Upregulation of the MYB2 Transcription Factor Is Associated with Increased Accumulation of Anthocyanin in the Leaves of Dendrobium bigibbum.

Orchids with colorful leaves and flowers have significant ornamental value. Here, we used γ-irradiation-based mutagenesis to produce a Dendrobium bigibbum mutant that developed purple instead of the normal green leaves. RNA sequencing of the mutant plant identified 2513 differentially expressed genes, including 1870 up- and 706 downregulated genes. The purple leaf color of mutant leaves was associated with increased expression of genes that encoded key biosynthetic enzymes in the anthocyanin biosynthetic pathway. In addition, the mutant leaves also showed increased expression of several families of transcription factors including the MYB2 gene. Transient overexpression of D. biggibumMYB2 in Nicotiana benthamiana was associated with increased expression of endogenous anthocyanin biosynthesis genes. Interestingly, transient overexpression of orthologous MYB2 genes from other orchids did not upregulate expression of endogenous anthocyanin biosynthesis genes. Together, these results suggest that the purple coloration of D. biggibum leaves is at least associated with increased expression of the MYB2 gene, and the MYB2 orthologs from orchids likely function differently, regardless of their high level of similarity.

Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat.

Because high-molecular-weight glutenin subunits (HMW-GS) are important contributors to wheat end-use quality, there is a need for high-throughput identification of HMW-GS in wheat genetic resources and breeding lines. We developed an optimized method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to distinguish individual HMW-GS by considering the effects of the alkylating reagent in protein extraction, solvent components, dissolving volume, and matrix II components. Using the optimized method, 18 of 22 HMW-GS were successfully identified in standard wheat cultivars by differences in molecular weights or by their associations with other tightly linked subunits. Interestingly, 1Bx7 subunits were divided into 1Bx7 group 1 and 1Bx7 group 2 proteins with molecular weights of about 82,400 and 83,000 Da, respectively. Cultivars containing the 1Bx7 group 2 proteins were distinguished from those containing 1Bx7OE using well-known DNA markers. HMW-GS 1Ax2* and 1Bx6 and 1By8 and 1By8*, which are difficult to distinguish due to very similar molecular weights, were easily identified using RP-HPLC. To validate the method, HMW-GS from 38 Korean wheat varieties previously evaluated by SDS-PAGE combined with RP-HPLC were analyzed by MALDI-TOF-MS. The optimized MALDI-TOF-MS method will be a rapid, high-throughput tool for selecting lines containing desirable HMW-GS for breeding efforts.

Predictive Factors for Efficacy of AST-120 Treatment in Diabetic Nephropathy: a Prospective Single-Arm, Open-Label, Multi-Center Study.

BACKGROUND: Removal of uremic toxins such as indoxyl sulfate by AST-120 is known to improve renal function and delay the initiation of dialysis in patients with advanced chronic kidney disease. However, it is unclear whether the addition of AST-120 to conventional treatments is effective in delaying the progression of renal dysfunction in patients with diabetic nephropathy. METHODS: A total of 100 patients with type 2 diabetes and renal dysfunction (serum creatinine levels ranging from 1.5 to 3.0 mg/dL) were recruited from eight centers in Korea and treated with AST-120 (6 g/day) for 24 weeks. The primary endpoint was improvement in renal function measured as the gradient of the reciprocal serum creatinine level (1/sCr) over time (i.e., the ratio of 1/sCr time slope for post- to pre-AST-120 therapy). A response was defined as a ratio change of the regression coefficient of 1/sCr ≤ 0.90. RESULTS: Renal function improved in 80.3% of patients (61/76) after 24 weeks of AST-120 treatment. There were no differences between responder and non-responder groups in baseline characteristics except for diastolic blood pressure (73.5 ± 9.5 mmHg in the responder group vs. 79.3 ± 11.1 mmHg in the non-responder group; P = 0.046). Serum lipid peroxidation level decreased significantly in the responder group (from 2.25 ± 0.56 µol/L to 1.91 ± 0.72 µol/L; P = 0.002) but not in the non-responder group. CONCLUSION: The addition of AST-120 to conventional treatments may delay the progression of renal dysfunction in diabetic nephropathy. The antioxidant effect of AST-120 might contribute to improvement in renal function.

