Adaptive responses of carbon and nitrogen metabolisms to nitrogen-deficiency in Citrus sinensis seedlings.

BACKGROUND: In China, nitrogen (N)-deficiency often occurs in Citrus orchards, which is one of the main causes of yield loss and fruit quality decline. Little information is known about the adaptive responses of Citrus carbon (C) and N metabolisms to N-deficiency. Seedlings of 'Xuegan' (Citrus sinensis (L.) Osbeck) were supplied with nutrient solution at an N concentration of 0 (N-deficiency), 5, 10, 15 or 20 mM for 10 weeks. Thereafter, we examined the effects of N supply on the levels of C and N in roots, stems and leaves, and the levels of organic acids, nonstructural carbohydrates, NH4+-N, NO3--N, total soluble proteins, free amino acids (FAAs) and derivatives (FAADs), and the activities of key enzymes related to N assimilation and organic acid metabolism in roots and leaves. RESULTS: N-deficiency elevated sucrose export from leaves to roots, C and N distributions in roots and C/N ratio in roots, stems and leaves, thus enhancing root dry weight/shoot dry weight ratio and N use efficiency. N-deficient leaves displayed decreased accumulation of starch and total nonstructural carbohydrates (TNC) and increased sucrose/starch ratio as well as a partitioning trend of assimilated C toward to sucrose, but N-deficient roots displayed elevated accumulation of starch and TNC and reduced sucrose/starch ratio as well as a partitioning trend of assimilated C toward to starch. N-deficiency reduced the concentrations of most FAADs and the ratios of total FAADs (TFAADs)/N in leaves and roots. N-deficiency reduced the demand for C skeleton precursors for amino acid biosynthesis, thus lowering TFAADs/C ratio in leaves and roots. N-deficiency increased (decreased) the relative amounts of C-rich (N-rich) FAADs, thus increasing the molar ratio of C/N in TFAADs in leaves and roots. CONCLUSIONS: Our findings corroborated our hypothesis that C and N metabolisms displayed adaptive responses to N-deficiency in C. sinensis seedlings, and that some differences existed between roots and leaves in N-deficiency-induced alterations of and C and N metabolisms.

Associations between Physical Activity Trajectories and Incident Hypertension.

Purpose: We aimed to characterize physical activity (PA) trajectories across adulthood and to estimate their association with incident hypertension risk. Methods: Data were obtained from the China Health and Nutrition Survey (CHNS) conducted during 2004-2011. Group-based trajectory modeling (GBTM) was used to identify distinct groups of PA trajectories. The Cox proportional hazards model was used to investigate the association. Results: A total of 11,162 participants whose PA was repeatedly estimated by self-report from questionnaires two to four times in the CHNS were included in our study. During the 5.4 years of follow-up, 3824 incident hypertension cases were identified. Five distinct PA trajectories were identified in men: light and slight decline, light and gradual decline then sharp rise, light to medium-heavy then decline, medium-heavy and gradual decline, and heavy and sharp decline. Two distinct PA trajectories were identified in women: light and stable, and medium and gradual decline. The PA trajectory of medium-heavy and gradual decline was significantly associated with decreased risk of hypertension in men, with the hazard ratios and 95% confidence intervals (CI) being 0.80 (0.63, 0.99), 0.74 (0.59, 0.93), 0.76 (0.60, 0.96), and 0.70 (0.55, 0.88) in models 1-4, respectively. Conclusions: Our study identified five distinct long-term PA trajectories in men and two distinct trajectories in women. The PA trajectory of medium-heavy PA in early adulthood followed by gradual decline was found to be significantly associated with a decreased risk of hypertension in later life in men.

Boron-mediated amelioration of copper-toxicity in sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] seedlings involved reduced damage to roots and improved nutrition and water status.

