SlPHL1 positively modulates acid phosphatase in response to phosphate starvation by directly activating the genes SlPAP10b and SlPAP15 in tomato.

Increased acid phosphatase (APase) activity is a prominent feature of tomato (Solanum lycopersicum) responses to inorganic phosphate (Pi) restriction. SlPHL1, a phosphate starvation response (PHR) transcription factor, has been identified as a positive regulator of low Pi (LP)-induced APase activity in tomato. However, the molecular mechanism underlying this regulation remains to be elucidated. Here, SlPHL1 was found to positively regulate the LP-induced expression of five potential purple acid phosphatase (PAP) genes, namely SlPAP7, SlPAP10b, SlPAP12, SlPAP15, and SlPAP17b. Furthermore, we provide evidence that SlPHL1 can stimulate transcription of these five genes by binding directly to the PHR1 binding sequence (P1BS) located on their promoters. The P1BS mutation notably weakened SlPHL1 binding to the promoters of SlPAP7, SlPAP12, and SlPAP17b but almost completely abolished SlPHL1 binding to the promoters of SlPAP10b and SlPAP15. As a result, the transcriptional activation of SlPHL1 on SlPAP10b and SlPAP15 was substantially diminished. In addition, not only did transient overexpression of either SlPAP10b or SlPAP15 in tobacco leaves increase APase activity, but overexpression of SlPAP15 in Arabidopsis and tomato also increased APase activity and promoted plant growth. Subsequently, two SPX proteins, SlSPX1 and SlSPX4, were shown to physically interact with SlPHL1. Moreover, SlSPX1 inhibited the transcriptional activation of SlPHL1 on SlPAP10b and SlPAP15 and negatively regulated the activity of APase. Taken together, these results demonstrate that SlPHL1-mediated LP signaling promotes APase activity by activating the transcription of SlPAP10b and SlPAP15, which may provide valuable insights into the mechanisms of tomato response to Pi-limited stress.

Genome-Wide Analysis of Cation/Proton Antiporter Family in Soybean (Glycine max) and Functional Analysis of GmCHX20a on Salt Response.

Monovalent cation proton antiporters (CPAs) play crucial roles in ion and pH homeostasis, which is essential for plant development and environmental adaptation, including salt tolerance. Here, 68 CPA genes were identified in soybean, phylogenetically dividing into 11 Na+/H+ exchangers (NHXs), 12 K+ efflux antiporters (KEAs), and 45 cation/H+ exchangers (CHXs). The GmCPA genes are unevenly distributed across the 20 chromosomes and might expand largely due to segmental duplication in soybean. The GmCPA family underwent purifying selection rather than neutral or positive selections. The cis-element analysis and the publicly available transcriptome data indicated that GmCPAs are involved in development and various environmental adaptations, especially for salt tolerance. Based on the RNA-seq data, twelve of the chosen GmCPA genes were confirmed for their differentially expression under salt or osmotic stresses using qRT-PCR. Among them, GmCHX20a was selected due to its high induction under salt stress for the exploration of its biological function on salt responses by ectopic expressing in Arabidopsis. The results suggest that the overexpression of GmCHX20a increases the sensitivity to salt stress by altering the redox system. Overall, this study provides comprehensive insights into the CPA family in soybean and has the potential to supply new candidate genes to develop salt-tolerant soybean varieties.

SlPHL1 is involved in low phosphate stress promoting anthocyanin biosynthesis by directly upregulation of genes SlF3H, SlF3'H, and SlLDOX in tomato.

Phosphate (Pi) deficiency is a common stress that limits plant growth and development. Plants exhibit a variety of Pi starvation responses (PSRs), including anthocyanin accumulation. The transcription factors of the PHOSPHATE STARVATION RESPONSE (PHR) family, such as AtPHR1 in Arabidopsis, play central roles in the regulation of Pi starvation signaling. Solanum lycopersicum PHR1-like 1 (SlPHL1) is a recently identified PHR involved in PSR regulation in tomato, but the detailed mechanism of its participation in Pi starvation-inducing anthocyanin accumulation remains unclear. Here we found that overexpression of SlPHL1 in tomato increases the expression of genes associated with anthocyanin biosynthesis, thereby promoting anthocyanin biosynthesis, but silencing SlPHL1 with Virus Induced Gene Silencing (VIGS) attenuated low phosphate (LP) stress-induced anthocyanin accumulation and expression of the biosynthesis-related genes. Notably, SlPHL1 is able to bind the promoters of genes Flavanone 3-Hydroxylase (SlF3H), Flavanone 3'-Hydroxylase (SlF3'H), and Leucoanthocyanidin Dioxygenase (SlLDOX) by yeast one-hybrid (Y1H) analysis. Furthermore, Electrophoretic Mobility Shift Assay (EMSA) and transient transcript expression assay showed that PHR1 binding t (sequence (P1BS) motifs located on the promoters of these three genes are critical for SlPHL1 binding and enhancing the gene transcription. Additionally, allogenic overexpression of SlPHL1 could promote anthocyanin biosynthesis in Arabidopsis under LP conditions through the similar mechanism to AtPHR1, suggesting that SlPHL1 might be functionally conserved with AtPHR1 in this process. Taken together, SlPHL1 positively regulates LP-induced anthocyanin accumulation by directly promoting the transcription of SlF3H, SlF3'H and SlLDOX. These findings will contribute to understanding the molecular mechanism of PSR in tomato.

