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Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane, and its activity accounts for nearly all biologically produced methane released into the atmosphere. The assembly of MCR is an intricate process involving the installation of a complex set of posttranslational modifications and the unique Ni-containing tetrapyrrole called coenzyme F430. Despite decades of research, details of MCR assembly remain largely unresolved. Here, we report the structural characterization of MCR in two intermediate states of assembly. These intermediate states lack one or both F430 cofactors and form complexes with the previously uncharacterized McrD protein. McrD is found to bind asymmetrically to MCR, displacing large regions of the alpha subunit and increasing active-site accessibility for the installation of F430-shedding light on the assembly of MCR and the role of McrD therein. This work offers crucial information for the expression of MCR in a heterologous host and provides targets for the design of MCR inhibitors.
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Atmósfera , MetanoRESUMEN
A stable but reversible phenotype switch from normal to persister state is advantageous to the intracellular pathogens to cause recurrent infections and to evade the host immune system. Staphylococcus aureus is a versatile opportunistic pathogen known to cause chronic infections with significant mortality. One of the notable features is the ability to switch to a per-sisters cell, which is found in planktonic and biofilm states. This phenotypic switch is always an open question to explore the hidden fundamental science that coheres with a calculated or fortuitous move. Toxin-antitoxin modules, nutrient stress, and an erroneous translation-enabled state of dormancy entail this persistent behaviour in S. aureus. It is paramount to get a clear picture of why the cell chooses to enter a persistent condition, as it would decide the course of treatment. Analyzing the exit from a persistent state to an active state and the subsequent repercussion of this transition is essential to determine its role in chronic infections. This review attempts to provide a constructed argument discussing the most widely accepted mechanisms and identifying the various attributes of persistence.
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Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.
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Biología Computacional/métodos , Enzimas/metabolismo , Péptidos/química , Péptidos/metabolismo , Ingeniería de Proteínas/métodos , Productos Biológicos/química , Productos Biológicos/clasificación , Productos Biológicos/metabolismo , Enzimas/química , Hidroxilación , Metilación , Péptidos/clasificación , Péptidos/genética , Fosforilación , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/fisiología , Ribosomas/metabolismoRESUMEN
Intracerebral hemorrhage (ICH) is a major public health problem and devastating subtype of stroke with high morbidity and mortality. Notably, there is no effective treatment for ICH. Neuroinflammation, a pathological hallmark of ICH, contributes to both brain injury and repair and hence, it is regarded as a potential target for therapeutic intervention. Recent studies document that microRNAs, small non-coding RNA molecules, can regulate inflammatory brain response after ICH and are viable molecular targets to alter brain function. Therefore, there is an escalating interest in studying the role of microRNAs in the pathophysiology of ICH. Herein, we provide, for the first time, an overview of the microRNAs that play roles in ICH-induced neuroinflammation and identify the critical knowledge gap in the field, as it would help design future studies.
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Hemorragia Cerebral/metabolismo , Encefalitis/metabolismo , MicroARNs/metabolismo , Transducción de Señal/genética , Accidente Cerebrovascular/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Lesiones Encefálicas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Microglía/metabolismo , Microglía/patología , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
TSPO (18 kDa translocator protein) was identified decades ago in a search for peripheral tissue binding sites for benzodiazepines, and was formerly called the peripheral benzodiazepine receptor. TSPO is a conserved protein throughout evolution and it is implicated in the regulation of many cellular processes, including inflammatory responses, oxidative stress, and mitochondrial homeostasis. TSPO, apart from its broad expression in peripheral tissues, is highly expressed in neuroinflammatory cells, such as activated microglia. In addition, emerging studies employing the ligands of TSPO suggest that TSPO plays an important role in neuropathological settings as a biomarker and therapeutic target. However, the precise molecular function of this protein in normal physiology and neuropathology remains enigmatic. This review provides an overview of recent advances in our understanding of this multifaceted molecule and identifies the knowledge gap in the field for future functional studies.
