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1.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445145

RESUMO

The main goal of growing plants under various photoperiods is to optimize photosynthesis for using the effect of day length that often acts on plants in combination with biotic and/or abiotic stresses. In this study, Brassica juncea plants were grown under four different day-length regimes, namely., 8 h day/16 h night, 12 h day/12 h night, 16 h day/8 h night, and continuous light, and were infected with a necrotrophic fungus Alternaria brassicicola. The development of necroses on B. juncea leaves was strongly influenced by leaf position and day length. The largest necroses were formed on plants grown under a 16 h day/8 h night photoperiod at 72 h post-inoculation (hpi). The implemented day-length regimes had a great impact on leaf morphology in response to A. brassicicola infection. They also influenced the chlorophyll and carotenoid contents and photosynthesis efficiency. Both the 1st (the oldest) and 3rd infected leaves showed significantly higher minimal fluorescence (F0) compared to the control leaves. Significantly lower values of other investigated chlorophyll a fluorescence parameters, e.g., maximum quantum yield of photosystem II (Fv/Fm) and non-photochemical quenching (NPQ), were observed in both infected leaves compared to the control, especially at 72 hpi. The oldest infected leaf, of approximately 30% of the B. juncea plants, grown under long-day and continuous light conditions showed a 'green island' phenotype in the form of a green ring surrounding an area of necrosis at 48 hpi. This phenomenon was also reflected in changes in the chloroplast's ultrastructure and accelerated senescence (yellowing) in the form of expanding chlorosis. Further research should investigate the mechanism and physiological aspects of 'green islands' formation in this pathosystem.


Assuntos
Alternaria/patogenicidade , Mostardeira/microbiologia , Mostardeira/fisiologia , Necrose/microbiologia , Necrose/patologia , Fotossíntese/fisiologia , Doenças das Plantas/microbiologia , Carotenoides/metabolismo , Clorofila/metabolismo , Clorofila A/metabolismo , Fluorescência , Mostardeira/metabolismo , Necrose/metabolismo , Fotoperíodo , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia
2.
Chem Biol Interact ; 347: 109605, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34333021

RESUMO

Cell injury is a necessary and critical event during CaOx kidney stone formation. Sirt1 exerts a number of pleiotropic effects, protecting against renal cell injury. This study aims to explore the relationship between Sirt1 and CaOx kidney stone formation and the underlying mechanism. Sirt1 expression in renal tissues or HK-2 cells was detected by Western blot, immunohistochemistry and immunofluorescence. Apoptosis in renal tissues was examined by TUNEL staining. Renal pathological changes and the crystals deposition were detected by hematoxylin-eosin and Von Kossa staining. Crystal-cell adhesion and cell injury in HK-2 cells were assessed by atomic absorption spectrometry and flow cytometry, respectively. Sirt1 expression in nephrolithiasis patients was downregulated and the level of apoptosis was increased. Further study found that Sirt1 expression was decreased in both in vivo and in vitro models. Interestingly, the levels of cell injury were elevated in vivo and in vitro models. Suppressing Sirt1 expression promoted COM-induced crystal-cell adhesion and exacerbated cell injury. In contrast, increasing the expression of Sirt1 by lentivirus transfection in vitro and resveratrol administration in vivo, alleviated crystal deposition and cell damage. Our findings suggest that Sirt1 could inhibit kidney stone formation, at least in part, through attenuating CaOx -induced cell injury.


Assuntos
Oxalato de Cálcio/efeitos adversos , Cálculos Renais/metabolismo , Sirtuína 1/metabolismo , Idoso , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Oxalato de Cálcio/química , Oxalato de Cálcio/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular , Cristalização , Feminino , Inativação Gênica , Glioxilatos , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Cálculos Renais/induzido quimicamente , Cálculos Renais/tratamento farmacológico , Cálculos Renais/patologia , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Necrose/induzido quimicamente , Necrose/metabolismo , Resveratrol/uso terapêutico , Sirtuína 1/genética
3.
Ecotoxicol Environ Saf ; 221: 112423, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34146985