PHR1 positively regulates phosphate starvation-induced anthocyanin accumulation through direct upregulation of genes F3'H and LDOX in Arabidopsis.

MAIN CONCLUSION: Phosphate deficiency promotes anthocyanin accumulation in Arabidopsis through direct binding of PHR1 to the P1BS motifs on the promoters of F3'H and LDOX and thereby upregulating their expression. Phosphorus is one of the essential elements for plants, and plants mainly absorb inorganic phosphate (Pi) from soil. But Pi deficiency is a common factor limiting plant growth and development. Anthocyanin accumulation in green tissues (such as leaves) is one of the characteristics of many plants in response to Pi starvation. However, little is known about the mechanism by which Pi starvation induces anthocyanin accumulation. Here, we found that the mutation of the gene PHOSPHATE STARVATION RESPONSE1 (PHR1), which encodes a key factor involved in Pi starvation signaling in Arabidopsis, significantly attenuates anthocyanin accumulation under Pi-limiting conditions. Moreover, the expression of several Pi deficiency-upregulated genes that are involved in anthocyanin biosyntheses, such as flavanone 3'-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and production of anthocyanin pigment 1 (PAP1), was significantly lower in the phr1-1 mutant than in the wild type (WT). Both yeast one-hybrid (Y1H) analysis and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) showed that PHR1 can interact with the promoters of F3'H and LDOX, but not DFR and PAP1. By electrophoretic mobility shift assay (EMSA), it was further confirmed that the PHR1-binding sequence (P1BS) motifs located on the F3'H and LDOX promoters are required for the PHR1 bindings. Also, in Arabidopsis protoplasts, PHR1 enhanced the transcriptional activity of the F3'H and LDOX promoters, but these effects were markedly impaired when the P1BS motifs were mutated. Taken together, these results indicate that PHR1 positively regulates Pi starvation-induced anthocyanin accumulation in Arabidopsis, at least in part, by directly binding the P1BS motifs located on the promoters to upregulate the transcription of anthocyanin biosynthetic genes F3'H and LDOX.

SlPHL1, a MYB-CC transcription factor identified from tomato, positively regulates the phosphate starvation response.

Inorganic phosphate (Pi) deficiency is a major limiting factor for plant growth and development. Previous reports have demonstrated that PHOSPHATE STARVATION RESPONSE 1 (PHR1) and OsPHR2 play central roles in Pi-starvation signaling in Arabidopsis and rice, respectively. However, the Pi-starvation signaling network in tomato (Solanum lycopersicum) is still not fully understood. In this work, SlPHL1, a homolog of AtPHR1 and OsPHR2, was identified from tomato. It was found that SlPHL1 contains the MYB and coiled-coil (CC) domains, localizes in the nucleus, and has transcriptional activity, indicating that it is a typical MYB-CC transcription factor (TF). Overexpression of SlPHL1 enhanced Pi-starvation responses both in Arabidopsis Col-0 and in tomato Micro-Tom, including elevated root hair growth, promoted APase activity, favored Pi uptake, and increased transcription of Pi starvation-inducing (PSI) genes. Besides, overexpressing SlPHL1 was able to compensate for the Pi-starvation response weakened by the AtPHR1 mutation. Notably, electrophoretic mobility shift assay (EMSA) showed that SlPHL1 could bind to the PHR1-binding sequence (P1BS, GNATATNC)-containing DNA fragments. Furthermore, SlPHL1 specifically interacted with the promoters of the tomato PSI genes SlPht1;2 and SlPht1;8 through the P1BS cis-elements. Taken these results together, SlPHL1 is a newly identified MYB-CC TF from tomato, which participates in Pi-starvation signaling by directly upregulating the PSI genes. These findings might contribute to the understanding of the Pi-starvation signaling in tomato.

PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) negatively regulates anthocyanin accumulation by inhibiting PAP1 transcription in Arabidopsis seedlings.

Anthocyanin accumulation is a striking symptom of plant environmental response and plays an important role in plant adaptation to adverse stimuli. PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) is a member of the PIFs family that directly interacts with light-activated phytochromes, and it can not only regulate various light responses but also optimize growth as a key integrator of multiple signaling pathways. However, the mechanism by which PIF4 participates in the regulation of anthocyanin accumulation remains to be elucidated. In this study, we found that anthocyanin accumulation was effectively induced by white light in Arabidopsis Col-0, but such an effect was impaired in the overexpression line PIF4OX. Consistently, the transcript level of PAP1 that encodes a key transcript factor involved in regulating anthocyanin biosynthesis was significantly decreased in PIF4OX compared with Col-0. Moreover, the expression of PAP1 was markedly lower in pap1-D/PIF4OX than pap1-D, as a result, the phenotype that highly accumulates anthocyanins in leaves of pap1-D caused by PAP1 overexpressing was almost eliminated in pap1-D/PIF4OX. Analyses through chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) revealed that PIF4 could directly bind to the G-box motif present in the promoter of PAP1. Furthermore, transient transcriptional expression analysis showed that PIF4 could weaken the transcriptional activity of the PAP1 promoter, and the G-box motif is necessary for the effect of PIF4. Subsequently, when the seedlings shifted from darkness to light and grew under constant red light and short-day photoperiod, it was found that the PAP1 transcription level and anthocyanin content in pif4-2/pap1-D were significantly higher than pap1-D, implying that PIF4 mutation can strengthen PAP1's effect on anthocyanin biosynthesis under these conditions. Taken together, the results indicate that PIF4 negatively regulates anthocyanin accumulation in Arabidopsis through transcriptional suppression of PAP1 by directly binding to the G-box motif of the promoter.

A continuous responder algorithm to optimize clinical management of small-cell lung cancer with progastrin-releasing peptide as a simple blood test.

This study aimed to investigate whether changes in progastrin-releasing peptide (ProGRP) levels correlate with treatment response and can be used to optimize clinical management of patients with small-cell lung cancer. Patients with small-cell lung cancer (any stage) receiving chemotherapy were eligible. ProGRP was measured in serum/plasma at baseline and after each chemotherapy cycle using the Elecsys® ProGRP assay (Roche Diagnostics). Treatment response was assessed by computed tomography scan. The primary objective was to examine whether changes in ProGRP levels correlated with computed tomography scan results after two cycles of chemotherapy. The prognostic value of ProGRP among patients receiving first-line chemotherapy was also assessed. Overall, 261 patients from six centers were eligible. Among patients with elevated baseline ProGRP (>100 pg/mL), a ProGRP decline after Cycle 2 was associated with nonprogression (area under the curve: 84%; 95% confidence interval: 72.8-95.1; n = 141). ProGRP changes from baseline to end of Cycle 1 were predictive of response, as determined by computed tomography scan 3 weeks later (area under the curve: 87%; 95% confidence interval: 74.1-99.2; n = 137). This was enhanced by repeat measurements, with a 92% area under the curve (95% confidence interval: 85.3-97.8) among patients with ProGRP data after both Cycles 1 and 2 (n = 123); if a patient experienced a ≥25% decline in ProGRP after Cycle 1, and ProGRP remained stable or decreased after Cycle 2, the probability of finding progression on the interim computed tomography scan at the end of Cycle 2 was almost zero (sensitivity: 100%, specificity: 71%). Both ProGRP levels at baseline and at the end of first-line chemotherapy were prognostic; the latter provided a moderately improved hazard ratio of 2.43 (95% confidence interval: 1.33-4.46; n = 110) versus 1.87 (95% confidence interval: 1.04-3.37; n = 216). In summary, for patients with small-cell lung cancer and elevated baseline ProGRP levels, ProGRP may be a simple, reliable, and repeatable tool for monitoring response to chemotherapy and provide valuable prognostic information.

Case study of an autoantibody panel for early detection of lung cancer and ground-glass nodules.