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Receptores de GABA/genética , Receptores de GABA/metabolismo , Animales , Secuencia Conservada , Susceptibilidad a Enfermedades , Evolución Molecular , Humanos , Ligandos , Mitocondrias/genética , Mitocondrias/metabolismo , Terapia Molecular Dirigida , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Receptores de GABA/química , Esteroides/biosíntesisRESUMEN
BACKGROUND: Intracerebral hemorrhage (ICH) is a potentially fatal stroke subtype accounting for 10-15 % of all strokes. Despite neurosurgical intervention and supportive care, the 30-day mortality rate remains 30-50 % with ICH survivors frequently displaying neurological impairment and requiring long-term assisted care. Although accumulating evidence demonstrates the role of neuroinflammation in secondary brain injury and delayed fatality after ICH, the molecular regulators of neuroinflammation remain poorly defined after ICH. METHODS: In the present study, ICH was induced in CD1 male mice by collagenase injection method and given the emerging role of TSPO (18-kDa translocator protein) in neuroinflammation, immunofluorescence staining of brain sections was performed to characterize the temporal expression pattern and cellular and subcellular localization of TSPO after ICH. Further, both genetic and pharmacological studies were employed to assess the functional role of TSPO in neuroinflammation. RESULTS: The expression of TSPO was found to be increased in the peri-hematomal brain region 1 to 7 days post-injury, peaking on day 3 to day 5 in comparison to sham. Further, the TSPO expression was mostly observed in microglia/macrophages, the inflammatory cells of the central nervous system, suggesting an unexplored role of TSPO in neuroinflammatory responses after ICH. Further, the subcellular localization studies revealed prominent perinuclear expression of TSPO after ICH. Moreover, both genetic and pharmacological studies revealed a regulatory role of TSPO in the release of pro-inflammatory cytokines in a macrophage cell line, RAW 264.7. CONCLUSIONS: Altogether, the data suggest that TSPO induction after ICH could be an intrinsic mechanism to prevent an exacerbated inflammatory response and raise the possibility of targeting TSPO for the attenuation of secondary brain injury after ICH.
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Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/patología , Receptores de GABA/biosíntesis , Animales , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Hemorragia Cerebral/genética , Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Receptores de GABA/genéticaRESUMEN
Retinopathy of prematurity adversely affects premature infants because of oxygen-induced damage of the immature retinal vasculature, resulting in pathological neovascularization (NV). Our pilot studies using the mouse model of oxygen-induced retinopathy (OIR) showed marked increases in angiogenic mediators, including endothelins and endothelin receptor (EDNR) A. We hypothesized that activation of the endothelin system via EDNRA plays a causal role in pathological angiogenesis and up-regulation of angiogenic mediators, including vascular endothelial growth factor A (VEGFA) in OIR. Mice were exposed to 75% oxygen from post-natal day P7 to P12, treated with either vehicle or EDNRA antagonist BQ-123 or EDNRB antagonist BQ-788 on P12, and kept at room air from P12 to P17 (ischemic phase). RT-PCR analysis revealed increased levels of EDN2 and EDNRA mRNA, and Western blot analysis revealed increased EDN2 expression during the ischemic phase. EDNRA inhibition significantly increased vessel sprouting, resulting in enhanced physiological angiogenesis and decreased pathological NV, whereas EDNRB inhibition modestly improved vascular repair. OIR triggered significant increases in VEGFA protein and mRNA for delta-like ligand 4, apelin, angiopoietin-2, and monocyte chemoattractant protein-1. BQ-123 treatment significantly reduced these alterations. EDN2 expression was localized to retinal glia and pathological NV tufts of the OIR retinas. EDN2 also induced VEGFA protein expression in cultured astrocytes. In conclusion, inhibition of the EDNRA during OIR suppresses pathological NV and promotes physiological angiogenesis.
