Fertilization controls tiller numbers via transcriptional regulation of a MAX1-like gene in rice cultivation.

Fertilization controls various aspects of cereal growth such as tiller number, leaf size, and panicle size. However, despite such benefits, global chemical fertilizer use must be reduced to achieve sustainable agriculture. Here, based on field transcriptome data from leaf samples collected during rice cultivation, we identify fertilizer responsive genes and focus on Os1900, a gene orthologous to Arabidopsis thaliana MAX1, which is involved in strigolactone biosynthesis. Elaborate genetic and biochemical analyses using CRISPR/Cas9 mutants reveal that Os1900 together with another MAX1-like gene, Os5100, play a critical role in controlling the conversion of carlactone into carlactonoic acid during strigolactone biosynthesis and tillering in rice. Detailed analyses of a series of Os1900 promoter deletion mutations suggest that fertilization controls tiller number in rice through transcriptional regulation of Os1900, and that a few promoter mutations alone can increase tiller numbers and grain yields even under minor-fertilizer conditions, whereas a single defective os1900 mutation does not increase tillers under normal fertilizer condition. Such Os1900 promoter mutations have potential uses in breeding programs for sustainable rice production.

A bifurcated palea mutant infers functional differentiation of WOX3 genes in flower and leaf morphogenesis of barley.

Barley (Hordeum vulgare) is the fourth most highly produced cereal in the world after wheat, rice and maize and is mainly utilized as malts and for animal feed. Barley, a model crop of the tribe Triticeae, is important in comparative analyses of Poaceae. However, molecular understanding about the developmental processes is limited in barley. Our previous work characterized one of two WUSCHEL-RELATED HOMEOBOX 3 (WOX3) genes present in the barley genome: NARROW LEAFED DWARF1 (NLD1). We demonstrated that NLD1 plays a pivotal role in the development of lateral organs. In the present study, we describe a bifurcated palea (bip) mutant of barley focusing on flower and leaf phenotypes. The palea in the bip mutant was split into two and develop towards inside the lemma surrounding the carpels and anthers. The bip mutant is devoid of lodicules, which develop in a pair at the base of the stamen within the lemma in normal barley. bip also exhibited malformations in leaves, such as narrow leaf due to underdeveloped leaf-blade width, and reduced trichome density. Map-based cloning and expression analysis indicated that BIP is identical to another barley WOX3 gene, named HvWOX3. The bip nld1 double mutant presented a more severe reduction in leaf-blade width and number of trichomes. By comparing the phenotypes and gene expression patterns of various WOX3 mutants, we concluded that leaf bilateral outgrowth and trichome development are promoted by both NLD1 and HvWOX3, but that HvWOX3 serves unique and pivotal functions in barley development that differ from those of NLD1.

NARROW AND DWARF LEAF 1, the Ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN, Mediates Leaf Development and Maintenance of the Shoot Apical Meristem in Oryza sativa L.

The molecular basis for leaf development, a major focus in developmental biology, remains unclear in the monocotyledonous grass, rice (Oryza sativa). Here, we performed a mutant screen in rice and identified an AP2-type transcription factor family protein, NARROW AND DWARF LEAF1 (NDL1). NDL1 is the ortholog of Arabidopsis thaliana (subsequently called Arabidopsis) ENHANCER OF SHOOT REGENERATION1 (ESR1)/DORNRÖSCHEN (DRN) and mediates leaf development and maintenance of the shoot apical meristem (SAM). Loss of function of NDL1 results in bladeless leaves and SAMs that are flat, rather than dome-shaped, and lack cell proliferation activity. This loss of function also causes reduced auxin signaling. Moreover, as is the case with Arabidopsis ESR1/DRN, NDL1 plays crucial roles in shoot regeneration. Importantly, we found that NDL1 is not expressed in the SAM but is expressed in leaf primordia. We propose that NDL1 cell autonomously regulates leaf development, but non-cell autonomously regulates SAM maintenance in rice.

NARROW AND DWARF LEAF 1, the Ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN, Mediates Leaf Development and Maintenance of the Shoot Apical Meristem in Oryza sativa L.

