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BACKGROUND: Renal denervation (RDN) can lower blood pressure (BP) in patients with hypertension in both the presence and absence of medication. This is the first sham-controlled trial investigating the safety and efficacy of RDN in China. METHODS: This prospective, multicenter, randomized, patient- and outcome-assessor-blinded, sham-controlled trial investigated radiofrequency RDN in patients with hypertension on standardized triple antihypertensive therapy. Eligible patients were randomized 1:1 to undergo RDN using a multi-electrode radiofrequency catheter (Iberis; AngioCare, Shanghai, China) or a sham procedure. The primary efficacy outcome was the between-group difference in baseline-adjusted change in mean 24-hour ambulatory systolic BP from randomization to 6 months. RESULTS: Of 217 randomized patients (mean age, 45.3±10.2 years; 21% female), 107 were randomized to RDN and 110 were randomized to sham control. At 6 months, there was a greater reduction in 24-hour systolic BP in the RDN (-13.0±12.1 mm Hg) compared with the sham control group (-3.0±13.0 mm Hg; baseline-adjusted between-group difference, -9.4 mm Hg [95% CI, -12.8 to -5.9]; P<0.001). Compared with sham, 24-hour diastolic BP was lowered by -5.0 mm Hg ([95% CI, -7.5 to -2.4]; P<0.001) 6 months after RDN, and office systolic and diastolic BP was lowered by -6.4 mm Hg ([95% CI, -10.5 to -2.3]; P=0.003) and -5.1 mm Hg ([95% CI, -8.2 to -2.0]; P=0.001), respectively. One patient in the RDN group experienced an access site complication (hematoma), which resolved without sequelae. No other major device- or procedure-related safety events occurred through follow-up. CONCLUSIONS: In this trial of Chinese patients with uncontrolled hypertension on a standardized triple pharmacotherapy, RDN was safe and reduced ambulatory and office BP at 6 months compared with sham. REGISTRATION: URL: https://clinicaltrials.gov; Unique identifier: NCT02901704.
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Aging is a significant risk factor associated with the progression of CNS neurodegenerative diseases including multiple sclerosis (MS). Microglia, the resident macrophages of the CNS parenchyma, are a major population of immune cells that accumulate in MS lesions. While they normally regulate tissue homeostasis and facilitate the clearance of neurotoxic molecules including oxidized phosphatidylcholines (OxPCs), their transcriptome and neuroprotective functions are reprogrammed by aging. Thus, determining the factors that instigate aging associated microglia dysfunction can lead to new insights for promoting CNS repair and for halting MS disease progression. Through single-cell RNA sequencing (scRNAseq), we identified Lgals3, which encodes for galectin-3 (Gal3), as an age upregulated gene by microglia responding to OxPC. Consistently, excess Gal3 accumulated in OxPC and lysolecithin-induced focal spinal cord white matter (SCWM) lesions of middle-aged mice compared with young mice. Gal3 was also elevated in mouse experimental autoimmune encephalomyelitis (EAE) lesions and more importantly in MS brain lesions from two male and one female individuals. While Gal3 delivery alone into the mouse spinal cord did not induce damage, its co-delivery with OxPC increased cleaved caspase 3 and IL-1ß within white matter lesions and exacerbated OxPC-induced injury. Conversely, OxPC-mediated neurodegeneration was reduced in Gal3-/- mice compared with Gal3+/+ mice. Thus, Gal3 is associated with increased neuroinflammation and neurodegeneration and its overexpression by microglia/macrophages may be detrimental for lesions within the aging CNS.SIGNIFICANCE STATEMENT Aging accelerates the progression of neurodegenerative diseases such as multiple sclerosis (MS). Understanding the molecular mechanisms of aging that increases the susceptibility of the CNS to damage could lead to new strategies to manage MS progression. Here, we highlight that microglia/macrophage-associated galectin-3 (Gal3) was upregulated with age exacerbated neurodegeneration in the mouse spinal cord white matter (SCWM) and in MS lesions. More importantly, co-injection of Gal3 with oxidized phosphatidylcholines (OxPCs), which are neurotoxic lipids found in MS lesions, caused greater neurodegeneration compared with injection of OxPC alone, whereas genetic loss of Gal3 reduced OxPC damage. These results demonstrate that Gal3 overexpression is detrimental to CNS lesions and suggest its deposition in MS lesions may contribute to neurodegeneration.