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Endotelinas/metabolismo , Retina/metabolismo , Neovascularización Retiniana/metabolismo , Retinopatía de la Prematuridad/metabolismo , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Modelos Animales de Enfermedad , Antagonistas de los Receptores de Endotelina/farmacología , Ratones , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Piperidinas/farmacología , Retina/efectos de los fármacos , Retina/patología , Neovascularización Retiniana/patología , Retinopatía de la Prematuridad/patología , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Malignant glioma is one of the most devastating tumors in adults with poor patient prognosis. Notably, glioma often exhibits resistance to conventional chemotherapeutic approaches, complicating patient treatments. However, the molecular mediators involved in tumor chemoresistance remain poorly defined, creating a barrier to the successful management of glioma. In the present study, we hypothesized that the antioxidant transcription factor, Nrf2 (nuclear factor erythroid-derived 2 like 2), attenuates glioma cytotoxicity to Carmustine (BCNU), a widely used chemotherapeutic agent known to modulate cellular oxidative balance. METHODS: To test the hypothesis, we employed human malignant glioma cell line, U87MG and overexpression of Nrf2 in glioma cells was achieved using both pharmacological and genetic approaches. RESULTS: Notably, induction of Nrf2 was associated with increased expression of heme oxygenase-1 (HO-1), a stress inducible enzyme involved in anti-oxidant defense. In addition, over expression of Nrf2 in U87MG cells significantly attenuated the cytotoxicity of Carmustine as evidenced by both cellular viability assay and flow cytometry analysis. Consistent with this, antioxidants such as glutathione and N-acetyl cysteine significantly reduced Carmustine mediated glioma cytotoxicity. CONCLUSIONS: Taken together, these data strongly implicate an unexplored role of Nrf2 in glioma resistance to Carmustine and raise the possible use of Nrf2 inhibitors as adjunct to Carmustine for the treatment of malignant glioma.
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Antineoplásicos Alquilantes/farmacología , Carmustina/farmacología , Resistencia a Antineoplásicos/genética , Expresión Génica , Glioma/genética , Factor 2 Relacionado con NF-E2/genética , Antineoplásicos Alquilantes/toxicidad , Antioxidantes/farmacología , Carmustina/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Glioma/metabolismo , Humanos , Hidroquinonas/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide. Cerebral edema, a life-threatening medical complication, contributes to elevated intracranial pressure (ICP) and a poor clinical prognosis after TBI. Unfortunately, treatment options to reduce post-traumatic edema remain suboptimal, due in part, to a dearth of viable therapeutic targets. Herein, we tested the hypothesis that cerebral innate immune responses contribute to edema development after TBI. Our results demonstrate that high-mobility group box protein 1 (HMGB1) was released from necrotic neurons via a NR2B-mediated mechanism. HMGB1 was clinically associated with elevated ICP in patients and functionally promoted cerebral edema after TBI in mice. The detrimental effects of HMGB1 were mediated, at least in part, via activation of microglial toll-like receptor 4 (TLR4) and the subsequent expression of the astrocytic water channel, aquaporin-4 (AQP4). Genetic or pharmacological (VGX-1027) TLR4 inhibition attenuated the neuroinflammatory response and limited post-traumatic edema with a delayed, clinically implementable therapeutic window. Human and rodent tissue culture studies further defined the cellular mechanisms demonstrating neuronal HMGB1 initiates the microglial release of interleukin-6 (IL-6) in a TLR4 dependent mechanism. In turn, microglial IL-6 increased the astrocytic expression of AQP4. Taken together, these data implicate microglia as key mediators of post-traumatic brain edema and suggest HMGB1-TLR4 signaling promotes neurovascular dysfunction after TBI.