'Xuegan' (Citrus sinensis) seedlings were fertilized 6 times weekly for 24 weeks with 0.5 or 350 µM CuCl2 and 2.5, 10 or 25 µM H3BO3. Cu-toxicity increased Cu uptake per plant (UPP) and Cu concentrations in leaves, stems and roots, decreased water uptake and phosphorus, nitrogen, calcium, magnesium, potassium, sulfur, boron and iron UPP, and increased the ratios of magnesium, potassium, calcium and sulfur UPP to phosphorus UPP and the ratios of leaf magnesium, potassium and calcium concentrations to leaf phosphorus concentration. Many decaying and dead fibrous roots occurred in Cu-toxic seedlings. Cu-toxicity-induced alterations of these parameters and root damage decreased with the increase of boron supply. These results demonstrated that B supplementation lowered Cu uptake and its concentrations in leaves, stems and roots and subsequently alleviated Cu-toxicity-induced damage to root growth and function, thus improving plant nutrient (decreased Cu uptake and efficient maintenance of the other nutrient homeostasis and balance) and water status. Further analysis indicated that the improved nutrition and water status contributed to the boron-mediated amelioration of Cu-toxicity-induced inhibition of seedlings, decline of leaf pigments, large reduction of leaf CO2 assimilation and impairment of leaf photosynthetic electron transport chain revealed by greatly altered chlorophyll a fluorescence (OJIP) transients, reduced maximum quantum yield of primary photochemistry (Fv/Fm), quantum yield for electron transport (ETo/ABS) and total performance index (PIabs,total), and elevated dissipated energy per reaction center (DIo/RC). To conclude, our findings corroborate the hypothesis that B-mediated amelioration of Cu-toxicity involved reduced damage to roots and improved nutrient and water status. Principal component analysis showed that Cu-toxicity-induced changes of above physiological parameters generally decreased with the increase of B supply and that B supply-induced alterations of above physiological parameters was greater in 350 µM Cu-treated than in 0.5 µM Cu-treated seedlings. B and Cu had a significant interactive influence on C. sinensis seedlings.

High pH Alleviated Sweet Orange (Citrus sinensis) Copper Toxicity by Enhancing the Capacity to Maintain a Balance between Formation and Removal of Reactive Oxygen Species and Methylglyoxal in Leaves and Roots.

The contribution of reactive oxygen species (ROS) and methylglyoxal (MG) formation and removal in high-pH-mediated alleviation of plant copper (Cu)-toxicity remains to be elucidated. Seedlings of sweet orange (Citrus sinensis) were treated with 0.5 (non-Cu-toxicity) or 300 (Cu-toxicity) µM CuCl2 × pH 4.8, 4.0, or 3.0 for 17 weeks. Thereafter, superoxide anion production rate; H2O2 production rate; the concentrations of MG, malondialdehyde (MDA), and antioxidant metabolites (reduced glutathione, ascorbate, phytochelatins, metallothioneins, total non-protein thiols); and the activities of enzymes (antioxidant enzymes, glyoxalases, and sulfur metabolism-related enzymes) in leaves and roots were determined. High pH mitigated oxidative damage in Cu-toxic leaves and roots, thereby conferring sweet orange Cu tolerance. The alleviation of oxidative damage involved enhanced ability to maintain the balance between ROS and MG formation and removal through the downregulation of ROS and MG formation and the coordinated actions of ROS and MG detoxification systems. Low pH (pH 3.0) impaired the balance between ROS and MG formation and removal, thereby causing oxidative damage in Cu-toxic leaves and roots but not in non-Cu-toxic ones. Cu toxicity and low pH had obvious synergistic impacts on ROS and MG generation and removal in leaves and roots. Additionally, 21 (4) parameters in leaves were positively (negatively) related to the corresponding root parameters, implying that there were some similarities and differences in the responses of ROS and MG metabolisms to Cu-pH interactions between leaves and roots.

Capilliposide from Lysimachia capillipes inhibits AKT activation and restores gefitinib sensitivity in human non-small cell lung cancer cells with acquired gefitinib resistance.