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Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Masculino , Femenino , Ratones , Animales , Esclerosis Múltiple/patología , Galectina 3/genética , Fosfatidilcolinas , Encefalomielitis Autoinmune Experimental/patología , Médula Espinal , Microglía/fisiologíaRESUMEN
Motor disturbances predominantly characterize hypoxic-ischemic encephalopathy (HIE). Among its intervention methods, environmental enrichment (EE) is strictly considered a form of sensory intervention. However, limited research uses EE as a single sensory input intervention to validate outcomes postintervention. A Sprague-Dawley rat model subjected to left common carotid artery ligation and exposure to oxygen-hypoxic conditions is used in this study. EE was achieved by enhancing the recreational and stress-relief items within the cage, increasing the duration of sunlight, colorful items exposure, and introducing background music. JZL184 (JZL) was administered as neuroprotective drugs. EE was performed 21 days postoperatively and the rats were randomly assigned to the standard environment and EE groups, the two groups were redivided into control, JZL, and vehicle injection subgroups. The Western blotting and behavior test indicated that EE and JZL injections were efficacious in promoting cognitive function in rats following HIE. In addition, the motor function performance in the EE-alone intervention group and the JZL-alone group after HIE was significantly improved compared with the control group. The combined EE and JZL intervention group exhibited even more pronounced improvements in these performances. EE may enhance motor function through sensory input different from the direct neuroprotective effect of pharmacological treatment.NEW & NOTEWORTHY Rarely does literature assess motor function, even though it is common after hypoxia ischemic encephalopathy (HIE). Previously used environmental enrichment (EE) components have not been solely used as sensory inputs. Physical factors were minimized in our study to observe the effects of purely sensory inputs.
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Hipoxia-Isquemia Encefálica , Ratas Sprague-Dawley , Animales , Hipoxia-Isquemia Encefálica/terapia , Hipoxia-Isquemia Encefálica/fisiopatología , Ratas , Modelos Animales de Enfermedad , Fármacos Neuroprotectores/farmacología , Masculino , Ambiente , Recuperación de la Función/fisiología , Actividad Motora/fisiologíaRESUMEN
In this Letter, an omni-directional reflector (ODR) with a thin hybrid dielectric layer (hybrid-ODR) is proposed to enhance the light extraction efficiency (LEE) for inclined-sidewall-shaped AlGaN-based deep ultraviolet light-emitting diode (DUV LED) by inserting a thin diamond with high refraction index into a conventional Al/Al2O3-based ODR. The three-dimensional finite-difference time-domain (3D FDTD) simulation results show that the LEE of TM-polarized light for the DUV LED with hybrid-ODR is enhanced by 18.5% compared with Al/Al2O3-based ODR. It is because the diamond can transform the evanescent wave in Al2O3 into the propagating light wave in diamond, thereby preventing effective excitation of the surface plasmon polariton (SPP) on the surface of the metal Al. Moreover, the Brewster's angle effect causes the TM-polarized light in diamond to propagate effectively into AlGaN. Furthermore, decreasing the total thickness of the dielectric layer also improves the scattering effect of the inclined sidewall. However, the utilization of hybrid-ODR results in a slight reduction in the LEE for transverse electric (TE) polarized light because the light is confined to the diamond layer and eventually absorbed by the metal Al.