G-749, a novel FLT3 kinase inhibitor, can overcome drug resistance for the treatment of acute myeloid leukemia.

Aberrant activations of Fms-like tyrosine receptor kinase (FLT) 3 are implicated in the pathogenesis of 20% to 30% of patients with acute myeloid leukemia (AML). G-749 is a novel FLT3 inhibitor that showed potent and sustained inhibition of the FLT3 wild type and mutants including FLT3-ITD, FLT3-D835Y, FLT3-ITD/N676D, and FLT3-ITD/F691L in cellular assays. G-749 retained its inhibitory potency in various drug-resistance milieus such as patient plasma, FLT3 ligand surge, and stromal protection. Furthermore, it displayed potent antileukemic activity in bone marrow blasts from AML patients regardless of FLT3 mutation status, including those with little or only minor responses to AC220 or PKC412. Oral administration of G-749 yielded complete tumor regression and increased life span in animal models. Thus, G-749 appears to be a promising next-generation drug candidate for the treatment of relapsed and refractory AML patients with various FLT3-ITD/FLT3-TKD mutants and further shows the ability to overcome drug resistance.

Highly potent and selective pyrazolylpyrimidines as Syk kinase inhibitors.

A series of pyrazolylpyrimidine scaffold based Syk inhibitors were synthesized and evaluated for their biological activities and selectivity. Lead optimization efforts provided compounds with potent Syk inhibition in both enzymatic and TNF-α release assay.

Radioactive iodine ablation does not prevent recurrences in patients with papillary thyroid microcarcinoma.

OBJECTIVE: Although papillary thyroid microcarcinoma (PTMC) has a favourable long-term prognosis, disease recurrence after initial treatment remains a problem and controversy exists regarding the role of radioactive iodine (RAI) ablation in PTMC. We performed this study to evaluate the effect of RAI ablation on disease recurrence in patients with PTMC. PATIENTS AND METHODS: Between 1994 and 2004, 2579 patients underwent thyroid surgery for thyroid cancer at Samsung Medical Center. Among these patients, 704 patients with PTMC presumed disease-free after initial treatment were followed up for disease recurrence (median, 64 months; range, 1-185 months). Patients with PTMC with microscopic extrathyroidal extension, cervical lymph node metastases or multifocality were considered to be in the intermediate-risk group for recurrence. RESULTS: Disease recurrence was found in six patients at a median of 29 months (range, 10-70 months) after initial treatment; all six patients with recurrent tumours had received RAI treatment after total thyroidectomy. Disease-related mortality was not observed, even after recurrence. Based on a Cox regression model considering the standardized inverse probability of treatment weight (IPTW) within each propensity score stratum of patients with a similar likelihood of having received RAI ablation, the likelihood ratio for recurrence did not differ between the RAI ablation group and no RAI group (P = 0·17). When we performed a subgroup analysis considering only patients with PTMC at intermediate-risk for recurrence, RAI ablation again did not have a significant effect on recurrence (P = 0·79). CONCLUSIONS: Radioactive iodine ablation after total thyroidectomy in low- and intermediate-risk patients with PTMC did not prevent recurrent tumours. Future randomized, controlled, multicenter prospective trials involving a larger sample of patients followed-up for a longer duration are warranted to confirm our findings.

Associations between body mass index and clinico-pathological characteristics of papillary thyroid cancer.