RESUMO

Tetrabromodiphenyl ether (BDE-47) is widely used as commercial flame retardants that can be released into the environment and finally enter human body through the food chain. It has been identified to generate neurotoxicity, but little is known about auditory damage and the underlying mechanism following BDE-47 exposure. This study aimed to assess the cell viability with BDE-47 concentration ranging from 0 to 150 µM in mouse organ of Corti-derived cell lines (HEI-OC1). Aryl hydrocarbon receptor (AhR) as an environmental sensor, reactive oxygen species (ROS), NLRP3 inflammasome and p38 MAPK pathways were detected. Results: (1) BDE-47 inhibited the viability in a time- and dose-dependent way in HEI-OC1 cells. Cell cycle was arrested in G1 phase by BDE-47; (2) Elevated intracellular ROS, LDH levels and necrosis were found, which was alleviated by pretreatment with ROS scavenger N-acetylcysteine (NAC); (3) AhR plays an essential role in ligand-regulated transcription factor activation by exogenous environmental compounds. We found increased expression of AhR and decreased downstream targets of CYP 1A1 and CYP 1B1 in BDE-47-treated HEI-OC1 cells, which was reversed by the AhR antagonist CH-223191 for 2 h before BDE-47 exposure. No significant change was detected in CYP 2B; (4) Enhanced expressions of NLRP3 and caspase-1 were induced by BDE-47, with up-regulations of both pro-inflammatory factors for IL-1ß, IL-6 and TNF-α, and anti-inflammatory factors for IL-4, IL-10 and IL-13, but down-regulation for IL-1α; (5) Additionally, the p38 MAPK signaling pathway was activated with increased phosphorylation levels of MKK/3/6, p38 MAPK and NF-kB. Overall, our findings illustrate a role of AhR in ROS-induced necrosis of cochlear hair cells by BDE-47 exposure, in which NLRP3 inflammasome and p38 MAPK signaling pathways are activated. The current study first elucidates the sense of hearing damage induced by BDE-47, and cell-specific or mixture exposures in vivo or human studies are needed to confirm this association.


Assuntos
Retardadores de Chama/toxicidade , Células Ciliadas Auditivas/efeitos dos fármacos , Éteres Difenil Halogenados/toxicidade , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Inflamassomos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose/induzido quimicamente , Necrose/metabolismo , Necrose/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
4.
Int J Biol Macromol ; 182: 1150-1160, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33865895

RESUMO

In the current study, the treatment efficacy of ECHCAH was evaluated in vitro studies using cell viability and flow cytometry in human TNBCs. The results here showed significant gradual reduction in growth of TNBCs (MDA-231cell lines) after their exposure to serial concentrations for hydrogel assembly (5 µg/mL to 25 µg/mL) for 24 and 48 h, representing (86 ± 1% to 45 ± 1.5% p < 0.001) and (79 ± 1.5% to 35 ± 2.5% p < 0.001) respectively. The flow cytometry showed significant increase in the present of late apoptotic and necrotic cells (64% ± 1.2 and 27% ± 0.3 p < 0.001) after 48 h incubation compared to untreated cells (1.13% ± 0.3 and 4% ± 0.2 p < 0.001) respectively. It can be summarized that ECHCA inside targeted hydrogel assemblies can inhibit proliferation of cancer cells.


Assuntos
Carotenoides/química , Quitosana/química , Clorofila/química , Apoptose/fisiologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Feminino , Citometria de Fluxo , Humanos , Hidrogéis/química , Necrose/metabolismo
5.
Biomolecules ; 11(5)2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924766

RESUMO

Chronic inflammatory disorders are characterised by aberrant and exaggerated inflammatory immune cell responses. Modes of extrinsic cell death, apoptosis and necroptosis, have now been shown to be potent drivers of deleterious inflammation, and mutations in core repressors of these pathways underlie many autoinflammatory disorders. The receptor-interacting protein (RIP) kinases, RIPK1 and RIPK3, are integral players in extrinsic cell death signalling by regulating the production of pro-inflammatory cytokines, such as tumour necrosis factor (TNF), and coordinating the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, which underpin pathological inflammation in numerous chronic inflammatory disorders. In this review, we firstly give an overview of the inflammatory cell death pathways regulated by RIPK1 and RIPK3. We then discuss how dysregulated signalling along these pathways can contribute to chronic inflammatory disorders of the joints, skin, and gastrointestinal tract, and discuss the emerging evidence for targeting these RIP kinases in the clinic.


