RESUMO
Chemotherapy is an important method for the treatment of lung cancer, but multidrug resistance (MDR) greatly reduces the efficacy. The superfamily of ATP-binding cassette (ABC) transport proteins is related to MDR. As a subfamily of ABC proteins, ABCG2/BCRP (breast cancer resistance protein, BCRP) is considered a major player in the development of cancer MDR. For the stratification of chemotherapeutic choices, we constructed Cy5.5- or 89Zr-labeled ABCG2-targeted monoclonal antibody (mAb) ABCG2-PKU1 for noninvasive evaluation of ABCG2 expression in lung cancer xenograft models. ABCG2 expression was screened in H460/MX (mitoxantrone resistant), H460, and H1299 human lung cancer cell lines using Western blotting. ELISA, flow cytometry, and cell immunofluorescent staining were used to evaluate the binding ability of ABCG2-PKU1 to ABCG2 antigen. Lung cancer murine xenograft models were built for in vivo experiments. ABCG2-PKU1 was labeled with Cy5.5 (Cy5.5-ABCG2) for fluorescent imaging and radiolabeled with 89Zr (89Zr-DFO-ABCG2) for immunoPET imaging following the conjugation with p-SCN-deferoxamine (DFO). In vivo imaging was performed in lung cancer models at 2, 24, 48, 72, 96, 120, 144, and 168 h postinjection. Ex vivo biodistribution was conducted after the terminal time point of imaging. Finally, tissue immunohistochemical staining was used to evaluate the tumor expression of ABCG2. Western blotting showed that the H460/MX cells had a high ABCG2 expression level whereas H460 and H1299 had moderate and low levels. ELISA, flow cytometry, and cell immunofluorescent staining results validated the good binding affinity between ABCG2-PKU1 and ABCG2. The H460/MX and H460 cells were used to build positive lung cancer models, and H1299 cells were used to build negative models. The fluorescent imaging showed that the tumor average radiant efficiency of Cy5.5-ABCG2 reached the maximum at 72 and 120 h in H460/MX and H460 respectively (n = 3, P < 0.01). The tumor uptake of Cy5.5-ABCG2 in H1299 (n = 3) was significantly lower than H460/MX and H460 (P < 0.01). ImmunoPET imaging showed that the tumor uptake of 89Zr-DFO-ABCG2 in H460/MX was significantly higher than H460, with a maximum of 4.15 ± 0.41 %ID/g and 2.81 ± 0.24 %ID/g at 168 and 144 h, respectively (n = 5, P < 0.01). The H1299 tumors showed significantly lower uptake than H460/MX and H460 (n = 5, P < 0.01). The radioactive uptake of 89Zr-DFO-ABCG2 among three groups in the heart, liver, and kidney gradually decreased over time. Ex vivo biodistribution verified the differential tumor uptake among the three groups (P < 0.01). Immunohistochemical staining revealed that the H460/MX tumor had the highest expression of ABCG2, whereas H460 and H1299 had the moderate and lowest expression, respectively. Therefore, in this study, fluorescent and immunoPET imaging of lung cancer MDR models using Cy5.5-ABCG2 and 89Zr-DFO-ABCG2 noninvasively evaluated the differential expression of ABCG2, which are expected to be used for the diagnosis and the selection for clinical treatment options for lung cancer MDR patients in future applications.
Assuntos
Neoplasias Pulmonares , Mitoxantrona , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Carbocianinas , Linhagem Celular Tumoral , Desferroxamina , Modelos Animais de Doenças , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Xenoenxertos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Proteínas de Neoplasias/metabolismo , Distribuição TecidualRESUMO
AIMS: Aortic aneurysm and dissection (AAD) are high-risk cardiovascular diseases with no effective cure. Macrophages play an important role in the development of AAD. As succinate triggers inflammatory changes in macrophages, we investigated the significance of succinate in the pathogenesis of AAD and its clinical relevance. METHODS AND RESULTS: We used untargeted metabolomics and mass spectrometry to determine plasma succinate concentrations in 40 and 1665 individuals of the discovery and validation cohorts, respectively. Three different murine AAD models were used to determine the role of succinate in AAD development. We further examined the role of oxoglutarate dehydrogenase (OGDH) and its transcription factor cyclic adenosine monophosphate-responsive element-binding protein 1 (CREB) in the context of macrophage-mediated inflammation and established p38αMKOApoe-/- mice. Succinate was the most upregulated metabolite in the discovery cohort; this was confirmed in the validation cohort. Plasma succinate concentrations were higher in patients with AAD compared with those in healthy controls, patients with acute myocardial infarction (AMI), and patients with pulmonary embolism (PE). Moreover, succinate administration aggravated angiotensin II-induced AAD and vascular inflammation in mice. In contrast, knockdown of OGDH reduced the expression of inflammatory factors in macrophages. The conditional deletion of p38α decreased CREB phosphorylation, OGDH expression, and succinate concentrations. Conditional deletion of p38α in macrophages reduced angiotensin II-induced AAD. CONCLUSION: Plasma succinate concentrations allow to distinguish patients with AAD from both healthy controls and patients with AMI or PE. Succinate concentrations are regulated by the p38α-CREB-OGDH axis in macrophages.