OBJECTIVE: Epidemiological studies have demonstrated that excess weight increases the risk of thyroid cancer. However, the associations between excess weight and prognostic factors for thyroid cancer are uncertain. We evaluated the relationships between body mass index (BMI) and clinico-pathological features and outcomes of papillary thyroid cancer (PTC). PATIENTS AND METHODS: Retrospective analysis of 2057 patients with PTC was performed. Patients were grouped according to BMI (underweight, normal weight, overweight and obesity)-based World Health Organization standardized categories. Logistic regression models were used to assess the relationships between BMI and clinico-pathological features of PTC. A Cox proportional hazards model was used to examine the association between BMI and disease recurrence. RESULTS: A 5-kg/m(2) increase in BMI was associated with PTC tumours larger than 1 cm [odds ratio (OR) 1.31, P < 0.001], with microscopic extrathyroidal invasion (OR 1.23, P = 0.006), and with advanced tumour-node-metastasis (TNM) stage (OR, 1.30, P = 0.003), which is independent of confounding variables such as gender, age, serum TSH, total cholesterol and fasting glucose level. The multivariate-adjusted OR [95% confidence intervals (CI)] in the overweight (25.0-29.9 kg/m(2)) and obese (BMI ≥ 30) groups for tumours larger than 1 cm were 1.41 (1.10-1.81) and 2.17 (1.23-3.82), respectively, compared to the normal weight group (BMI 18.5-24.9). The multivariate-adjusted OR (95% CI) for microscopic extrathyroidal extension in the obesity group was 1.88 (1.06-3.32), and the OR for advanced TNM stage in the overweight group was 1.35 (1.02-1.79) compared to the normal weight group. During follow-up (median, 84 month; range, 1-185), 43 patients (2.1%) experienced recurrence. There were no significant differences in recurrence of PTCs among BMI groups. CONCLUSIONS: Higher BMI was strongly associated with larger tumour size, extrathyroidal invasion and advanced TNM stage of PTCs. However, there was no difference in recurrence rate among BMI groups. This study suggests that excess weight is associated with aggressive features of PTCs. Further studies with long-term follow-up are needed to confirm this finding.

OsPRA1 plays a significant role in targeting of OsRab7 into the tonoplast via the prevacuolar compartment during vacuolar trafficking in plant cells.

In yeast and mammals, the Yip/PRA1 family of proteins has been reported to facilitate the delivery of Rab GTPases to the membrane by dissociating the Rab-GDI complex during vesicle trafficking. Recently, we identified OsPRA1, a plant Yip/PRA1 homolog, as an OsRab7-interacting protein that localizes to the prevacuolar compartment, which suggests that it plays a role in vacuolar trafficking of plant cells. Here, we show that OsPRA1 is essential for vacuolar trafficking and that it has molecular properties that are typical of the Yip/PRA1 family of proteins. A trafficking assay using Arabidopsis protoplasts showed that the point mutant OsPRA1((Y94A)) strongly inhibits the vacuolar trafficking of cargo proteins, but has no inhibitory effect on the plasma membrane trafficking of H(+)-ATPase-GFP, suggesting its specific involvement in vacuolar trafficking. Moreover, OsPRA1 was shown to be an integral membrane protein, suggesting that its two hydrophobic domains may mediate membrane integration, and its cytoplasmic N- and C-terminal regions were found to be important for binding to OsRab7. OsPRA1 also interacted with OsVamp3, implying its involvement in vesicle fusion. Finally, we used a yeast expression system to show that OsPRA1 opposes OsGDI2 activity and facilitates the delivery of OsRab7 to the target membrane. Taken together, our results support strongly that OsPRA1 targets OsRab7 to the tonoplast during vacuolar trafficking.

Inhibition of histone deacetylase activity down-regulates urokinase plasminogen activator and matrix metalloproteinase-9 expression in gastric cancer.

