RESUMO
BACKGROUND: The spinal cord is a crucial part of the vertebrate CNS, controlling movements and receiving and processing sensory information from the trunk and limbs. However, there is much we do not know about how this essential organ develops. Here, we describe expression of 21 transcription factors and one transcriptional regulator in zebrafish spinal cord. RESULTS: We analyzed the expression of aurkb, foxb1a, foxb1b, her8a, homeza, ivns1abpb, mybl2b, myt1a, nr2f1b, onecut1, sall1a, sall3a, sall3b, sall4, sox2, sox19b, sp8b, tsc22d1, wdhd1, zfhx3b, znf804a, and znf1032 in wild-type and MIB E3 ubiquitin protein ligase 1 zebrafish embryos. While all of these genes are broadly expressed in spinal cord, they have distinct expression patterns from one another. Some are predominantly expressed in progenitor domains, and others in subsets of post-mitotic cells. Given the conservation of spinal cord development, and the transcription factors and transcriptional regulators that orchestrate it, we expect that these genes will have similar spinal cord expression patterns in other vertebrates, including mammals and humans. CONCLUSIONS: Our data identify 22 different transcriptional regulators that are strong candidates for playing different roles in spinal cord development. For several of these genes, this is the first published description of their spinal cord expression.
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Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by the modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.
Assuntos
Apolipoproteína L1/genética , Variação Genética , Glomerulosclerose Segmentar e Focal/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Podócitos/metabolismo , Negro ou Afro-Americano/genética , Animais , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/fisiopatologia , Homeostase , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/fisiologia , Podócitos/fisiologia , Proteinúria , Triglicerídeos/metabolismoRESUMO
The lack of tools for early detection of pancreatic ductal adenocarcinoma (PDAC) is directly correlated with the abysmal survival rates in patients. In addition to several potential detection tools under active investigation, we tested the gut microbiome and its metabolic complement as one of the earliest detection tools that could be useful in patients at high risk for PDAC. We used a combination of 16s rRNA pyrosequencing and whole-genome sequencing of gut fecal microbiota in a genetically engineered PDAC murine model (KRASG12DTP53R172HPdxCre or KPC). Metabolic reconstruction of microbiome was done using the HUMAnN2 pipeline. Serum polyamine levels were measured from murine and patient samples using chromogenic assay. Our results showed a Proteobacterial and Firmicutes dominance in gut microbiota in early stages of PDAC development. Upon in silico reconstruction of active metabolic pathways within the altered microbial flora, polyamine and nucleotide biosynthetic pathways were significantly elevated. These metabolic products are known to be actively assimilated by the host and eventually utilized by rapidly dividing cells for proliferation validating their importance in the context of tumorigenesis. In KPC mice, as well as PDAC patients, we show significantly elevated serum polyamine concentrations. Therefore, at the early stages of tumorigenesis, there is a strong correlation between microbial changes and release of metabolites that foster host tumorigenesis, thereby fulfilling the 'vicious cycle hypothesis' of the role of microbiome in health and disease states. Our results provide a potential, precise, noninvasive tool for early detection of PDAC, which may result in improved outcomes.
Assuntos
Carcinoma Ductal Pancreático/diagnóstico , Disbiose/complicações , Detecção Precoce de Câncer , Microbioma Gastrointestinal , Neoplasias Pancreáticas/diagnóstico , Poliaminas/metabolismo , Animais , Carcinogênese , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/microbiologia , Carcinoma Ductal Pancreático/patologia , Disbiose/microbiologia , Fezes/microbiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/microbiologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Resistance to therapy is one of the major factors that contribute to dismal survival statistics in pancreatic cancer. While there are many tumor intrinsic and tumor microenvironment driven factors that contribute to therapy resistance, whether pre-existing metabolic diseases like type 2 diabetes (T2D) contribute to this has remained understudied. It is well accepted that hyperglycemia associated with type 2 diabetes changes the gut microbiome. Further, hyperglycemia also enriches for a "stem-like" population within the tumor. In the current study, we observed that in a T2D mouse model, the microbiome changed significantly as the hyperglycemia developed in these animals. Our results further showed that, tumors implanted in the T2D mice responded poorly to gemcitabine/paclitaxel (Gem/Pac) standard of care compared to those in the control group. A metabolomic reconstruction of the WGS of the gut microbiota further revealed that an enrichment of bacterial population involved in drug metabolism in the T2D group. Additionally, we also observed an increase in the CD133+ tumor cells population in the T2D model. These observations indicated that in an animal model for T2D, microbial dysbiosis is associated with increased resistance to chemotherapeutic compounds.