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Edema Encefálico/etiología , Lesiones Encefálicas/complicaciones , Proteína HMGB1/metabolismo , Microglía/metabolismo , Neuronas/metabolismo , Receptor Toll-Like 4/metabolismo , Acetatos/farmacología , Animales , Edema Encefálico/patología , Lesiones Encefálicas/líquido cefalorraquídeo , Células Cultivadas , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Factores Inmunológicos/farmacología , Masculino , Ratones , Ratones Endogámicos C3H , Microglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Oxazoles/farmacología , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Receptor Toll-Like 4/genéticaRESUMEN
Histone deacetylases (HDACs) are critical regulators of inflammatory gene expression, and the efficacy of pan-HDAC inhibitors has been implicated in various disease conditions. However, it remains largely unclear how HDACs precisely regulate inflammation. To this end, evaluating the isoform-specific function of HDACs is critical, and the isoform-specific targeting could also circumvent the off-target effects of pan-HDAC inhibitors. This review provides an overview of the roles of HDAC3, a class I HDAC isoform, in modulating inflammatory responses and discusses the molecular mechanisms by which HDAC3 regulates inflammation associated with brain pathology, arthritis, cardiovascular diseases, lung pathology, allergic conditions, and kidney disorders. The articles also identify knowledge gaps in the field for future studies. Despite some conflicting reports, the selective inhibition of HDAC3 has been demonstrated to play a beneficial role in various inflammatory pathologies. Exploring the potential of HDAC3 inhibition to improve disease prognosis is a promising avenue requiring further investigation.
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Inhibidores de Histona Desacetilasas , Histona Desacetilasas , Inflamación , Humanos , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genética , Inflamación/inmunología , Animales , Inhibidores de Histona Desacetilasas/uso terapéutico , Inhibidores de Histona Desacetilasas/farmacologíaRESUMEN
The radical S-adenosylmethionine (rSAM) superfamily has become a wellspring for discovering new enzyme chemistry, especially regarding ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we report a compendium of nearly 15,000 rSAM proteins with high-confidence involvement in RiPP biosynthesis. While recent bioinformatics advances have unveiled the broad sequence space covered by rSAM proteins, the significant challenge of functional annotation remains unsolved. Through a combination of sequence analysis and protein structural predictions, we identified a set of catalytic site proximity residues with functional predictive power, especially among the diverse rSAM proteins that form sulfur-to-α carbon thioether (sactionine) linkages. As a case study, we report that an rSAM protein from Streptomyces sparsogenes (StsB) shares higher full-length similarity with MftC (mycofactocin biosynthesis) than any other characterized enzyme. However, a comparative analysis of StsB to known rSAM proteins using "catalytic site proximity" predicted that StsB would be distinct from MftC and instead form sactionine bonds. The prediction was confirmed by mass spectrometry, targeted mutagenesis, and chemical degradation. We further used "catalytic site proximity" analysis to identify six new sactipeptide groups undetectable by traditional genome-mining strategies. Additional catalytic site proximity profiling of cyclophane-forming rSAM proteins suggests that this approach will be more broadly applicable and enhance, if not outright correct, protein functional predictions based on traditional genomic enzymology principles.
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Stroke is one of the most common diseases that leads to brain injury and mortality in patients, and intracerebral hemorrhage (ICH) is the most devastating subtype of stroke. Though the prevalence of ICH increases with aging, the effect of aging on the pathophysiology of ICH remains largely understudied. Moreover, there is no effective treatment for ICH. Recent studies have demonstrated the potential of circulating microRNAs as non-invasive diagnostic and prognostic biomarkers in various pathological conditions. While many studies have identified microRNAs that play roles in the pathophysiology of brain injury, few demonstrated their functions and roles after ICH. Given this significant knowledge gap, the present study aims to identify microRNAs that could serve as potential biomarkers of ICH in the elderly. To this end, sham or ICH was induced in aged C57BL/6 mice (18-24 months), and 24 h post-ICH, serum microRNAs were isolated, and expressions were analyzed. We identified 28 significantly dysregulated microRNAs between ICH and sham groups, suggesting their potential to serve as blood biomarkers of acute ICH. Among those microRNAs, based on the current literature, miR-124-3p, miR-137-5p, miR-138-5p, miR-219a-2-3p, miR-135a-5p, miR-541-5p, and miR-770-3p may serve as the most promising blood biomarker candidates of ICH, warranting further investigation.