PURPOSE: Increasing lung cancer incidence in China with a high death rate due to late diagnosis highlights the need for biomarkers, such as panels of autoantibodies (AAbs), for prediction and early lung cancer diagnosis. We conducted a study to further evaluate the clinical performance of an AAb diagnostic kit. METHODS: Using enzyme-linked immunosorbent assay, levels of seven AAbs in serum samples from 121 patients with newly diagnosed lung cancer, 84 controls (34 healthy individuals and 50 patients with benign lung disease), and 100 indeterminate solid nodules, were measured. Participants were followed up until 6 months after a positive test result to confirm lung cancer diagnosis. RESULTS: The seven AAb concentration was significantly higher in lung cancer patients than in controls (P < 0.05). The seven AAb sensitivity and specificity for newly diagnosed lung cancer were 45.5% and 85.3%, respectively, while the seven AAb combined area under the curve (in lung cancer patients versus controls) was 0.660. Of the 28 patients with solid nodules with positive test results, 8 and 3 were diagnosed with lung cancer and benign lung disease, respectively, during follow-up. The positive predictive value of the experiment was 72.7%. CONCLUSION: Positive AAb test results were associated with a high risk of lung cancer. The seven-AAb panel also had a high predictive value for detecting lung cancer in patients with solid nodules. Our seven lung cancer autoantibody types can provide an important early warning sign in the clinical setting.

Novel circulating protein biomarkers for thyroid cancer determined through data-independent acquisition mass spectrometry.

BACKGROUND: Distinguishing between different types of thyroid cancers (TC) remains challenging in clinical laboratories. As different tumor types require different clinical interventions, it is necessary to establish new methods for accurate diagnosis of TC. METHODS: Proteomic analysis of the human serum was performed through data-independent acquisition mass spectrometry for 29 patients with TC (stages I-IV): 13 cases of papillary TC (PTC), 10 cases of medullary TC (MTC), and six cases follicular TC (FTC). In addition, 15 patients with benign thyroid nodules (TNs) and 10 healthy controls (HCs) were included in this study. Subsequently, 17 differentially expressed proteins were identified in 291 patients with TC, including 247 with PTC, 38 with MTC, and six with FTC, and 69 patients with benign TNs and 176 with HC, using enzyme-linked immunosorbent assays. RESULTS: In total, 517 proteins were detected in the serum samples using an Orbitrap Q-Exactive-plus mass spectrometer. The amyloid beta A4 protein, apolipoprotein A-IV, gelsolin, contactin-1, gamma-glutamyl hydrolase, and complement factor H-related protein 1 (CFHR1) were selected for further analysis. The median serum CFHR1 levels were significantly higher in the MTC and FTC groups than in the PTC and control groups (P < 0.001). CFHR1 exhibited higher diagnostic performance in distinguishing patients with MTC from those with PTC (P < 0.001), with a sensitivity of 100.0%, specificity of 85.08%, area under the curve of 0.93, and detection cut-off of 0.92 ng/mL. CONCLUSION: CFHR1 may serve as a novel biomarker to distinguish PTC from MTC with high sensitivity and specificity.

Gibberellins play dual roles in response to phosphate starvation of tomato seedlings, negatively in shoots but positively in roots.

Gibberellins (GAs), a group of plant hormones, and phosphate (Pi), a macronutrient, are essential for numerous aspects of plant growth and development. During Pi starvation, plants develop many adaptive strategies to cope. However, the detailed roles of GAs in Pi deficiency responses of plants are largely unclear. In the present work, we found that low Pi (LP) treatment caused many responses in tomato (Solanum lycopersicum), including anthocyanin accumulation, upregulation of genes encoding high-affinity Pi transporters, and a striking induction of primary root growth. Application of exogeneous GA3 in the wild-type Micro-Tom (MT) significantly impaired LP-induced shoot anthocyanin accumulation and the upregulation of several key biosynthetic genes, including SlCHS, SlDFR, and SlF3'H. Meanwhile, LP-induced primary root elongation, upregulated SlPT2 and SlPT7 (genes encoding high-affinity Pi transporters), and favored Pi uptake were obviously attenuated in GA biosynthetic mutant gib3. Moreover, LP treatment obviously decreased the content of endogenous GA4 (a main form of GAs in tomato) in shoots but increased it in roots of MT seedlings. Additionally, in pro, a tomato mutant of DELLA protein, the LP-induced anthocyanin accumulation and expression of SlCHS, SlDFR, and SlF3'H were impaired, whereas the LP-induced primary root growth, expression of genes SlPT2 and SlPT7, and Pi uptake were more enhanced compared with the wild-type MT. Taking these data together, GAs play dual roles in the Pi starvation response of tomato seedlings, negatively in shoots but positively in roots. In addition, the GA-PRO system may play an important role in responding to Pi starvation in tomato.