Most gefitinib-treated patients with non-small cell lung cancer (NSCLC) would eventually develop resistance. Lysimachia capillipes (LC) capilliposide extracts from LC Hemsl. show both in vitro and in vivo anti-cancer effects. In this study we investigated whether LC capilliposide in combination with gefitinib could overcome the resistance of NSCLC cells to gefitinib and identified the signaling pathways involved. Treatment with LC capilliposide alone inhibited the growth of a panel of NSCLC cell lines (PC-9, H460, H1975, H1299 and PC-9-GR) sensitive or resistant to gefitinib with IC50 values in the range of µg/mL. In the gefitinib-resistant PC-9-GR cells (which have a T790M EGFR mutation), LC capilliposide (at the IC30, i.e.1.2 µg/mL) markedly enhanced the inhibitory effects of gefitinib with its IC50 value being decreased from 6.80±1.00 to 0.77±0.12 µmol/L. By using the median effect analysis we showed that combination treatment of LC capilliposide and gefitinib could restore gefitinib sensitivity in PC-9-GR cells. Furthermore, LC capilliposide (1.2 µg/mL) significantly increased the apoptotic responses to gefitinib (0.77 µmol/L) in PC-9-GR cells, but did not affect gefitinib-induced G0/G1 arrest. Moreover, LC capilliposide (1.2 µg/mL) in combination with gefitinib (0.77, 1.0 µmol/L) markedly decreased the phosphorylation of the EGFR downstream signaling molecule AKT, which neither LC capilliposide nor gefitinib alone affected. In PC-9-GR cells with siRNA knockdown of AKT, addition of LC capilliposide was unable to increase gefitinib sensitivity. In a PC-9-GR xenograft mouse model, combination treatment with LC capilliposide (15 mg·kg-1·d-1, ip) and gefitinib (50 mg·kg-1·d-1, ip) dramatically enhanced tumor growth suppression (with a TGI of 109.3%), compared with TGIs of 22.6% and 56.6%, respectively, in mice were treated with LC capilliposide or gefitinib alone. LC capilliposide can restore the cells' sensitivity to gefitinib through modulation of pAKT levels, suggesting that a combination of LC capilliposide and gefitinib may be a promising therapeutic strategy to overcome gefitinib resistance in NSCLCs with a T790M mutation.

Hospital­acquired pneumonia caused by Kodamaea ohmeri during extracorporeal membrane oxygenation treatment: A case report and literature review.

Kodamaea ohmeri (K. ohmeri) is an ascosporogenic species of yeast that belongs to the genus Ascosporogenous and the family of Saccharomycetaceae. It has recently been found to cause various types of infections, particularly in critically ill immunocompromised patients. The present study describes a case of hospital-acquired pneumonia caused by K. ohmeri during veno-arterial extracorporeal membrane oxygenation. The fungal culture turned negative after the administration of caspofungin and amphotericin B. Extracorporeal membrane oxygenation (ECMO) is an adjunctive medical technique that provides temporary cardiopulmonary support for patients. Previous observations have suggested that the immune function of patients will typically decline during the use of ECMO, rendering infection to be one of the main complications of ECMO. K. ohmeri is a rare pathogenic fungus, particularly in immunocompromised individuals with vascular catheters, while amphotericin B is the most common antifungal therapy administered to treat K. ohmeri infections. It is important to raise awareness of rare fungal infections and actively treat them.

Boron Reduced Copper Excess-Induced Oxidative Damage in Citrus sinensis by Modulating Reactive Oxygen Species and Methylglyoxal Formation and Their Detoxification Systems.

Citrus is mainly cultivated in acid soil with low boron (B) and high copper (Cu). In this study, Citrus sinensis seedlings were submitted to 0.5 (control) or 350 µM Cu (Cu excess or Cu exposure) and 2.5, 10, or 25 µM B for 24 weeks. Thereafter, H2O2 production rate (HPR), superoxide production rate (SAPR), malondialdehyde, methylglyoxal, and reactive oxygen species (ROS) and methylglyoxal detoxification systems were measured in leaves and roots in order to test the hypothesis that B addition mitigated Cu excess-induced oxidative damage in leaves and roots by reducing the Cu excess-induced formation and accumulation of ROS and MG and by counteracting the impairments of Cu excess on ROS and methylglyoxal detoxification systems. Cu and B treatments displayed an interactive influence on ROS and methylglyoxal formation and their detoxification systems. Cu excess increased the HPR, SAPR, methylglyoxal level, and malondialdehyde level by 10.9% (54.3%), 38.9% (31.4%), 50.3% (24.9%), and 312.4% (585.4%), respectively, in leaves (roots) of 2.5 µM B-treated seedlings, while it only increased the malondialdehyde level by 48.5% (97.8%) in leaves (roots) of 25 µM B-treated seedlings. Additionally, B addition counteracted the impairments of Cu excess on antioxidant enzymes, ascorbate-glutathione cycle, sulfur metabolism-related enzymes, sulfur-containing compounds, and methylglyoxal detoxification system, thereby protecting the leaves and roots of Cu-exposed seedlings against oxidative damage via the coordinated actions of ROS and methylglyoxal removal systems. Our findings corroborated the hypothesis that B addition alleviated Cu excess-induced oxidative damage in leaves and roots by decreasing the Cu excess-induced formation and accumulation of ROS and MG and by lessening the impairments of Cu excess on their detoxification systems. Further analysis indicated that the pathways involved in the B-induced amelioration of oxidative stress caused by Cu excess differed between leaves and roots.

