peak areas for both 4-vinylphenol and indole were weak in aromatic rice. Volatile odor-active compounds were detected in a sensitive and time-resolved manner; therefore, the proposed method could be useful for differentiating varieties of cooked rice from the viewpoints of cooking conditions, freshness, and cultivar types.

Prognostic impact of circulating tumor DNA status post-allogeneic hematopoietic stem cell transplantation in AML and MDS.

This study was performed to assess the utility of tumor-derived fragmentary DNA, or circulating tumor DNA (ctDNA), for identifying high-risk patients for relapse of acute myeloid leukemia and myelodysplastic syndrome (AML/MDS) after undergoing myeloablative allogeneic hematopoietic stem cell transplantation (alloSCT). We retrospectively collected tumor and available matched serum samples at diagnosis and 1 and 3 months post-alloSCT from 53 patients with AML/MDS. After identifying driver mutations in 51 patients using next-generation sequencing, we designed at least 1 personalized digital polymerase chain reaction assay per case. Diagnostic ctDNA and matched tumor DNA exhibited excellent correlations with variant allele frequencies. Sixteen patients relapsed after a median of 7 months post-alloSCT. Both mutation persistence (MP) in bone marrow (BM) at 1 and 3 months post-alloSCT and corresponding ctDNA persistence (CP) in the matched serum (MP1 and MP3; CP1 and CP3, respectively) were comparably associated with higher 3-year cumulative incidence of relapse (CIR) rates (MP1 vs non-MP1, 72.9% vs 13.8% [P = .0012]; CP1 vs non-CP1, 65.6% vs 9.0% [P = .0002]; MP3 vs non-MP3, 80% vs 11.6% [P = .0002]; CP3 vs non-CP3, 71.4% vs 8.4% [P < .0001]). We subsequently evaluated whether subset analysis of patients with 3 genes associated with clonal hematopoiesis, DNMT3A, TET2, and ASXL1 (DTA), could also be helpful in relapse prediction. As a result, CP based on DTA gene mutations also had the prognostic effect on CIR. These results, for the first time, support the utility of ctDNA as a noninvasive prognostic biomarker in patients with AML/MDS undergoing alloSCT.

Cell-lineage level-targeted sequencing to identify acute myeloid leukemia with myelodysplasia-related changes.

Acute myeloid leukemia (AML) is a clonal myeloid neoplasm that typically arises de novo; however, some cases evolve from a preleukemic state, such as myelodysplastic syndrome (MDS). Such secondary AMLs and those with typical MDS-related clinical features are known as AMLs with myelodysplasia-related changes (AML-MRC). Because patients with AML-MRC have poor prognosis, more accurate diagnostic approaches are required. In this study, we performed targeted sequencing of 54 genes in 3 cell populations (granulocyte, blast, and T-cell fractions) using samples from 13 patients with MDS, 16 patients with clinically diagnosed AML-MRC, 4 patients with suspected AML-MRC but clinically diagnosed as AML not otherwise specified (AML-NOS), and 11 patients with de novo AML. We found that overlapping mutations, defined as those shared at least by the blast and granulocyte fractions, were significantly enriched in patients with MDS and AML-MRC, including those with suspected AML-MRC, indicating a substantial history of clonal hematopoiesis. In contrast, blast-specific nonoverlapping mutations were significantly enriched in patients with de novo AML. Furthermore, the presence of overlapping mutations, excluding DNMT3A, TET2, and ASXL1, effectively segregated patients with MDS and AML-MRC or suspected AML-MRC from patients with de novo AML. Additionally, the presence of ≥3 mutations in the blast fraction was useful for distinguishing patients with AML-MRC from those with MDS. In conclusion, our approach is useful for classifying clinically diagnosable AML-MRC and identifying clinically diagnosed AML-NOS as latent AML-MRC. Additional prospective studies are needed to confirm the utility of this approach.

Circulating tumor DNA dynamically predicts response and/or relapse in patients with hematological malignancies.