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Diabetes Mellitus Tipo 2/microbiologia , Resistencia a Medicamentos Antineoplásicos , Disbiose/microbiologia , Hiperglicemia/microbiologia , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Microbioma Gastrointestinal , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/microbiologia , Gencitabina , Neoplasias PancreáticasRESUMO
BACKGROUND: Autoimmune diseases have been associated with changes in the gut microbiome. In this study, the gut microbiome was evaluated in individuals with dry eye and bacterial compositions were correlated to dry eye (DE) measures. We prospectively included 13 individuals with who met full criteria for Sjögren's (SDE) and 8 individuals with features of Sjögren's but who did not meet full criteria (NDE) for a total of 21 cases as compared to 21 healthy controls. Stool was analyzed by 16S pyrosequencing, and associations between bacterial classes and DE symptoms and signs were examined. RESULTS: Results showed that Firmicutes was the dominant phylum in the gut, comprising 40-60% of all phyla. On a phyla level, subjects with DE (SDE and NDE) had depletion of Firmicutes (1.1-fold) and an expansion of Proteobacteria (3.0-fold), Actinobacteria (1.7-fold), and Bacteroidetes (1.3-fold) compared to controls. Shannon's diversity index showed no differences between groups with respect to the numbers of different operational taxonomic units (OTUs) encountered (diversity) and the instances these unique OTUs were sampled (evenness). On the other hand, Faith's phylogenetic diversity showed increased diversity in cases vs controls, which reached significance when comparing SDE and controls (13.57 ± 0.89 and 10.96 ± 0.76, p = 0.02). Using Principle Co-ordinate Analysis, qualitative differences in microbial composition were noted with differential clustering of cases and controls. Dimensionality reduction and clustering of complex microbial data further showed differences between the three groups, with regard to microbial composition, association and clustering. Finally, differences in certain classes of bacteria were associated with DE symptoms and signs. CONCLUSIONS: In conclusion, individuals with DE had gut microbiome alterations as compared to healthy controls. Certain classes of bacteria were associated with DE measures.
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Actinobacteria/metabolismo , Bacteroidetes/metabolismo , Disbiose/metabolismo , Microbioma Gastrointestinal , Síndrome de Sjogren/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Ulcerative colitis (UC) is a Th2 inflammatory bowel disease characterized by increased IL-5 and IL-13 expression, eosinophilic/neutrophilic infiltration, decreased mucus production, impaired epithelial barrier, and bacterial dysbiosis of the colon. Acetylcholine and nicotine stimulate mucus production and suppress Th2 inflammation through nicotinic receptors in lungs but UC is rarely observed in smokers and the mechanism of the protection is unclear. METHODS: In order to evaluate whether acetylcholine can ameliorate UC-associated pathologies, we employed a mouse model of dextran sodium sulfate (DSS)-induced UC-like conditions, and a group of mice were treated with Pyridostigmine bromide (PB) to increase acetylcholine availability. The effects on colonic tissue morphology, Th2 inflammatory factors, MUC2 mucin, and gut microbiota were analyzed. RESULTS: DSS challenge damaged the murine colonic architecture, reduced the MUC2 mucin and the tight-junction protein ZO-1. The PB treatment significantly attenuated these DSS-induced responses along with the eosinophilic infiltration and the pro-Th2 inflammatory factors. Moreover, PB inhibited the DSS-induced loss of commensal Clostridia and Flavobacteria, and the gain of pathogenic Erysipelotrichia and Fusobacteria. CONCLUSIONS: Together, these data suggest that in colons of a murine model, PB promotes MUC2 synthesis, suppresses Th2 inflammation and attenuates bacterial dysbiosis therefore, PB has a therapeutic potential in UC.