The molecular basis for leaf development, a major focus in developmental biology, remains unclear in the monocotyledonous grass, rice (Oryza sativa). Here, we performed a mutant screen in rice and identified an AP2-type transcription factor family protein, NARROW AND DWARF LEAF1 (NDL1). NDL1 is the ortholog of Arabidopsis thaliana (subsequently called Arabidopsis) ENHANCER OF SHOOT REGENERATION1 (ESR1)/DORNRÖSCHEN (DRN) and mediates leaf development and maintenance of the shoot apical meristem (SAM). Loss of function of NDL1 results in bladeless leaves and SAMs that are flat, rather than dome-shaped, and lack cell proliferation activity. This loss of function also causes reduced auxin signaling. Moreover, as is the case with Arabidopsis ESR1/DRN, NDL1 plays crucial roles in shoot regeneration. Importantly, we found that NDL1 is not expressed in the SAM but is expressed in leaf primordia. We propose that NDL1 cell autonomously regulates leaf development, but non-cell autonomously regulates SAM maintenance in rice.

Regulation of the plastochron by three many-noded dwarf genes in barley.

The plastochron, the time interval between the formation of two successive leaves, is an important determinant of plant architecture. We genetically and phenotypically investigated many-noded dwarf (mnd) mutants in barley. The mnd mutants exhibited a shortened plastochron and a decreased leaf blade length, and resembled previously reported plastochron1 (pla1), pla2, and pla3 mutants in rice. In addition, the maturation of mnd leaves was accelerated, similar to pla mutants in rice. Several barley mnd alleles were derived from three genes-MND1, MND4, and MND8. Although MND4 coincided with a cytochrome P450 family gene that is a homolog of rice PLA1, we clarified that MND1 and MND8 encode an N-acetyltransferase-like protein and a MATE transporter-family protein, which are respectively orthologs of rice GW6a and maize BIGE1 and unrelated to PLA2 or PLA3. Expression analyses of the three MND genes revealed that MND1 and MND4 were expressed in limited regions of the shoot apical meristem and leaf primordia, but MND8 did not exhibit a specific expression pattern around the shoot apex. In addition, the expression levels of the three genes were interdependent among the various mutant backgrounds. Genetic analyses using the double mutants mnd4mnd8 and mnd1mnd8 indicated that MND1 and MND4 regulate the plastochron independently of MND8, suggesting that the plastochron in barley is controlled by multiple genetic pathways involving MND1, MND4, and MND8. Correlation analysis between leaf number and leaf blade length indicated that both traits exhibited a strong negative association among different genetic backgrounds but not in the same genetic background. We propose that MND genes function in the regulation of the plastochron and leaf growth and revealed conserved and diverse aspects of plastochron regulation via comparative analysis of barley and rice.

Genome-wide analysis of spatiotemporal expression patterns during rice leaf development.

BACKGROUND: Rice leaves consist of three distinct regions along a proximal-distal axis, namely the leaf blade, sheath, and blade-sheath boundary region. Each region has a unique morphology and function, but the genetic programs underlying the development of each region are poorly understood. To fully elucidate rice leaf development and discover genes with unique functions in rice and grasses, it is crucial to explore genome-wide transcriptional profiles during the development of the three regions. RESULTS: In this study, we performed microarray analysis to profile the spatial and temporal patterns of gene expression in the rice leaf using dissected parts of leaves sampled in broad developmental stages. The dynamics in each region revealed that the transcriptomes changed dramatically throughout the progress of tissue differentiation, and those of the leaf blade and sheath differed greatly at the mature stage. Cluster analysis of expression patterns among leaf parts revealed groups of genes that may be involved in specific biological processes related to rice leaf development. Moreover, we found novel genes potentially involved in rice leaf development using a combination of transcriptome data and in situ hybridization, and analyzed their spatial expression patterns at high resolution. We successfully identified multiple genes that exhibit localized expression in tissues characteristic of rice or grass leaves. CONCLUSIONS: Although the genetic mechanisms of leaf development have been elucidated in several eudicots, direct application of that information to rice and grasses is not appropriate due to the morphological and developmental differences between them. Our analysis provides not only insights into the development of rice leaves but also expression profiles that serve as a valuable resource for gene discovery. The genes and gene clusters identified in this study may facilitate future research on the unique developmental mechanisms of rice leaves.

Effects of intensive exercise combined with dapagliflozin on body composition in patients with type 2 diabetes: a randomized controlled trial.

