In Vitro Propagation Technology for the Endangered Aquatic Species Nymphoides coronata.

Nymphoides coronata is an endangered aquatic plant species with significant medicinal and ecological importance. To preserve N. coronata from going extinct, we need to provide seedlings and efficient multiplication techniques so that it can be extensively studied. This study aimed to identify the most suitable sterilization treatment, growth medium, and substrate for the cultivation and propagation of N. coronata. Ethanol sterilization, fungicide treatment, and sterile water washing were the most important sterilization steps. A combination of 6-benzylaminopurine (6-BA) and indoleacetic acid (IAA) was the most suitable medium for bud induction and shoot proliferation. The use of α-naphthaleneacetic acid (NAA) increased the rooting rate and rooting time compared to indole-3-butyric acid (IBA). Increasing the concentration of NAA from 0.5 to 1.0 mg/L increased the rooting rate from 78 to 100% and reduced the rooting time from 7 to 5 days. The survival rate of N. coronata seedlings was 100% in a mixture of red soil and sand (1:1, w/w). As a result, the procedure mentioned above could potentially be used to safely propagate this rare species on a large scale. These findings provide valuable insights into the optimal conditions for the successful cultivation and propagation of N. coronata, which can contribute to the conservation and sustainable use of this important rare plant species.

Digital assessment of tertiary lymphoid structures and therapeutic responses in gastric cancer: A multi-centric retrospective study.

BACKGROUND: Tertiary lymphoid structures (TLSs) are associated with favorable prognosis and enhanced response to anti-cancer therapy. A digital assessment of TLSs could provide an objective alternative that mitigates variability inherent in manual evaluation. This study aimed to develop and validate a digital gene panel based on biological prior knowledge for assessment of TLSs, and further investigate its associations with survival and multiple anti-cancer therapies. MATERIALS AND METHODS: The present study involved 1,704 patients with gastric cancer from seven cancer centers. TLSs were identified morphologically through hematoxylin-and-eosin staining. We further developed a digital score based on targeted gene expression profiling to assess TLSs status, recorded as gene signature of tertiary lymphoid structures (gsTLS). For enhanced interpretability, we employed the SHapley Additive exPlanation (SHAP) analysis to elucidate its contribution to the prediction. We next evaluated the signature's associations with prognosis, and investigated its predictive accuracy for multiple anti-cancer therapies, including adjuvant chemotherapy and immunotherapy. RESULTS: The gsTLS panel with nine gene features achieved high accuracies in predicting TLSs status in the training, internal and external validation cohorts (area under the curve, range: 0.729-0.791). In multivariable analysis, gsTLS remained an independent predictor of disease-free and overall survival (hazard ratio, range: 0.346-0.743, all P < 0.05) after adjusting for other clinicopathological variables. SHAP analysis highlighted gsTLS as the strongest predictor of TLSs status compared with clinical features. Importantly, patients with high gsTLS (but not those with low gsTLS) exhibited substantial benefits from adjuvant chemotherapy (P < 0.05). Furthermore, we found that the objective response rate to anti-programmed cell death protein 1 (anti-PD-1) immunotherapy was significantly higher in the high-gsTLS group (40.7%) versus the low-gsTLS group (5.6%, P = 0.036), and the diagnosis was independent from Epstein-Barr virus (EBV), tumor mutation burden (TMB), and programmed cell death-ligand 1 (PD-L1) expression. CONCLUSION: The gsTLS digital panel enables accurate assessment of TLSs status, and provides information regarding prognosis and responses to multiple therapies for gastric cancer.

Physiological and transcriptomic comparisons shed light on the cold stress response mechanisms of Dendrobium spp.