Assuntos
Aneurisma Aórtico , Animais , Biomarcadores , Dissecação , Humanos , Metabolômica , Camundongos , Ácido SuccínicoRESUMO
Rationale: The constrained mitochondria in cardiomyocytes communicate with each other, through mitochondrial kissing or nanotunneling, forming a dynamically continuous network to share content and transfer signals. However, the molecular mechanism of cardiac inter-mitochondrial communication is unclear. Objective: To determine the molecular mechanism underlying the robust inter-mitochondrial communication and its pathophysiological relevance in the heart. Methods and Results: By mitochondria-targeted expressing the photoactivatable green fluorescent protein, we revealed that most mitochondrial nanotubes bridge communicating mitochondrial pairs were associated with microtubules. Miro2 (mitochondrial Rho GTPase), the outer mitochondrial membrane protein which usually mediates mitochondrial transport within cells, accompanied with mitochondrial nanotubes along microtubules in adult cardiomyocytes. Adenovirus mediated expression of Miro2 in cardiomyocytes accelerated inter-mitochondrial communication through increasing mitochondrial nanotunneling and mitochondrial kissing between adjacent mitochondrial pairs. In transverse aortic constriction-induced hypertrophic mouse hearts Miro2 protein was declined, accompanied with decreased inter-mitochondrial communication. Miro2 transgenic mice showed ameliorated cardiac function, increased mitochondrial nanotube formation and inter-mitochondrial communication, and improved mitochondrial function after transverse aortic constriction. E3 ubiquitin ligase Parkin was increased in transverse aortic constriction mouse hearts and phenylephrine stimulation-induced hypertrophic cardiomyocytes. Inhibition of proteasome blocked phenylephrine-induced decrease of Miro2, and Parkin overexpression led to the decrease of Miro2. Conclusions: Mitochondrial Miro2 expression levels regulate inter-mitochondrial communication along microtubules in adult cardiomyocytes, and degradation of Miro2 through Parkin-mediated ubiquitination contributes to impaired inter-mitochondrial communication and cardiac dysfunction during hypertrophic heart diseases.Visual Overview: An online visual overview is available for this article.
Assuntos
Cardiomegalia/metabolismo , Microtúbulos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Cardiomegalia/etiologia , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fenilefrina/toxicidade , Proteólise , Ratos , Ratos Sprague-Dawley , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Accumulating evidence shows that agents targeting gut dysbiosis are effective for improving symptoms of irritable bowel syndrome (IBS). However, the potential mechanisms remain unclear. In this study we investigated the effects of berberine on the microbiota-gut-brain axis in two rat models of visceral hypersensitivity, i.e., specific pathogen-free SD rats subjected to chronic water avoidance stress (WAS) and treated with berberine (200 mg· kg-1 ·d-1, ig, for 10 days) as well as germ-free (GF) rats subjected to fecal microbiota transplantation (FMT) from a patient with IBS (designated IBS-FMT) and treated with berberine (200 mg· kg-1 ·d-1, ig, for 2 weeks). Before the rats were sacrificed, visceral sensation and depressive behaviors were evaluated. Then colonic tryptase was measured and microglial activation in the dorsal lumbar spinal cord was assessed. The fecal microbiota was profiled using 16S rRNA sequencing, and short chain fatty acids (SCFAs) were measured. We showed that berberine treatment significantly alleviated chronic WAS-induced visceral hypersensitivity and activation of colonic mast cells and microglia in the dorsal lumbar spinal cord. Transfer of fecal samples from berberine-treated stressed donors to GF rats protected against acute WAS. FMT from a patient with IBS induced visceral hypersensitivity and pro-inflammatory phenotype in microglia, while berberine treatment reversed the microglial activation and altered microbial composition and function and SCFA profiles in stools of IBS-FMT rats. We demonstrated that berberine did not directly influence LPS-induced microglial activation in vitro. In both models, several SCFA-producing genera were enriched by berberine treatment, and positively correlated to the morphological parameters of microglia. In conclusion, activation of microglia in the dorsal lumbar spinal cord was involved in the pathogenesis of IBS caused by dysregulation of the microbiota-gut-brain axis, and the berberine-altered gut microbiome mediated the modulatory effects of the agent on microglial activation and visceral hypersensitivity, providing a potential option for the treatment of IBS.