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During the perinatal period, dairy cows undergo negative energy balance, resulting in elevated circulating levels of nonesterified fatty acids (NEFA). Although increased blood NEFA concentrations are a physiological adaptation of early lactation, excessive NEFA in dairy cows is a major cause of fatty liver. Aberrant lipid metabolism leads to hepatic lipid accumulation and subsequently the development of fatty liver. Both inositol-requiring enzyme 1α (IRE1α) and c-Jun N-terminal kinase (JNK) have been validated for their association with hepatic lipid accumulation, including their regulatory functions in calf hepatocyte insulin resistance, oxidative stress, and apoptosis. Meanwhile, both IRE1α and JNK are involved in lipid metabolism in nonruminants. Therefore, the aim of this study was to investigate how IRE1α and JNK regulate lipid metabolism in bovine hepatocytes. An experiment was conducted on randomly selected 10 healthy cows (hepatic triglyceride [TG] content <1%) and 10 cows with fatty liver (hepatic TG content >5%). Liver tissue and blood samples were collected from experimental cows. Serum concentrations of NEFA and ß-hydroxybutyrate (BHB) were greater, whereas serum concentrations of glucose and milk production were lower in cows with fatty liver. The western blot results revealed that dairy cows with fatty liver had higher phosphorylation levels of JNK, c-Jun, and IRE1α in the liver tissue. Three in vitro experiments were conducted using primary calf hepatocytes isolated from 5 healthy calves (body weight: 30-40 kg; 1 d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h, which showed that the phosphorylated levels of JNK, c-Jun, and IRE1α increased in both linear and quadratic effects. In the second experiment, hepatocytes were treated with high concentrations of NEFA (1.2 mM) for 12 h with or without SP600125, a canonical inhibitor of JNK. Western blot results showed that SP600125 treatment could decrease the expression of lipogenesis-associated proteins (PPARγ and SREBP-1c) and increase the expression of fatty acid oxidation (FAO)-associated proteins (CPT1A and PPARα) in NEFA-treated hepatocytes. The perturbed expression of lipogenesis-associated genes (FASN, ACACA, and CD36) and FAO-associated gene ACOX1 were also recovered by JNK inhibition, indicating that JNK reduced excessive NEFA-induced lipogenesis and FAO dysregulation in calf hepatocytes. Third, short hairpin RNA targeting IRE1α (sh-IRE1α) was transfected into calf hepatocytes to silence IRE1α, and KIRA6 was used to inhibit the kinase activity of IRE1α. The blockage of IRE1α could at least partially suppressed NEFA-induced JNK activation. Moreover, the blockage of IRE1α downregulated the expression of lipogenesis genes and upregulated the expression of FAO genes in NEFA-treated hepatocytes. In conclusion, these findings indicate that targeting the IRE1α-JNK axis can reduce NEFA-induced lipid accumulation in bovine hepatocytes by modulating lipogenesis and FAO. This may offer a prospective therapeutic target for fatty liver in dairy cows.
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Mitochondrial dysfunction has been reported to occur in the mammary gland of dairy cows suffering from ketosis. Prohibitin 2 (PHB2) plays a crucial role in regulating mitophagy, which clears impaired mitochondria to maintain normal mitochondrial function. Therefore, the current study aimed to investigate how PHB2 mediates mitophagy, thereby influencing mitochondrial function in the bovine mammary epithelial cell MAC-T. First, mammary gland tissue and blood samples were collected from healthy cows (control; n = 15, BHB <0.6 mM) and cows with clinical ketosis (CK; n = 15, BHB >3.0 mM). Compared with the control group, the CK group exhibited lower dry matter intake (DMI), milk production, milk protein, milk lactose, and serum glucose. In contrast, milk fat, serum nonesterified fatty acids (NEFA) and BHB were greater in CK group. The protein abundance of PHB2, peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α), mitofusin 2 (MFN2) in whole cell lysates (WCL), as well as PHB2, sequestosome-1 (SQSTM1, also called p62), microtubule-associated protein 1 light chain 3-II (LC3-II), and ubiquitinated proteins in mitochondrial fraction were significantly lower in the CK group. ATP content of mammary gland tissue in CK group was lower than that of healthy cows. Second, MAC-T were cultured and treated with NEFA (0, 0.3, 0.6, 1.2 mM). MAC-T treated with 1.2 mM NEFA displayed decreased protein abundance of PHB2, PGC-1α, MFN2 in WCL, as well as protein abundance of PHB2, p62, LC3-II, and ubiquitinated proteins in mitochondrial fraction. The content of ATP and JC-1 aggregates in 1.2 mM NEFA group were lower than in the 0 mM NEFA group. Additionally, 1.2 mM NEFA disrupted the fusion between mitochondria and lysosomes. MAC-T were then pretreated with 100 nM rapamycin, followed by treatment with or without NEFA. Rapamycin alleviated impaired mitophagy and mitochondria dysfunction induced by 1.2 mM NEFA. Third, MAC-T were transfected with small interfering RNA to silence PHB2 or a plasmid for overexpression of PHB2, followed by treatment with or without NEFA. The silencing of PHB2 aggravated 1.2 mM NEFA induced impaired mitophagy and mitochondrial dysfunction, whereas the overexpression of PHB2 alleviated these effects. Overall, this study provides evidence that PHB2, in regulation of mitophagy, is a mechanism for bovine mammary epithelial cells to counteract NEFA-induced mitochondrial dysfunction.