This study was aimed to evaluate the effects of intensive exercise in addition to the administration of sodium-glucose cotransporter 2 inhibitor dapagliflozin (DAPA) on body composition, including fat-free mass, in type 2 diabetes. We randomly assigned 146 patients to 24 weeks of treatment with intensive exercise, including resistance training, plus 5 mg (up to 10 mg) of DAPA daily (IT group) or DAPA alone (CT group). The primary endpoint was the difference in the change in fat-free mass from baseline to 24 weeks between the groups. The skeletal muscle mass index (SMI); metabolic profile, including HbA1c; and regional fat mass were also determined. ANCOVA was used for the group comparison, with least squares mean (LSM) differences and 95% confidence interval (CI). There was no significant difference in the change in fat-free mass (LSM difference -0.1 kg (95% CI: -0.5 to 0.4) and SMI (LSM difference -0.1 kg (95% CI: -0.2 to 0.1) between the groups. In contrast, the reduction of trunk fat mass was significantly higher in the IT group than in the CT group ((LSM difference -0.5 kg [95% CI -0.9 to -0.1]). Higher adherence to the resistance training tended to be associated with changes in HbA1c and high-sensitivity CRP levels. Our study suggests that intensive exercise do not prevent the reduction of fat-free mass after administration of SGLT2 inhibitors but can increase the reduction in abdominal fat, presumably leading to further improvements of hyperglycemia and chronic inflammation than DAPA alone in type 2 diabetes patients.

Effects of Nutritional Treatment on the Frequency of Pneumonia in Bedridden Patients Receiving Oral Care.

Objective Pneumonia develops in bedridden patients, even in those receiving oral care, and malnutrition is associated with the development of pneumonia. We examined the effects of nutritional treatment on the prevention of pneumonia. Patients and Methods We retrospectively examined the effects of nutritional treatment on the prevention of pneumonia by analyzing the records of bedridden patients (n=68; mean age: 68.0 years) who stayed in a hospital for 2 years or longer. Results Among the analyzed patients, pneumonia developed in 52 (76%) patients, and the mean frequency of pneumonia was 1.6 times per year during the first year of stay. In a multivariate analysis, the serum albumin level at admission in the pneumonia group was lower than that in the non-pneumonia group. The frequency of pneumonia during the second year of stay was lower than that during the first year of stay. Serum levels of albumin and total protein (TP) at one year after admission were higher than those at admission in all analyzed patients, and in all patients (n=52) and elderly (≥65 years) patients (n=31) in the pneumonia group. The proportions of patients with hypoalbuminemia (<3.5 g/dL) and hypoproteinemia (<6.5 g/dL) at one year after admission were lower than those at admission. The increases in the proportions of patients presenting a reduced frequency of pneumonia were correlated with increases in the proportions of patients presenting increased levels of albumin and/or TP. Conclusion Nutritional treatment may reduce the frequency of pneumonia by improving malnutrition in bedridden patients receiving oral care.

Specification of basal region identity after asymmetric zygotic division requires mitogen-activated protein kinase 6 in rice.

Asymmetric cell division is a key step in cellular differentiation in multicellular organisms. In plants, asymmetric zygotic division produces the apical and basal cells. The mitogen-activated protein kinase (MPK) cascade in Arabidopsis acts in asymmetric divisions such as zygotic division and stomatal development, but whether the effect on cellular differentiation of this cascade is direct or indirect following asymmetric division is not clear. Here, we report the analysis of a rice mutant, globular embryo 4 (gle4). In two- and four-cell-stage embryos, asymmetric zygotic division and subsequent cell division patterns were indistinguishable between the wild type and gle4 mutants. However, marker gene expression and transcriptome analyses showed that specification of the basal region was compromised in gle4 We found that GLE4 encodes MPK6 and that GLE4/MPK6 is essential in cellular differentiation rather than in asymmetric zygotic division. Our findings provide a new insight into the role of MPK in plant development. We propose that the regulation of asymmetric zygotic division is separate from the regulation of cellular differentiation that leads to apical-basal polarity.

BLADE-ON-PETIOLE genes temporally and developmentally regulate the sheath to blade ratio of rice leaves.