BACKGROUND: Dendrobium spp. comprise a group of tropical orchids with ornamental and medicinal value. Dendrobium spp. are sensitive to low temperature, and the underlying cold response regulatory mechanisms in this group are unclear. To understand how these plants respond to cold stress, we compared the transcriptomic responses of the cold-tolerant cultivar 'Hongxing' (HX) and the cold-sensitive cultivar 'Sonia Hiasakul' (SH) to cold stress. RESULTS: Chemometric results showed that the physiological response of SH in the later stages of cold stress is similar to that of HX throughout the cold treatment. Orthogonal partial least squares discriminant analysis (OPLS-DA) revealed that soluble protein content and peroxidase activity are key physiological parameters for assessing the cold tolerance of these two Dendrobium spp. cultivars. Additionally, weighted gene co-expression network analysis (WGCNA) results showed that many cold response genes and metabolic pathways significantly associated with the physiological indices were enriched in the 12 detected modules. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) enrichment analyses of the 105 hub genes showed that Dendrobium spp. adapt to cold stress by regulating signal transduction, phytohormones, transcription factors, protein translation and modification, functional proteins, biosynthesis and metabolism, cell structure, light, and the circadian clock. Hub genes of the cold stress response network included the remorin gene pp34, the abscisic acid signaling pathway-related genes PROTEIN PHOSPATASE 2 C (PP2C), SNF1-RELATED PROTEIN KINASE 2 (SnRK2), ABRE-BINDING FACTOR 1 (ABF1) and SKI-INTERACTING PROTEIN 17 (SKIP17), the Ca2+ signaling-related GTP diphosphokinase gene CRSH1, the carbohydrate-related gene STARCH SYNTHASE 2 (SS2), the cell wall biosynthesis gene CINNAMYL ALCOHOL DEHYDROGENASE (CAD7), and the endocytosis-related gene VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 52 A (VPS52A). CONCLUSIONS: The cold-responsive genes and metabolic pathways of Dendrobium spp. revealed in this study provide important insight to enable the genetic enhancement of cold tolerance in Dendrobium spp., and to facilitate cold tolerance breeding in related plants.

A High-Resolution Linkage Map Construction and QTL Analysis for Morphological Traits in Anthurium (Anthurium andraeanum Linden).

Anthurium andraeanum Linden is a prominent ornamental plant belonging to the family Araceae and is cultivated worldwide. The morphology characteristics are crucial because they significantly impact ornamental values, commercial properties, and the efficiency of space utilization in production. However, only a few related investigations have been conducted in anthurium to date. In this study, an F1 genetic segregation population containing 160 progenies was generated through hybridization between potted and cut anthurium varieties. Fifteen morphological traits were assessed and revealed substantial levels of genetic variation and widespread positive correlation. Based on specific length amplified fragment (SLAF) sequencing technology, 8171 single nucleotide polymorphism (SNP) markers were developed, and the high-density linkage map of 2202.27 cM in length distributed on 15 linkage groups was constructed successfully, with an average distance of 0.30 cM. Using the inclusive composite interval mapping (ICIM) method, 59 QTLs related to 15 key morphological traits were successfully identified, which explained phenotypic variance (PVE) ranging from 6.21% to 17.74%. Thirty-three of those associated with 13 traits were designated as major QTLs with PVE > 10%. These findings offer valuable insights into the genetic basis of quantitative traits and are beneficial for molecular marker-assisted selection (MAS) in anthurium breeding.

DhMYB2 and DhbHLH1 regulates anthocyanin accumulation via activation of late biosynthesis genes in Phalaenopsis-type Dendrobium.

Phalaenopsis-type Dendrobium is a popular orchid with good ornamental and market value. Despite their popularity, molecular regulation of anthocyanin biosynthesis during flower development remains poorly understood. In this study, we systematically investigated the regulatory roles of the transcription factors DhMYB2 and DhbHLH1 in anthocyanins biosynthesis. Gene expression analyses indicated that both DhMYB2 and DhbHLH1 are specifically expressed in flowers and have similar expression patterns, showing high expression in purple floral tissues with anthocyanin accumulation. Transcriptomic analyses showed 29 differentially expressed genes corresponding to eight enzymes in anthocyanin biosynthesis pathway have similar expression patterns to DhMYB2 and DhbHLH1, with higher expression in the purple lips than the yellow petals and sepals of Dendrobium 'Suriya Gold'. Further gene expression analyses and Pearson correlation matrix analyses of Dendrobium hybrid progenies revealed expression profiles of DhMYB2 and DhbHLH1 were positively correlated with the structural genes DhF3'H1, DhF3'5'H2, DhDFR, DhANS, and DhGT4. Yeast one-hybrid and dual-luciferase reporter assays revealed DhMYB2 and DhbHLH1 can bind to promoter regions of DhF3'H1, DhF3'5'H2, DhDFR, DhANS and DhGT4, suggesting a role as transcriptional activators. These results provide new evidence of the molecular mechanisms of DhMYB2 and DhbHLH1 in anthocyanin biosynthesis in Phalaenopsis-type Dendrobium.