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Radiotherapy and chemotherapy remain the mainstay of treatment for colorectal cancer (CRC), although their efficacy is limited. A detailed understanding of the molecular mechanisms underlying CRC progression could lead to the development of new therapeutic strategies. Although it has been established that MYC signaling is dysregulated in various human cancers, direct targeting MYC remains challenging due to its "undruggable" protein structure. Post-translational modification of proteins can affect their stability, activation, and subcellular localization. Hence, targeting the post-translational modification of MYC represents a promising approach to disrupting MYC signaling. Herein, we revealed that NEK8 positively regulates CRC progression by phosphorylating c-MYC protein at serine 405, which exhibited enhanced stability via polyubiquitination. Our findings shed light on the role of NEK8/MYC signaling in CRC progression, offering a novel and helpful target for colorectal cancer treatment. Video Abstract.
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Neoplasias Colorrectales , Quinasas Relacionadas con NIMA , Proteínas Proto-Oncogénicas c-myc , Humanos , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-myc/genética , Serina , Transducción de SeñalRESUMEN
The metabolic score for insulin resistance (METS-IR) is a recently developed parameter for screening of metabolic disorder. However, the association between METS-IR and risk of hypertension in general adult population remains not fully determined. A meta-analysis was therefore performed. Observational studies evaluating the association between METS-IR and hypertension in adults were retrieved by searching PubMed, Embase, and Web of Science databases from inception to October 10, 2022. A random-effects model, which incorporates the potential influence of heterogeneity, was used to pool the results. Eight studies with 305 341 adults were included in the meta-analysis, and 47 887 (15.7%) of them had hypertension. Pooled results showed that a higher METS-IR was associated with hypertension after adjusting for multiple conventional risk factors [relative risk (RR) for highest versus lowest category of METS-IR: 1.67, 95% confidence interval (CI): 1.53 to 1.83, p<0.001, I2=8%]. The results were consistent in subgroup analyses according to study design, source of the cohort, age, sex, body mass index of the participants, and quality scores of the study (p for subgroup difference all>0.05). Results of meta-analysis with METS-IR analyzed in continuous variables also showed that METS-IR was associated with the risk of hypertension (RR for 1-unit increment of METS-IR: 1.15, 95% CI: 1.08 to 1.23, p<0.001, I2=79%). In conclusion, a high METS-IR is associated with hypertension in general adult population. Measuring METS-IR may be useful for screening participants at high risk of hypertension.
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Hipertensión , Resistencia a la Insulina , Síndrome Metabólico , Adulto , Humanos , Síndrome Metabólico/epidemiología , Factores de Riesgo , Hipertensión/epidemiología , Índice de Masa CorporalRESUMEN
Alleviating vascular injury improves the prognosis of atherosclerosis. Semaphorin-3a (Sema3A) is a special membrane-associated secreted protein with various biological properties, like pro-inflammation, anti-tumor and et al. This study aims to investigate the effects of inhibition of Sema3A on lipopolysaccharide (LPS)-induced vascular injury in mice. The mice were randomized into three groups: control, LPS, and LPS + siRNA. Mice in the combined group were given siRNA through fast tail vein injection, then LPS was injected intraperitoneally 7 days later, finally the mice were euthanized 24 h later. Vascular function and structure were assessed by vascular injury biomarkers and relevant stainings. LPS-induced vascular dysfunction and pathological injury were substantially improved by inhibition of Sema3A. Western blot and quantitative real-time polymerase chain reaction assays were used for investigating molecular pathways. The relevant proteins of vascular endothelial cells activation, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), increased after LPS stimulation, while these effects were reversed by inhibition of Sema3A. The levels of inflammatory cytokines (IL-1ß, IL-6 and NLRP3) were upregulated after LPS stimulation, however, inhibition of Sema3A reversed it through NF-κB and MAPKs signaling pathways involvement. Moreover, inhibition of Sema3A alleviated LPS-induced oxidative stress, evidenced by a decrease in total reactive oxygen species and an increase in antioxidant protein of SOD-1. The results showed that inhibition of Sema3A protects against LPS-induced vascular injury by suppressing vascular endothelial cells activation, vascular inflammation, and vascular oxidative stress, implying that inhibition of Sema3A might be used as a therapeutic strategy for septic vascular injury or atherosclerosis.