Assuntos
Berberina/uso terapêutico , Eixo Encéfalo-Intestino/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Microglia/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Dor Visceral/tratamento farmacológico , Animais , Berberina/farmacologia , Eixo Encéfalo-Intestino/fisiologia , Linhagem Celular , Transplante de Microbiota Fecal/métodos , Microbioma Gastrointestinal/fisiologia , Humanos , Síndrome do Intestino Irritável/tratamento farmacológico , Síndrome do Intestino Irritável/metabolismo , Masculino , Camundongos , Microglia/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/metabolismo , Dor Visceral/metabolismoRESUMO
Spinal cord injury (SCI) often leads to permanent loss of motor, sensory, and autonomic functions. We have previously shown that neurotrophin3 (NT3)-loaded chitosan biodegradable material allowed for prolonged slow release of NT3 for 14 weeks under physiological conditions. Here we report that NT3-loaded chitosan, when inserted into a 1-cm gap of hemisectioned and excised adult rhesus monkey thoracic spinal cord, elicited robust axonal regeneration. Labeling of cortical motor neurons indicated motor axons in the corticospinal tract not only entered the injury site within the biomaterial but also grew across the 1-cm-long lesion area and into the distal spinal cord. Through a combination of magnetic resonance diffusion tensor imaging, functional MRI, electrophysiology, and kinematics-based quantitative walking behavioral analyses, we demonstrated that NT3-chitosan enabled robust neural regeneration accompanied by motor and sensory functional recovery. Given that monkeys and humans share similar genetics and physiology, our method is likely translatable to human SCI repair.
Assuntos
Quitosana/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Neurotrofina 3/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Axônios/efeitos dos fármacos , Imagem de Tensor de Difusão/métodos , Feminino , Haplorrinos , Neurônios Motores/efeitos dos fármacos , Tratos Piramidais/efeitos dos fármacos , Medula Espinal/efeitos dos fármacosRESUMO
Photostable and bright organic dyes emitting in the near-infrared region are highly desirable for long-term dynamic bioimaging. Herein, we report a synthetic approach to build novel methoxy modified Si-rhodamine (SiRMO) dyes by introducing the methoxybenzene on the xanthene moiety. The brightness of SiRMO increased from 2300 M-1 cm-1 (SiRMO-0) to 49000 M-1 cm-1 (SiRMO-2) when the substituent 2,5-dimethoxybenzene was replaced with 2,6-dimethoxybenzene. Moreover, the stability of SiRMO-2 was significantly improved due to the steric hindrance protection of the two methoxy groups on the ninth carbon atom of the xanthene. After fast cellular uptake, the SiRMO dyes selectively stained the mitochondria with a low background in live cultured cells and primary neurons. The high brightness and stability of SiRMO-2 significantly improved the capability of monitoring mitochondria dynamic processes in living cells under super-resolution conditions. Moreover, with the fluorescence nanoscopy techniques, we observed the structure of mitochondrial cristae and mitochondria fission, fusion, and apoptosis with a high temporal resolution. Under two-photon illumination, SiRMO-2 showed also enhanced two-photon brightness and stability, which are important for imaging in thick tissue.