The underlying mechanisms by which boron mitigated copper toxicity in Citrus sinensis leaves revealed by integrated analysis of transcriptome, metabolome, and physiology.

Both copper (Cu) excess and boron (B) deficiency are often observed in some citrus orchard soils. The molecular mechanisms by which B alleviates excessive Cu in citrus are poorly understood. Seedlings of sweet orange (Citrus sinensis (L.) Osbeck cv. Xuegan) were treated with 0.5 (Cu0.5) or 350 (Cu350 or Cu excess) µM CuCl2 and 2.5 (B2.5) or 25 (B25) µM HBO3 for 24 weeks. Thereafter, this study examined the effects of Cu and B treatments on gene expression levels revealed by RNA-Seq, metabolite profiles revealed by a widely targeted metabolome, and related physiological parameters in leaves. Cu350 upregulated 564 genes and 170 metabolites, and downregulated 598 genes and 58 metabolites in leaves of 2.5 µM B-treated seedlings (LB2.5), but it only upregulated 281 genes and 100 metabolites, and downregulated 136 genes and 40 metabolites in leaves of 25 µM B-treated seedlings (LB25). Cu350 decreased the concentrations of sucrose and total soluble sugars, and increased the concentrations of starch, glucose, fructose, and total nonstructural carbohydrates (TNC) in LB2.5, but it only increased the glucose concentration in LB25. Further analysis demonstrated that B addition reduced the oxidative damage and alterations in primary and secondary metabolisms caused by Cu350; and alleviated the impairment of Cu350 to photosynthesis and cell wall metabolism, thus improving leaf growth. LB2.5 exhibited some adaptive responses to Cu350 to meet the increasing need for the dissipation of excessive excitation energy (EEE) and the detoxification of reactive oxygen species (reactive aldehydes) and Cu. Cu350 increased photorespiration, xanthophyll cycle-dependent thermal dissipation, nonstructural carbohydrate accumulation, and secondary metabolite biosynthesis and abundances; and upregulated tryptophan metabolism and related metabolite abundances, and some antioxidant-related gene expression, and some antioxidant abundances. Additionally, this study identified some metabolic pathways, metabolites, and genes that might lead to Cu tolerance in leaves.

Boron-mediated amelioration of copper toxicity in Citrus sinensis seedlings involved reduced concentrations of copper in leaves and roots and their cell walls rather than increased copper fractions in their cell walls.

Little information is available on how boron (B) supplementation affects plant cell wall (CW) remodeling under copper (Cu) excess. 'Xuegan' (Citrus sinensis) seedlings were submitted to 0.5 or 350 µM Cu × 2.5 or 25 µM B for 24 weeks. Thereafter, we determined the concentrations of CW materials (CWMs) and CW components (CWCs), the degree of pectin methylation (DPM), and the pectin methylesterase (PME) activities and PME gene expression levels in leaves and roots, as well as the Cu concentrations in leaves and roots and their CWMs (CWCs). Additionally, we analyzed the Fourier transform infrared (FTIR) and X-ray diffraction (XRD) spectra of leaf and root CWMs. Our findings suggested that adding B reduced the impairment of Cu excess to CWs by reducing the Cu concentrations in leaves and roots and their CWMs and maintaining the stability of CWs, thereby improving leaf and root growth. Cu excess increased the Cu fractions in leaf and root pectin by decreasing DPM due to increased PME activities, thereby contributing to citrus Cu tolerance. FTIR and XRD indicated that the functional groups of the CW pectin, hemicellulose, cellulose, and lignin could bind and immobilize Cu, thereby reducing Cu cytotoxicity in leaves and roots.

Citrus sinensis manganese tolerance: Insight from manganese-stimulated secretion of root exudates and rhizosphere alkalization.

