Clathrin light chains negatively regulate plant immunity by hijacking the autophagy pathway.

The crosstalk between clathrin-mediated endocytosis (CME) and the autophagy pathway has been reported in mammals; however, the interconnection of CME with autophagy has not been established in plants. Here, we report that the Arabidopsis CLATHRIN LIGHT CHAIN (CLC) subunit 2 and 3 double mutant, clc2-1 clc3-1, phenocopies Arabidopsis AUTOPHAGY-RELATED GENE (ATG) mutants in both autoimmunity and nutrient sensitivity. Accordingly, the autophagy pathway is significantly compromised in the clc2-1 clc3-1 mutant. Interestingly, multiple assays demonstrate that CLC2 directly interacts with ATG8h/ATG8i in a domain-specific manner. As expected, both GFP-ATG8h/GFP-ATG8i and CLC2-GFP are subjected to autophagic degradation, and degradation of GFP-ATG8h is significantly reduced in the clc2-1 clc3-1 mutant. Notably, simultaneous knockout of ATG8h and ATG8i by CRISPR-Cas9 results in enhanced resistance against Golovinomyces cichoracearum, supporting the functional relevance of the CLC2-ATG8h/8i interactions. In conclusion, our results reveal a link between the function of CLCs and the autophagy pathway in Arabidopsis.

Nomogram for the prediction of infected pancreatic necrosis in moderately severe and severe acute pancreatitis.

OBJECTIVES: As a serious complication of moderately severe acute pancreatitis (MSAP) and severe acute pancreatitis (SAP), infected pancreatic necrosis (IPN) can lead to a prolonged course of interventional therapy. Most predictive models designed to identify such patients are complex or lack validation. The aim of this study was to develop a predictive model for the early detection of IPN in MSAP and SAP. METHODS: A total of 594 patients with MSAP or SAP were included in the study. To reduce dimensionality, least absolute shrinkage and selection operator regression analysis was used to screen potential predictive variables, a nomogram was then constructed using logistic regression analysis. The receiver operating characteristic (ROC) curve, calibration curve, and decision curve analysis (DCA) were used to evaluate the discrimination, accuracy, and clinical efficacy of the model. External data were also obtained to further validate the constructed model. RESULTS: There were 476, 118, and 82 patients in the training, internal validation, and external validation cohorts, respectively. Platelet count, hematocrit, albumin/globulin, severity of acute pancreatitis, and modified computed tomography severity index score were independent factors for predicting IPN in MSAP and SAP. The area under the ROC curves were 0.923, 0.940, and 0.817, respectively, in the three groups. There was a good consistency between the actual probabilities and the predicted probabilities. DCA revealed excellent clinical utility. CONCLUSION: The constructed nomogram is a simple and feasible model that has good clinical predictive value and efficacy in clinical decision-making for IPN in MSAP and SAP.

Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects.

The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5-7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.

Knocking out NtSARD1a/1b/1c/1d by CRISPR/CAS9 technology reduces the biosynthesis of salicylic acid (SA) and compromises immunity in tetraploid Nicotiana tabacum.