Axis formation is a fundamental issue in developmental biology. Axis formation and patterning in plant leaves is crucial for morphology and crop productivity. Here, we reveal the basis of proximal-distal patterning in rice leaves, which consist of a proximal sheath, a distal blade, and boundary organs formed between these two regions. Analysis of the three rice homologs of the Arabidopsis BLADE-ON-PETIOLE1 (BOP1) gene indicates that OsBOPs activate proximal sheath differentiation and suppress distal blade differentiation. Temporal expression changes of OsBOPs are responsible for the developmental changes in the sheath:blade ratio. We further identify that the change in the sheath:blade ratio during the juvenile phase is controlled by the miR156/SPL pathway, which modifies the level and pattern of expression of OsBOPs. OsBOPs are also essential for differentiation of the boundary organs. We propose that OsBOPs, the main regulators of proximal-distal patterning, control temporal changes in the sheath:blade ratio of rice leaves.

[A case of mixed adenoneuroendocrine carcinoma of the distal bile duct].

We diagnosed distal cholangiocarcinoma in a 76-year-old woman who was then treated by subtotal stomach-preserving pancreaticoduodenectomy. Histopathological examination revealed a well-differentiated tubular adenocarcinoma on the side of the bile duct, and a neuroendocrine carcinoma in an area outside the bile duct where the tumor had infiltrated. Immunohistochemical staining identified homology between cytokeratins and MUC, indicating a similar origin. This report discusses problems associated with diagnosis and treatment by summarizing 22 patients who underwent curative resection and subsequently had a confirmed prognosis.

Conserved functional control, but distinct regulation, of cell proliferation in rice and Arabidopsis leaves revealed by comparative analysis of GRF-INTERACTING FACTOR 1 orthologs.

Regulation of cell proliferation is crucial for establishing the shape of plant leaves. We have identified MAKIBA3 (MKB3), a loss-of-function mutant of which exhibits a narrowed- and rolled-leaf phenotype in rice. MKB3 was found to be an ortholog of Arabidopsis ANGUSTIFOLIA3 (AN3), which positively regulates cell proliferation. The reduced leaf size of mkb3 plants with enlarged cells and the increased size of MKB3-overexpressing leaves with normal-sized cells indicate that MKB3 is a positive regulator of leaf proliferation and that mkb3 mutation triggers a compensation syndrome, as does Arabidopsis an3 Expression analysis revealed that MKB3 is predominantly expressed on the epidermis of leaf primordia, which is different from the location of AN3 A protein movement assay demonstrated that MKB3 moves from an MKB3-expressing domain to a non-expressing domain, which is required for normal leaf development. Our results suggest that rice MKB3 and Arabidopsis AN3 have conserved functions and effects on leaf development. However, the expression pattern of MKB3 and direction of protein movement are different between rice and Arabidopsis, which might reflect differences in leaf primordia development in these two species.

Rice leaf hydrophobicity and gas films are conferred by a wax synthesis gene (LGF1) and contribute to flood tolerance.

Floods impede gas (O2 and CO2 ) exchange between plants and the environment. A mechanism to enhance plant gas exchange under water comprises gas films on hydrophobic leaves, but the genetic regulation of this mechanism is unknown. We used a rice mutant (dripping wet leaf 7, drp7) which does not retain gas films on leaves, and its wild-type (Kinmaze), in gene discovery for this trait. Gene complementation was tested in transgenic lines. Functional properties of leaves as related to gas film retention and underwater photosynthesis were evaluated. Leaf Gas Film 1 (LGF1) was identified as the gene determining leaf gas films. LGF1 regulates C30 primary alcohol synthesis, which is necessary for abundant epicuticular wax platelets, leaf hydrophobicity and gas films on submerged leaves. This trait enhanced underwater photosynthesis 8.2-fold and contributes to submergence tolerance. Gene function was verified by a complementation test of LGF1 expressed in the drp7 mutant background, which restored C30 primary alcohol synthesis, wax platelet abundance, leaf hydrophobicity, gas film retention, and underwater photosynthesis. The discovery of LGF1 provides an opportunity to better understand variation amongst rice genotypes for gas film retention ability and to target various alleles in breeding for improved submergence tolerance for yield stability in flood-prone areas.

Pneumonia risks in bedridden patients receiving oral care and their screening tool: Malnutrition and urinary tract infection-induced inflammation.