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Intracerebral hemorrhage (ICH) is a devastating subtype of stroke with high rates of mortality and morbidity. ICH patients often suffer devastating and debilitating neurological impairments, from which the majority of victims are unable to fully recover to functional independence. Unfortunately, there is no established medical therapy for ICH, which is partly attributed to the lack of understanding of the complex pathology of the disorder. Despite advanced age being a major risk factor of ICH, most preclinical studies on ICH employed young animal subjects. Due to this discrepancy, the molecular level changes in the aging brain after ICH are largely unknown, limiting the translation of preclinical studies into potential human treatments. The purpose of this review is to highlight the effects of advanced age on ICH- induced brain injury and recovery and to draw attention to current knowledge gaps, which warrant further investigation.
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Human natural anti-α-galactoside (anti-Gal) and anti-ß-glucoside (ABG) antibodies were previously reported to recognize the serine- and threonine-rich peptide sequences (STPS) of albumin-associated O-glycoproteins (AOP1 and AOP2) as surrogate antigens, forming anti-Gal/ABG-AOP1/AOP2-albumin triplet immune complexes in plasma. Since antibodies in these triplets still possessed unoccupied binding sites, the presence of triplets on human platelets that abound in surface O-glycoproteins was examined. Upon treatment with α-galactosides and ß-glucosides, normal platelets freshly isolated from young healthy individuals released triplets identical with plasma triplets according to ELISA results. The resulting denuded platelets, unless pre-treated with fibrinogen or the O-glycan-binding lectin jacalin, recaptured these sugar-extracted triplets in the absence of antibody-specific sugars, suggesting that the triplet antibodies recognized the STPS of O-glycosylated receptors on platelets. Molecular weight of the dominant jacalin-binding subunit on triplet-free platelet membrane was 116 kDa, close to the ~120 kDa reported for the IIb subunit of the most abundant fibrinogen-binding platelet O-glycoprotein, GPIIb/IIIa. Denuded, but not native, platelets underwent slow spontaneous aggregation and rapid ADP-mediated GPIIb/IIIa-dependent aggregation according to spectrophotometric assay. Pre-treatment of denuded platelets with jacalin significantly reduced their ADP-mediated aggregation. Amyloid ß (Aß-42 monomer) was reported to bind triplet O-glycoproteins through their STPS but not to albumin or the antibodies. This peptide bound to the triplets on normal platelets and to surface membrane O-glycoproteins on denuded platelets, suggesting that the surface O-glycoproteins on the normal platelets were engaged and masked by the triplets. The ABG-specific sugar glucose denuded the platelets at concentrations typically reached in diabetic sera, since anti-Gal specific or ABG-specific sugar released the triplets of both the antibodies from the platelets. In conclusion, the present study offered rationale for the presence of anti-Gal/ABG-O-glycoprotein-albumin triplets on normal platelets, for the role of triplets in platelet physiology amidst circulating platelet-activating factors such as ADP, and for platelet vulnerability during diabetes.
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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a promising source of new antimicrobials in the face of rising antibiotic resistance. Here, we report a scalable platform that combines high-throughput bioinformatics with automated biosynthetic gene cluster refactoring for rapid evaluation of uncharacterized gene clusters. As a proof of concept, 96 RiPP gene clusters that originate from diverse bacterial phyla involving 383 biosynthetic genes are refactored in a high-throughput manner using a biological foundry with a success rate of 86%. Heterologous expression of all successfully refactored gene clusters in Escherichia coli enables the discovery of 30 compounds covering six RiPP classes: lanthipeptides, lasso peptides, graspetides, glycocins, linear azol(in)e-containing peptides, and thioamitides. A subset of the discovered lanthipeptides exhibit antibiotic activity, with one class II lanthipeptide showing low µM activity against Klebsiella pneumoniae, an ESKAPE pathogen. Overall, this work provides a robust platform for rapidly discovering RiPPs.