We used manganese (Mn)-tolerant 'Xuegan' (Citrus sinensis) seedlings as materials and examined the characterization of Mn uptake and Mn-activated-release of root exudates under hydroponic conditions. We observed that root and shoot Mn bioaccumulation factor (BCF) reduced with the increase of Mn supply, and that Mn transfer factor (Tf) reduced greatly as Mn supply increased from 0 to 500 µM, beyond which Tf slightly increased with increasing Mn supply, suggesting that Mn supply reduced the ability to absorb and accumulate Mn in roots and shoots, as well as root-to-shoot Mn translocation. Without Mn, roots alkalized the solution pH from 5.0 to above 6.2, while Mn supply reduced root-induced alkalization. As Mn supply increased from 0 to 2000 µM, the secretion of root total phenolics (TPs) increased, while the solution pH decreased. Mn supply did not alter the secretion of root total free amino acids, total soluble sugars, malate, and citrate. Mn-activated-release of TPs was inhibited by low temperature and anion channel inhibitors, but not by protein biosynthesis inhibitor. Using widely targeted metabolome, we detected 48 upregulated [35 upregulated phenolic compounds + 13 other secondary metabolites (SMs)] and three downregulated SMs, and 39 upregulated and eight downregulated primary metabolites (PMs). These findings suggested that reduced ability to absorb and accumulate Mn in roots and shoots and less root-to-shoot Mn translocation in Mn-toxic seedlings, rhizosphere alkalization, and Mn-activated-release of root exudates (especially phenolic compounds) contributed to the high Mn tolerance of C. sinensis seedlings.

Spatial multiomics reveals a subpopulation of fibroblasts associated with cancer stemness in human hepatocellular carcinoma.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are the prominent cell type in the tumor microenvironment (TME), and CAF subsets have been identified in various tumors. However, how CAFs spatially coordinate other cell populations within the liver TME to promote cancer progression remains unclear. METHODS: We combined multi-region proteomics (6 patients, 24 samples), 10X Genomics Visium spatial transcriptomics (11 patients, 25 samples), and multiplexed imaging (92 patients, 264 samples) technologies to decipher the expression heterogeneity, functional diversity, spatial distribution, colocalization, and interaction of fibroblasts. The newly identified CAF subpopulation was validated by cells isolated from 5 liver cancer patients and in vitro functional assays. RESULTS: We identified a liver CAF subpopulation, marked by the expression of COL1A2, COL4A1, COL4A2, CTGF, and FSTL1, and named F5-CAF. F5-CAF is preferentially located within and around tumor nests and colocalizes with cancer cells with higher stemness in hepatocellular carcinoma (HCC). Multiplexed staining of 92 patients and the bulk transcriptome of 371 patients demonstrated that the abundance of F5-CAFs in HCC was associated with a worse prognosis. Further in vitro experiments showed that F5-CAFs isolated from liver cancer patients can promote the proliferation and stemness of HCC cells. CONCLUSIONS: We identified a CAF subpopulation F5-CAF in liver cancer, which is associated with cancer stemness and unfavorable prognosis. Our results provide potential mechanisms by which the CAF subset in the TME promotes the development of liver cancer by supporting the survival of cancer stem cells.

A distinct tumor microenvironment makes anaplastic thyroid cancer more lethal but immunotherapy sensitive than papillary thyroid cancer.

Both anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) originate from thyroid follicular epithelial cells, but ATC has a significantly worse prognosis and shows resistance to conventional therapies. However, clinical trials found that immunotherapy works better in ATC than late-stage PTC. Here, we used single-cell RNA sequencing (scRNA-Seq) to generate a single-cell atlas of thyroid cancer. Differences in ATC and PTC tumor microenvironment components (including malignant cells, stromal cells, and immune cells) leading to the polarized prognoses were identified. Intriguingly, we found that CXCL13+ T lymphocytes were enriched in ATC samples and might promote the development of early tertiary lymphoid structure (TLS). Last, murine experiments and scRNA-Seq analysis of a treated patient's tumor demonstrated that famitinib plus anti-PD-1 antibody could advance TLS in thyroid cancer. We displayed the cellular landscape of ATC and PTC, finding that CXCL13+ T cells and early TLS might make ATC more sensitive to immunotherapy.

Roles of Hormones in Elevated pH-Mediated Mitigation of Copper Toxicity in Citrus sinensis Revealed by Targeted Metabolome.