AIM: Pneumonia develops in bedridden patients even when they are receiving oral care. However, the pneumonia risk in bedridden patients remains unclear, and no screening tool has been developed to assess this risk by using daily hospital data. METHODS: We retrospectively examined pneumonia risk factors by analyzing the records of 102 bedridden patients receiving oral care. RESULTS: Body mass index, peripheral blood hemoglobin, and serum concentrations of total protein, albumin, total cholesterol and uric acid in the pneumonia group (n = 51; mean age 73.4 years) were lower than those in the non-pneumonia group (n = 51; mean age 68.1 years). In the univariate analysis, body mass index; leukocytosis; high C-reactive protein; low levels of hemoglobin, total protein and albumin (<3.5 g/dL); and urine bacteria were associated with the development of pneumonia. Furthermore, in the multivariate analysis, low levels of albumin and urine bacteria were independently associated with pneumonia. We developed a bedridden patient pneumonia risk (BPPR) score using these two risk factors to assess pneumonia risk. We applied scores of zero (0) or one (1) according to the absence or presence of the two risk factors and summed the scores in each patient. The proportion of pneumonia patients increased with increasing BPPR score when the patients were divided into three groups - low, moderate and high risk - according to the BPPR score (0, 1 or 2, respectively). CONCLUSIONS: Malnutrition, urinary tract infection-induced inflammation and anemia were associated with pneumonia in bedridden patients. BPPR scoring might be useful for assessing pneumonia risk and managing affected patients. Geriatr Gerontol Int 2018; 18: 714-722.

LEAF LATERAL SYMMETRY1, a Member of the WUSCHEL-RELATED HOMEOBOX3 Gene Family, Regulates Lateral Organ Development Differentially from Other Paralogs, NARROW LEAF2 and NARROW LEAF3 in Rice.

In several eudicot species, one copy of each member of the WUSCHEL-RELATED HOMEOBOX (WOX) gene family, WOX1 and WOX3, is redundantly or differentially involved in lateral leaf outgrowth, whereas only the WOX3 gene regulating the lateral domain of leaf development has been reported in grass. In this study, we show that a WOX3 gene, LEAF LATERAL SYMMETRY1 (LSY1), regulates lateral leaf development in a different manner ftom that of other duplicated paralogs of WOX3, NARROW LEAF2 (NAL2)/NAL3, in rice. A loss-of-function mutant of LSY1 exhibited an asymmetrical defect from early leaf development, which is different from a symmetric defect in a double loss-of-function mutant of NAL2/3, whereas the expression of both genes was observed in a similar domain in the margins of leaf primordia. Unlike NAL2/3, overexpression of LSY1 produced malformed leaves whose margins were curled adaxially. Expression domains and the level of adaxial/abaxial marker genes were affected in the LSY1-overexpressing plants, indicating that LSY1 is involved in regulation of adaxial-abaxial patterning at the margins of the leaf primordia. Additive phenotypes in some leaf traits of lsy1 nal2/3 triple mutants and the unchanged level of NAL2/3 expression in the lsy1 background suggested that LSY1 regulates lateral leaf development independently of NAL2/3. Our results indicated that all of the rice WOX3 genes are involved in leaf lateral outgrowth, but the functions of LSY1 and NAL2/3 have diverged. We propose that the function of WOX3 and the regulatory mode of leaf development in rice are comparable with those of WOX1/WOX3 in eudicot species.

Clinicopathological significance of ganglioside DSGb5 expression in renal cell carcinoma.