Assuntos
Inflamação/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose/fisiologia , Morte Celular/imunologia , Doença Crônica , Citocinas/metabolismo , Humanos , Inflamassomos/metabolismo , Inflamação/fisiopatologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Necrose/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
6.
Cancer Chemother Pharmacol ; 88(2): 203-209, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33907881

RESUMO

PURPOSE: As critical parameter after extravasation of cytotoxic vesicants, anthracyclines were determined in removed tissue from patients requiring surgical intervention due to tissue necrosis. We monitored their distribution within the affected lesion to establish a possible dose-toxicity relation. METHODS: From six patients scheduled for surgery, removed tissue flaps were systematically analysed by HPLC (epirubicin: 5 subjects; doxorubicin: 1 subject). RESULTS: After extravasation, tissue concentrations were highly variable with an individual anthracycline distribution pattern ranging from a few nanograms up to 17 µg per 100 mg tissue, which indicated a substantial difference in tissue sensitivity among patients. The resection borders coincided with the extension of the erythema and guided the surgical intervention after demarcation of the lesion, which occurred usually 2 or 3 weeks after extravasation. At that time, drug was hardly detected at the resection borders. Wound drains were negative for the extravasated drugs while showing a time profile of vascular growth factors and inflammatory cytokines, which was highly similar to routine surgery. In all six patients, surgical debridement with immediate wound closure led to healing within approximately 2 weeks, when therapy was resumed in all patients with reasonable time delay. CONCLUSION: Surgical intervention after demarcation of the extravasation lesion allows for almost uninterrupted continuation of treatment independent of the amount of extravasated anthracycline. As even minor amounts of the vesicants may trigger tissue necrosis, preventive measures merit the highest priority.


Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Antibióticos Antineoplásicos/farmacocinética , Epirubicina/efeitos adversos , Epirubicina/farmacocinética , Distribuição Tecidual/fisiologia , Idoso , Antraciclinas/efeitos adversos , Antraciclinas/farmacocinética , Antraciclinas/uso terapêutico , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Citocinas/metabolismo , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacocinética , Doxorrubicina/uso terapêutico , Epirubicina/uso terapêutico , Feminino , Humanos , Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Necrose/induzido quimicamente , Necrose/metabolismo , Dermatopatias/tratamento farmacológico , Dermatopatias/metabolismo , Retalhos Cirúrgicos/patologia , Cicatrização/efeitos dos fármacos
7.
Int J Mol Sci ; 22(7)2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33808256

RESUMO

Histiocytic sarcomas refer to highly aggressive tumors with a poor prognosis that respond poorly to conventional treatment approaches. Oncolytic viruses, which have gained significant traction as a cancer therapy in recent decades, represent a promising option for treating histiocytic sarcomas through their replication and/or by modulating the tumor microenvironment. The live attenuated canine distemper virus (CDV) vaccine strain Onderstepoort represents an attractive candidate for oncolytic viral therapy. In the present study, oncolytic virotherapy with CDV was used to investigate the impact of this virus infection on tumor cell growth through direct oncolytic effects or by virus-mediated modulation of the tumor microenvironment with special emphasis on angiogenesis, expression of selected MMPs and TIMP-1 and tumor-associated macrophages in a murine xenograft model of canine histiocytic sarcoma. Treatment of mice with xenotransplanted canine histiocytic sarcomas using CDV induced overt retardation in tumor progression accompanied by necrosis of neoplastic cells, increased numbers of intratumoral macrophages, reduced angiogenesis and modulation of the expression of MMPs and TIMP-1. The present data suggest that CDV inhibits tumor growth in a multifactorial way, including direct cell lysis and reduction of angiogenesis and modulation of MMPs and their inhibitor TIMP-1, providing further support for the concept of its role in oncolytic therapies.


Assuntos
Sarcoma Histiocítico/metabolismo , Neoplasias/metabolismo , Terapia Viral Oncolítica/métodos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Cinomose/metabolismo , Cinomose/virologia , Vírus da Cinomose Canina/patogenicidade , Doenças do Cão/imunologia , Cães , Feminino , Xenoenxertos , Sarcoma Histiocítico/veterinária , Sarcoma Histiocítico/virologia , Metaloendopeptidases/metabolismo , Camundongos , Camundongos SCID , Necrose/metabolismo , Neoplasias/virologia , Neovascularização Patológica/metabolismo , Vírus Oncolíticos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Microambiente Tumoral/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Front Immunol ; 12: 631821, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33746968