A reference interval for serum IgG subclasses in Chinese children.

BACKGROUND: Reference intervals (RIs) for serum IgG subclasses vary greatly among different geographical regions. The present study aimed to establish RIs for serum IgG subclasses in Chinese children, which is essential for interpretation of laboratory findings and making clinical decisions. METHODS: This study was performed in accordance with guideline C28-A3, proposed by the International Federation of Clinical Chemistry and the Clinical and Laboratory Standards Institute. In total, 607 apparently healthy Chinese children were enrolled, and serum levels of IgG subclasses were measured. Individuals were stratified by age and the RIs were determined through statistical analysis. RESULTS: Following were the median values of RIs for serum IgG subclasses in Chinese children: IgG1, 2.78 g/L; IgG2, 0.85 g/L; IgG3, 0.13 g/L; IgG4, 0.06 g/L at 1-6 months of age; IgG1, 3.64 g/L; IgG2, 0.73 g/L; IgG3, 0.19 g/L; IgG4, 0.03 g/L at 6-12 months of age; IgG1, 5.15 g/L; IgG2, 0.87 g/L; IgG3, 0.19 g/L; IgG4, 0.07 g/L at 1-2 years of age; IgG1, 5.26 g/L; IgG2, 1.23 g/L; IgG3, 0.14 g/L; IgG4, 0.11 g/L at 2-3 years of age; IgG1, 6.33 g/L; IgG2, 1.8 g/L; IgG3, 0.2 g/L; IgG4, 0.21 g/L at age 3-4 years; IgG1, 7.05 g/L; IgG2, 1.87 g/L; IgG3, 0.25 g/L; IgG4, 0.29 g/L at 4-6 years of age; IgG1, 6.19 g/L; IgG2, 1.93 g/L; IgG3, 0.2 g/L; IgG4, 0.28 g/L at 6-9 years of age; IgG1, 6.76 g/L; IgG2, 2.29 g/L; IgG3, 0.27 g/L; IgG4, 0.37 g/L at 10-12 years of age; IgG1, 7.45 g/L; IgG2, 2.92 g/L; IgG3, 0.28 g/L; IgG4, 0.38 g/L at 13-16 years of age. CONCLUSION: To our knowledge, this study is the first to establish RIs for serum IgG subclasses exclusively in Chinese children.

DELLA proteins negatively regulate dark-induced senescence and chlorophyll degradation in Arabidopsis through interaction with the transcription factor WRKY6.

KEY MESSAGE: DELLA proteins' negative regulation of dark-induced senescence and chlorophyll degradation in Arabidopsis is through interaction with WRKY6 and thus repression of its transcriptional activities on senescence-related genes. Senescence is an intricate and highly orchestrated process regulated by numerous endogenous and environmental signals. Gibberellins (GAs) and their signaling components DELLA proteins have been known to participate in the regulation of senescence. However, the mechanism of the GA-DELLA system involved in the senescence process remains largely unclear. Darkness is a known environmental factor that induces plant senescence. In this study, exogenous GA3 (an active form of GA) accelerated but paclobutrazol (a specific GA biosynthesis inhibitor) retarded dark-induced leaf yellowing in Arabidopsis. Moreover, the dark-triggered decrease in chlorophyll content, increase in cell membrane leakage, and upregulation of senescence-associated genes were notably impaired in both endogenous GA-decreased mutants ga3ox1/ga3ox2 and ga20ox1/ga20ox2 compared with those in wild-type Col-0. These effects of darkness were enhanced in the quintuple mutant of DELLA genes gai-t6/rga-t2/rgl1-1/rgl2-1/rgl3-1 and conversely attenuated in the gain-of-function mutant gai and transgenic plant 35S::TAP-RGAd17 compared with wild-type Ler. Subsequently, RGA interacted with the transcription factor WRKY6 in a yeast two-hybrid assay, as confirmed by bimolecular fluorescence complementation and pull-down analyses. In addition, mutation and overexpression of WRKY6 retarded and accelerated dark-induced senescence, respectively. Furthermore, transient expression assays in Arabidopsis protoplasts indicated that RGA and GAI weakened the transcriptional activities of WRKY6 on its downstream senescence-related genes, including SAG13 and SGR. Taken together, these results suggest that GAs positively and DELLAs negatively regulate dark-induced senescence and chlorophyll degradation in Arabidopsis. DELLAs function in this process, at least in part, by interacting with WRKY6.