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Acetilcolinesterase/metabolismo , Anti-Inflamatórios/farmacologia , Inibidores da Colinesterase/farmacologia , Colite Ulcerativa/tratamento farmacológico , Colo/efeitos dos fármacos , Disbiose , Microbioma Gastrointestinal , Brometo de Piridostigmina/farmacologia , Animais , Colite Ulcerativa/enzimologia , Colite Ulcerativa/microbiologia , Colite Ulcerativa/patologia , Colo/enzimologia , Colo/microbiologia , Colo/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Proteínas Ligadas por GPI/metabolismo , Mediadores da Inflamação/metabolismo , Mucina-2/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/metabolismoRESUMO
It is now well appreciated that the human microbiome plays a significant role in a number of processes in the body, significantly affecting its metabolic, inflammatory, and immune homeostasis. Recent research has revealed that almost every mucosal surface in the human body is associated with a resident commensal microbiome of its own. While the gut microbiome and its role in regulation of host metabolism along with its alteration in a disease state has been well studied, there is a lacuna in understanding the resident microbiota of other mucosal surfaces. Among these, the scientific information on the role of lung microbiota in pulmonary diseases is currently severely limited. Historically, lungs have been considered to be sterile and lung diseases have only been studied in the context of bacterial pathogenesis. Recently however, studies have revealed a resilient microbiome in the upper and lower respiratory tracts and there is increased evidence on its central role in respiratory diseases. Knowledge of lung microbiome and its metabolic fallout (local and systemic) is still in its nascent stages and attracting immense interest in recent times. In this review, we will provide a perspective on lung-associated metabolic disorders defined for lung diseases (e.g., chronic obstructive pulmonary disease, asthma, and respiratory depression due to infection) and correlate it with lung microbial perturbation. Such perturbations may be due to altered biochemical or metabolic stress as well. Finally, we will draw evidence from microbiome and classical microbiology literature to demonstrate how specific lung morbidities associate with specific metabolic characteristics of the disease, and with the role of microbiome in this context. © 2018 IUBMB Life, 71(1):152-165, 2019.
Assuntos
Asma/metabolismo , Fibrose Cística/metabolismo , Neoplasias Pulmonares/metabolismo , Pneumonia Bacteriana/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Actinobacteria/imunologia , Actinobacteria/metabolismo , Actinobacteria/patogenicidade , Asma/imunologia , Asma/microbiologia , Asma/patologia , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Firmicutes/imunologia , Firmicutes/metabolismo , Firmicutes/patogenicidade , Homeostase/imunologia , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/microbiologia , Neoplasias Pulmonares/patologia , Microbiota/imunologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/patologia , Proteobactérias/imunologia , Proteobactérias/metabolismo , Proteobactérias/patogenicidade , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/microbiologia , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologia , Mucosa Respiratória/patologiaRESUMO