RESUMO

Neutrophils or polymorphonuclear leukocytes (PMN) are key participants in the innate immune response for their ability to execute different effector functions. These cells express a vast array of membrane receptors that allow them to recognize and eliminate infectious agents effectively and respond appropriately to microenvironmental stimuli that regulate neutrophil functions, such as activation, migration, generation of reactive oxygen species, formation of neutrophil extracellular traps, and mediator secretion, among others. Currently, it has been realized that activated neutrophils can accomplish their effector functions and simultaneously activate mechanisms of cell death in response to different intracellular or extracellular factors. Although several studies have revealed similarities between the mechanisms of cell death of neutrophils and other cell types, neutrophils have distinctive properties, such as a high production of reactive oxygen species (ROS) and nitrogen species (RNS), that are important for their effector function in infections and pathologies such as cancer, autoimmune diseases, and immunodeficiencies, influencing their cell death mechanisms. The present work offers a synthesis of the conditions and molecules implicated in the regulation and activation of the processes of neutrophil death: apoptosis, autophagy, pyroptosis, necroptosis, NETosis, and necrosis. This information allows to understand the duality encountered by PMNs upon activation. The effector functions are carried out to eliminate invading pathogens, but in several instances, these functions involve activation of signaling cascades that culminate in the death of the neutrophil. This process guarantees the correct elimination of pathogenic agents, damaged or senescent cells, and the timely resolution of the inflammation that is essential for the maintenance of homeostasis in the organism. In addition, they alert the organism when the immunological system is being deregulated, promoting the activation of other cells of the immune system, such as B and T lymphocytes, which produce cytokines that potentiate the microbicide functions.


Assuntos
Morte Celular/imunologia , Neutrófilos/patologia , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/imunologia , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Radicais Livres/metabolismo , Humanos , Necroptose/imunologia , Necrose/imunologia , Necrose/metabolismo , Ativação de Neutrófilo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose/imunologia , Piroptose/imunologia , Receptores de Morte Celular/metabolismo
9.
PLoS Genet ; 17(2): e1009066, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33571185

RESUMO

Intracellular Ca2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an "eat-me" signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca2+ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca2+ pool. Our findings indicate that high levels of cytoplasmic Ca2+ are necessary and sufficient for PS exposure. They further reveal two Ca2+-dependent, necrosis-specific pathways that promote PS exposure, a "two-step" pathway initiated by a modest influx of Ca2+ and further boosted by the release of Ca2+ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca2+ pathways promote PS externalization through activating ANOH-1.


Assuntos
Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Neurônios/metabolismo , Fosfatidilserinas/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/genética , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Citoplasma/metabolismo , Dantroleno/farmacologia , Canais de Sódio Degenerina/genética , Canais de Sódio Degenerina/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Inibidores Enzimáticos/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Relaxantes Musculares Centrais/farmacologia , Necrose/genética , Necrose/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Canais de Sódio/genética , Canais de Sódio/metabolismo , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Tapsigargina/farmacologia
10.
Am J Physiol Renal Physiol ; 320(4): F578-F595, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33615890

RESUMO

Regulated cell death (RCD), distinct from accidental cell death, refers to a process of well-controlled programmed cell death with well-defined pathological mechanisms. In the past few decades, various terms for RCDs were coined, and some of them have been implicated in the pathogenesis of various types of acute kidney injury (AKI). Cisplatin is widely used as a chemotherapeutic drug for a broad spectrum of cancers, but its usage was hampered because of being highly nephrotoxic. Cisplatin-induced AKI is commonly seen clinically, and it also serves as a well-established prototypic model for laboratory investigations relevant to acute nephropathy affecting especially the tubular compartment. Literature reports over a period of three decades have indicated that there are multiple types of RCDs, including apoptosis, necroptosis, pyroptosis, ferroptosis, and mitochondrial permeability transition-mediated necrosis, and some of them are pertinent to the pathogenesis of cisplatin-induced AKI. Interestingly, myo-inositol metabolism, a vital biological process that is largely restricted to the kidney, seems to be relevant to the pathogenesis of certain forms of RCDs. A comprehensive understanding of RCDs in cisplatin-induced AKI and their relevance to myo-inositol homeostasis may yield novel therapeutic targets for the amelioration of cisplatin-related nephropathy.