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Corantes Fluorescentes/química , Microscopia de Fluorescência por Excitação Multifotônica , Mitocôndrias/química , Rodaminas/química , Silício/química , Animais , Células Cultivadas , Chlorocebus aethiops , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Estrutura Molecular , Imagem ÓpticaRESUMO
BACKGROUND: During the outbreak of coronavirus disease 2019 (COVID-19), consistent and considerable differences in disease severity and mortality rate of patients treated in Hubei province compared to those in other parts of China have been observed. We sought to compare the clinical characteristics and outcomes of patients being treated inside and outside Hubei province, and explore the factors underlying these differences. METHODS: Collaborating with the National Health Commission, we established a retrospective cohort to study hospitalised COVID-19 cases in China. Clinical characteristics, the rate of severe events and deaths, and the time to critical illness (invasive ventilation or intensive care unit admission or death) were compared between patients within and outside Hubei. The impact of Wuhan-related exposure (a presumed key factor that drove the severe situation in Hubei, as Wuhan is the epicentre as well the administrative centre of Hubei province) and the duration between symptom onset and admission on prognosis were also determined. RESULTS: At the data cut-off (31 January 2020), 1590 cases from 575 hospitals in 31 provincial administrative regions were collected (core cohort). The overall rate of severe cases and mortality was 16.0% and 3.2%, respectively. Patients in Hubei (predominantly with Wuhan-related exposure, 597 (92.3%) out of 647) were older (mean age 49.7 versus 44.9â years), had more cases with comorbidity (32.9% versus 19.7%), higher symptomatic burden, abnormal radiologic manifestations and, especially, a longer waiting time between symptom onset and admission (5.7 versus 4.5â days) compared with patients outside Hubei. Patients in Hubei (severe event rate 23.0% versus 11.1%, death rate 7.3% versus 0.3%, HR (95% CI) for critical illness 1.59 (1.05-2.41)) have a poorer prognosis compared with patients outside Hubei after adjusting for age and comorbidity. However, among patients outside Hubei, the duration from symptom onset to hospitalisation (mean 4.4 versus 4.7â days) and prognosis (HR (95%) 0.84 (0.40-1.80)) were similar between patients with or without Wuhan-related exposure. In the overall population, the waiting time, but neither treated in Hubei nor Wuhan-related exposure, remained an independent prognostic factor (HR (95%) 1.05 (1.01-1.08)). CONCLUSION: There were more severe cases and poorer outcomes for COVID-19 patients treated in Hubei, which might be attributed to the prolonged duration of symptom onset to hospitalisation in the epicentre. Future studies to determine the reason for delaying hospitalisation are warranted.
Assuntos
Infecções por Coronavirus/mortalidade , Hospitalização , Pneumonia Viral/mortalidade , Adulto , Idoso , Betacoronavirus , COVID-19 , Doenças Cardiovasculares/epidemiologia , China , Estudos de Coortes , Comorbidade , Infecções por Coronavirus/complicações , Infecções por Coronavirus/diagnóstico por imagem , Tosse/etiologia , Diabetes Mellitus/epidemiologia , Surtos de Doenças , Dispneia/etiologia , Fadiga/etiologia , Feminino , Febre/etiologia , Geografia , Humanos , Hipertensão/epidemiologia , Unidades de Terapia Intensiva/estatística & dados numéricos , Pulmão/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Pandemias , Faringite/etiologia , Pneumonia Viral/complicações , Pneumonia Viral/diagnóstico por imagem , Prognóstico , Modelos de Riscos Proporcionais , Respiração Artificial/estatística & dados numéricos , Estudos Retrospectivos , SARS-CoV-2 , Índice de Gravidade de Doença , Fatores de Tempo , Tempo para o Tratamento/estatística & dados numéricos , Tomografia Computadorizada por Raios XRESUMO
OBJECTIVE: The cerebral ischemia leads to brain dysfunction with neuron degeneration and responses from astrocytes and vessels. The aim of this study was to study the changes of astrocyte and microvessel in modified BCCAO mice. METHODS: Adult transgenic Tie2-GFP mice were subjected to modified BCCAO operation and cranial window implantation. CBF and neurological injury were examined after ischemia. Astrocytes and vessels were investigated by two-photon laser-scanning microscope and confocal laser-scanning microscope in vivo. RESULTS: The CBF decreased to approximately 40% of the baseline in the ischemic mice (P<.05). The neuron damage was explicit after the cerebral ischemia (P<.05), while no significant impairment of the motor and cognitive function was detected (P>.05). The density of astrocyte and volume of the astrocyte soma was increased significantly after ischemia (P<.01). Meanwhile, the mean distance between the penetrating artery and the nearest astrocyte soma decreased significantly (P<.01). Besides, the increased diameter of capillary and change of vessel arrangement were observed. CONCLUSION: The cerebral ischemia was successfully induced by this modified BCCAO model. Astrocyte activation and the capillary remodeling, including dilution of capillary and tortuosity, were observed in this model.