A growing body of evidence suggests that tumor-derived fragmentary DNA, known as circulating tumor DNA (ctDNA), has the potential to serve as a non-invasive biomarker for disease monitoring. However, in the setting of hematological malignancy, few published studies support the utility of ctDNA. We retrospectively investigated ctDNA levels of 17 patients with various hematological malignancies who had achieved remission after first-line therapy. We identified somatic driver mutations by next-generation sequencing, and designed droplet digital PCR assays for each mutation to measure ctDNA. Variant allele frequencies of ctDNA changed in association with clinical response in all patients. Eight patients clinically relapsed after a median of 297 days post-first-line therapy (termed, "relapsed group"); the remaining nine patients remained disease-free for a median of 332 days (termed, "remission group"). Among patients in the relapsed group, ctDNA levels increased more than twofold at paired serial time points. In marked contrast, ctDNA levels of all patients in the remission group remained undetectable or stable during clinical remission. Notably, ctDNA-based molecular relapse demonstrated a median 30-day lead time over clinical relapse. In summary, ctDNA monitoring may help identify hematologic cancer patients at risk for relapse in advance of established clinical parameters.

Koshihikari: a premium short-grain rice cultivar - its expansion and breeding in Japan.

Koshihikari, a Japanese short-grain rice cultivar, was developed in 1956, more than 60 years ago. Despite its age, it has been the most widely grown cultivar in Japan for more than 35 years, making it the most important rice for the Japanese people. In its early days, there was no reason to predict that Koshihikari would become so widely disseminated. However, since the end of the post-World War II food shortage in the 1960s, Japanese preferences changed from high productivity to good eating quality. This triggered wide expansion of Koshihikari cultivation due to the cultivar's excellent taste and texture. With increasing cultivation of Koshihikari in Japan, several good agronomic characteristics beyond its high eating quality became apparent, such as its good adaptation to different environments, tolerance to pre-harvest sprouting, and cold tolerance during the booting stage. These characteristics outweigh drawbacks such as its low blast resistance and low lodging resistance. The popularity of Koshihikari influenced subsequent rice breeding trends at regional agricultural experimental stations, and the characteristics of newly developed rice cultivars in Japan are usually rated relative to Koshihikari, which is used as the benchmark. Koshihikari was the first japonica rice cultivar whose whole genome has been sequenced by means of next-generation sequencing. Furthermore, comparison of the genomes of Koshihikari and Nipponbare has provided detailed insights into the genetic diversity of Japanese rice cultivars relative to that in rice populations elsewhere in the world. Further progress in rice genomics is gradually unlocking the mechanisms that underlie the agronomic characteristics of Koshihikari. To support both research and the development of novel rice cultivars, a series of isogenic and near-isogenic lines in the Koshihikari genetic background have been continuously developed. These new findings and materials will facilitate genomics-assisted rice breeding, eventually leading to superior cultivars.

Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering.

The development of an automated cell culture system would allow stable and economical cell processing for wider clinical applications in the field of regenerative medicine. However, it is crucial to determine whether the cells obtained by automated culture are comparable to those generated by manual culture. In the present study, we focused on the primary culture process of bone marrow stromal cells (BMSCs) for bone tissue engineering and investigated the feasibility of its automation using a commercially available automated cell culture system in a clinical setting. A comparison of the harvested BMSCs from manual and automated cultures using clinically acceptable protocols showed no differences in cell yields, viabilities, surface marker expression profiles, and in vivo osteogenic abilities. Cells cultured with this system also did not show malignant transformation and the automated process was revealed to be safe in terms of microbial contamination. Taken together, the automated procedure described in this report provides an approach to clinical bone tissue engineering.

A new method for evaluation of the resistance to rice kernel cracking based on moisture absorption in brown rice under controlled conditions.

We developed and evaluated the effectiveness of a new method to detect differences among rice cultivars in their resistance to kernel cracking. The method induces kernel cracking under laboratory controlled condition by moisture absorption to brown rice. The optimal moisture absorption conditions were determined using two japonica cultivars, 'Nipponbare' as a cracking-resistant cultivar and 'Yamahikari' as a cracking-susceptible cultivar: 12% initial moisture content of the brown rice, a temperature of 25°C, a duration of 5 h, and only a single absorption treatment. We then evaluated the effectiveness of these conditions using 12 japonica cultivars. The proportion of cracked kernels was significantly correlated with the mean 10-day maximum temperature after heading. In addition, the correlation between the proportions of cracked kernels in the 2 years of the study was higher than that for values obtained using the traditional late harvest method. The new moisture absorption method could stably evaluate the resistance to kernel cracking, and will help breeders to develop future cultivars with less cracking of the kernels.

Detection and verification of QTLs associated with heat-induced quality decline of rice (Oryza sativa L.) using recombinant inbred lines and near-isogenic lines.