Assuntos
Injúria Renal Aguda/induzido quimicamente , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Inositol/metabolismo , Necroptose/efeitos dos fármacos , Injúria Renal Aguda/patologia , Animais , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Camundongos Endogâmicos C57BL , Necrose/metabolismo , Necrose/patologia , Morte Celular Regulada/efeitos dos fármacos
11.
Int J Mol Sci ; 22(3)2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33494299

RESUMO

Innate immune receptors initiate a host immune response, or inflammatory response, upon detecting pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Among the innate immune receptors, nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play a pivotal role in detecting cytosolic PAMPs and DAMPs. Some NLRs can form a multiprotein cytosolic complex known as the inflammasome. Inflammasome activation triggers caspase-1-mediated cleavage of the pore-forming protein gasdermin D (GSDMD), which drives a form of inflammatory cell death called pyroptosis. Parallelly, activated caspase-1 cleaves immature cytokines pro-IL-1ß and pro-IL-18 into their active forms, which can be released via GSDMD membrane pores. The NLR family apoptosis inhibitory proteins (NAIP)-NLR family caspase-associated recruitment domain-containing protein 4 (NLRC4) inflammasome is important for mounting an immune response against Gram-negative bacteria. NLRC4 is activated through NAIPs sensing type 3 secretion system (T3SS) proteins from Gram-negative bacteria, such as Salmonella Typhimurium. Mutations in NAIPs and NLRC4 are linked to autoinflammatory disorders in humans. In this review, we highlight the role of the NAIP/NLRC4 inflammasome in host defense, autoinflammatory diseases, cancer, and cell death. We also discuss evidence pointing to a role of NLRC4 in PANoptosis, which was recently identified as a unique inflammatory programmed cell death pathway with important physiological relevance in a range of diseases. Improved understanding of the NLRC4 inflammasome and its potential roles in PANoptosis paves the way for identifying new therapeutic strategies to target disease.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Inflamassomos/metabolismo , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Autoimunidade , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas de Ligação ao Cálcio/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Necrose/genética , Necrose/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Neoplasias/patologia , Piroptose/genética
12.
Theranostics ; 11(6): 2876-2891, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33456578

RESUMO

Rationale: Structural remodeling or damage as a result of disease or injury is often not evenly distributed throughout a tissue but strongly depends on localization and extent of damaging stimuli. Skeletal muscle as a mechanically active organ can express signs of local or even systemic myopathic damage, necrosis, or repair. Conventionally, muscle biopsies (patients) or whole muscles (animal models) are mechanically sliced and stained to assess structural alterations histologically. Three-dimensional tissue information can be obtained by applying deep imaging modalities, e.g. multiphoton or light-sheet microscopy. Chemical clearing approaches reduce scattering, e.g. through matching refractive tissue indices, to overcome optical penetration depth limits in thick tissues. Methods: Here, we optimized a range of different clearing protocols. We find aqueous solution-based protocols employing (20-80%) 2,2'-thiodiethanol (TDE) to be advantageous over organic solvents (dibenzyl ether, cinnamate) regarding the preservation of muscle morphology, ease-of-use, hazard level, and costs. Results: Applying TDE clearing to a mouse model of local cardiotoxin (CTX)-induced muscle necrosis, a complete loss of myosin-II signals was observed in necrotic areas with little change in fibrous collagen or autofluorescence (AF) signals. The 3D aspect of myofiber integrity could be assessed, and muscle necrosis in whole muscle was quantified locally via the ratios of detected AF, forward- and backward-scattered Second Harmonic Generation (fSHG, bSHG) signals. Conclusion: TDE optical clearing is a versatile tool to study muscle architecture in conjunction with label-free multiphoton imaging in 3D in injury/myopathy models and might also be useful in studying larger biofabricated constructs in regenerative medicine.


