Die to Survive - Functional Analysis of Grapevine Metacaspases Responsive to Effector - Triggered Immunity (ETI)-Related Cell Death

Der programmierte Zelltod (programmed cell death, PCD) ist ein unverzichtbarer Vorgang, welcher sowohl bei Pflanzen als auch bei Tieren durch ein intrazelluläres Programm vermittelt wird. Vor allem im pflanzlichen Immunitätssystem ist er als letztes Mittel eine unentbehrliche Strategie, um im Rahmen einer sogenannten hypersensitiven Antwort (hypersensitive response, HR) die Ausbreitung von biotrophen Pathogenen zu unterbinden. Als fortgeschrittener Zelltodmechanismus erscheint die hypersensitive Antwort häufig als Gipfelpunkt des zweiten Immunitätsniveaus einer Pflanze, die als Effektor-vermittelte Immunität (effector-triggered immunity, ETI) bezeichnet wird und ein Zeichen höherer Resistenz darstellt. Unsere bisherige Arbeit hat bereits eine Cystein-abhängige Protease-Genfamilie in der Weinrebe identifiziert, eine so genannte Metacaspase (MC), die eine wesentliche Rolle bei Prozessen des programmierten Zelltods in der Entwicklung von Pflanzen spielt. Ziel dieser Doktorarbeit ist es, zentrale MC-Genkandidaten zu screenen, die auf den ETI-induzierten HR-Prozess reagieren und die zelluläre Funktion und den entsprechenden Regulationsmechanismus der Kandidaten zu verstehen. Die Doktorarbeit besteht wie folgt aus drei progressiven Teilen. Wir verwendeten ein typisches biotrophes Pathogenisolat, Plasmopara viticola, als HR-Elicitor auf Blattscheibenbioassays, um die Unterschiede der nekrotischen Zone während der Infektion von neun Genotypen auf Weinrebenblätter zu beobachten. Zwei gegensätzliche Genotypen wurden untersucht: Das Kultivar "Müller Thurgau" steht für einen anfälligen Genotyp, wohingegen Vitis rupestris für einen HR-resistenten Genotyp steht. Eine weitere Genexpressionsanalyse zeigte, dass nach der Infektion nur VrMC2 und VrMC5 von V. rupestris zunehmend exprimiert wurden, was 24 h vor dem Auftreten der hypersensitive Antwort erfolgte. Daher wurden diese beiden Gene als Kandidaten ausgewählt. In einer Suspensionszelllinie wurde Harpin als ein wirksamer, die hypersensitive Antwort auslösender Elicitor eingesetzt, der den Zelltod spezifisch für V. rupestris und “Pinot Noir”induzieren konnte. Die Genexpressionsanalyse zeigte, dass nach 24 h mit 18 µg / ml Harpinbehandlung VrMC2 und VrMC5 entsprechend hochreguliert wurden. Dieses Ergebnis entsprach unseren früheren Ergebnissen auf Blattscheiben. Bezüglich ihrer zellulären Funktionen wurden VrMC2 und VrMC5 durch heterologe Expression in BY-2 Tabakzellen charakterisiert. Die subzellulären Lokalisierungsstudien zeigten, dass sich VrMC2 am endoplasmatischen Retikulum (ER) um die Kern herum und VrMC5 im Zytoplasma und dem Kernbereich befand. Dies legt nahe, dass in der Weinrebe zwei verschiedene Typen von Metacaspasen wahrscheinlich die Rolle des programmierten Zelltods in verschiedenen subzellulären Orten in der Zelle übernehmen. Darüber hinaus zeigte ein Zellmortalitätsassay, dass beide VrMC2- und VrMC5-BY-2-Überexpressionslinien stark auf den durch Harpin-induzierten Zelltod reagierten. Darüber hinaus ist diese Mortalität signalabhängig, was durch die Zugabe von exogenem Methyljasmonat (MeJA), ein wichtiger Überträger der basalen Immunität, oder Diphenyleniodonium (DPI), einem Inhibitor von Nicotinamid-Adenin- Dinukleotid-Phosphat-Wasserstoff (NADPH) Oxidasen, welche apoplastisches Superoxid erzeugen, vermindert werden konnte. Die 5\u27-Upstream-Sequenz-Promotoren von MC2 (pMC2) und MC5 (pMC5) wurden aus beiden Kultivaren, "Müller Thurgau" und V. rupestris, kloniert, um den durch Genexpression regulierten Mechanismus weiter zu untersuchen. Die Verteilung der auf die Abwehrreaktion bezogenen cis-Elemente von pMC2 und pMC5 wurde in den zwei Genotypen weiter analysiert. Die Ergebnisse zeigten, dass es fünf verschiedene Arten cis-Elementen gibt, die sowohl auf pMC2 als auch auf pMC5 verteilt sind, wie z. B. die W-Box, die TC-reiche Region und das GT-1-Motiv. Die Gesamtelementanzahl von pVrMC2 und pVrMC5 ist höher als die Anzahl von pVvMC2 und pVvMC5. Nicht zuletzt wurde mit Hilfe eines Dual-Luciferase-Systems zum Nachweis der Promotoraktivität von pVrMC2 und pVrMC5 gezeigt, dass nach der Harpinbehandlung sowohl die Aktivität von pVrMC2 als auch die von pVrMC5 zumeist verdoppelt wurden. Jedoch zeigte pVrMC2 keine Antwort auf MeJA. Außerdem verringerte sich sogar die pVrMC5-Aktivität durch Zugabe von MeJA. Diese Ergebnisse bestätigten, dass die VrMC2- und VrMC5-Genexpressionsmuster teilweise durch ihre entsprechenden Promotoraktivitäten reguliert wurden