Salicylic acid (SA) is a key phyto-hormone that is essential for plant immunity. SARD1 (SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1), a member of the CBP60 (CALMODULIN-BINDING PROTEIN60) gene family, is one of the major transcription factors regulating the expression of the genes in SA biosynthesis. SARD1 has been extensively studied in model plant Arabidopsis. However, the function of SARD1 homologues in SA biosynthesis and immune responses have rarely been investigated in other plant species. In this study, the CRISPR/CAS9 (Clustered Regularly Interspersed Short Palindromic Repeats/CAS9) technology was used in creating transgenic tobacco mutant lines with 6-8 alleles of four NtSARD1 homologous genes (NtSARD1a/1b/1c/1d) knocked out. No significant difference in morphological phenotype was observed between the transgenic knockout lines and the wild type tobacco plants, indicating that knocking out NtSARD1s does not affect the growth and development in tobacco. However, knocking out or partially knocking out of NtSARD1a/b/c/d resulted in a significantly reduced expression of NtICS1, the key gene in SA biosynthesis pathway, and thus the subsequently decreased SA/SAG accumulations in response to Pst DC3000 (Pseudomonas syrangae pv.tomato DC3000) infection, indicating a key role of NtSARD1 genes in SA biosynthesis in tobacco. As a consequence of reduced SA/SAG accumulation, the Pst DC3000-induced expression of NtPR genes as well as the resistance to Pst DC3000 were both significantly reduced in these knockout lines compared with the wild type tobacco plants. Interestingly, the reductions in the SA/SAG level, NtPR gene induction and Pst DC3000 resistance were positively correlated with the number of alleles being knocked out. Furthermore, LUC reporter gene driven by the promoter of NtICS1 containing two G(A/T)AATT(T/G) motifs could be activated by NtSARD1a, suggesting that NtSARD1a could bind to the core G(A/T)AATT(T/G) motifs and thus activate the expression of LUC reporter. Taken together, our results demonstrated that the NtSARD1 proteins play essential roles in SA biosynthesis and immune responses in tobacco. Our results also demonstrated that the CRISPR/CAS9 technology can overcome gene redundancy and is a powerful tool to study gene functions in polyploid plant species.

Physiological and transcription analyses reveal regulatory pathways of 6-benzylaminopurine delaying leaf senescence and maintaining quality in postharvest Chinese flowering cabbage.

The regulatory role of cytokinins (CTKs) in leaf senescence has been documented in different species, including Chinese flowering cabbage. However, its physiological and molecular basis relatively remains unknown. In this study, exogenous application of a CTK analogue 6-benzylaminopurine (6-BA) significantly retarded leaf senescence of Chinese flowering cabbage, with less chlorophyll degradation and lower accumulation of reactive oxygen species (ROS) and malondialdehyde compared with the control group. Meanwhile, higher levels of soluble sugars and proteins, flavonoids, cellulose, amino acids, total phenols, procanthocyanins, and vitamin C were retained in 6-BA-treated leaves. 6-BA treatment also prevented the decline in endogenous CTK content and the increase in ethylene, abscisic acid, and jasmonic acid contents. Moreover, the comparative transcriptome analysis revealed that a total of 21,895 differently expressed genes (DEGs) were identified from four comparisons of 6-BA treatment versus the control during senescence. Further analysis showed that most of the identified DEGs were enriched in ROS, respiratory metabolism, and phytohormone pathways, and a total of 50 classes of transcription factors that were possibly involved in modulating these DEGs were obtained. The transcriptional levels of 18 DEGs were verified by Quantitative real-time PCR (qRT-PCR), which confirmed the accuracy of the transcriptomic data. Overall, these findings and data provide a comprehensive view of physiological and molecular events concerning with the CTK-mediated leaf senescence and -maintained quality in economical leafy vegetables.

Evaluation and Clinical Implication of Zhenwu Decoction on Seven Cytochrome P450 Enzyme in Chronic Heart Failure Rats.