Assuntos
Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Músculo Esquelético/metabolismo , Necrose/diagnóstico , Animais , Cardiotoxinas/farmacologia , Colágeno/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Imageamento Tridimensional/métodos , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Miofibrilas/metabolismo , Miosina Tipo II/metabolismo , Necrose/induzido quimicamente , Necrose/metabolismo , Compostos de Sulfidrila/farmacologia
13.
Lab Invest ; 101(3): 381-395, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33483597

RESUMO

Real-time tissue classifiers based on molecular patterns are emerging tools for fast tumor diagnosis. Here, we used rapid evaporative ionization mass spectrometry (REIMS) and multivariate statistical analysis (principal component analysis-linear discriminant analysis) to classify tissues with subsequent comparison to gold standard histopathology. We explored whether REIMS lipid patterns can identify human liver tumors and improve the rapid characterization of their underlying metabolic features. REIMS-based classification of liver parenchyma (LP), hepatocellular carcinoma (HCC), and metastatic adenocarcinoma (MAC) reached an accuracy of 98.3%. Lipid patterns of LP were more similar to those of HCC than to those of MAC and allowed clear distinction between primary and metastatic liver tumors. HCC lipid patterns were more heterogeneous than those of MAC, which is consistent with the variation seen in the histopathological phenotype. A common ceramide pattern discriminated necrotic from viable tumor in MAC with 92.9% accuracy and in other human tumors. Targeted analysis of ceramide and related sphingolipid mass features in necrotic tissues may provide a new classification of tumor cell death based on metabolic shifts. Real-time lipid patterns may have a role in future clinical decision-making in cancer precision medicine.


Assuntos
Lipídeos/análise , Neoplasias Hepáticas , Fígado , Necrose , Adulto , Estudos de Coortes , Humanos , Fígado/química , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/química , Neoplasias Hepáticas/classificação , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Necrose/classificação , Necrose/metabolismo , Necrose/patologia , Análise de Componente Principal , Espectrometria de Massas por Ionização por Electrospray
14.
Cell Prolif ; 54(3): e12992, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33506613

RESUMO

Spinal cord injury (SCI) always leads to functional deterioration due to a series of processes including cell death. In recent years, programmed cell death (PCD) is considered to be a critical process after SCI, and various forms of PCD were discovered in recent years, including apoptosis, necroptosis, autophagy, ferroptosis, pyroptosis and paraptosis. Unlike necrosis, PCD is known as an active cell death mediated by a cascade of gene expression events, and it is crucial for elimination unnecessary and damaged cells, as well as a defence mechanism. Therefore, it would be meaningful to characterize the roles of PCD to not only enhance our understanding of the pathophysiological processes, but also improve functional recovery after SCI. This review will summarize and explore the most recent advances on how apoptosis, necroptosis, autophagy, ferroptosis, pyroptosis and paraptosis are involved in SCI. This review can help us to understand the various functions of PCD in the pathological processes of SCI, and contribute to our novel understanding of SCI of unknown aetiology in the near future.


Assuntos
Apoptose/genética , Morte Celular/efeitos dos fármacos , Necrose/tratamento farmacológico , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Autofagia/efeitos dos fármacos , Morte Celular/fisiologia , Humanos , Necroptose/efeitos dos fármacos , Necrose/metabolismo , Traumatismos da Medula Espinal/patologia
15.
Am J Transplant ; 21(4): 1440-1452, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32978875

RESUMO

Inhibition of mitochondrial reactive oxygen species (ROS) and subsequent damage-associated molecular patterns (DAMPs)-induced inflammatory responses could be a novel target in clinical islet transplantation. We investigated the protective effects of NecroX-7, a novel clinical-grade necrosis inhibitor that specifically targets mitochondrial ROS, against primary islet graft failure. Islets from heterozygote human islet amyloid polypeptide transgenic (hIAPP+/- ) mice and nonhuman primates (NHPs) were isolated or cultured with or without NecroX-7 in serum-deprived medium. Supplementation with NecroX-7 during hIAPP+/- mouse islet isolation markedly increased islet viability and adenosine triphosphate content, and attenuated ROS, transcription of c-Jun N-terminal kinases, high mobility group box 1, interleukin-1beta (IL-1 ß ), IL-6, and tumor necrosis factor-alpha. Supplementation of NecroX-7 during serum-deprived culture also protected hIAPP+/- mouse and NHP islets against impaired viability, serum deprivation-induced ROS, proinflammatory response, and accumulation of toxic IAPP oligomer. Supplementation with NecroX-7 during isolation or serum-deprived culture of hIAPP+/- mouse and NHP islets also improved posttransplant glycemia in the recipient streptozotocin-induced diabetic hIAPP-/- mice and BALB/c-nu/nu mice, respectively. In conclusion, pretransplant administration of NecroX-7 during islet isolation and serum-deprived culture suppressed mitochondrial ROS injury, generation of DAMPs-induced proinflammatory responses, and accumulation of toxic IAPP oligomers ex vivo, and improved posttransplant glycemia in vivo.