Genetic inter-relationships among Chinese wild grapes based on SRAP marker analyses

Sequence-Related Amplified Polymorphism (SRAP) markers were used to assess genetic inter-relationships among 39 grape genotypes. These included 22 indigenous Chinese grape species/varieties, the north American V. riparia and the European V. vinifera L. 'Thompson seedless' and 'Pinot noir'. Of the 72 SRAP primer combinations tested, 25 primers generated 135 reliable bands, with an average of 5.52 bands per primer pair. Further analysis shows that 106 of 135 bands were generated by 25 polymorphic primer pairs, with a polymorphism efficiency of 79 %. The similarity coefficients of SRAP polymorphism varied from 0.463 to 0.981 among the genotypes analysed. A dendrogram analysis divided the 39 Vitis accessions into 21 groups with similarity coefficients of 0.83. It reveals broadly similar genetic relationships among the genotypes examined to those previously determined using classical taxonomic methods. Our results define V. heyneana subsp. ficifolia and V. baihensis as subspecies of V. heyneana and V. bashanica, respectively. We question the placement of V. davidii var. cyanocarpa and V. davidii var. ningqiangensis as varieties in V. davidii

A mixed integer linear programming model for minimum backbone grid

Developing a minimum backbone grid in the power system planning is beneficial to improve the power system’s resilience. To obtain a minimum backbone grid, a mixed integer linear programming (MILP) model with network connectivity constraints for a minimum backbone grid is proposed. In the model, some constraints are presented to consider the practical application requirements. Especially, to avoid islands in the minimum backbone grid, a set of linear constraints based on single-commodity flow formulations is proposed to ensure connectivity of the backbone grid. The simulations on the IEEE-39 bus system and the French 1888 bus system show that the proposed model can be solved with higher computational efficiency in only about 30 min for such a large system and the minimum backbone grid has a small scale only 52% of the original grid. Compared with the improved fireworks method, the minimum backbone grid from the proposed method has fewer lines and generators

Survival and Clinicopathological Significance of SIRT1 Expression in Cancers: A Meta-Analysis

Background: Silent information regulator 2 homolog 1 (SIRT1) is an evolutionarily conserved enzymes with nicotinamide adenine dinucleotide (NAD)+-dependent deacetylase activity. SIRT1 is involved in a large variety of cellular processes, such as genomic stability, energy metabolism, senescence, gene transcription, and oxidative stress. SIRT1 has long been recognized as both a tumor promoter and tumor suppressor. Its prognostic role in cancers remains controversial.Methods: A meta-analysis of 13,138 subjects in 63 articles from PubMed, EMBASE, and Cochrane Library was performed to evaluate survival and clinicopathological significance of SIRT1 expression in various cancers.Results: The pooled results of meta-analysis showed that elevated expression of SIRT1 implies a poor overall survival (OS) of cancer patients [Hazard Ratio (HR) = 1.566, 95% CI: 1.293–1.895, P < 0.0001], disease free survival (DFS) (HR = 1.631, 95% CI: 1.250–2.130, P = 0.0003), event free survival (EFS) (HR = 2.534, 95% CI: 1.602–4.009, P = 0.0001), and progress-free survival (PFS) (HR = 3.325 95% CI: 2.762–4.003, P < 0.0001). Elevated SIRT1 level was associated with tumor stage [Relative Risk (RR) = 1.299, 95% CI: 1.114–1.514, P = 0.0008], lymph node metastasis (RR = 1.172, 95% CI: 1.010–1.360, P = 0.0363), and distant metastasis (RR = 1.562, 95% CI: 1.022–2.387, P = 0.0392). Meta-regression and subgroup analysis revealed that ethnic background has influence on the role of SIRT1 expression in predicting survival and clinicopathological characteristics of cancers. Overexpression of SIRT1 predicted a worse OS and higher TNM stage and lymphatic metastasis in Asian population especially in China.Conclusion: Our data suggested that elevated expression of SIRT1 predicted a poor OS, DFS, EFS, PFS, but not for recurrence-free survival (RFS) and cancer-specific survival (CCS). SIRT1 overexpression was associated with higher tumor stage, lymph node metastasis, and distant metastasis. SIRT1-mediated molecular events and biological processes could be an underlying mechanism for metastasis and SIRT1 is a therapeutic target for inhibiting metastasis, leading to good prognosis

Nanoparticle-Based Drug Delivery Systems Targeting Tumor Microenvironment for Cancer Immunotherapy Resistance: Current Advances and Applications