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Aterosclerosis , Lesiones del Sistema Vascular , Animales , Células Endoteliales/metabolismo , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/prevención & control , Lipopolisacáridos/toxicidad , Ratones , FN-kappa B , ARN Interferente Pequeño , Semaforina-3A/genética , Semaforina-3A/metabolismoRESUMEN
This study was to provide a theoretical basis for effective treatment of myocardial ischemia-reperfusion injury (I/R injury) and explore the effect of cerium oxide (CeO2) nanoparticles on myocardial cell apoptosis induced by I/R injury. In this study, 50 healthy male Sprague Dawley (SD) rats were selected and divided into five groups according to the random table method: a sham operation group, an I/R group, a 1 - 10 nm CeO2 nanoparticle group (CeO2-1 group), a 10 - 25 nm CeO2 nanoparticle group (CeO2-2 group), and a 50 nm CeO2 nanoparticle group (CeO2-3 group). Rats in different groups were injected with phosphate buffer solution (PBS) and CeO2 nanoparticles with different diameters, respectively. The rat models of I/R injury were prepared to explore and analyze the superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, glutathione peroxidase (GSH-Px) activity, and myocardial cell apoptosis of rats with I/R injury by CeO2 nanoparticles. The results showed that the cardiomyocyte necrosis, SOD activity, MDA content, GSH-Px activity, and apoptosis index of the three groups of rats injected with CeO2 nanoparticles were much better than those in the I/R group. The effects on SOD activity, MDA content, GSH-Px activity, and apoptosis index of cardiomyocytes in the CeO2-2 group were significantly better than those in the CeO2-1 and CeO2-3 groups, showing statistically great differences (P< 0.05); and effects on SOD activity, MDA content, and GSH-Px activity of cardiomyocytes in CeO2-1 group were better obviously than those in the CeO2-3 groups, showing statistically observable differences (P< 0.05). In addition, the difference between the CeO2-1 group and CeO2-3 on the apoptosis index of cardiomyocytes was not statistically remarkable (P> 0.05). It can be considered that the CeO2 nanoparticles can effectively alleviate the effects of myocardial I/R injury, showing reliable clinical significance.
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Daño por Reperfusión Miocárdica , Nanopartículas , Daño por Reperfusión , Animales , Antioxidantes/farmacología , Apoptosis , Cerio , Masculino , Malondialdehído , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Superóxido DismutasaRESUMEN
We compared antioxidant activity of the synthetic peptide Val-Leu-Leu-Tyr-Gln-Asp-His-Cys-His (VLLYQDHCH), sea cucumber peptide Val-Leu-Leu-Tyr (VLLY) and pine seed peptide Gln-Asp-His-Cys-His (QDHCH). The structure-activity relationship was analyzed based on radical scavenging ability and Raman, circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). Based on RP-HPLC, the contents of peptides in simulated gastrointestinal tract and digestive juices in rat intestinal sac were determined, and their absorption stability were explored. These results showed that the DPPH clearance rate of VLLYQDHCH was 45.90% higher than the sum of VLLY and QDHCH at 3 mmol/L. The α-helix, ß-sheet and random coil of VLLYQDHCH increased, ß-turn decreased, and the active hydrogen site shifted. After simulated digestion and absorption, the retention rate of VLLYQDHCH was 80.86 ± 0.88% in simulated stomach and 45.75 ± 0.97% in simulated intestine. There was no significant difference in the absorption rates of the three peptides (P > 0.05). This research provided a new idea for the development of safe and green food-derived animal-plant protein antioxidant peptides.
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BACKGROUND: Chondroitin sulfate proteoglycans (CSPGs) are potent inhibitors of axonal regrowth and remyelination. More recently, they have also been highlighted as a modulator of macrophage infiltration into the central nervous system in experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis. METHODS: We interrogated results from single nucleotide polymorphisms (SNPs) lying in or close to genes regulating CSPG metabolism in the summary results from two publicly available systematic studies of multiple sclerosis (MS) genetics. A demyelinating injury model in the spinal cord of exostosin-like 2 deficient (EXTL2-/-) mice was used to investigate the effects of dysregulation of EXTL2 on remyelination. Cell cultures of bone marrow-derived macrophages and primary oligodendrocyte precursor cells and neurons were supplemented with purified CSPGs or conditioned media to assess potential mechanisms of action. RESULTS: The strongest evidence for genetic association was seen for SNPs mapping to the region containing the glycosyltransferase exostosin-like 2 (EXTL2), an enzyme that normally suppresses CSPG biosynthesis. Six of these SNPs showed genome-wide significant evidence for association in one of the studies with concordant and nominally significant effects in the second study. We then went on to show that a demyelinating injury to the spinal cord of EXTL2-/- mice resulted in excessive deposition of CSPGs in the lesion area. EXTL2-/- mice had exacerbated axonal damage and myelin disruption relative to wild-type mice, and increased representation of microglia/macrophages within lesions. In tissue culture, activated bone marrow-derived macrophages from EXTL2-/- mice overproduce tumor necrosis factor α (TNFα) and matrix metalloproteinases (MMPs). CONCLUSIONS: These results emphasize CSPGs as a prominent modulator of neuroinflammation and they highlight CSPGs accumulating in lesions in promoting axonal injury.