Assuntos
Arteriopatias Oclusivas/patologia , Astrócitos/metabolismo , Capilares/metabolismo , Artéria Carótida Primitiva/patologia , Animais , Arteriopatias Oclusivas/fisiopatologia , Isquemia Encefálica , Artéria Carótida Primitiva/fisiopatologia , Circulação Cerebrovascular , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Receptor TIE-2/genéticaRESUMO
Arabidopsis thaliana respiratory burst oxidase homolog D (RbohD) functions as an essential regulator of reactive oxygen species (ROS). However, our understanding of the regulation of RbohD remains limited. By variable-angle total internal reflection fluorescence microscopy, we demonstrate that green fluorescent protein (GFP)-RbohD organizes into dynamic spots at the plasma membrane. These RbohD spots have heterogeneous diffusion coefficients and oligomerization states, as measured by photobleaching techniques. Stimulation with ionomycin and calyculin A, which activate the ROS-producing enzymatic activity of RbohD, increases the diffusion and oligomerization of RbohD. Abscisic acid and flg22 treatments also increase the diffusion coefficient and clustering of GFP-RbohD. Single-particle analysis in clathrin heavy chain2 mutants and a Flotillin1 artificial microRNA line demonstrated that clathrin- and microdomain-dependent endocytic pathways cooperatively regulate RbohD dynamics. Under salt stress, GFP-RbohD assembles into clusters and then internalizes into the cytoplasm. Dual-color fluorescence cross-correlation spectroscopy analysis further showed that salt stress stimulates RbohD endocytosis via membrane microdomains. We demonstrate that microdomain-associated RbohD spots diffuse at the membrane with high heterogeneity, and these dynamics closely relate to RbohD activity. Our results provide insight into the regulation of RbohD activity by clustering and endocytosis, which facilitate the activation of redox signaling pathways.
RESUMO
Understanding the enrichment and intracellular trafficking of substances is centrally important to the biological systems. Here, employing an amphiphilic molecule (denoted by TPE-11) bearing tetraphenylethene moiety, known for aggregation induced emission property, we demonstrated its localization shifting in Hela cells after prolonged incubation. Through a set of delicately designed experiments, we found that one type of cytoskeleton, i.e., microtubule, is responsible for the intracellular transportation regardless of the sources of fluorogens, via endocytosis pathways or not. As the polymerization of microtubules was blocked, the TPE-11 fluorogens were hindered to move to the inner cytoplasm, but scattered in the cells. On the contrary, blocking the polymerization of microfilament has no such effect. We assume that the dynamic polymerization of microtubules should be responsible to the transportation of TPE-11. More importantly, we found that the interaction between TPE-11 and microtubule proteins also happens during process of polymerization in vitro. The intracellular trafficking of TPE-11 by microtubules may be generalized to other amphiphilic molecules as well as endocytosis pathway, and serves as references in designing functional molecules involved in the intracellular transportation.
