Slow anion channels (SLAC/SLAH) are efflux channels previously shown to be critical for stomatal regulation. However, detailed analysis using the β-glucuronidase reporter gene showed that members of the SLAC/SLAH gene family are predominantly expressed in roots, in addition to stomatal guard cells, implicating distinct function(s) of SLAC/SLAH in the roots. Comprehensive mutant analyses of all slac/slah mutants indicated that slah3 plants showed a greater growth defect than wild-type plants when ammonium was supplied as the sole nitrogen source. Ammonium toxicity was mimicked by acidic pH in nitrogen-free external medium, suggesting that medium acidification by ammonium-fed plants may underlie ammonium toxicity. Interestingly, such toxicity was more severe in slah3 mutants and, particularly in wild-type plants, was alleviated by supplementing the media with micromolar levels of nitrate. These data thus provide evidence that SLAH3, a nitrate efflux channel, plays a role in nitrate-dependent alleviation of ammonium toxicity in plants.

We elucidated the extracellular ATP (eATP) signalling cascade active in programmed cell death (PCD) using cell cultures of Populus euphratica. Millimolar amounts of eATP induced a dose-and time-dependent reduction in viability, and the agonist-treated cells displayed hallmark features of PCD. eATP caused an elevation of cytosolic Ca 2+ levels, resulting in Ca 2+ uptake by the mitochondria and subsequent H2O2 accumulation. P. euphratica exhibited an increased mitochondrial transmembrane potential, and cytochrome c was released without opening of the permeability transition pore over the period of ATP stimulation. Moreover, the eATP-induced increase of intracellular ATP, essential for the activation of caspase-like proteases and subsequent PCD, was found to be related to increased mitochondrial transmembrane potential. NO is implicated as a downstream component of the cytosolic Ca 2+ concentration but plays a negligible role in eATP-stimulated cell death. We speculate that ATP binds purinoceptors in the plasma membrane, leading to the induction of downstream intermediate signals, as the proposed sequence of events in PCD signalling was terminated by the animal P2 receptor antagonist suramin.

The plant elicitor peptides (Peps), a family of damage/danger-associated molecular patterns (DAMPs), are perceived by two receptors, PEPR1 and PEPR2, and contribute to plant defense against pathogen attack and abiotic stress. Here, we show that the Peps-PEPR signaling pathway functions in stomatal immunity by activating guard cell anion channels in The mutant plants lacking both and () displayed enhanced bacterial growth after being sprayed with pv () DC3000, but not after pathogen infiltration into leaves, implicating PEPR function in stomatal immunity. Indeed, synthetic Arabidopsis Peps (Peps) effectively induced stomatal closure in wild-type but not mutant leaves, suggesting that thePeps-PEPR signaling pathway triggers stomatal closure. Consistent with this finding, patch-clamp recording revealed Pep1-induced activation of anion channels in the guard cells of wild-type but not mutant plants. We further identified two guard cell-expressed anion channels, SLOW ANION CHANNEL1 (SLAC1) and its homolog SLAH3, as functionally overlapping components responsible for Pep1-induced stomatal closure. The double mutant, but not or single mutants, failed to respond to Pep1 in stomatal closure assays. Interestingly, disruption of (), an essential gene for abscisic acid-triggered stomatal closure, did not affect the Pep1-induced anion channel activity and stomatal response. Together, these results illustrate a DAMP-triggered signaling pathway that, unlike the flagellin22-FLAGELLIN-SENSITIVE2 pathway, triggers stomata immunity through an OST1-independent mechanism.

Plant elicitor peptides (Peps) are damage/danger-associated molecular patterns that are perceived by the receptor-like kinases, PEPR1 and PEPR2, to enhance innate immunity and to inhibit root growth in Arabidopsis (Arabidopsis thaliana). Here, we show that Arabidopsis Pep1 inhibits root growth in a PEPR2-dependent manner, which is accompanied by swelling epidermal and cortex cells and root hair formation in the transition zone (TZ). These Pep1-induced changes were mimicked by exogenous auxin application and were suppressed in the auxin perception mutants transport inhibitor response1 (tir1) and tir1 afb1 afb2. Pep1-induced auxin accumulation in the TZ region preceded cell expansion in roots. Because local auxin distribution depends on PIN-type auxin transporters, we examined Pep1-PEPR-induced root growth inhibition in several pin mutants and found that pin2 was highly sensitive but pin3 was less sensitive to Pep1. The pin2 pin3 double mutant was as sensitive to Pep1 treatment as wild-type plants. Pep1 reduced the abundance of PIN2 in the plasma membrane through activating endocytosis while increasing PIN3 expression in the TZ, leading to changes in local auxin distribution and inhibiting root growth. These results suggest that Pep-PEPR signaling undergoes crosstalk with auxin accumulation to control cell expansion and differentiation in roots during immune responses.