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Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Enfermedades Desmielinizantes/patología , Proteínas de la Membrana/metabolismo , Esclerosis Múltiple/patología , N-Acetilglucosaminiltransferasas/metabolismo , Animales , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/metabolismo , Femenino , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Macrófagos/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , N-Acetilglucosaminiltransferasas/genética , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: CYP2C19*17 (rs12248560) is a functional single nucleotide polymorphism (SNP) in the CYP2C19 gene. It has been shown that CYP2C19*17 is associated with the clinical outcome of some drugs metabolized by CYP2C19 and a decreased risk of some diseases. The aim of this study was to develop a reliable and simple method to detect this polymorphism. METHODS: Tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) was used to detect the CYP2C19*17 polymorphism. A total of 93 samples were screened by this method, and the results of T-ARMS-PCR were validated by DNA sequencing. RESULTS: There were 91 samples with the CC genotype (97.8%) and two samples with the CT genotype (2.2%). The frequency of the C allele was 98.9%, and the frequency of the T allele was 1.1%. The DNA sequencing results were completely concordant with the T-ARMS-PCR results. CONCLUSION: T-ARMS-PCR can detect the CYP2C19*17 polymorphism with high accuracy, low costs, and a simple process.
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Alelos , Citocromo P-450 CYP2C19/genética , Mutación/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , HumanosRESUMEN
The process of autophagy is conserved among all eukaryotes from yeast to humans and is mainly responsible for bulk degradation of cellular contents and nutrient recycling during starvation. Autophagy has been suggested to play a role in the pathogenesis of the opportunistic human fungal pathogen Cryptococcus neoformans, potentially through a contribution to the export of virulence factors. In this study, we showed that deletion of each of the ATG1, ATG7, ATG8, and ATG9 genes in C. neoformans leads to autophagy-related phenotypes, including impaired amino acid homeostasis under nitrogen starvation. In addition, the atgΔ mutants were hypersensitive to inhibition of the ubiquitin-proteasome system, a finding consistent with a role in amino acid homeostasis. Although each atgΔ mutant was not markedly impaired in virulence factor production in vitro, we found that all four ATG genes contribute to C. neoformans virulence in a murine inhalation model of cryptococcosis. Interestingly, these mutants displayed significant differences in their ability to promote disease development. A more detailed investigation of virulence for the atg1Δ and atg8Δ mutants revealed that both strains stimulated an exaggerated host immune response, which, in turn, contributed to disease severity. Overall, our results suggest that different ATG genes are involved in nonautophagic functions and contribute to C. neoformans virulence beyond their core functions in autophagy.
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Proteínas Relacionadas con la Autofagia/genética , Autofagia , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidad , Proteínas Fúngicas/genética , Factores de Virulencia/genética , Aminoácidos/metabolismo , Animales , Proteína 7 Relacionada con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/inmunología , Criptococosis/inmunología , Cryptococcus neoformans/inmunología , Modelos Animales de Enfermedad , Femenino , Proteínas Fúngicas/inmunología , Eliminación de Gen , Genes Fúngicos , Homeostasis , Ratones , Ratones Endogámicos C57BL , Virulencia/genética , Factores de Virulencia/inmunologíaRESUMEN
Vitellogenin (Vtg) is a sensitive biomarker for environmental estrogens. In this study, an immunosensor for quantifying zebrafish Vtg was developed using the Octet system. First, Protein A sensors were immobilized with purified anti-lipovitellin (Lv) antibody that demonstrated specificity to Vtg. Then, antibody-coated biosensors were immersed into zebrafish Lv standards and diluted samples. The Octet system measured and recorded kinetic parameters between antigens and captured antibody within 5 min. Sample Vtg concentrations were automatically calculated by interpolating relative binding rates observed with each sample and the immobilized anti-Lv antibody into the developed standard curve. The sensor arrays exhibited a wide linear range from 78 to 5000 ng/mL, and the inter-assay coefficient of variation was 0.66-1.97%. Furthermore, the performance of the immunosensor in detecting Vtg was evaluated by quantifying Vtg induction in juvenile zebrafish exposed to 17ß-estradiol (E2). Compared with conventional immunoassay techniques, the Vtg immunosensor developed based on the Octet system was much simpler and less time-consuming, allowing rapid Vtg quantification within 15 min. Moreover, Protein A sensors could be reused many times to ensure that the assays have high reproducibility. Therefore, we suggest that immunosensors based on the Octet system are an easily operated detection method for ecotoxicological research.