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Danazol , Ribosomas , Antibacterianos/metabolismo , Antibacterianos/farmacología , Danazol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Familia de Multigenes , Péptidos/química , Procesamiento Proteico-Postraduccional , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Intracerebral hemorrhage (ICH) or hemorrhagic stroke is a major public health problem with no effective treatment. Given the emerging role of epigenetic mechanisms in the pathophysiology of ICH, we tested the hypothesis that a class 1 histone deacetylase inhibitor (HDACi), Entinostat, attenuates neurodegeneration and improves neurobehavioral outcomes after ICH. To address this, we employed a preclinical mouse model of ICH and Entinostat was administered intraperitoneally one-hour post induction of ICH. Entinostat treatment significantly reduced the number of degenerating neurons and TUNEL-positive cells after ICH in comparison to vehicle-treated controls. Moreover, Entinostat treatment significantly reduced hematoma volume, T2-weighted hemorrhagic lesion volume and improved acute neurological outcomes after ICH. Further, Entinostat significantly reduced the hemin-induced release of proinflammatory cytokines in vitro. Consistently, the expression of proinflammatory microglial/macrophage marker, CD16/32, was remarkably reduced in Entinostat treated group after ICH in comparison to control. Altogether, data implicates the potential of class 1 HDACi, Entinostat, in improving acute neurological function after ICH warranting further investigation.
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Benzamidas/administración & dosificación , Hematoma/patología , Hematoma/prevención & control , Inhibidores de Histona Desacetilasas/administración & dosificación , Hemorragias Intracraneales/complicaciones , Piridinas/administración & dosificación , Animales , Conducta Animal/efectos de los fármacos , Hematoma/etiología , Hemorragias Intracraneales/patología , Masculino , RatonesRESUMEN
Intracerebral hemorrhage (ICH) is a major public health problem characterized by cerebral bleeding. Despite recent advances in preclinical studies, there is no effective treatment for ICH making it the deadliest subtype of stroke. The lack of effective treatment options partly attributes to the complexity as well as poorly defined pathophysiology of ICH. The emerging evidence indicates the potential of targeting secondary brain damage and hematoma resolution for improving neurological outcomes after ICH. Herein, we provide an overview of our understanding of the functional roles of activated microglia and brain-infiltrating monocyte-derived macrophages in brain injury and repair after ICH. The clinical and preclinical aspects that we discuss in this manuscript are related to ICH that occurs in adults, but not in infants. Also, we attempt to identify the knowledge gap in the field for future functional studies given the potential of targeting microglia and brain-infiltrating macrophages for therapeutic intervention after ICH.
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Lesiones Encefálicas/metabolismo , Encéfalo/metabolismo , Hemorragia Cerebral/metabolismo , Macrófagos/metabolismo , Microglía/metabolismo , Adulto , Animales , Encéfalo/inmunología , Lesiones Encefálicas/inmunología , Lesiones Encefálicas/terapia , Hemorragia Cerebral/inmunología , Hemorragia Cerebral/terapia , Humanos , Macrófagos/inmunología , Microglía/inmunologíaRESUMEN
Graspetides are a class of ribosomally synthesized and post-translationally modified peptide natural products featuring ATP-grasp ligase-dependent formation of macrolactones/macrolactams. These modifications arise from serine, threonine, or lysine donor residues linked to aspartate or glutamate acceptor residues. Characterized graspetides include serine protease inhibitors such as the microviridins and plesiocin. Here, we report an update to Rapid ORF Description and Evaluation Online (RODEO) for the automated detection of graspetides, which identified 3,923 high-confidence graspetide biosynthetic gene clusters. Sequence and co-occurrence analyses doubled the number of graspetide groups from 12 to 24, defined based on core consensus sequence and putative secondary modification. Bioinformatic analyses of the ATP-grasp ligase superfamily suggest that extant graspetide synthetases diverged once from an ancestral ATP-grasp ligase and later evolved to introduce a variety of ring connectivities. Furthermore, we characterized thatisin and iso-thatisin, two graspetides related by conformational stereoisomerism from Lysobacter antibioticus. Derived from a newly identified graspetide group, thatisin and iso-thatisin feature two interlocking macrolactones with identical ring connectivity, as determined by a combination of tandem mass spectrometry (MS/MS), methanolytic, and mutational analyses. NMR spectroscopy of thatisin revealed a cis conformation for a key proline residue, while molecular dynamics simulations, solvent-accessible surface area calculations, and partial methanolytic analysis coupled with MS/MS support a trans conformation for iso-thatisin at the same position. Overall, this work provides a comprehensive overview of the graspetide landscape, and the improved RODEO algorithm will accelerate future graspetide discoveries by enabling open-access analysis of existing and emerging genomes.