The effects of copper (Cu)-pH interactions on the levels of hormones and related metabolites (HRMs) in Citrus sinensis leaves and roots were investigated. Our findings indicated that increased pH mitigated Cu toxicity-induced alterations of HRMs, and Cu toxicity increased low-pH-induced alterations of HRMs. Increased pH-mediated decreases in ABA, jasmonates, gibberellins, and cytokinins, increases in (±)strigol and 1-aminocyclopropanecarboxylic acid, and efficient maintenance of salicylates and auxins homeostasis in 300 µM Cu-treated roots (RCu300); as well as efficient maintenance of hormone homeostasis in 300 µM Cu-treated leaves (LCu300) might contribute to improved leaf and root growth. The upregulation of auxins (IAA), cytokinins, gibberellins, ABA, and salicylates in pH 3.0 + 300 µM Cu-treated leaves (P3CL) vs. pH 3.0 + 0.5 µM Cu-treated leaves (P3L) and pH 3.0 + 300 µM Cu-treated roots (P3CR) vs. pH 3.0 + 0.5 µM Cu-treated roots (P3R) might be an adaptive response to Cu toxicity, so as to cope with the increased need for reactive oxygen species and Cu detoxification in LCu300 and RCu300. Increased accumulation of stress-related hormones (jasmonates and ABA) in P3CL vs. P3L and P3CR vs. P3R might reduce photosynthesis and accumulation of dry matter, and trigger leaf and root senescence, thereby inhibiting their growth.

A resting-state functional magnetic resonance imaging study of altered functional brain activity in cardiac arrest survivors with good neurological outcome.

Purpose: To investigate the spontaneous brain activity alterations in survivors of cardiac arrest (CA) with good neurological outcome using resting-state functional magnetic resonance imaging (rs-fMRI) with amplitude of low-frequency fluctuation (ALFF) and regional homogeneity (ReHo) methods. Materials and methods: Thirteen CA survivors with favorable neurological outcomes and 13 healthy controls (HCs) were recruited and underwent rs-fMRI scans. The ALFF and ReHo methods were applied to assess the regional intensity and synchronization of spontaneous brain activity. Correlation analyses were performed to explore the relationships between the mean ALFF and ReHo values in significant clusters and clinical parameters. Results: The survivors of CA showed significantly decreased ALFF values in the left postcentral gyrus and precentral gyrus and increased ALFF values in the left hippocampus and parahippocampal gyrus than HCs. Significantly decreased ReHo values were observed in the left inferior occipital gyrus and middle occipital gyrus in the patients. Mean ALFF values in the left hippocampus and parahippocampal gyrus were positively correlated with the time to return of spontaneous circulation (r = 0.794, p = 0.006) in the patient group. Conclusion: Functional activity alterations in the brain areas corresponding to known cognitive and physical impairments were observed in CA survivors with preserved neurological function. Our results could advance the understanding of the neurological mechanisms underlying the residual deficits in those patients.

Genome-wide methylation profiling of diagnostic tumor specimens identified DNA methylation markers associated with metastasis among men with untreated localized prostate cancer.

BACKGROUND: We used a genome-wide discovery approach to identify methylation markers associated with metastasis in men with localized prostate cancer (PCa), as better identification of those at high risk of metastasis can inform treatment decision-making. METHODS: We identified men with localized PCa at Kaiser Permanente California (January 1, 1997-December 31, 2006) who did not receive curative treatment and followed them for 10 years to determine metastasis status. Cases were chart review-confirmed metastasis, and controls were matched using density sampling. We extracted DNA from the cancerous areas in the archived diagnostic tissue blocks. We used Illumina's Infinium MethylationEPIC BeadChip for methylation interrogation. We used conditional logistic regression and Bonferroni's correction to identify methylation markers associated with metastasis. In a separate validation cohort (2007), we evaluated the added predictive utility of the methylation score beyond clinical risk score. RESULTS: Among 215 cases and 404 controls, 31 CpG sites were significantly associated with metastasis status. Adding the methylation score to the clinical risk score did not meaningfully improve the c-statistic (0.80-0.81) in the validation cohort, though the score itself was statistically significant (p < 0.01). In the validation cohort, both clinical risk score alone and methylation marker score alone are well calibrated for predicted 10-year metastasis risks. Adding the methylation score to the clinical risk score only marginally improved predictive risk calibration. CONCLUSION: Our findings do not support the use of these markers to improve clinical risk prediction. The methylation markers identified may inform novel hypothesis in the roles of these genetic regions in metastasis development.

Regulation on copper-tolerance in Citrus sinensis seedlings by boron addition: Insights from root exudates, related metabolism, and gene expression.