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Anticuerpos Antiidiotipos/aislamiento & purificación , Técnicas Biosensibles , Vitelogeninas/aislamiento & purificación , Animales , Anticuerpos Antiidiotipos/inmunología , Proteínas del Huevo/inmunología , Estradiol/farmacología , Proteína Estafilocócica A/química , Vitelogeninas/inmunología , Pez Cebra/inmunologíaRESUMEN
Although classical dendritic cells (DCs) arise from distinct progenitors in the bone marrow, the origin of inflammatory DCs and the distinction between monocyte-derived DCs and macrophages is less clear. In vitro culture of mouse bone marrow cells with GM-CSF is a well-established method to generate DCs, but GM-CSF has also been used to generate bone marrow-derived macrophages. In this article, we identify a distinct subpopulation of cells within the GM-CSF bone marrow-derived DC culture based on their ability to bind hyaluronan (HA), a major component of the extracellular matrix and ligand for CD44. HA identified a morphologically distinct subpopulation of cells within the immature DC population (CD11c(+) MHC II(mid/low)) that were CCR5(+)/CCR7(-) and proliferated in response to GM-CSF, but, unlike immature DCs, did not develop into mature DCs expressing CCR7 and high levels of MHC II, even after stimulation with LPS. The majority of these cells produced TNF-α in response to LPS but were unable to activate naive T cells, whereas the majority of mature DCs produced IL-12 and activated naive T cells. This HA binding population shared many characteristics with alveolar macrophages and was retained in the alveolar space after lung instillation even after LPS stimulation, whereas the MHC II(high) mature DCs were found in the draining lymph node. Thus, HA binding in combination with MHC II expression can be used to identify alveolar-like macrophages from GM-CSF-treated bone marrow cultures, which provides a useful in vitro model to study alveolar macrophages.
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Células de la Médula Ósea/citología , Linaje de la Célula/inmunología , Células Dendríticas/citología , Ácido Hialurónico/metabolismo , Macrófagos Alveolares/citología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Antígeno CD11c/genética , Antígeno CD11c/inmunología , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Receptores de Hialuranos/genética , Receptores de Hialuranos/inmunología , Ácido Hialurónico/farmacología , Lipopolisacáridos/farmacología , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Activación de Linfocitos/efectos de los fármacos , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cultivo Primario de Células , Receptores CCR5/genética , Receptores CCR5/inmunología , Receptores CCR7/deficiencia , Receptores CCR7/genética , Receptores CCR7/inmunología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated "steady-state" normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.
Asunto(s)
Células Epiteliales/clasificación , Células Epiteliales/metabolismo , Glutatión/metabolismo , Glándulas Mamarias Humanas/citología , Estrés Oxidativo/fisiología , Western Blotting , Daño del ADN/fisiología , Citometría de Flujo , Humanos , Consumo de Oxígeno/fisiología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Madre/metabolismoRESUMEN
Refuse leachate is likely an important source of androgens. However, common in vitro bioassays underestimate the potential androgenic activity of leachate, owing to non-receptor-mediated mechanisms that modify the balance of sex hormones and promote the accumulation of endogenous androgens. This study aimed to develop an in vivo assay by using multiple biomarkers related to testosterone synthesis and conversion for assessing the potential androgenic activity of refuse leachate sampled from a municipal solid waste treatment plant in Qingdao, China. The results indicated that exposure to leachate increased the levels of testosterone and luteinizing hormone, but decreased those of 17ß-estradiol in both male and female goldfish (Carassius auratus), suggesting a potential androgenic activity. Further, Leydig cell hyperplasia and decreased gonadal P450 aromatase mRNA levels were observed; these alterations might promote the biosynthesis of testosterone and hinder the conversion of testosterone to 17ß-estradiol, which in turn enhance testosterone accumulation. Exposure to leachate also resulted in reproductive impairments, including decreased gonadosomatic index and plasma vitellogenin levels of female goldfish, as well as decreased testicular enzyme activities in male goldfish. The integrated use of biochemical, molecular, and histological markers not only improved our understanding of the androgenic effects of leachate but also verified the reliability and validity of the results. Therefore, the in vivo bioassay described in this study might allow the investigation of the androgenic effects of other complex contaminant mixtures in the future.