Disialosyl globopentaosylceramide (DSGb5) is a ganglioside originally isolated from tissue extracts of renal cell carcinoma (RCC) with metastasis. Previous in vitro experiments have suggested that DSGb5 promotes metastasis by enhancing the migration of RCC cells and downregulating NK cell cytotoxicity against RCC cells. In this study, we investigated the clinicopathological significance of DSGb5 expression in RCC and outcomes of RCC patients. A total of 156 RCC patients who underwent surgical treatments at our hospital from January 2007 through December 2012 were analyzed in this study. The expression of DSGb5 in RCC specimens was examined by immunohistochemical staining with monoclonal antibody 5F3. The immunostaining intensity of RCC tissues was assessed in comparison with that in benign renal tubules as an internal positive control. The relationship between DSGb5 expression and clinicopathological characteristics was investigated and recurrence free survival following surgery was evaluated. Microvascular invasion was observed in 68% (n = 19/28) and in 45% (n = 58/128) of the DSGb5 high expression group and low expression group, respectively (p = 0.031). Of 156 patients with a median follow up of 51 months, 18 patients (12%) developed metastasis following surgery. Patients in the DSGb5 high expression group showed significantly lower recurrence-free survival as compared with those in the DSGb5 low expression group (log-rank P = 0.047). In the present study, DSGb5 expression was associated with microvascular invasion in RCC tissues, and patients with DSGb5 high expression showed significantly lower recurrence-free survival rates. These findings suggest that DSGb5 expressed in RCC is correlated with metastasis and is a potential predictor for identifying patients who experience metastasis after surgery.

Specific niches for lung-resident memory CD8+ T cells at the site of tissue regeneration enable CD69-independent maintenance.

CD8+ tissue-resident memory T cells (TRM cells) reside permanently in nonlymphoid tissues and provide a first line of protection against invading pathogens. However, the precise localization of CD8+ TRM cells in the lung, which physiologically consists of a markedly scant interstitium compared with other mucosa, remains unclear. In this study, we show that lung CD8+ TRM cells localize predominantly in specific niches created at the site of regeneration after tissue injury, whereas peripheral tissue-circulating CD8+ effector memory T cells (TEM cells) are widely but sparsely distributed in unaffected areas. Although CD69 inhibited sphingosine 1-phosphate receptor 1-mediated egress of CD8+ T cells immediately after their recruitment into lung tissues, such inhibition was not required for the retention of cells in the TRM niches. Furthermore, despite rigid segregation of TEM cells from the TRM niche, prime-pull strategy with cognate antigen enabled the conversion from TEM cells to TRM cells by creating de novo TRM niches. Such damage site-specific localization of CD8+ TRM cells may be important for efficient protection against secondary infections by respiratory pathogens.

Significance of Expression of Complement C4d in Esophageal Squamous Cell Carcinoma.

BACKGROUND/AIM: Esophageal squamous cell carcinoma (ESCC) is one of the most difficult malignancies to cure. C4d is a degradation product of the classical complement pathway and is suggested as an early diagnostic marker for other SCCs. The purpose of this study was to clarify the association of complement C4d with ESCC. PATIENTS AND METHODS: Immunohistochemical staining for C4d was performed on surgical specimens obtained from 114 patients with ESCC. RESULTS: Positive C4d expression was observed in 70 (61.4%) cases and negative expression in 44 (38.6%) cases. There was a significant inverse correlation between C4d expression and depth of tumor invasion (p=0.0001), lymph node metastasis (p=0.011), lymphatic invasion (p=0.033), and TNM stage (p=0.0021). Kaplan-Meier analysis showed that negative C4d expression tended to lead to shorter overall survival (p=0.232). CONCLUSION: C4d expression in ESCC might be useful in developing treatment strategies or suppression of ESCC.

Jasmonate regulates juvenile-to-adult phase transition in rice.

Juvenile-to-adult phase transition is an important shift for the acquisition of adult vegetative characteristics and subsequent reproductive competence. We identified a recessive precocious (pre) mutant exhibiting a long leaf phenotype in rice. The long leaf phenotype is conspicuous in the second to the fourth leaves, which are juvenile and juvenile-to-adult transition leaves. We found that morphological and physiological traits, such as midrib formation, shoot meristem size, photosynthetic rate and plastochron, in juvenile and juvenile-to-adult transition stages of the pre mutant have precociously acquired adult characteristics. In agreement with these results, expression patterns of miR156 and miR172, which are microRNAs regulating phase change, support the accelerated juvenile-to-adult phase change in the pre mutant. The mutated gene encodes an allene oxide synthase (OsAOS1), which is a key enzyme for the biosynthesis of jasmonic acid (JA). The pre mutant showed a low level of JA and enhanced sensitivity to gibberellic acid, which promotes the phase change in some plant species. We also show that prolonged plastochron in the pre mutant is caused by accelerated PLASTOCHRON1 (PLA1) function. The present study reveals a substantial role of JA as a negative regulator of vegetative phase change.