Decline in the apparent quality of rice (Oryza sativa L.) grain due to high temperatures during ripening recently became a major concern in many areas in Japan. The occurrence of white-back kernels (WBK) is one of the main problems of heat-induced quality decline. We identified QTLs associated with the occurrence of WBK using recombinant inbred lines (RILs) and verified their effects using near-isogenic lines (NILs). The QTL analysis used F7 and F8 RILs derived from 'Hana-echizen' (HE), which is tolerant to high temperature, × 'Niigata-wase' (NW), which is sensitive to high temperature. Four QTLs were identified on chromosomes 3, 4, 6, and 9 (qWB3, qWB4, qWB6 and qWB9). To verify the effects of qWB6 and qWB9, we developed two NILs in which qWB6 or both were introduced from HE into the NW background. The HE allele at qWB6 significantly decreased WBK under multiple environments. The combination of qWB6 and qWB9 in an F2 population derived from a cross between a NIL and NW showed that the NW allele at qWB9 significantly decreased WBK if the qWB6 allele was HE. These results will be of value in marker-assisted selection for the breeding of rice with tolerance to heat-induced quality decline.

Distribution pattern of urine albumin creatinine ratio and the prevalence of high-normal levels in untreated asymptomatic non-diabetic hypertensive patients.

BACKGROUND: Even high-normal albuminuria is reportedly associated with cardiovascular events. OBJECTIVE: We determined the urine albumin creatinine ratio (UACR) in spot urine samples and analyzed the UACR distribution and the prevalence of high-normal levels. PATIENTS AND METHODS: The UACR was determined using immunoturbidimetry in 332 untreated asymptomatic non-diabetic Japanese patients with hypertension and in 69 control subjects. The microalbuminuria and macroalbuminuria levels were defined as a UCAR ≥30 and <300 µg/mg·creatinine and a UCAR ≥300 µg/mg·creatinine, respectively. RESULTS: The distribution patterns showed a highly skewed distribution for the lower levels, and a common logarithmic transformation produced a close fit to a Gaussian distribution with median, 25th and 75th percentile values of 22.6, 13.5 and 48.2 µg/mg·creatinine, respectively. When a high-normal UACR was set at >20 to <30 µg/mg·creatinine, 19.9% (66/332) of the hypertensive patients exhibited a high-normal UACR. Microalbuminuria and macroalbuminuria were observed in 36.1% (120/336) and 2.1% (7/332) of the patients, respectively. UACR was significantly correlated with the systolic and diastolic blood pressures and the pulse pressure. A stepwise multivariate analysis revealed that these pressures as well as age were independent factors that increased UACR. CONCLUSION: The UACR distribution exhibited a highly skewed pattern, with approximately 60% of untreated, non-diabetic hypertensive patients exhibiting a high-normal or larger UACR. Both hypertension and age are independent risk factors that increase the UACR. The present study indicated that a considerable percentage of patients require anti-hypertensive drugs with antiproteinuric effects at the start of treatment.

[Upper airway obstruction caused by a floppy epiglottis--report of two cases of amyotrophic lateral sclerosis (ALS)].

The motor system is extensively affected in amyotrophic lateral sclerosis (ALS). Rapid disease progression almost certainly ensures that about half of thsese cases will experience respiratory muscle paralysis to breathe within about five years. We report two cases of ALS involving upper airway obstruction. Fiberoptic laryngoscopy showed floppy epiglottis, tilted posteriorly and horizontally and impacting against the posterior pharyngeal wall during inspiration. Several months later, airway obstruction grew exceedingly worse, and the tilted epiglottis did not return to its vertical resting position. Tracheostomy was conducted during this period. We found that laryngoscopy may be useful in the evaluation of upper airway obstruction, and it may be safer to avoid continuous positive airway pressure.

Megalin and nonmuscle myosin heavy chain IIA interact with the adaptor protein Disabled-2 in proximal tubule cells.