BACKGROUND: Opioids such as morphine are widely used for the management of pain associated with acute pancreatitis. Interestingly, opioids are also known to affect the immune system and modulate inflammatory pathways in non-pancreatic diseases. However, the impact of morphine on the progression of acute pancreatitis has never been evaluated. In the current study, we evaluated the impact of morphine on the progression and severity of acute pancreatitis. METHODS: Effect of morphine treatment on acute pancreatitis in caerulein, L-arginine and ethanol-palmitoleic acid models was evaluated after induction of the disease. Inflammatory response, gut permeability and bacterial translocation were compared. Experiments were repeated in mu (µ) opioid receptor knockout mice (MORKO) and in wild-type mice in the presence of opioid receptor antagonist naltrexone to evaluate the role of µ-opioid receptors in morphine's effect on acute pancreatitis. Effect of morphine treatment on pathways activated during pancreatic regeneration like sonic Hedgehog and activation of embryonic transcription factors like pdx-1 and ptf-1 were measured by immunofluorescence and quantitative PCR. RESULTS: Histological data show that treatment with morphine after induction of acute pancreatitis exacerbates the disease with increased pancreatic neutrophilic infiltration and necrosis in all three models of acute pancreatitis. Morphine also exacerbated acute pancreatitis-induced gut permeabilisation and bacteraemia. These effects were antagonised in the MORKO mice or in the presence of naltrexone suggesting that morphine's effect on severity of acute pancreatitis are mediated through the µ-opioid receptors. Morphine treatment delayed macrophage infiltration, sonic Hedgehog pathway activation and expression of pdx-1 and ptf-1. CONCLUSION: Morphine treatment worsens the severity of acute pancreatitis and delays resolution and regeneration. Considering our results, the safety of morphine for analgesia during acute pancreatitis should be re-evaluated in future human studies.
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Analgésicos Opioides/efeitos adversos , Morfina/efeitos adversos , Pâncreas/patologia , Pancreatite/diagnóstico , Doença Aguda , Analgésicos Opioides/administração & dosagem , Animais , Arginina , Ceruletídeo , Modelos Animais de Doenças , Progressão da Doença , Ácidos Graxos Monoinsaturados , Camundongos , Camundongos Knockout , Morfina/administração & dosagem , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Índice de Gravidade de Doença , Fatores de TempoRESUMO
Neural connectivity between the spinal cord and paired appendages is key to the superior locomotion of tetrapods and aquatic vertebrates. In contrast to nerves that innervate axial muscles, those innervating appendages converge at a specialized structure, the plexus, where they topographically reorganize before navigating towards their muscle targets. Despite its importance for providing appendage mobility, the genetic program that drives nerve convergence at the plexus, as well as the functional role of this convergence, are not well understood. Here, we show that in zebrafish the transcription factor foxc1a is dispensable for trunk motor nerve guidance but is required to guide spinal nerves innervating the pectoral fins, equivalent to the tetrapod forelimbs. In foxc1a null mutants, instead of converging with other nerves at the plexus, pectoral fin nerves frequently bypass the plexus. We demonstrate that foxc1a expression in muscle cells delineating the nerve path between the spinal cord and the plexus region restores convergence at the plexus. By labeling individual fin nerves, we show that mutant nerves bypassing the plexus enter the fin at ectopic positions, yet innervate their designated target areas, suggesting that motor axons can select their appropriate fin target area independently of their migration through the plexus. Although foxc1a mutants display topographically correct fin innervation, mutant fin muscles exhibit a reduction in the levels of pre- and postsynaptic structures, concomitant with reduced pectoral fin function. Combined, our results reveal foxc1a as a key player in the development of connectivity between the spinal cord and paired appendages, which is crucial for appendage mobility.