Adult reference intervals for IgG subclasses with Siemens immunonephelometric assays in Chinese population.

OBJECTIVE: To determine the adult reference intervals for the Siemens IgG subclass reagents. METHODS: 636 blood samples of healthy adults were analyzed to determine the level of IgG subclass using the reagents of Siemens immunonephelometric assay with molecular biology kits. RESULTS: IgGSc reference intervals were as follows: IgG1 4.45-9.76 g/L, IgG2 2.07-8.57 g/L, IgG3 0.08-0.80 g/L and IgG4 0.05-1.54 g/L. There was an excellent correlation between the total IgG and the sum of the IgG subclasses. No significant gender and age differences were observed. CONCLUSIONS: Our data provide the missing reference intervals and enable the use of the nephelometric IgG subclass reagents in Chinese. The study can offer reference on clinic diagnose.

GA-DELLA pathway is involved in regulation of nitrogen deficiency-induced anthocyanin accumulation.

KEY MESSAGE: DELLA proteins positively regulate nitrogen deficiency-induced anthocyanin accumulation through directly interaction with PAP1 to enhance its transcriptional activity on anthocyanin biosynthetic gene expressions. Plants can survive a limiting nitrogen supply by undergoing adaptive responses, including induction of anthocyanin production. However, the detailed mechanism is still unclear. In this study, we found that this process was impaired and enhanced, respectively, by exogenous GA3 (an active form of GAs) and paclobutrazol (PAC, a specific GA biosynthesis inhibitor) in Arabidopsis seedlings. Consistently, the nitrogen deficiency-induced transcript levels of several key genes involved in anthocyanin biosynthesis, including F3'H, DFR, LDOX, and UF3GT, were decreased and enhanced by exogenous GA3 and PAC, respectively. Moreover, the nitrogen deficiency-induced anthocyanin accumulation and biosynthesis gene expressions were impaired in the loss-of-function mutant gai-t6/rga-t2/rgl1-1/rgl2-1/rgl3-1 (della) but enhanced in the GA-insensitive mutant gai, suggesting that DELLA proteins, known as repressors of GA signaling, are necessary for fully induction of nitrogen deficiency-driven anthocyanin biosynthesis. Using yeast two-hybrid (Y2H) assay, pull-down assay, and luciferase complementation assay, it was found that RGA, a DELLA of Arabidopsis, could strongly interact with PAP1, a known regulatory transcription factor positively involved in anthocyanin biosynthesis. Furthermore, transient expression assays indicated that RGA and GAI could enhance the transcriptional activities of PAP1 on its downstream genes, including F3'H and DFR. Taken together, this study suggests that DELLAs are necessary regulators for nitrogen deficiency-induced anthocyanin accumulation through interaction with PAP1 and enhancement of PAP1's transcriptional activity on its target genes. GA-DELLA-involved anthocyanin accumulation is important for plant adaptation to nitrogen deficiency.

Characteristics of Allergic Pulmonary Inflammation in CXCR3Knockout Mice Sensitized and Challenged with House Dust Mite Protein.

Chemokine C-X-C motif receptor 3 (CXCR3) is a chemokine receptor that is mainly expressed by activated T lymphocytes. T cells play important roles in allergic pulmonary inflammation, which is a hallmark of asthma and elicits the localized accumulation of activated T cells in the lung. In China, a marked increase in the incidence rate of chronic allergic pulmonary inflammation has made it a major public health threat. In the present study, we investigated the role of CXCR3 and its ligands in airway inflammation induced by house dust mite protein (HDMP) in a CXCR3 knockout (CXCR3KO) asthma mouse model. Pathological manifestations in the lung, cell counts and bronchoalveolar lavage fluid (BALF) classifications were studied using hematoxylin and eosin (H&E) staining. The levels of IL-4 and IFN-γ in the BALF and splenocyte supernatants were measured using ELISA. CD4+ and CD8+ T cells in the lung and spleen were analyzed by flow cytometry. RT-PCR was applied to measure the mRNA transcript levels of monokines induced by IFN-γ(CXCL9) and IFN-γ inducible protein 10(CXCL10). The total cell counts, eosinophil counts, and IL-4 levels in the BALF and cultured splenocyte supernatants were significantly increased, while the levels of IFN-γ were reduced in the HDMP groups(P<0.01). Changes in the total cell counts, eosinophil counts, and lymphocyte counts, as well as the total protein levels in the BALF, the levels of IL-4 in splenocyte supernatants, and the pathological manifestations in the lung, were all greater in CXCR3KO mice than in C57BL/6 wild-type mice. Furthermore, the expression levels of CXCL9 and CXCL10 mRNA transcripts in the lungs of CXCR3KO mice were lower than those in C57BL/6 wild-type mice (P<0.05). CXCR3 and its ligands (i.e., CXCL9 and CXCL10) may play anti-inflammatory roles in this animal model. Promoting the expression of CXCR3 and its ligands may represent a novel therapeutic approach for preventing and curing asthma.