BACKGROUND: Recently, the combination of Traditional Chinese Medicine (TCM) formulae and other drugs has been used frequently in clinical practice, while the possibility of herb-drug interaction (HDI) risk remains a challenge. Since metabolic enzymes mediate the majority of drug interactions, evaluating the effects of formulae on metabolic enzymes is therefore instructive for the rational formulation of drug delivery plans. OBJECTIVE: Herein, we are devoted to estimating the effects of Zhenwu detection (ZWD) on activities and mRNA expression of 7 cytochrome P450 (CYP450) isoenzymes in chronic heart failure (CHF) rats. METHODS: The CHF rats were replicated by coronary artery ligation and were randomly divided into sham operation group, model group, ZWD low- (2.188 g/kg), middle- (4.375 g/kg), and high- (8.750 g/kg) dose groups, n=6. After 8 weeks, rats were administrated with ZWD and normal saline (NS) for four weeks, and the mixed solution of 7 probe drugs (1 mL/kg) was subsequently injected into 30 rats through the caudal vein after the last administration. Pharmacokinetic parameters and mRNA expression of 7 probe drugs were measured by using UPLC-MS/MS and RT-qPCR, respectively. RESULTS: Activities and mRNA expression of CYP1A2, CYP2B1, CYP2C6, CYP2C11, CYP3A1 were inhibited in CHF rats, and ZWD could reverse this effect except for CYP2B1. CONCLUSION: Overall, these findings underscore that for CHF patients, the HDI should be taken into consideration when ZWD is used on its own or combined with drugs meditated by CYP1A2 (CYP1A2 in rats), CYP2C9 (CYP2C6 in rats), CYP2C19 (CYP2C11 in rats) and CYP3A4 (CYP3A1 in rats). Furthermore, since the apparent volume of distribution (Vd) of amodiaquine, dextromethorphan and bupropion has been proved to be far greater than the total volume of body fluids, we speculate that the dose adjustment and potential organotoxicity of these substrates may need further consideration.

Melatonin delays leaf senescence of postharvest Chinese flowering cabbage through ROS homeostasis.

Reactive oxygen species (ROS) trigger and accelerate leaf senescence. Melatonin, a low molecular compound with several biological functions in plants, is known to delay leaf senescence in different species, including Chinese flowering cabbage. However, the mechanism(s) underpinning melatonin-delayed leaf senescence remains unclear. Here, we found that melatonin lowered the expression of chlorophyll catabolic genes (BrPAO and BrSGR1) and senescence-associated genes (BrSAG12 and BrSEN4), decreased chlorophyll loss, minimized the alteration in Fv/Fm ratio and remarkably delayed senescence of Chinese flowering cabbage after harvest. Moreover, the over-accumulation of O2•-, hydrogen peroxide (H2O2) and malondialdehyde contents and the expression of respiratory burst oxidase homologues (RBOH) genes (BrRbohB, BrRbohC, BrRbohD, BrRbohD2 and BrRbohE) were significantly inhibited by melatonin treatment. Melatonin-treated cabbages also showed higher O2•-, OH• and DPPH radical scavenging capacity and enhanced activities of peroxidase (POD), superoxide dismutase (SOD) and their gene expressions. Up-regulation of key components of ascorbate-glutathione (AsA-GSH) cycle, the metabolic pathway that detoxify H2O2, was also observed in melatonin-treated cabbages. These findings suggest that melatonin-delayed postharvest leaf senescence of postharvest Chinese flowering cabbage may be mediated, at least in part, by maintaining ROS homeostasis through restraining RBOHs-catalyzed ROS production and enhancing the activity of ROS-scavenging system including major antioxidant enzymes and AsA-GSH cycle.

Multi-center investigation of the clinical and pathological characteristics of inflammatory breast cancer based on Chinese Society of Breast Surgery (CSBrs-007).

BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive type of cancer with poor prognosis and outcomes. This study aimed to investigate clinicopathological features, molecular characteristics, and treatments among Chinese patients diagnosed with IBC. METHODS: We collected data of 95 patients with IBC who were treated by members of the Chinese Society of Breast Surgery, from January 2017 to December 2018. The data, including demographic characteristics, pathological findings, surgical methods, systemic treatment plans, and follow-up, were obtained using a uniform electronic questionnaire. The clinicopathological features of different molecular types in patients without distant metastases were compared using the Kruskal-Wallis (H) test followed by post hoc analyses. RESULTS: Lymph node metastasis was noted in 75.8% of all patients, while distant metastasis was noted in 21.4%. Pathological findings indicated invasive ductal and lobular carcinomas in 86.8% and 5.3% of cases, respectively. Hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) (41.5%) and HR-/HER2+ (20.1%) were the most common biologic subtypes, followed by HR+/HER2+ (19.1%) and HR-/HER2- (19.1%). Stage III IBC was treated via pre-operative neoadjuvant chemotherapy in 87.7% of the cases, predominantly using anthracycline and taxanes. A total of 91.9% of patients underwent surgical treatment. Among them, 77.0% of the patients underwent modified radical mastectomy, 8.1% of whom also underwent immediate breast reconstruction. The Kruskal-Wallis test revealed that the efficacy of chemotherapy significantly differed among those with HR+/HER2- and HR-/HER2- tumors (adjusted P = 0.008), and Ki-67 expression significantly differed in HR-/HER2+ and HR+/HER2+ molecular subtypes (adjusted P = 0.008). CONCLUSION: Our study provides novel insight into clinicopathological characteristics and treatment status among patients with IBC in China, and might provide a direction and basis for further studies. TRIAL REGISTRATION: chictr.org.cn, No. ChiCTR1900027179; http://www.chictr.org.cn/showprojen.aspx?proj=45030.