Assuntos
Diabetes Mellitus Tipo 2 , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Amiloide/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Inflamação/metabolismo , Inflamação/prevenção & controle , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Necrose/metabolismo , Estresse Oxidativo
16.
Biochim Biophys Acta Mol Cell Res ; 1868(2): 118911, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33227312

RESUMO

In this work, the effect of an early oxidative stress on human endothelial cells induced by menadione was studied using a combined methodology of label-free Raman imaging and fluorescence staining. Menadione-induced ROS-dependent endothelial inflammation in human aorta endothelial cells (HAEC) was studied with focus on changes in cytochrome, proteins, nucleic acids and lipids content and their distribution in cells. Fluorescence staining (ICAM-1, VCAM-1, vWF, LipidTox, MitoRos and DCF) was used to confirm endothelial inflammation and ROS generation. The results showed that short time, exposure to menadione did not cause their apoptosis or necrosis (Annexin V Apoptosis Detection Kit) within the 3 h timescale of measurement. On the other hand, 3 h of incubation, did result in endothelial inflammation (ICAM-1, VCAM-1, vWF) that was associated with an increased ROS formation (MitoRos and DCF) suggesting the oxidative stress-mediated inflammation. Chemometric analysis of spectral data enabled the determination of spectroscopic markers of menadione-induced oxidative stress-mediated endothelial inflammation including a decrease of the bands intensity of cytochrome (604, 750, 1128, 1315 and 1585 cm-1), nucleic acids bands (785 cm-1), proteins (1005 cm-1) and increased intensity of lipid bands (722, 1085, 1265, 1303, 1445 and 1660 cm-1), without changes in the spectroscopic signature of the cell nucleus. In conclusion, oxidative stress resulting in endothelial inflammation was featured by significant alterations in the number of biochemical changes in mitochondria and other cellular compartments detected by Raman spectroscopy. Most of these, coexisted with results from fluorescence imaging, and most importantly occurred earlier than the detection of increased ROS or markers of endothelial inflammation.


Assuntos
Aorta/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Análise Espectral Raman/métodos , Vitamina K 3/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Necrose/metabolismo , Imagem Óptica/métodos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Viruses ; 12(10)2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-33007975

RESUMO

Grapevine leafroll-associated virus 1 (GLRaV-1) is a major pathogen associated with grapevine leafroll disease. However, the molecular mechanisms underlying GLRaV-1 interactions with plant cells are unclear. Using Agrobacterium infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24G1) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24G1 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24G1 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24G1 interaction with importin α1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24G1 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24G1 may be a factor in pathogenesis. Furthermore, p24G1 function in pathogenesis required its RSS activity, dimerization and nuclear localization. In addition, the region of amino acids 122-139 played a crucial role in the nuclear import, siRNA binding, silencing suppression and pathogenic activity of p24G1. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.


Assuntos
Closteroviridae/genética , Necrose/metabolismo , Interferência de RNA/fisiologia , Tabaco/virologia , Agrobacterium/genética , Closteroviridae/patogenicidade , Necrose/virologia , Doenças das Plantas/virologia , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Potexvirus/genética , RNA Interferente Pequeno/metabolismo
18.
Ann Clin Lab Sci ; 50(5): 611-624, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33067207