Asunto(s)
Andrógenos/toxicidad , Bioensayo , Carpa Dorada/metabolismo , Residuos Sólidos , Testosterona/metabolismo , Contaminantes Químicos del Agua/toxicidad , Animales , Aromatasa/genética , Biomarcadores/metabolismo , China , Estradiol/metabolismo , Femenino , Carpa Dorada/sangre , Gónadas/efectos de los fármacos , Gónadas/metabolismo , Hormona Luteinizante/metabolismo , Masculino , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Reproducción/efectos de los fármacos , Vitelogeninas/sangreRESUMEN
Semicarbazide (SMC) is a carcinogenic and genotoxic substance that has been found in aquatic systems. SMC may also cause thyroid follicular epithelial cell injury in rats, but the thyroid-disrupting properties of SMC and its potential mechanisms remain unclear. In this study, we exposed fertilized eggs of Japanese flounder (Paralichthys olivaceus) to 1, 10, 100, and 1000µg/L SMC for 55 d to assess the impact of SMC exposure on the thyroid system. The number of larvae in each metamorphic stage was counted, the concentrations of whole-body thyroid hormones (THs) 3,5,3'-triiodothyronine (T3) and thyroxine (T4) were measured, and the transcription levels of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis and gamma-aminobutyric acid (GABA) synthesis were quantified. The results showed that 10µg/L SMC significantly increased whole-body T3 levels, and 100 and 1000µg/L SMC markedly enhanced whole-body T4 and T3 levels. Furthermore, 100µg/L SMC exposure shortened the time it took for flounder larvae to complete metamorphosis by 2 d as compared to the control group. Thus, this study demonstrated that SMC exerted thyroid-disrupting effects on Japanese flounder. SMC-mediated stimulation of TH levels was primarily related to transcriptional alterations of pituitary-derived thyroid stimulating hormone ß-subunit (tshß) and hepatic deiodinase (id). In the 10 and 100µg/L SMC exposure groups, the increased TH levels may have resulted from inhibition of TH metabolism caused by down-regulation of id3 mRNA expression, while at 1000µg/L SMC-exposed group, up-regulation of tshß and id1 transcripts was expected to enhance the synthesis of T4 and the conversion of T4 to T3 and, consequently, result in higher T4 and T3 levels. In addition, 1000µg/L SMC-induced down-regulation in glutamic acid decarboxylase gad65 and gad67 transcription may have also contributed to the increased TH levels. The thyroid-disrupting effects of 10 and 100µg/L SMC indicated that environmentally relevant concentrations of SMC posed potential environmental risks to aquatic organisms. Overall, our results demonstrated for the first time that SMC exhibited thyroid-disrupting properties by affecting the HPT axis and GABA synthesis, providing theoretical support for environmental risk assessment.
Asunto(s)
Disruptores Endocrinos/toxicidad , Lenguado/metabolismo , Semicarbacidas/toxicidad , Glándula Tiroides/efectos de los fármacos , Hormonas Tiroideas/metabolismo , Contaminantes Químicos del Agua/toxicidad , Animales , Regulación hacia Abajo , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/metabolismo , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , Larva/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Metamorfosis Biológica/efectos de los fármacos , Glándula Tiroides/metabolismo , Hormonas Tiroideas/genética , Regulación hacia Arriba , Ácido gamma-Aminobutírico/biosíntesisRESUMEN
OBJECTIVE: To report our experience of using image-guided computer navigation for bone bridge resection in children. METHODS: Between August 2008 and March 2011 at our hospital, computed tomography (CT)-based navigation system for bone bridge resection was used in 42 children with lower extremity deformities due to partial physeal growth arrest. There were 26 males and 16 females with an average age of 9.8 (5.0-13.0) years. RESULTS: Six patients were lost to follow-up. In other 36 patients, the average duration of radiographic and clinical follow-up was 25 (15-44) months. Among them, 13 were corrected to neutral while 15 had insufficient correction. The average correction angle of deformities was 14 (8-25) degrees. CONCLUSION: The image-guided computer navigation system enhances the accuracy and safety of bone bridge resection in children.