Nonalcoholic fatty liver disease (NAFLD) has become a major risk factor for hepatocellular carcinoma (HCC) worldwide. However, the underlying mechanism remains insufficiently elucidated. The expression of Collagen I, an important component of extracellular matrix (ECM), was increased during the progression from simple steatosis to NASH. The purpose of this study was to investigate the role of Collagen I in NAFLD-related HCC. To study this, the decellularized liver matrix, which preserves the pathological changes of ECM, was prepared from the human fatty liver (FLM) and human normal liver (NLM). HepG2 cells cultured in FLM had a higher proliferation rate than those in NLM. SMMC-7721 and HepG2 cells cultured on Collagen I-coated plates grew faster than those on either Collagen IV- or fibronectin-coated plates. This effect was dose-dependent and associated with elevated integrin β1 expression and activation of downstream phospho-FAK. Knocking down the expression of integrin β1 significantly decreased the proliferation of HCC cells. Additionally, an orthotopic tumor model was established in NAFLD mice at different stages. The over-expressed Collagen I in the mice liver increased the expression of integrin β1 and downstream phospho-FAK, resulting in the proliferation of HCC cells. This proliferation could be inhibited by blocking the integrin β1/FAK pathway. In summary, our study demonstrated that Collagen I promoted HCC cell proliferation by regulating the integrin β1/FAK pathway. Decellularized liver matrix can be used as a platform to three-dimensionally culture HCC cells and reproduce the impact of changed ECM on the progression of NAFLD-related HCC.

ABSTRACT. Background and Aims: Using decellularized scaffold to reengineer liver tissue is a promising alternative therapy for end-stage liver diseases. Though the decellularized human liver matrix is the ideal scaffold for reconstruction of the liver theoretically, the shortage of liver donors is still an obstacle for potential clinical application. Therefore, an appropriate alternative scaffold is needed. In the present study, we used a tissue engineering approach to prepare a rat decellularized spleen matrix (DSM) and evaluate the effectiveness of this DSM for primary rat hepatocytes culture. Methods: Rat decellularized spleen matrix (DSM) was prepared by perfusion of a series of detergents through spleen vasculature. DSM was characterized by residual DNA and specific extracellular matrix distribution. Thereafter, primary rat hepatocytes were cultured in the DSM in a 3-dimensional dynamic culture system, and liver cell survival and biological functions were evaluated by comparison with 3-dimensional sandwich culture and also with cultured in decellularized liver matrix (DLM). Results: Our research found that DSM did not exhibit any cellular components, but preserved the main extracellular matrix and the intact vasculature evaluated by DNA detection, histology, immunohistochemical staining, vessel corrosion cast and upright metallurgical microscope. *Correspondence to: Yi Lv; Email: luyi169@126.com Received July 7, 2014; Revised September 10, 2014; Accepted January 18, 2015.16 Organogenesis, 11:16-29, 2015 Ó 2015 Taylor & Francis Group, LLC ISSN: 1547-6278 print / 1555-8592 online DOI: 10.1080/15476278.2015 Moreover, the method of DSM preparation procedure was relatively simple with high success rate (100%). After seeding primary hepatocytes in DSM, the cultured hepatocytes survived inside DSM with albumin synthesis and urea secretion within 10 d. Additionally, hepatocytes in dynamic culture medium had better biological functions at day 10 than that in sandwich culture. Albumin synthesis was 85.67 § 6.34 mg/10 7 cell/24h in dynamic culture in DSM compared to 62.43 § 4.59 mg/10 7 cell/ 24h in sandwich culture (P < 0.01) and to 87.54 § 5.25 mg/10 7 cell/24h in DLM culture (P > 0.05); urea release was 32.14 § 8.62 mg/10 7 cell/24h in dynamic culture in DSM compared to 20.47 § 4.98 mg/10 7 cell/24h in sandwich culture (P < 0.05) and to 37.38 § 7.29 mg/10 7 cell/24h cultured in DLM (P > 0.05). Conclusion: The present study demonstrates that DSM can be prepared successfully using a tissue engineering approach. The DSM is an appropriate scaffold for primary hepatocytes culture.

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Liver fibrosis and cirrhosis is associated with the prognosis of patients with hepatocellular carcinoma (HCC) after treatment. The γ-glutamyl transpeptidase to platelet ratio (GPR) is reported to predict significant liver fibrosis and cirrhosis. The aim of this study was to investigate the predictive value of preoperative GPR on the recurrence and survival of patients with HCC who underwent curative hepatectomy.A retrospective review of demographics, medical records, and prognosis of patients with hepatitis B virus (HBV)–related HCC was performed. Overall survival (OS) and disease-free survival (DFS) were evaluated using Kaplan–Meier method, and the log-rank test was used to analyze differences in recurrence and survival. Univariate and multivariate analyses were used for significance of prognostic factor.A total of 357 patients with HBV-related HCC were included in this analysis. The preoperative GPR was associated with recurrence and survival rates, independent of HCC progression or tumor marker levels, in a multivariate analysis. OS was higher in patients with a GPR <0.84 versus ≥084 (5-year survival rates 58.6% vs. 38.5%; P < 0.001). DFS was also worse in patients with a GPR ≥0.84 than in those with GPR <0.84 (5-year recurrence rates 42.8% vs. 22.8%; P < 0.001).GPR score of ≥0.84 represents a major risk factor for the poor prognosis for HBV-related HCC after hepatic resection, and GPR served as an independent predictive factor for HBV-related HCC OS.