RESUMO

OBJECTIVE: Patients with epithelial ovarian cancers experience the highest fatality rates among all gynecological malignancies which require development of novel treatment strategies. Tumor cell necrosis was previously reported in a number of cancer cell lines following treatment with a p53-derived anti-cancer peptide called PNC-27. This peptide induces necrosis by transmembrane pore formation with HDM-2 protein that is expressed in the cancer cell membrane. We aimed to extend these studies further by investigating expression of membrane HDM-2 protein in ovarian cancer as it relates to susceptibility to PNC-27. PROCEDURES: Herein, we measured HDM-2 membrane expression in two ovarian cancer cell lines (SKOV-3 and OVCAR-3) and a non-transformed control cell line (HUVEC) by flow cytometric and western blot analysis. Immunofluorescence was used to visualize colocalization of PNC-27 with membrane HDM-2. Treatment effects with PNC-27 and control peptide were assessed using a MTT cell proliferation assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers; annexin V and caspase-3. RESULTS: HDM-2 protein was highly expressed and frequently detected in the membranes of SKOV-3 and OVCAR-3 cells; a prominent 47.6 kDa HDM-2 plasma membrane isoform was present in both cell lines whereas 25, 29, and 30 kDa isoforms were preferentially expressed in OVCAR-3. Notably, PNC-27 colocalized with HDM-2 in the membranes of both cancer cell lines that resulted in rapid cellular necrosis. In contrast, no PNC-27 colocalization and cytotoxicity was observed with non-transformed HUVEC demonstrating minimal expression of membrane HDM-2. CONCLUSIONS: Our results suggest that HDM-2 is highly expressed in the membranes of these ovarian cancer cell lines and colocalizes with PNC-27. We therefore conclude that the association of PNC-27 with preferentially expressed membrane HDM-2 isoforms results in the proposed model for the formation of transmembrane pores and epithelial ovarian cancer tumor cell necrosis, as previously described in a number of solid tissue and hematologic malignancies.


Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/farmacologia , Anexina A5/análise , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Epitelial do Ovário/metabolismo , Caspase 3/análise , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , L-Lactato Desidrogenase/análise , Necrose/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo
19.
JCI Insight ; 5(19)2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32941183

RESUMO

We determined that renal proximal tubular (PT) NF-κB essential modulator (NEMO) plays a direct and critical role in ischemic acute kidney injury (AKI) using mice lacking renal PT NEMO and by targeted renal PT NEMO inhibition with mesoscale nanoparticle-encapsulated NEMO binding peptide (NBP MNP). We subjected renal PT NEMO-deficient mice, WT mice, and C57BL/6 mice to sham surgery or 30 minutes of renal ischemia and reperfusion (IR). C57BL/6 mice received NBP MNP or empty MNP before renal IR injury. Mice treated with NBP MNP and mice deficient in renal PT NEMO were protected against ischemic AKI, having decreased renal tubular necrosis, inflammation, and apoptosis compared with control MNP-treated or WT mice, respectively. Recombinant peptidylarginine deiminase type 4 (rPAD4) targeted kidney PT NEMO to exacerbate ischemic AKI in that exogenous rPAD4 exacerbated renal IR injury in WT mice but not in renal PT NEMO-deficient mice. Furthermore, rPAD4 upregulated proinflammatory cytokine mRNA and NF-κB activation in freshly isolated renal proximal tubules from WT mice but not from PT NEMO-deficient mice. Taken together, our studies suggest that renal PT NEMO plays a critical role in ischemic AKI by promoting renal tubular inflammation, apoptosis, and necrosis.


Assuntos
Injúria Renal Aguda/genética , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Necrose/genética , Peptídeos/farmacologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Apoptose/genética , Proteínas de Transporte , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Composição de Medicamentos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Necrose/metabolismo , Necrose/patologia , Proteína-Arginina Desiminase do Tipo 4/farmacologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
20.
Nat Cell Biol ; 22(9): 1042-1048, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32868903

RESUMO

Ferroptosis is a regulated form of necrotic cell death that is caused by the accumulation of oxidized phospholipids, leading to membrane damage and cell lysis1,2. Although other types of necrotic death such as pyroptosis and necroptosis are mediated by active mechanisms of execution3-6, ferroptosis is thought to result from the accumulation of unrepaired cell damage1. Previous studies have suggested that ferroptosis has the ability to spread through cell populations in a wave-like manner, resulting in a distinct spatiotemporal pattern of cell death7,8. Here we investigate the mechanism of ferroptosis execution and discover that ferroptotic cell rupture is mediated by plasma membrane pores, similarly to cell lysis in pyroptosis and necroptosis3,4. We further find that intercellular propagation of death occurs following treatment with some ferroptosis-inducing agents, including erastin2,9 and C' dot nanoparticles8, but not upon direct inhibition of the ferroptosis-inhibiting enzyme glutathione peroxidase 4 (GPX4)10. Propagation of a ferroptosis-inducing signal occurs upstream of cell rupture and involves the spreading of a cell swelling effect through cell populations in a lipid peroxide- and iron-dependent manner.


Assuntos
Ferroptose/fisiologia , Osmose/fisiologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Ferro/metabolismo , Células MCF-7 , Necrose/metabolismo , Necrose/patologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Células U937
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