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Extremidades/embriologia , Fatores de Transcrição Forkhead/metabolismo , Neurônios Motores/citologia , Proteínas de Peixe-Zebra/metabolismo , Nadadeiras de Animais/embriologia , Animais , Axônios/fisiologia , Fatores de Transcrição Forkhead/genética , Neurogênese/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: The opioid epidemic is a growing concern, and emerging evidence suggests that morphine use may be associated with sepsis. Enteric glial cells (EGCs) are the most numerous cell type in the enteric nervous system and regulate gastrointestinal function through the production of trophic factors, including glial-derived neurotrophic factor (GDNF). We sought to determine the effect of morphine on enteric glia and hypothesized that morphine contributes to EGC dysfunction and increased gut permeability. MATERIALS AND METHODS: Rat intestinal epithelial cells (IECs) and EGC lines were purchased from ATCC. Immunocytochemistry was used to evaluate the impact of EGCs on IEC barrier proteins and detect the µ-opioid receptor. Co-culture assays were used to determine the effect of EGCs, GDNF, and morphine on barrier integrity. Quantitative polymerase chain reaction and western blotting were performed to determine the impact of morphine in GDNF production. Transepithelial resistance of IEC-6 cell monolayers was measured in the presence of EGC-conditioned media (EGC-CM) and morphine treated EGC-CM using electrical cell impedance sensing. RESULTS: EGC-CM enhanced tight junction organization in IECs. IEC barrier integrity was enhanced when co-cultured with unstimulated EGCs or with GDNF alone; this barrier protective effect was lost with morphine-treated EGCs. GDNF RNA and protein expression were decreased by morphine treatment. Transepithelial resistance was decreased in IEC confluent monolayers when exposed to morphine-treated EGC-CM compared with control. CONCLUSIONS: Morphine compromises intestinal epithelial cell barrier function through a mechanism which appears to involve GDNF. Further studies are warranted to delineate the role of enteric glial cell function in opioid signaling and sepsis.
Assuntos
Analgésicos Opioides/efeitos adversos , Mucosa Intestinal/efeitos dos fármacos , Morfina/efeitos adversos , Neuroglia/efeitos dos fármacos , Animais , Linhagem Celular , Fatores Neurotróficos Derivados de Linhagem de Célula Glial/metabolismo , Neuroglia/química , Neuroglia/metabolismo , Ratos , Receptores Opioides mu/análiseRESUMO
BACKGROUND: Elevated levels of oncostatin M (OSM), an interleukin-6 cytokine family member, have been observed in HIV-1-associated neurocognitive disorders (HAND) and Alzheimer's disease. However, the function of OSM in these disease conditions is unclear. Since deficient glutamate uptake by astrocytes is instrumental in HAND-associated neurotoxicity, we hypothesized that OSM impairs glutamate uptake in astrocytes and thereby promotes neuronal excitotoxicity. METHODS: Primary cultures of mouse cortical astrocytes, neurons, microglia, and BV2 cell line were used. The expression of glutamate transporters (GLAST/EAAT1 and GLT-1/EAAT2) was investigated using real-time PCR and Western blot, and their activity was assessed by measuring (3)H-D-aspartate uptake. Neuronal toxicity was measured using the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide) assay and immunocytochemistry. A chimeric HIV-1 that infects murine cells (EcoHIV/NL4-3-GFP virus (EcoHIV)) was used to investigate whether the virus induces OSM, OSM receptor (OSMR)-ß, glycoprotein 130 (gp130), GLT-1, GLAST (mRNA and protein), and OSM release (ELISA) in cultured BV2 cells, primary microglia, or astrocytes. Statistical analyses of the data were performed using one-way ANOVA (to allow multiple comparisons) and two-tailed Student's t test. RESULTS: OSM treatment (10 ng/mL) time-dependently reduced GLAST and GLT-1 expression and inhibited (3)H-D-aspartate uptake in cultured astrocytes in a concentration-dependent manner, an effect prevented by the Janus kinase (JAK)/signal transducers and activators of transcription (STAT)3 inhibitor AG490. Down-regulation of astrocytic glutamate transport by OSM resulted in NMDA receptor-dependent excitotoxicity in cortical neurons. Infection with EcoHIV induced OSM gene expression and protein release in BV2 cells and microglia, but not in astrocytes. Conversely, EcoHIV caused a fivefold increase in OSMR-ß mRNA (but not gp130) and protein in astrocytes, but not in microglia, which did not express OSMR-ß protein. Finally, astrocytic expression of GLAST gene was unaffected by EcoHIV, whereas GLT-1 mRNA was increased by twofold. CONCLUSIONS: We provide first evidence that activation of JAK/STAT3 signaling by OSM inhibits glutamate uptake in astrocytes, which results in neuronal excitotoxicity. Our findings with EcoHIV suggest that targeting OSMR-ß signaling in astrocytes might alleviate HIV-1-associated excitotoxicity.