Phytochrome-interacting factors PIF4 and PIF5 negatively regulate anthocyanin biosynthesis under red light in Arabidopsis seedlings.

Light is an important environmental factor inducing anthocyanin accumulation in plants. Phytochrome-interacting factors (PIFs) have been shown to be a family of bHLH transcription factors involved in light signaling in Arabidopsis. Red light effectively increased anthocyanin accumulation in wild-type Col-0, whereas the effects were enhanced in pif4 and pif5 mutants but impaired in overexpression lines PIF4OX and PIF5OX, indicating that PIF4 and PIF5 are both negative regulators for red light-induced anthocyanin accumulation. Consistently, transcript levels of several genes involved in anthocyanin biosynthesis and regulatory pathway, including CHS, F3'H, DFR, LDOX, PAP1 and TT8, were significantly enhanced in mutants pif4 and pif5 but decreased in PIF4OX and PIF5OX compared to in Col-0, indicating that PIF4 and PIF5 are transcriptional repressor of these gene. Transient expression assays revealed that PIF4 and PIF5 could repress red light-induced promoter activities of F3'H and DFR in Arabidopsis protoplasts. Furthermore, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) test and electrophoretic mobility shift assay (EMSA) showed that PIF5 could directly bind to G-box motifs present in the promoter of DFR. Taken together, these results suggest that PIF4 and PIF5 negatively regulate red light-induced anthocyanin accumulation through transcriptional repression of the anthocyanin biosynthetic genes in Arabidopsis.

PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) positively regulates dark-induced senescence and chlorophyll degradation in Arabidopsis.

Darkness is a known environmental factor that induces plant senescence. Here, Phytochrome-Interacting Factors (PIFs), several bHLH transcription factors involved in plant skotomorphogenesis, were examined for their roles in the regulation of dark-induced senescence and chlorophyll breakdown in Arabidopsis thaliana. After light-grown seedlings were transferred to darkness, green leaves turned yellow, and chlorophyll contents decreased, but membrane lipid peroxidation and cell death increased in wild-type Col-0. These responses were enhanced in overexpression line PIF5OX but decreased in mutant pif5-3. Darkness significantly induced expression of several genes involved in chlorophyll breakdown, including SGR, NYC1, NOL, and PAO, as well as genes encoding for transcription factors that have been shown to be required for dark-induced senescence, including WRKY22, NAP, EIN3, EIL1, and ORE1. These effects on gene expression were also enhanced in PIF5OX but decreased in pif5-3 relative to Col-0. Further analyses using ChIP-qPCR, EMSA, and protoplast transient assays indicated that PIF5 binds to the G-box motifs in the promoters of SGR, NYC1, and ORE1 genes and stimulate their expression. Collectively, our data indicate that PIF5 is a key factor that positively regulates dark-induced senescence upstream of ORE1 and regulates chlorophyll breakdown upstream of SGR and NYC1.

Brassinosteroid is required for sugar promotion of hypocotyl elongation in Arabidopsis in darkness.

MAIN CONCLUSION: Brassinosteroid is necessary for sugar promotion of Arabidopsis hypocotyl elongation in darkness, and sugar positively regulates BRASSINAZOLE RESISTANT1 (BZR1) at both transcription and protein levels. Sugar has the ability to induce Arabidopsis hypocotyl elongation in the dark, but the detailed mechanisms remain not well understood. Here, we report that the steroidal phytohormone brassinosteroid (BR) is involved in sugar promotion of hypocotyl elongation in the dark. Sugar-induced hypocotyl elongation was significantly repressed in the BR-deficient mutant det2-1, BR-insensitive mutant bri1-5, and wild-type plants (Col-0), but not in the BR-hypersensitive mutants bzr1-1D and bes1-D treated with the BR biosynthetic inhibitor brassinazole (BRZ). Sugar also up-regulated the expression of genes that are related to cell elongation in a BR-dependent manner, and this effect was more remarkable in bzr1-1D and bes1-D than in their corresponding wild types in the presence of BRZ, suggesting an important role of BZR1 and bri1-ems-suppressor 1 (BES1) in this process. Sugar treatment seems to have little effect on BR biosynthesis, but enhances the expression of BZR1 and BES1, two transcription factors in BR signaling, in the dark. Furthermore, sugar treatment maintains higher BZR1 protein levels in plants grown in the dark. Collectively, our results indicate that BR is required for sugar promotion of hypocotyl elongation in darkness in Arabidopsis.