Boron (B) can alleviate Citrus copper (Cu)-toxicity. However, the underlying mechanism by which B mitigates Cu-toxicity is unclear. 'Xuegan' (Citrus sinensis) seedlings were exposed to 0.5 (control) or 350 (Cu-toxicity) µM Cu and 2.5 or 25 µM B for 24 weeks. Thereafter, we investigated the secretion of low molecular weight compounds [LMWCs; citrate, malate, total soluble sugars (TSS), total phenolics (TP), and total free amino acids (TFAA)] by excised roots and their concentrations in roots and leaves, as well as related enzyme gene expression and activities in roots and leaves. Cu-stress stimulated root release of malate and TFAA, which might contribute to citrus Cu-tolerance. However, B-mediated-mitigation of Cu-stress could not be explained in this way, since B addition failed to further stimulate malate and TFAA secretion. Indeed, B addition decreased Cu-stimulated-secretion of malate. Further analysis suggested that Cu-induced-exudation of malate and TFAA was not regulated by their levels in roots. By contrast, B addition increased malate, citrate, and TFAA concentrations in Cu-toxic roots. Cu-toxicity increased TP concentration in 25 µM B-treated leaves, but not in 2.5 µM B-treated leaves. Our findings suggested that the internal detoxification of Cu by LMWCs played a role in B-mediated-alleviation of Cu-toxicity.

Characterization of copper-induced-release of exudates by Citrus sinensis roots and their possible roles in copper-tolerance.

Copper (Cu) excess is often observed in old Citrus orchards. Little information is available on the characterization of Cu-induced-release of root exudates and their possible roles in plant Cu-tolerance. Using sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] seedlings as materials, we investigated the impacts of 0, 0.5, 25, 150, 350, 550, 1000, 2000 or 5000 µM CuCl2 (pH 4.8) on Cu uptake, root exudates [malate, citrate, total phenolics (TP), total soluble sugars (TSS) and total free amino acids (TFAA)], electrolyte leakage and malondialdehyde, and solution pH under hydroponic conditions; the time-course of root exudates and solution pH in response to Cu; and the impacts of protein synthesis and anion-channel inhibitors, and temperature on Cu-induced-secretion of root exudates and solution pH. About 70% of Cu was accumulated in 0 and 0.5 µM Cu-exposed roots, while over 97% of Cu was accumulated in ≥25 µM Cu-exposed roots. Without Cu, the seedlings could alkalize the solution pH from 4.8 to above 6.0. Cu-stimulated-secretion of root exudates elevated with the increment of Cu concentration from 0 to 1000 µM, then decreased or remained unchanged with the further increment of Cu concentration, while root electrolyte leakage and malondialdehyde (root-induced alkalization) increased (lessened) with the increment of Cu concentration from 0 to 5000 µM. Further analysis indicated that Cu-stimulated-secretion of root exudates was an energy-dependent process and could repressed by inhibitors, and that there was no discernible delay between the onset of exudate release and the addition of Cu. To conclude, both root-induced alkalization and Cu-stimulated-release of root exudates played a key role in sweet orange Cu-tolerance via increasing root Cu accumulation and reducing Cu uptake and phytotoxicity.

[Inhibitory acting mechanism of psoralen-osthole on bone metastasis of breast cancer--an expatiation viewing from OPG/RANKL/RANK system].

OBJECTIVE: To find the optimal proportion of Composite Fructus Psoralea and Fructus Cnidii (CFPC) for inhibiting the bone metastasis of breast cancer by way of exploring its acting mechanism viewing from OPG/RANKL/RANK system. METHODS: The human bone metastasis of breast cancer model was established by injecting tumor cells of MDA-MB-231BO cell line into the left cardiac ventricle of nude mice. The modeled mice were randomly divided into seven groups: the blank group administered with normal saline by gastrogavage, the positive control group with zoledronic acid via peritoneal injection, and the 5 tested group with CFPC in different proportions of Fructus Psoralea and Fructus Cnidii, i.e., (A, 4:0; B, 3:1; C, 1:1; D, 1:3, and E 0:4), given by gastric infusion. The treatment started from 1 week after modeling and lasted for six weeks. By the end of the experiment, the metastatic foci in bone were imaged by radionuclide tracing method and X-ray photograph, and separated for detecting gene and protein expressions of osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB ligand (RANKL), interleukin-8 (IL-8), parathyroid hormone-related protein (PTHrP), macrophage colony stimulating factor (MCSF) by Real-time PCR and Western blot respectively. RESULTS: Inhibition of bone metastasis gene was displayed to some extent in all the tested groups treated with CFPC, showing an increased level of OPG mRNA expression (It was 60.343 +/- 6.274 in the tested group C), and decreased mRNA expressions of IL-8, PTHrP, MCSF, RANKL (218.010 +/- 12.802, 232.399 +/- 14.354, 319.831 +/- 5.322, and 195.701 +/- 4. 862, respectively in the tested group C). The optimal effect was shown in the tested group C, showing significant difference to that in the blank group (P < 0.01). Meanwhile, the OPG in the bone metastatic foci could be up-regulated and protein expressions of RANKL/IL-8/PTHrP/MCSF down-regulated in all the tested groups. The optimal effect was shown in the tested group C, with significant difference from those of the normal saline group. CONCLUSION: CFPC could inhibit the bone metastasis of breast cancer through activating OPG/RANKL/RANK pathway. Among different proportions of Fructus Psoralea and Fructus Cnidii, 1:1 was the best one.