AaMYB3 interacts with AabHLH1 to regulate proanthocyanidin accumulation in Anthurium andraeanum (Hort.)-another strategy to modulate pigmentation.

Proanthocyanidins (PAs), also known as "condensed tannins", are colorless metabolites produced through the flavonoid pathway that are involved in stress resistance in plants. Because PAs are involved in the anthocyanin biosynthetic pathway, they play a role in the modification of pigmentation conferred by anthocyanins in ornamental organs. In this study, we isolated the gene and functionally characterized an R2R3-MYB transcription factor (TF), AaMYB3, and a basic helix-loop-helix TF, AabHLH1, from Anthurium andraeanum (Hort.), a typical tropical flower. AaMYB3 is primarily expressed in the spathe and negatively correlates with anthocyanin accumulation. A complementation test in an Arabidopsis tt8 mutant showed that AabHLH1 successfully restores the PA-deficient seed coat phenotype. The ectopic overexpression of AaMYB3 alone or its coexpression with AabHLH1 in transgenic tobacco resulted in light pink or even pale-pink corolla limbs by reducing their anthocyanin levels and greatly enhancing their accumulation of PAs. This overexpression of the anthurium TF genes upregulated the late anthocyanin enzyme-encoding genes (NtDFR and NtANS) and the key PA genes (NtLAR and NtANR) in transgenic tobacco. The interaction between AaMYB3 and the AabHLH1 protein was confirmed using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. In the developing red spathes of the cultivars "Vitara" and "Tropical", the expression of AaMYB3 was closely linked to PA accumulation, and AaMYB3 was coexpressed with AaCHS, AaF3H, AaDFR, AaANS, AaLAR, and AaANR. The expression pattern of AabHLH1 was similar to that of AaF3'H. Our results suggest that AaMYB3 and AabHLH1 are involved in the regulation of PA biosynthesis in anthurium and could potentially be used to metabolically engineer PA biosynthesis in plants.

Determinants of hospitalization in Chinese patients with type 2 diabetes receiving a peer support intervention and JADE integrated care: the PEARL randomised controlled trial.

BACKGROUND: In a randomized controlled trial of 628 Chinese patients with type 2 diabetes receiving multidisciplinary care in the Joint Asia Diabetes Evaluation (JADE) Progam, 372 were randomized to receive additional telephone-based peer support (Peer Empowerment And Remote communication Linked by information technology, PEARL) intervention. After 12 months, all-cause hospitalization was reduced by half in the PEARL group especially in those with high Depression Anxiety and Stress Scale (DASS) scores. METHODS: We used stratified analyses, negative binomial regression, and structural equation modelling (SEM) to examine the inter-relationships between emotions, self-management, cardiometabolic risk factors, and hospitalization. RESULTS: Hospitalized patients were older, more likely to have heart or kidney disease, and negative emotions than those without hospitalization. Patients with high DASS score who did not receive peer support had the highest hospitalization rates. After adjustment for confounders, peer support reduced the frequency of hospitalizations by 48% with a relative risk of 0.52 (95% CI 0·35-0·79;p = 0·0018). Using SEM, improvement of negative emotions reduced treatment nonadherence (Est = 0.240, p = 0.034) and hospitalizations (Est=-0.218, p = 0.001). The latter was also reduced by an interactive term of peer support and chronic kidney disease (Est = 0.833, p = < 0.001) and that of peer support and heart disease (Est = 0.455, p = 0.001). CONCLUSIONS: In type 2 diabetes, improvement of negative emotions and peer support reduced hospitalizations, especially in those with comorbidities, in part mediated through improving treatment nonadherence. Integrating peer support is feasible and adds value to multidisciplinary care, augmented by information technology, especially in patients with comorbidities. TRIAL REGISTRATION: NCT00950716 Registered July 31, 2009.

Anthocyanin biosynthesis regulation of DhMYB2 and DhbHLH1 in Dendrobium hybrids petals.