Evaluation of Zhenwu Decoction Effects on CYP450 Enzymes in Rats Using a Cocktail Method by UPLC-MS/MS.

This thesis is aimed at shedding light on the effects of the Zhenwu decoction (ZWD) on the activities and mRNA expressions of seven CYP450 isoenzymes. In the first step, we determined the main chemical compounds of ZWD by high-performance liquid chromatography (HPLC). Next, 48 male (SD) rats were randomly divided into the normal saline (NS) group and the ZWD low- (2.1875 g/kg), medium- (4.375 g/kg), and high- (8.75 g/kg) dose groups (12 per group). All rats were gavaged once daily for 28 consecutive days. A mixed solution of seven probe drugs was injected into 24 rats through the caudal vein after the last intragastric administration. Lastly, a validated cocktail method and real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) were used to detect pharmacokinetic parameters and mRNA expressions, respectively. Compared with the NS group, ZWD at medium- and high-dose groups could significantly induce CYP2C6 (P < 0.05) activity, while the mRNA expression (P < 0.05) increased only in the high-dose group. Additionally, CYP2C11 activity was induced and consistent with mRNA expression (P < 0.05). Moreover, ZWD could induce the activity of CYP3A1 (P < 0.05), but the mRNA expression showed no significant differences except in high-dose groups. Additionally, ZWD has no effects on CYP1A2, CYP2B1, CYP2C7, and CYP2D2. In conclusion, the significant inductive effects of ZWD on three CYP450 isoenzymes indicated that when ZWD was coadministrated with drugs mediated by these enzymes, not only should the potential herb-drug interactions (HDIs) be observed, but the dosage adjustment and tissue drug concentration should also be considered. Furthermore, the approach described in this article can be applied to study the importance of gender, age, and disease factors to HDI prediction.

GJA8 missense mutation disrupts hemichannels and induces cell apoptosis in human lens epithelial cells.

Autosomal dominant congenital cataract (ADCC), the most common hereditary disease, is a major cause of eye disease in children. Due to its high genetic and clinical heterogeneity, the identification of ADCC-associated gene mutations is essential for the development of molecular therapies. In this study, we examined a four-generation Chinese pedigree with ADCC and identified putative mutations in ADCC candidate genes via next-generation sequencing (NGS) followed by Sanger sequencing. A novel missense mutation in GJA8 (c.T217C) in ADCC patients causes a serine-to-proline substitution at residue 73 of connexin 50 (Cx50); no mutation was found in unaffected family members and unrelated healthy individuals. Functional analysis revealed that this missense mutation disrupts protein function in human lens epithelial cells (HLEpiCs), which fails to form calcium-sensitive hemichannels. Furthermore, mutant Cx50 leads to decreased ROS scavenging by inhibiting G6PD expression and thus induces cell apoptosis via aberrant activation of the unfolded protein response (UPR). In conclusion, we report a novel GJA8 heterozygous mutation in a Chinese family with a vital role in ADCC, broadening the genetic spectrum of this disease.