Intestinal Macrophage Autophagy and its Pharmacological Application in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), comprised of Crohn's disease (CD) and ulcerative colitis (UC), is a group of chronic inflammatory disorders. IBD is regarded as a severe healthcare problem worldwide, with high morbidity and lethality. So far, despite of numerous studies on this issue, the specific mechanisms of IBD still remain unclarified and ideal treatments are not available for IBD. The intestinal mucosal barrier is vital for maintaining the function of the intestinal self-defensive system. Among all of the components, macrophage is an important one in the intestinal self-defensive system, normally protecting the gut against exotic invasion. However, the over-activation of macrophages in pathological conditions leads to the overwhelming induction of intestinal inflammatory and immune reaction, thus damaging the intestinal functions. Autophagy is an important catabolic mechanism. It has been proven to participate the regulation of various kinds of inflammation- and immune-related disorders via the regulation of inflammation in related cells. Here in this paper, we will review the role and mechanism of intestinal macrophage autophagy in IBD. In addition, several well-studied kinds of agents taking advantage of intestinal macrophage autophagy for the treatment of IBD will also be discussed. We aim to bring novel insights in the development of therapeutic strategies against IBD.

Molecular and physiological mechanisms underlying magnesium-deficiency-induced enlargement, cracking and lignification of Citrus sinensis leaf veins.

Little is known about the physiological and molecular mechanisms underlying magnesium (Mg)-deficiency-induced enlargement, cracking and lignification of midribs and main lateral veins of Citrus leaves. Citrus sinensis (L.) Osbeck seedlings were irrigated with nutrient solution at a concentration of 0 (Mg-deficiency) or 2 (Mg-sufficiency) mM Mg(NO3)2 for 16 weeks. Enlargement, cracking and lignification of veins occurred only in lower leaves, but not in upper leaves. Total soluble sugars (glucose + fructose + sucrose), starch and cellulose concentrations were less in Mg-deficiency veins of lower leaves (MDVLL) than those in Mg-sufficiency veins of lower leaves (MSVLL), but lignin concentration was higher in MDVLL than that in MSVLL. However, all four parameters were similar between Mg-deficiency veins of upper leaves (MDVUL) and Mg-sufficiency veins of upper leaves (MSVUL). Using label-free, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we identified 1229 and 492 differentially abundant proteins (DAPs) in MDVLL vs MSVLL and MDVUL vs MSVUL, respectively. Magnesium-deficiency-induced alterations of Mg, nonstructural carbohydrates, cell wall components, and protein profiles were greater in veins of lower leaves than those in veins of upper leaves. The increased concentration of lignin in MDVLL vs MSVLL might be caused by the following factors: (i) repression of cellulose and starch accumulation promoted lignin biosynthesis; (ii) abundances of proteins involved in phenylpropanoid biosynthesis pathway, hormone biosynthesis and glutathione metabolism were increased; and (iii) the abundances of the other DAPs [viz., copper/zinc-superoxide dismutase, ascorbate oxidase (AO) and ABC transporters] involved in lignin biosynthesis were elevated. Also, the abundances of several proteins involved in cell wall metabolism (viz., expansins, Rho GTPase-activating protein gacA, AO, monocopper oxidase-like protein and xyloglucan endotransglucosylase/hydrolase) were increased in MDVLL vs MSVLL, which might be responsible for the enlargement and cracking of leaf veins.