Dendrobium hybrids orchid are popular throughout the world. They have various floral color and pigmentation patterns that are mainly caused by anthocyanins. It is well established that anthocyanin biosynthesis is regulated by the interplay between MYB and bHLH transcription factors (TF) in most plants. In this study, we identified one R2R3-MYB gene, DhMYB2, and one bHLH gene, DhbHLH1, from a Dendrobium hybrid. Their expression profiles were related to anthocyanin pigmentation in Dendrobium petals. Transient over-expression of these two TF genes showed that both DhMYB2 and DhbHLH1 resulted in anthocyanin production in white petals. The interaction between the two TFs was observed in vitro. In different Dendrobium hybrids petals with various pigmentations, DhMYB2 and DhbHLH1 were co-expressed with DhDFR and DhANS, which are regarded as potential regulatory targets of the two TFs. In flowers with distinct purple lips but white or yellow petals/sepals, the expression of DhbHLH1 was only related to anthocyanin accumulation in the lips. Taken together, DhMYB2 interacted with DhbHLH1 to regulate anthocyanin production in Dendrobium hybrid petals. DhbHLH1 was also responsible for the distinct anthocyanin pigmentation in lip tissues. The functional characterization of DhMYB2 and DhbHLH1 will improve understanding of anthocyanin biosynthesis modulation in Dendrobium orchids.

Prevention and Care Programs Addressing the Growing Prevalence of Diabetes in China.

According to a 2010 national survey, 11 % of adults in China have diabetes, affecting 109.6 million individuals. The high prevalence of diabetes has been attributed to the aging of the population, the rapid adoption of energy-dense foods, and a reduction in physical activity. Collectively, these secular changes have created an obesogenic environment that can unmask diabetes in subjects with a genetic predisposition. The growing prevalence of maternal obesity, gestational diabetes, childhood obesity, and early-onset disease can lead to premature morbidity and mortality. Rising to meet these public health challenges, researchers in China have conducted randomized studies to demonstrate the benefits of lifestyle modification in preventing diabetes (the Da Qing Study), as well as that of team-based integrated care, using multiple strategies including peer support and information technology, in order to reduce hospitalizations, cardiovascular-renal complications, and premature deaths. With growing evidence supporting the benefits of these diabetes prevention and management programs, the next challenge is to use policies and systems to scale up the implementation of these programs through raising awareness, building capacity, and providing resources to reduce the human and socioeconomic burden of diabetes.

Isolation and characterization of a R2R3-MYB transcription factor gene related to anthocyanin biosynthesis in the spathes of Anthurium andraeanum (Hort.).

KEY MESSAGE: A R2R3-MYB gene AaMYB2 was isolated from Anthurium andraeanum (Hort.) and was functionally characterized to be a positive transcriptional regulator for anthocyanin biosynthesis in the spathes and leaves. Spathe coloration is an important Anthurium andraeanum (Hort.) characteristic, which is mainly contributed by anthocyanins. R2R3-MYB transcription factors (TFs) are important regulators of anthocyanin biosynthesis in plants. Here we describe the identification and characterization of AaMYB2, a member in subgroup 6 of the R2R3-MYB TFs family, which correlated with anthocyanin biosynthesis in A. andraeanum. AaMYB2 was a nuclear-localization protein with positive transcriptional activity, and prominently expressed in the red spathes. Ectopic expression of AaMYB2 in tobacco led to anthocyanin accumulation and up-regulation of the early and late anthocyanin pathway genes, particularly NtDFR, NtANS, and NtUFGT, and the endogenous TF genes NtAn2 and NtAn1 in leaves. In the developing red spathes of 'Tropical' and 'Vitara', the expression of AaMYB2 was closely linked to anthocyanin accumulation, and co-expressed with AaCHS, AaF3H, and AaANS, the latter two of which were regarded as the potential targets of the R locus encoding a TF controlling spathe colors inheritance in anthurium. In addition, the transcription level of AaMYB2 in various cultivars with different color phenotypes showed that AaMYB2 was drastically expressed in the spathes from the red, pink, and purple cultivars, but hardly detected in the spathes from the white and green ones. Besides, AaMYB2 also showed higher expression in newly developmental leaves when anthocyanin was actively biosynthesized. Taken together, AaMYB2 positively related to anthocyanin biosynthesis in anthurium spathes and leaves, and appeared to regulate the expression of AaF3H, AaANS, and possibly AaCHS.