Recent advances in metal-organic frameworks for adsorption of common aromatic pollutants.

This review (with 85 refs.) summarizes the recent literature on the adsorption of common aromatic pollutants by using modified metal-organic frameworks (MOFs). Four kinds of aromatic pollutants are discussed, namely benzene homologues, polycyclic aromatic hydrocarbons (PAHs), organic dyes and their intermediates, and pharmaceuticals and personal care products (PPCPs). MOFs are shown to be excellent adsorbents that can be employed to both the elimination of pollutants and to their extraction and quantitation. Adsorption mechanisms and interactions between aromatic pollutants and MOFs are discussed. Finally, the actual challenges of existence and the perspective routes towards future improvements in the field are addressed. Graphical abstract Recent advance on adsorption of common aromatic pollutants including benzene series, polycyclic aromatic hydrocarbons, organic dyes and their intermediates, pharmaceuticals and personal care products by metal-organic frameworks.

Two novel mutations identified in ADCC families impair crystallin protein distribution and induce apoptosis in human lens epithelial cells.

Congenital cataract (CC) is a clinical and genetically heterogeneous eye disease that primarily causes lens disorder and even amblyopic blindness in children. As the mechanism underlying CC is genetically inherited, identification of CC-associated gene mutations and their role in protein distribution are topics of both pharmacological and biological research. Through physical and ophthalmic examinations, two Chinese pedigrees with autosomal dominant congenital cataract (ADCC) were recruited for this study. Mutation analyses of CC candidate genes by next-generation sequencing (NGS) and Sanger sequencing revealed a novel missense mutation in CRYBB2 (p.V146L) and a deletion mutation in CRYAA (p.116_118del). Both mutations fully co-segregated were not observed in unaffected family members or in 100 unrelated healthy controls. The CRYBB2 missense mutation disrupts the distribution of CRYBB2 in human lens epithelial cells (HLEpiCs), and the CRYAA deletion mutation causes hyperdispersion of CRYAA. Furthermore, these two crystallin mutations result in aberrant expression of unfolded protein response (UPR) marker genes as well as apoptosis in HLEpiCs. Collectively, these findings broaden the genetic spectrum of ADCC.

Surgical treatment and visual outcomes of cataract with persistent hyperplastic primary vitreous.

AIM: To evaluate the surgical treatment and visual outcomes of eyes with cataract and persistent hyperplastic primary vitreous (PHPV). METHODS: This retrospective study included patients with cataract and PHPV treated with various strategies. Anterior PHPV was treated using phacoemulsification with underwater electric coagulation on posterior capsule neovascularization, posterior capsulotomy, anterior vitrectomy, and intraocular lens (IOL) implantation. Posterior PHPV was treated with lensectomy, posterior vitrectomy, retinal photocoagulation, and IOL implantation or silicone oil tamponade. Visual acuity (VA), pattern visual evoked potential (P-VEP), anatomic recovery, postoperative complications, and amblyopia outcome were examined. Subjects were followed-up for 3-48mo after surgery. RESULTS: Of the 30 patients (33 eyes) with congenital cataract and PHPV included (average age, 39.30±35.47mo), 9 eyes had anterior PHPV and 24 had posterior PHPV. Thirty-two eyes were surgically treated. Eyes with anterior PHPV received an IOL during one-stage (6 eyes) and two-stage (3 eyes) implantation. Postoperative complications included retinal detachment (1 eye) and recurrent anterior chamber hemorrhage (1 eye). In eyes with posterior PHPV, 6 and 11 eyes received IOLs in one- and two-stage procedures, respectively. Silicone oil was retained in 2 eyes, and IOLs were not implanted in 4 eyes. VA significantly improved in 25 eyes following operations and 3-48mo of amblyopia treatment. P-VEP P100 was improved following surgery in both PHPV types. CONCLUSION: Our surgical strategies are appropriate and effective for anterior and posterior PHPV. Early surgical intervention and amblyopia therapy result in positive treatment outcomes.