Assuntos
Antineoplásicos/efeitos adversos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Ácido Glutâmico/metabolismo , Oncostatina M/efeitos adversos , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Ácido Aspártico/metabolismo , Astrócitos/virologia , Células Cultivadas , Córtex Cerebral/citologia , Citocinas/genética , Citocinas/metabolismo , Embrião de Mamíferos , Agonistas de Aminoácidos Excitatórios/toxicidade , Transportador 2 de Aminoácido Excitatório/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , N-Metilaspartato/toxicidade , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oncostatina M/farmacologia , Subunidade beta de Receptor de Oncostatina M/metabolismo , Proteínas Oncogênicas de Retroviridae/toxicidade , Transdução de Sinais/efeitos dos fármacosRESUMO
Cigarette smoke (CS) is the strongest risk factor for emphysema. However, the mechanism of the disease is not clear. One reason is that each puff of CS is a complex mixture of approximately 4,000 chemicals, and it is yet to be known which of these chemical(s) are directly involved in the pathogenesis of lung injury in emphysema. The purpose of this study was to demonstrate that p-benzoquinone (p-BQ) produced in the lungs of CS-exposed guinea pigs is a causative factor for destruction of alveolar cells resulting in emphysema that is prevented by vitamin C. Vitamin C-restricted guinea pigs were subjected to whole-body CS exposure from five Kentucky research cigarettes (3R4F) per day or intramuscular injection of p-BQ in amounts approximately produced in the lung from CS exposure with and without oral supplementation of vitamin C. Progressive exposure of CS or p-BQ treatment caused progressive accumulation of p-BQ in the lung that was accompanied by destruction of alveolar cells and emphysema. The pathogenesis involved was arylation, oxidative stress, inflammation, and apoptosis. Vitamin C (30 mg/kg body weight/d), a potential antagonist of p-BQ, prevented accumulation of p-BQ in the lung and the pathogenesis of emphysema. Our study provides the first proof that inactivation of p-BQ, a causative factor of emphysema in CS-exposed lung, could constitute a novel and effective approach in the prevention of emphysema. We consider that a moderately high dose of vitamin C may be a simple preventive therapy for emphysema in chronic smokers.
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Ácido Ascórbico/farmacologia , Benzoquinonas/efeitos adversos , Benzoquinonas/metabolismo , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/tratamento farmacológico , Fumaça/efeitos adversos , Fumar/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Cobaias , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Enfisema Pulmonar/metabolismoRESUMO
Trunk neural crest cells delaminate from the dorsal neural tube as an uninterrupted sheet; however, they convert into segmentally organized streams before migrating through the somitic territory. These neural crest cell streams join the segmental trajectories of pathfinding spinal motor axons, suggesting that interactions between these two cell types might be important for neural crest cell migration. Here, we show that in the zebrafish embryo migration of both neural crest cells and motor axons is temporally synchronized and spatially restricted to the center of the somite, but that motor axons are dispensable for segmental neural crest cell migration. Instead, we find that muscle-specific receptor kinase (MuSK) and its putative ligand Wnt11r are crucial for restricting neural crest cell migration to the center of each somite. Moreover, we find that blocking planar cell polarity (PCP) signaling in somitic muscle cells also results in non-segmental neural crest cell migration. Using an F-actin biosensor we show that in the absence of MuSK neural crest cells fail to retract non-productive leading edges, resulting in non-segmental migration. Finally, we show that MuSK knockout mice display similar neural crest cell migration defects, suggesting a novel, evolutionarily conserved role for MuSK in neural crest migration. We propose that a Wnt11r-MuSK dependent, PCP-like pathway restricts neural crest cells to their segmental path.