Improvement of specific detection of circulating tumor cells using combined CD45 staining and fluorescence in situ hybridization.

BACKGROUND: Conventional cytokeratin (CK)-based methods to detect circulating tumor cells (CTCs) often present suboptimal sensitivities for decreased or non-expression of cytokeratin in some CTCs. We clinically investigated a new method that combines immunocytochemistry staining (ICC) of CD45 and fluorescence in situ hybridization (FISH). METHODS: Circulating epithelial cells from 141 subjects were enriched using an EpCAM-independent strategy and then identified by either a combination of FISH with chromosome 8 centromere probe (CEP8) and ICC staining of CD45 (CD45-FISH) or ICC staining of CK. RESULTS: For detecting CTCs enriched from lung cancers, CD45-FISH had larger areas under ROC curves of 0.963 (P=0.000) compared to ICC (0.653; P=0.031) using cut-off values of 2 and 1 cell/3.75ml blood with sensitivities of 83.3% and 43.3%, specificities of 98.6% and 89.5%, respectively. Moreover, CD45-FISH showed 76.2% sensitivity in detecting CTCs in ovarian cancers (P<0.001). Four of six ovarian cancers showed dramatical decrease in both CTCs and serum CA125 on the 7th day after surgery. CONCLUSION: CD45-FISH method had improved sensitivity and specificity in detecting CTCs of lung and ovarian cancers compared to ICC-CK. This combined detection strategy may be useful in detecting or monitoring CTCs after ovarian cancer surgery.

[Relationship among the expression of GSK3ß, PI3K/Akt, and IL-6 in chronic rhinosinusitis].

OBJECTIVE: To explore the relationship among the expression of GSK3ß, PI3K/Akt signaling transduction pathway,and cytokines IL6 in Chronic Rhinosinusitis. METHODS: We assayed mRNA and protein for GSK3ß, PI3K, Akt by using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), and measured the cytokines IL6 mRNA by using reverse transcription polymerase chain reaction (RT-PCR) in the nasal tissue from the patients with Chronic Rhinosinusitis with Nasal Polyps (CRSwNP), Chronic Rhinosinusitis without Nasal Polyps (CRSsNP) and control subjects. RESULTS: The relative expression levels of GSK3ß, PI3K, Akt and IL-6 in CRSwNP were 0.6254 ± 0.0584, 0.8239 ± 0.7186, 0.9369 ± 0.0823 and 0.8973 ± 0.0680. But the relative expression levels of GSK3ß, PI3K, Akt and IL-6 in control subjects were 0.2684 ± 0.0726, 0.3578 ± 0.0994, 0.6721 ± 0.0590, 0.5898 ± 0.0891. There were significant differences between the groups of CRSwNP and control subjects (t values were 2.358, 3.071, 2.764, and 2.239, respectively, all P < 0.05). There was no significant difference of GSK3ß, PI3K, Akt and GSK3ß between the groups of CRSwNP and CRSsNP (t values were 1.597, 1.842, 1.468 and 0.926, respectively, all P < 0.05). GSK3ß, PI3K, Akt were not only expressed in the cytoplasm of the epithelium and gland cells, but also in the cytoplasm of the inflammatory cell. GSK3ß, PI3K, Akt protein in CRS were detected at a higher rate than the normal nasal tissue (values were 16.42, 16.25 and 15.57,respectively, all P < 0.01). However there was no significant difference of GSK3ß, PI3K, Akt protein between the groups of CRSwNP and CRSsNP (values were 3.27, 2.85 and 2.46, respectively, all P > 0.05). There was a positive correlation trend among the expression of GSK3ß, PI3K and Akt, and IL-6 in CRS (r values were 0.645, 0.617 and 0.583,respectively, all P < 0.01). CONCLUSIONS: The abnormal expression of IL-6, PI3K, Akt and GSK3ß in the nasal mucosa of CRS may play a pro-inflammatory role in the occurrence and development of CRS.