Msp40 effector of root-knot nematode manipulates plant immunity to facilitate parasitism.

Root-knot nematodes (RKNs) are obligate biotrophic parasites that invade plant roots and engage in prolonged and intimate relationships with their hosts. Nematode secretions, some of which have immunosuppressing activity, play essential roles in successful parasitism; however, their mechanisms of action remain largely unknown. Here, we show that the RKN-specific gene MiMsp40, cloned from Meloidogyne incognita, is expressed exclusively in subventral oesophageal gland cells and is strongly upregulated during early parasitic stages. Arabidopsis plants overexpressing MiMsp40 were more susceptible to nematode infection than were wild type plants. Conversely, the host-derived MiMsp40 RNAi suppressed nematode parasitism and/or reproduction. Moreover, overexpression of MiMsp40 in plants suppressed the deposition of callose and the expression of marker genes for bacterial elicitor elf18-triggered immunity. Transient expression of MiMsp40 prevented Bax-triggered defence-related programmed cell death. Co-agroinfiltration assays indicated that MiMsp40 also suppressed macroscopic cell death triggered by MAPK cascades or by the ETI cognate elicitors R3a/Avr3a. Together, these results demonstrate that MiMsp40 is a novel Meloidogyne-specific effector that is injected into plant cells by early parasitic stages of the nematode and that plays a role in suppressing PTI and/or ETI signals to facilitate RKN parasitism.

SphK1 promotes tumor cell migration and invasion in colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Sphingosine kinase 1 (SphK1), which phosphorylates sphingosine to sphingosine-1-phosphate (S1P), is overexpressed in various types of cancers and may act as an oncogene in tumorigenesis. However, little is known about the role of SphK1 in CRC patients. We studied the expression of SphK1 in 85 cases of CRC tissues by immunohistochemistry, qRT-PCR, and western blot. We also evaluated the effect of SphK1 on cell proliferation and invasion by MTT and transwell invasion assay. SphK1 is overexpressed in CRC tissues and cell lines, and upregulation of SphK1 correlated significantly with the following parameters: lymph node metastasis, liver metastasis, and advanced TNM stage. SphK1 knockdown results in inhibition of cancer cell proliferation. Inhibition of CRC cell migration and invasion is also evident through reversal of EMT by increases in E-cadherin expression and decreases in vimentin expression. In conclusion, SphK1 is associated with the proliferation and invasiveness of CRC cells and the SphK1 gene may contribute to a novel therapeutic approach against CRC.

Gender, diabetes education, and psychosocial factors are associated with persistent poor glycemic control in patients with type 2 diabetes in the Joint Asia Diabetes Evaluation (JADE) program.

BACKGROUND: Factors associated with persistent poor glycemic control were explored in patients with type 2 diabetes under the Joint Asia Diabetes Evaluation (JADE) program. METHODS: Chinese adults enrolled in JADE with HbA1c ≥8% at initial comprehensive assessment (CA1) and repeat assessment were analyzed. The improved group was defined as those with a ≥1% absolute reduction in HbA1c, and the unimproved group was those with <1% reduction at the repeat CA (CA2). RESULTS: Of 4458 enrolled patients with HbA1c ≥8% at baseline, 1450 underwent repeat CA. After a median interval of 1.7 years (interquartile range[IQR] 1.1-2.2) between CA1 and CA2, the unimproved group (n = 677) had a mean 0.4% (95% confidence interval [CI] 0.3%, 0.5%) increase in HbA1c compared with a mean 2.8% reduction (95% CI -2.9, -2.6%) in the improved group (n = 773). The unimproved group had a female preponderance with lower education level, and was more likely to be insulin treated. Patients in the improved group received more diabetes education between CAs with improved self-care behaviors, whereas the unimproved group had worsening of health-related quality of life at CA2. Apart from female gender, long disease duration, low educational level, obesity, retinopathy, history of hypoglycemia, and insulin use, lack of education from diabetes nurses between CAs had the strongest association for persistent poor glycemic control. CONCLUSIONS: These results highlight the multidimensional nature of glycemic control, and the importance of diabetes education and optimizing diabetes care by considering psychosocial factors.