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Productos Biológicos/química , Biología Computacional/métodos , Ligasas/química , Péptidos/química , Inhibidores de Serina Proteinasa/química , Conformación Molecular , Familia de Multigenes , Procesamiento Proteico-Postraduccional , Ribosomas , Espectrometría de Masas en TándemRESUMEN
Intracerebral hemorrhage (ICH) induces neurovascular injury via poorly defined mechanisms. The aim of this study was to determine whether gliovascular communication may restrict hemorrhagic vascular injury. Hemin, a hemoglobin by-product, concentration- and time-dependently increased apoptotic cell death in mouse bEnd.3 cells and in primary human brain microvascular endothelial cells, at least in part, via a caspase-3 dependent pathway. Cell death was preceded by a NFκB-mediated increase in inflammatory gene expression, including upregulation of inducible nitric oxide synthase (iNOS) expression and activity. Functionally, inhibition of iNOS or the addition of a peroxynitrite decomposition catalyst reduced cell death. Interestingly, co-treatment with astrocyte-conditioned media (ACM) reversed hemin-induced NFκB activation, nitrotyrosine formation, and apoptotic cell death, at least in part, via the release of the endogenous antioxidant, reduced glutathione (GSH). Prior treatment of astrocytes with the GSH-depleting agent, DL-buthionine (S,R)-sulfoximine or direct addition of diethyl maleate, a thiol-depleting agent, to ACM reversed the observed protection. In contrast, neither exogenous GSH nor the GSH precursor, N-acetylcysteine, was protective in bEnd.3 cells. Together, these data support an important role for astrocyte-derived GSH in the maintenance of oxidative balance in the vasculature and suggest therapeutic targeting of the GSH system may reduce neurological injury following ICH.
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Apoptosis/efectos de los fármacos , Astrocitos/química , Glutatión/farmacología , Hemina/farmacología , Microvasos/citología , Acetilcisteína/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Encéfalo/citología , Caspasa 3/metabolismo , Células Cultivadas , Curcumina/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Glutatión/metabolismo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Ratones , Fármacos Neuroprotectores/farmacología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ácido Peroxinitroso/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de TiempoRESUMEN
Traumatic brain injury is a devastating neurological injury associated with significant morbidity and mortality. Medical therapies to limit cerebral edema, a cause of increased intracranial hypertension and poor clinical outcome, are largely ineffective, emphasizing the need for novel therapeutic approaches. In the present study, pre-treatment with curcumin (75, 150 mg/kg) or 30 min post-treatment with 300 mg/kg significantly reduced brain water content and improved neurological outcome following a moderate controlled cortical impact in mice. The protective effect of curcumin was associated with a significant attenuation in the acute pericontusional expression of interleukin-1beta, a pro-inflammatory cytokine, after injury. Curcumin also reversed the induction of aquaporin-4, an astrocytic water channel implicated in the development of cellular edema following head trauma. Notably, curcumin blocked IL-1beta-induced aquaporin-4 expression in cultured astrocytes, an effect mediated, at least in part, by reduced activation of the p50 and p65 subunits of nuclear factor kappaB. Consistent with this notion, curcumin preferentially attenuated phosphorylated p65 immunoreactivity in pericontusional astrocytes and decreased the expression of glial fibrillary acidic protein, a reactive astrocyte marker. As a whole, these data suggest clinically achievable concentrations of curcumin reduce glial activation and cerebral edema following neurotrauma, a finding which warrants further investigation.