Assuntos
Movimento Celular/fisiologia , Crista Neural/citologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteínas de Homeodomínio , Camundongos , Camundongos Knockout , Morfogênese/fisiologia , Crista Neural/fisiologia , Receptores Proteína Tirosina Quinases/genética , Proteínas Wnt/genética , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genéticaRESUMO
Background: The spinal cord is a crucial part of the vertebrate CNS, controlling movements and receiving and processing sensory information from the trunk and limbs. However, there is much we do not know about how this essential organ develops. Here, we describe expression of 21 transcription factors and one transcriptional regulator in zebrafish spinal cord. Results: We analyzed the expression of aurkb, foxb1a, foxb1b, her8a, homeza, ivns1abpb, mybl2b, myt1a, nr2f1b, onecut1, sall1a, sall3a, sall3b, sall4, sox2, sox19b, sp8b, tsc22d1, wdhd1, zfhx3b, znf804a, and znf1032 in wild-type and MIB E3 ubiquitin protein ligase 1 zebrafish embryos. While all of these genes are broadly expressed in spinal cord, they have distinct expression patterns from one another. Some are predominantly expressed in progenitor domains, and others in subsets of post-mitotic cells. Given the conservation of spinal cord development, and the transcription factors and transcriptional regulators that orchestrate it, we expect that these genes will have similar spinal cord expression patterns in other vertebrates, including mammals and humans. Conclusions: Our data identify 22 different transcriptional regulators that are strong candidates for playing different roles in spinal cord development. For several of these genes, this is the first published description of their spinal cord expression.
RESUMO
Immunotherapy, particularly targeting the PD-1/PD-L1 pathway, holds promise in cancer treatment by regulating the immune response and preventing cancer cells from evading immune destruction. Nonetheless, this approach poses a risk of unwanted immune system activation against healthy cells. To minimize this risk, our study proposes a strategy based on selective targeting of the PD-L1 pathway within the acidic microenvironment of tumors. We employed in silico methods, such as virtual screening, molecular mechanics, and molecular dynamics simulations, analyzing approximately 10,000 natural compounds from the MolPort database to find potential hits with the desired properties. The simulations were conducted under two pH conditions (pH = 7.4 and 5.5) to mimic the environments of healthy and cancerous cells. The compound MolPort-001-742-690 emerged as a promising pH-selective inhibitor, showing a significant affinity for PD-L1 in acidic conditions and lower toxicity compared to known inhibitors like BMS-202 and LP23. A detailed 1000 ns molecular dynamics simulation confirmed the stability of the inhibitor-PD-L1 complex under acidic conditions. This research highlights the potential of using in silico techniques to discover novel pH-selective inhibitors, which, after experimental validation, may enhance the precision and reduce the toxicity of immunotherapies, offering a transformative approach to cancer treatment.
RESUMO
Chronic drug users account for a third of all cases of AIDS in the United States and the progression to AIDS dementia is accelerated in opiate drug abusers. Clinically, microglial activation better correlates with HIV-associated neurocognitive disorders (HAND) than productive HIV-1 infection in the CNS. Moreover, pneumococcal pneumonia is the most common opportunistic infection in individuals with HAND. We show that coinfection with Streptococcus pneumoniae may be a contributing factor in the increased prevalence of HAND in the opioid-dependent population. To date, there have been no studies published implicating the Toll-like receptors (TLR) in the neurocognitive disorders associated with NeuroAIDS in the context of opportunistic infection. Our studies show for the first time, in a morphine-dependent model, synergistic increase and activation of TLR expression in the presence of HIV-1 protein TAT and S. pneumoniae with a significant increase in proinflammatory cytokines (IL-6, TNF-α) levels. Furthermore, concurrent increases in reactive oxygen species and nitric oxide production leading to increased caspase 3 activation are also observed in both murine and human microglial cells. These effects are recapitulated with TLR 2, 4, and 9 cognate ligands (Pam3CSK4, LPS, and CpG) and significantly attenuated in TLR 2 and 4 knock-out mice and TLR2/4 double knock-out mice. Therefor, our findings clearly suggest for the first time that activation of TLRs on microglia cells by morphine and TAT in the context of S. pneumoniae infection may be a potential mechanism for the increased prevalence of HAND in HIV-infected opioid-dependent patients.