Effects of Providing Peer Support on Diabetes Management in People With Type 2 Diabetes.

PURPOSE: We examined the effects of participating in a "train-the-trainer" program and being a peer supporter on metabolic and cognitive/psychological/behavioral parameters in Chinese patients with type 2 diabetes. METHODS: In response to our invitation, 79 patients with fair glycemic control (HbA1c <8%) agreed to participate in a "train-the-trainer" program to become peer supporters. Of the 59 who completed the program successfully, 33 agreed to be peer supporters ("agreed trainees") and were each assigned to support 10 patients for 1 year, with a voluntary extension period of 3 additional years, while 26 trainees declined to be supporters ("refused trainees"). A group of 60 patients with fair glycemic control who did not attend the training program and were under usual care were selected as a comparison group. The primary outcome was the change in average HbA1c levels for the 3 groups from baseline to 6 months. RESULTS: At 6 months, HbA1c was unchanged in the trainees (at baseline, 7.1 ± 0.3%; at 6 months, 7.1 ± 1.1%) but increased in the comparison group (at baseline, 7.1 ± 0.5%; at 6 months, 7.3 ± 1.1%. P = .02 for between-group comparison). Self-reported self-care activities including diet adherence and foot care improved in the trainees but not the comparison group. After 4 years, HbA1c remained stable among the agreed trainees (at baseline, 7.0 ± 0.2%; at 4 years: 7.2 ± 0.6%), compared with increases in the refused trainees (at baseline, 7.1 ± 0.4%; at 4 years, 7.8 ± 0.8%) and comparison group (at baseline, 7.1 ± 0.5%; at 4 years, 8.1 ± 0.6%. P = .001 for between-group comparison). CONCLUSIONS: Patients with diabetes who engaged in providing ongoing peer support to other patients with diabetes improved their self-care while maintaining glycemic control over 4 years.

De Novo characterization of a Cephalotaxus hainanensis transcriptome and genes related to paclitaxel biosynthesis.

Cephalotaxus hainanensis, an endangered plant, is known to contain several metabolites with anti-cancer activity. Despite its clinical impact, the alkaloid metabolism of this species has remained largely uncharacterized. The potential of Cephalotaxus for metabolic engineering of medically interesting compounds has, so far, not been exploited, due to the almost complete lack of molecular information. We have therefore performed a high throughput RNA-seq analysis and assembled the transcriptome de novo. Raw reads comprising 4.3 Gbp were assembled de novo into 39,416 unique sequences (unigenes) with a mean length of 1,089.8 bp and a total assembly size of 45.8 Mbp, which equals to more than 50 times the number of Cephalotaxaceae sequences currently deposited in the GenBank (as of August 2013). As proof of principle for medically interesting pathways, gene fragments related to paclitaxel biosynthesis were searched and detected. To verify their functionality, the metabolic product paclitaxel, and its precursor baccatin III, were identified in the leaves of C. hainanensis by HPLC, and shown to be induced by MeJA. This finding demonstrates exemplarily the potential of the annotated transcriptome as information resource for the biotechnological exploitation of plant secondary metabolism.

[Parent grouping of 31 elite inbred lines in hot pepper (Capsicum annuum L.)].

Genetic differences were examined among thirty-one elite inbred lines in Capsicum annuum L. Two types of analytic technologies, i.e. SRAP markers and genotypes of traits, were used, and their relative effectiveness was compared. 27 of 30 primer combinations could amplify 310 polymorphic bands among inbred lines, indicating SRAP marker was efficient to detect polymorphism among pepper inbred lines. A dendrogram of 31 inbred lines based on SRAP markers and Yule coefficients could basically separate lines of C. annuum var. grossum and C. annuum var. longum, and reveal the pedigrees of inbred lines. A dendrogram of 31 inbred lines based on genotypes of traits and standardized Euclidean coefficients could separate lines of C. annuum var. grossum and C. annuum var. longum. The SRAP marker genetic distances were correlated with distances based on the genotypes of traits. These results and their application in the development of hot pepper F1 hy-brids were also discussed.