Histone acetylation and deacetylaion play important roles in chromatin remodeling and gene expression. An imbalance of these reactions leads to aberrant behavior of the cells in the cell cycle, which in turn contributes to carcinogenesis. Histone deacetylase (HDAC) inhibitors have been shown to have anti-tumor effects in clinical trials. However, the exact mechanisms by which HDAC inhibitors exert anti-tumor effects and modulate gene expression are not completely understood, and remain a subject of intense investigation. In the current study, we determined whether HDACs regulate urokinase plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9), and tumor invasion. Using cDNA microarray analysis, we found that hepatocyte growth factor (HGF) induced HDAC5 expression in gastric cancer cell lines, NUGC-3 and MKN-28. TSA, a HDAC inhibitor, decreased HGF-induced HADC-5 expression and also repressed uPA and MMP-9 expression. TSA inhibited cell proliferation in both cell lines. In vitro Matrigel invasion assays showed that the HDAC inhibitor decreased cancer cell invasion. Furthermore, GO6976, a PKC inhibitor, significantly inhibited not only HGF-induced HDAC5 expression but also cell invasion. These results demonstrated that HDACs regulate HGF-induced uPA and MMP-9 expression through a PKC-dependent signal pathway in gastric cancer cells. Our data probably suggest that such activities serve as anti-tumor mechanisms of the HDAC inhibitor.

Hepatoma-derived growth factor regulates the bad-mediated apoptotic pathway and induction of vascular endothelial growth factor in stomach cancer cells.

Hepatoma-derived growth factor (HDGF) is highly expressed in tumor cells and may play an important role in the development and progression of carcinomas. However, the molecular mechanism by which HDGF participates in gastric carcinomatosis requires further analysis. In this study, we determined the role of HDGF in tumorigenesis and elucidated the mechanisms of action. To determine aggressive biological behavior, we knocked down HDGF expression with HDGF-specific shRNA in two gastric cancer cell lines. First, using cDNA microarrary, we showed that hepatocyte growth factor (HGF) induced HDGF and confirmed this by Western blotting. HGF increased HDGF in a dose-dependent manner. We also determined whether HDGF induces angiogenic factor, and found the vascular endothelial growth factor (VEGF) was induced by HDGF. Downregulation of HDGF resulted in a decrement of VEGF. HDGF knock-down was found to induce the expression of the proapoptotic protein, Bad, and also inactivate ERK, which in turn led to dephosphorylation of Bad at ser112 and ser136, and induced apoptosis. Transfection with HDGF-siRNA resulted in a decrement of cell proliferation, as determined with a MMT assay. In an in vitro invasion assay, significantly fewer cells transfected with HDGF-siRNA than control cells were able to invade across a Matrigel membrane barrier. Our results suggest that HDGF is involved in cell growth, cell invasion, and apoptosis. These qualities may contribute to the HDGF-associated aggressive biological behavior of gastric cancer and thus serve as a potential target for cancer therapy.

Correction to: MEAM-based MD calculations of melting temperature for Fe.

A Correction to this paper has been published: https://doi.org/10.1007/s00894-020-04609-9.

MEAM-based MD calculations of melting temperature for Fe.

The molecular dynamics (MD) simulations were applied to the melting transition of the BCC metal Fe using the modified embedded atom method (MEAM) potential proposed by Jin et al. [Appl. Phys. A120 (2015) 189], and the newly derived formulas were adopted to calculate the forces acting on atoms in the MD simulations. We first determined the structural and energetic properties of the effectively infinite solid with no boundaries, and then investigated the Fe samples with low-index surfaces, namely Fe(100), Fe(110), and Fe(111). The simulations show that as the temperature increases, the (111) surface firstly disorders, followed by the (100) surface, while the (110) surface remains stable up to the melting temperature. The disorder phenomenon diffuses from the surface to the entire block, and as the density of atoms on the surface decreases, the effect of the premelting phenomenon also increases, being most pronounced on Fe(111) which has the lowest surface density. This conclusion is in line with the behavior found for BCC metal V in the previous simulation study.

Dark/Light Treatments Followed by γ-Irradiation Increase the Frequency of Leaf-Color Mutants in Cymbidium.