Megalin plays a critical role in the endocytosis of albumin and other filtered low-molecular-weight proteins. Here we studied the interaction between megalin and Disabled-2 (Dab2), an adaptor protein that binds to the cytoplasmic domain of megalin and appears to control its trafficking. We co-immunoprecipitated megalin and Dab2 from cultured proximal tubule cells and identified the proteins by liquid chromatography and tandem mass spectrometry. We found two proteins associated with the megalin/Dab2 complex, nonmuscle myosin heavy chain IIA (NMHC-IIA) and beta-actin. Subcellular fractionation followed by sucrose velocity gradient separation showed that megalin, Dab2, and NMHC-IIA existed as a complex in the same endosomal fractions. In vitro pull-down assays demonstrated that NMHC-IIA was bound to the carboxyl-terminal region of Dab2, but not to megalin's cytoplasmic domain. We then transfected COS-7 cells with plasmids that induced the expression of Dab2, NMHC-IIA, and the megalin minireceptor, a truncated form of megalin. Co-immunoprecipitation studies showed that the minireceptor and NMHC-IIA co-immunoprecipitated only with Dab2. Furthermore, the uptake of (125)I-lactoferrin, an endocytic ligand of megalin, by rat yolk sac-derived megalin-expressing L2 cells was inhibited by blebbistatin, a specific inhibitor of nonmuscle myosin II. Our study shows that NMHC-IIA is functionally linked to megalin by interaction with Dab2 and is likely involved in megalin-mediated endocytosis in proximal tubule cells.

Regulation of megalin expression in cultured proximal tubule cells by angiotensin II type 1A receptor- and insulin-mediated signaling cross talk.

Impairment of proximal tubular endocytosis of glomerular-filtered proteins including albumin results in the development of proteinuria/albuminuria in patients with chronic kidney disease. However, the mechanisms regulating the proximal tubular function are largely unknown. This study aimed to investigate the role of angiotensin II type 1A receptor (AT(1A)R)- and insulin-mediated signaling pathways in regulating the expression of megalin, a multiligand endocytic receptor in proximal tubule cells (PTCs). Opossum kidney PTC-derived OK cells that stably express rat AT(1A)R but are deficient in endogenous angiotensin II receptors (AT(1A)R-OK cells) were used for this study. Treatment of the cells with angiotensin II suppressed mRNA and protein expression of megalin at 3- and 24-h incubation time points, respectively. Cellular uptake and degradation of albumin and receptor-associated protein, megalin's endocytic ligands were suppressed 24 h after angiotensin II treatment. The AT(1A)R-mediated decrease in megalin expression was partially prevented by ERK inhibitors. Insulin competed with the AT(1A)R-mediated ERK activation and decrease in megalin expression. Inhibitors of phosphatidylinositol 3-kinase (PI3K), a major component of insulin signaling, also suppressed megalin expression, and activation of the insulin receptor substrate (IRS)/PI3K system was prevented by angiotensin II. Collectively the AT(1A)R-mediated ERK signaling is involved in suppressing megalin expression in the OK cell line, and insulin competes with this pathway. Conversely, the insulin-IRS/PI3K signaling, with which angiotensin II competes, tends to stimulate megalin expression. In conclusion, there is AT(1A)R- and insulin-mediated competitive signaling cross talk to regulate megalin expression in cultured PTCs.

Crystallization and preliminary X-ray diffraction studies of an RNA aptamer in complex with the human IgG Fc fragment.

Aptamers, which are folded DNA or RNA molecules, bind to target molecules with high affinity and specificity. An RNA aptamer specific for the Fc fragment of human immunoglobulin G (IgG) has recently been identified and it has been demonstrated that an optimized 24-nucleotide RNA aptamer binds to the Fc fragment of human IgG and not to other species. In order to clarify the structural basis of the high specificity of the RNA aptamer, it was crystallized in complex with the Fc fragment of human IgG1. Preliminary X-ray diffraction studies revealed that the crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 83.7, b = 107.2, c = 79.0 A. A data set has been collected to 2.2 A resolution.

Megalin-mediated endocytosis of cystatin C in proximal tubule cells.

Serum levels of cystatin C, an endogenous cysteine proteinase inhibitor, are often used as an indicator of glomerular filtration rate. Although it is known that cystatin C is filtered by glomeruli and metabolized in proximal tubule cells (PTC), the precise molecular mechanism underlying this process is undetermined. Using quartz-crystal microbalance analyses, we demonstrate that cystatin C binds directly to megalin, an endocytic receptor in PTC, in a Ca(+)-dependent manner. We also find that cystatin C is endocytosed specifically via megalin in rat yolk sac epithelium-derived L2 cells which share a variety of characteristics with PTC. Finally, in vivo studies using kidney-specific megalin knockout mice provide evidence that megalin mediates proximal tubular uptake of cystatin C. We conclude that megalin is an endocytic receptor of cystatin C in PTC.