Cancer immunotherapy has shown impressive anti-tumor activity in patients with advanced and early-stage malignant tumors, thus improving long-term survival. However, current cancer immunotherapy is limited by barriers such as low tumor specificity, poor response rate, and systemic toxicities, which result in the development of primary, adaptive, or acquired resistance. Immunotherapy resistance has complex mechanisms that depend on the interaction between tumor cells and the tumor microenvironment (TME). Therefore, targeting TME has recently received attention as a feasibility strategy for re-sensitizing resistant neoplastic niches to existing cancer immunotherapy. With the development of nanotechnology, nanoplatforms possess outstanding features, including high loading capacity, tunable porosity, and specific targeting to the desired locus. Therefore, nanoplatforms can significantly improve the effectiveness of immunotherapy while reducing its toxic and side effects on non-target cells that receive intense attention in cancer immunotherapy. This review explores the mechanisms of tumor microenvironment reprogramming in immunotherapy resistance, including TAMs, CAFs, vasculature, and hypoxia. We also examined whether the application of nano-drugs combined with current regimens is improving immunotherapy clinical outcomes in solid tumors

Gene Cloning, Expression and Enzyme Activity of <i>Vitis vinifera</i> Vacuolar Processing Enzymes (<i>VvVPEs</i>)

<div><p>Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the <i>VPE</i> gene family, we identified 3 <i>Vitis vinifera VPE</i> genes (<i>VvβVPE</i>, <i>VvγVPE</i>, and <i>VvδVPE</i>) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘<i>Vitis vinifera</i> cv. Pinot Noir’ and ‘<i>Vitis vinifera</i> cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 <i>Arabidopsis thaliana</i> and 6 <i>Vitis vinifera</i> VPEs revealed that the 10 <i>VPE</i>s form 3 major branches. Furthermore, the 6 grapevine <i>VPE</i>s share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine <i>VPEs</i> are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the <i>Pichia Pastoris</i> expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that <i>VvδVPE</i> is only expressed in flowers, buds and ovules, that <i>VvγVPE</i> is expressed in various tissues, and that <i>VvβVPE</i> was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that <i>VvβVPE</i> in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that <i>VvβVPE</i> is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.</p></div

Relationship between social capital and heroin use behaviors among patients in methadone maintenance treatment in Sichuan Province, China: A cross-sectional study

Covertly using heroin during methadone maintenance treatment (MMT) is very common among heroin-dependent patients, which has posed threats to the physical health of heroin-dependent patients and social safety. Covertly using heroin may be influenced by many factors, especially social capital. Therefore, we aimed to investigate the relationship between behaviors of covertly using heroin during MMT and social capital heroin-dependent patients in Sichuan Province, China. A cross-sectional study was conducted between October and November 2018, with a total of 581 heroin-dependent patients participating in the study. In addition to socio-demographic characteristics and heroin use related behaviors, the questionnaire also included the measures of social capital: social network (SN), social support (SP), community participation (CP) and social trust (ST). Multivariate logistic regression analyses were used to estimate the association between different measures of social capital and heroin use. The prevalence of covertly using heroin of heroin during MMT was 31.0% among our participants in the 6 months before the study. After adjusting for socio-demographic factors and heroin-use related variables, SN (OR = 0.85, 95% CI: 0.76–0.95), SP (OR = 0.89, 95% CI: 0.83–0.95), and ST (OR = 0.88, 95% CI: 0.81–0.95) were significantly associated with heroin use. Results suggest that social capital may have a protective effect on behavior of covertly using heroin during MMT, which should be consider in the interventions for heroin-dependent patients, in order to reduce the incidence of heroin use during MMT as well as improve the compliance of MMT

Expression of <i>VPE</i> genes in different grapevine tissues.

<p>Expression of <i>VPE</i> genes analyzed by sqRT-PCR in root, stem, leaf, tendril, alabastrum, flowers, pericarp, pulp, and ovule tissues of <i>Vitis vinifera</i> cv. Pinot Noir.</p

Expression of <i>VvδVPE</i> gene in different development stages of ovule in seed and seedless grapes.

<p>Relative expression level of <i>VvδVPE</i> gene in different development stages (15, 20, 25, 30, 35, 40, and 45 DAF) of ovule in ‘<i>V</i>. <i>vinifera</i> cv. Thompson Seedless ‘, ‘<i>V</i>. <i>vinifera</i> cv. Youngle’, ‘<i>V</i>. <i>vinifera</i> cv. Pinot Noir’ and ‘<i>V</i>. <i>vinifera</i> cv.Flame Seedless’. DAF (days after full-bloom) (error bars indicate ±SD).</p

Multi-sequence alignment of <i>Vitis vinifera</i> and <i>Arabidopsis thaliana</i> VPE proteins.

<p>Amino acid sequences from 6 grapevine and 4 <i>Arabidopsis thaliana VPE</i> genes share similar catalytic dyad His (177), Cys (219) and the substrate binding pocket consisting of Arg (112), Arg (389), and Ser (395), with the exception of Ser (395) in VvγVPE, which was replaced with Ala (395).</p