Radiation randomly induces chromosomal mutations in plants. However, it was recently found that the frequency of flower-color mutants could be specifically increased by upregulating anthocyanin pathway gene expression before radiation treatments. The mechanisms of chlorophyll biosynthesis and degradation are active areas of plant study because chlorophyll metabolism is closely connected to photosynthesis. In this study, we determined the dark/light treatment conditions that resulted in upregulation of the expression levels of six chlorophyll pathway genes, uroporphyrinogen III synthase (HEMD), uroporphyrinogen III decarboxylase (HEME2), NADPH-protochlorophyllide oxidoreductase (POR) A (PORA), chlorophyll synthase (CHLG), chlorophyllase (CLH2), and red chlorophyll catabolite reductase (RCCR), and measured their effects on the γ-irradiation-induced frequencies of leaf-color mutants in two Cymbidium cultivars. To degrade chlorophyll in rhizomes, 60-75 days of dark treatment were required. To upregulate the expressions of chlorophyll pathway genes, 10 days of light treatment appeared to be optimal. Dark/light treatments followed by γ-irradiation increased chlorophyll-related leaf mutants by 1.4- to 2.0-fold compared with γ-ray treatment alone. Dark/light treatments combined with γ-irradiation increased the frequency of leaf-color mutants in Cymbidium, which supports the wider implementation of a plant breeding methodology that increases the mutation frequency of a target trait by controlling the expression of target trait-related genes.

Frequency, Spectrum, and Stability of Leaf Mutants Induced by Diverse γ-Ray Treatments in Two Cymbidium Hybrids.

Ionizing radiation combined with in vitro tissue culture has been used for development of new cultivars in diverse crops. The effects of ionizing radiation on mutation induction have been analyzed on several orchid species, including Cymbidium. Limited information is available on the comparison of mutation frequency and spectrum based on phenotypes in Cymbidium species. In addition, the stability of induced chimera mutants in Cymbidium is unknown. In this study, we analyzed the radiation sensitivity, mutation frequency, and spectrum of mutants induced by diverse γ-ray treatments, and analyzed the stability of induced chimera mutants in the Cymbidium hybrid cultivars RB003 and RB012. The optimal γ-irradiation conditions of each cultivar differed as follows: RB003, mutation frequency of 4.06% (under 35 Gy/4 h); RB012, 1.51% (20 Gy/1 h). Re-irradiation of γ-rays broadened the mutation spectrum observed in RB012. The stability of leaf-color chimera mutants was higher than that of leaf-shape chimeras, and stability was dependent on the chimera type and location of a mutation in the cell layers of the shoot apical meristem. These results indicated that short-term γ-irradiation was more effective to induce mutations in Cymbidium. Information on the stability of chimera mutants will be useful for mutation breeding of diverse ornamental plants.

Transcriptome analysis to identify candidate genes associated with the yellow-leaf phenotype of a Cymbidium mutant generated by γ-irradiation.

Leaf color is an important agronomic trait in flowering plants, including orchids. However, factors underlying leaf phenotypes in plants remain largely unclear. A mutant displaying yellow leaves was obtained by the γ-ray-based mutagenesis of a Cymbidium orchid and characterized using RNA sequencing. A total of 144,918 unigenes obtained from over 25 million reads were assigned to 22 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes database. In addition, gene ontology was used to classify the predicted functions of transcripts into 73 functional groups. The RNA sequencing analysis identified 2,267 differentially expressed genes between wild-type and mutant Cymbidium sp. Genes involved in the chlorophyll biosynthesis and degradation, as well as ion transport, were identified and assayed for their expression levels in wild-type and mutant plants using quantitative real-time profiling. No critical expression changes were detected in genes involved in chlorophyll biosynthesis. In contrast, seven genes involved in ion transport, including two metal ion transporters, were down-regulated, and chlorophyllase 2, associated with chlorophyll degradation, was up-regulated. Together, these results suggest that alterations in chlorophyll metabolism and/or ion transport might contribute to leaf color in Cymbidium orchids.