Assuntos
Coinfecção/metabolismo , Infecções por HIV/metabolismo , Microglia/efeitos dos fármacos , Morfina/farmacologia , Pneumonia Pneumocócica/metabolismo , Receptores Toll-Like/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Citocinas/metabolismo , Infecções por HIV/complicações , HIV-1 , Masculino , Camundongos , Camundongos Knockout , Microglia/metabolismo , Pneumonia Pneumocócica/complicações , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Streptococcus pneumoniae , Receptores Toll-Like/genéticaRESUMO
Alloy formation and chemisorption at bimetallic surfaces formed by vapor-depositing Zn on a Pt(111) single crystal were investigated primarily by using X-ray photoelectron diffraction (XPD), X-ray photoelectron spectroscopy (XPS), low-energy alkali ion scattering spectroscopy (ALISS), low electron energy diffraction (LEED), and temperature programmed desorption (TPD). A wide range of conditions were investigated to explore whether deposition and annealing of Zn films could produce well-defined, ordered alloy surfaces, similar to those encountered for Sn/Pt(111) surface alloys. These attempts were unsuccessful, although weak, diffuse (2 × 2) spots were observed under special conditions. The particular PtZn bimetallic alloy created by annealing one monolayer of Zn on Pt(111) at 600 K, which has a Zn composition in the surface layer of about 5 at. %, was investigated in detail by using XPD and ALISS. Only a diffuse (1 × 1) pattern was observed from this surface by LEED, suggesting that no long-range, ordered alloy structure was formed. Zn atoms were substitutionally incorporated into the Pt(111) crystal to form a near-surface alloy in which Zn atoms were found to reside primarily in the topmost and second layers. The alloyed Zn atoms in the topmost layer are coplanar with the Pt atoms in the surface layer, without any "buckling" of Zn, that is, displacement in the vertical direction. This result is expected because of the similar size of Pt and Zn, based on previous studies of bimetallic Pt alloys. Zn atoms desorb upon heating rather than diffusing deep into the bulk of the Pt crystal. Temperature programmed desorption (TPD) measurements show that both CO and NO have lower desorption energies on the PtZn alloy surface compared to that on the clean Pt(111) surface.
RESUMO
Venous leg ulcers (VLU) are the most common chronic wounds characterized by bacterial biofilms and perturbed microbiome. Staphylococcus epidermidis is primarily known as skin commensal beneficial for the host, however, some strains can form biofilms and cause infections. By employing shotgun metagenomic sequencing we show that genetic signatures of antimicrobial resistance, adhesion and biofilm formation in VLU isolates correlate with in vitro bacterial traits. We demonstrate that the capability of chronic wound isolates to form biofilms and elicit IL-8 and IL-1ß expression in human ex vivo wounds, correlates with the non-healing outcomes in patients with VLU. In contrast, commensal strains were incapable of surviving in the human ex vivo wounds. We show that major fitness traits of S. epidermis from VLU involve genes for resistance to methicillin and mupirocin, while the biofilm formation relied on the minimal number of genetic elements responsible for bacterial binding to fibronectin and fibrinogen. This underscores the importance of the emergence of treatment resistant virulent lineages in patients with non-healing wounds.
RESUMO
Background: V0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks, we FAC-sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 double mutants and wild-type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuronal expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.
RESUMO
BACKGROUND: V0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. METHODS: To identify candidate members of V0v gene regulatory networks, we FAC-sorted wild-type and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 double mutants and wild-type siblings. RESULTS: Our data reveal two molecularly distinct subtypes of zebrafish V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuron expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. CONCLUSIONS: This study identifies two molecularly distinct subsets of zebrafish V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.