Simultaneous adrenal pheochromocytoma and sarcoidosis.

A 48-year-old man presented for evaluation of general weakness. Because he had a history of excessive alcohol use, an abdominal computed tomography scan was obtained, which revealed a left adrenal mass. Hormonal evaluation showed elevated levels of urinary catecholamines. Bilateral hilar lymphadenopathy was detected on a chest radiograph. The suspected diagnosis was asymptomatic pheochromocytoma with sarcoidosis. We performed a mediastinoscopic lymph node biopsy, which was followed by endoscopic adrenalectomy. Histologic tissue analysis confirmed an adrenal pheochromocytoma and sarcoid granulomas in the mediastinal lymph nodes. This case highlights the difficulty in determining the appropriate work up of patients presenting with an adrenal incidentaloma and concomitant systemic disease.

[Hemoperitoneum due to ruptured gastric gastrointestinal stromal tumor].

The stomach is the most frequent site of gastrointestinal stromal tumor (GIST). The common clinical manifestation of GIST are melena and hematochezia caused by gastointestinal bleeding. However, hemoperitoneum due to GIST rupture is a very rare condition. We describe a 33-year-old man with gastric GIST causing hemoperitoneum. A preoperative CT scan demonstrated large amount of fluid collection and extraluminal mass lesion in gastric antral area. He underwent an emergent laparotomy. The antral mass was polypoid shaped and showed ruptured focus. We performed a distal gastrectomy. The tumor was revealed as GIST with intermediate malignant risk by pathologic examination. The patient had an uneventful postoperative course and remains well.

[Metachronous four primary malignancies in gastro-intestinal tract].

Multiple primary malignancy was reported firstly by Billroth in 1889. Recently, multiple primary malignancies are considered to increase due to improved survival rate of cancer patients, advanced diagnostic tools, and increased use of chemotherapy and radiotherapy. In Korea, several cases of triple primary malignancies were reported. However, four primary malignancies in gastro-intestinal tract was rarely reported. Recently, we experienced a 70 year-old male who was diagnosed with metachronous four primary malignancies in rectum, ascending colon, stomach, and ampulla of Vater. We report this rare case of metachronous four primary malignancies with a review of literature.

Role of Arabidopsis CHL27 protein for photosynthesis, chloroplast development and gene expression profiling.

In Chl biosynthesis, aerobic Mg-protoporphyrin IX monomethyl ester (MPE) cyclase is a key enzyme involved in the synthesis of protochlorophyllide a, and its membrane-bound component is known to be encoded by homologs of CHL27 in photosynthetic bacteria, green algae and plants. Here, we report that the Arabidopsis chl27-t knock-down mutant exhibits retarded growth and chloroplast developmental defects that are caused by damage to PSII reaction centers. The mutant contains a T-DNA insertion within the CHL27 promoter that dramatically reduces the CHL27 mRNA level. chl27-t mutant plants grew slowly with a pale green appearance, suggesting that they are defective in Chl biosynthesis. Chl fluorescence analysis showed significantly low photosynthetic activity in chl27-t mutants, indicating damage in their PSII reaction centers. The chl27-t mutation also conferred severe defects in chloroplast development, including the unstacking of thylakoid membranes. Microarray analysis of the chl27-t mutant showed repression of numerous nuclear genes involved in photosynthesis, including those encoding components of light-harvesting complex I (LHCI) and LHCII, and PSI and PSII, which accounts for the defects in photosynthetic activity and chloroplast development. In addition, the microarray data also revealed the significant repression of genes such as PORA and AtFRO6 for Chl biosynthesis and iron acquisition, respectively, and, furthermore, implied that there is cross-talk in the Chl biosynthetic pathway among the PORA, AtFRO6 and CHL27 proteins.