Assuntos
Microtúbulos , Transporte Biológico , Citoplasma , Células HeLa , Humanos , Espaço IntracelularRESUMO
BACKGROUND: Rebiopsy is highly recommended to identify the mechanism of acquired resistance to EGFR-TKIs in advanced lung cancer. Recent advances in multiplex genotyping based on circulating tumor DNA (ctDNA) provide a strong and non-invasive alternative for detection of the resistance mechanism. CASE PRESENTATION: Here we report a multiple metastatic NSCLC patient who was detected to have pure EGFR 19 exon deletion (negative for EML4-ALK and ROS1 in both IHC-based and sequencing assay) in the primary lesion and responded to first-line and second-line EGFR-TKI treatments (erlotinib then HY-15772). At 8 months, most lesions remained well controlled except for the liver metastases which presented dramatic progression. Considering the high risk of bleeding in rebiopsy of hepatic lesions, we conducted a multiplex genomic profiling with ctDNA. Results reported coexistence of EGFR mutation and EML4-ALK gene translocation in plasma which heavily indicated that ALK was the primary reason for progression of the liver lesions. This deduction was supported by the repeated response to ALK inhibitors (crizotinib then AP26113) of the hepatic metastases. CONCLUSIONS: This is the first report of the existence of ALK rearrangement in metastatic lesions in an EGFR mutated patient. It highlighted the feasibility and advantages of using ctDNA multiplex genotyping in identifying the heterogeneity across lesions and the resistance mechanism of targeted treatments.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , DNA/genética , Receptores ErbB/genética , Proteínas de Fusão Oncogênica/genética , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Crizotinibe , DNA/sangue , Resistencia a Medicamentos Antineoplásicos/genética , Cloridrato de Erlotinib/administração & dosagem , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Mutação , Proteínas de Fusão Oncogênica/sangue , Pirazóis/administração & dosagem , Piridinas/administração & dosagemRESUMO
Ammonium is a preferred source of nitrogen for plants but is toxic at high levels. Plant ammonium transporters (AMTs) play an essential role in NH4(+) uptake, but the mechanism by which AMTs are regulated remains unclear. To study how AMTs are regulated in the presence of ammonium, we used variable-angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy for single-particle fluorescence imaging of EGFP-tagged AMT1;3 on the plasma membrane of Arabidopsis root cells at various ammonium levels. We demonstrated that AMT1;3-EGFP dynamically appeared and disappeared on the plasma membrane as moving fluorescent spots in low oligomeric states under N-deprived and N-sufficient conditions. Under external high-ammonium stress, however, AMT1;3-EGFPs were found to amass into clusters, which were then internalized into the cytoplasm. A similar phenomenon also occurred in the glutamine synthetase mutant gln1;2 background. Single-particle analysis of AMT1;3-EGFPs in the clathrin heavy chain 2 mutant (chc2 mutant) and Flotllin1 artificial microRNA (Flot1 amiRNA) backgrounds, together with chemical inhibitor treatments, demonstrated that the endocytosis of AMT1;3 clusters induced by high-ammonium stress could occur mainly through clathrin-mediated endocytic pathways, but the contribution of microdomain-associated endocytic pathway cannot be excluded in the internalization. Our results revealed that the clustering and endocytosis of AMT1;3 provides an effective mechanism by which plant cells can avoid accumulation of toxic levels of ammonium by eliminating active AMT1;3 from the plasma membrane.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Endocitose , Compostos de Amônio Quaternário/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Western Blotting , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Microscopia de Vídeo/métodos , Mutação , Plantas Geneticamente Modificadas , Multimerização Proteica , Compostos de Amônio Quaternário/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Phospholipid transfer protein (PLTP) regulates lipid metabolism and plays an important role in oxidative stress. PLTP is highly expressed in blood-brain barrier (BBB), but the role of PLTP in BBB integrity is not clear. In this study, BBB permeability was detected with in vivo multiphoton imaging and Evans blue assay. We found that PLTP deficient mice exhibited increased BBB permeability, as well as decreased expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Cerebrovascular oxidative stress increased in PLTP deficient mice, including increased levels of reactive oxygen species (ROS) and lipid peroxidation marker 4-hydroxy-2-nonenal (HNE) and reduced superoxide dismutase (SOD) activity. Dietary supplementation of antioxidant vitamin E increased BBB integrity and tight junction proteins expression via reducing cerebrovascular oxidative stress. These findings indicated an essential role of PLTP in maintaining BBB integrity, possibly through its ability to transfer vitamin E, and modulate cerebrovascular oxidative stress.
Assuntos
Barreira Hematoencefálica/metabolismo , Estresse Oxidativo , Proteínas de Transferência de Fosfolipídeos/genética , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Encéfalo/irrigação sanguínea , Claudina-5/análise , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/análise , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Vitamina E/farmacologia , Proteína da Zônula de Oclusão-1/análiseRESUMO
Blood-brain barrier (BBB) dysfunction is a key event in the development of many central nervous system (CNS) diseases, such as septic encephalopathy and stroke. 4,4'-Diaminodiphenylsulfone (DDS, Dapsone) has displayed neuroprotective effect, but whether DDS has protective role on BBB integrity is not clear. This study was designed to examine the effect of DDS on lipopolysaccharide (LPS)-induced BBB disruption and oxidative stress in brain vessels. Using in vivo multiphoton imaging, we found that DDS administration significantly restored BBB integrity compromised by LPS. DDS also increased the expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Level of reactive oxygen species (ROS) was reduced by DDS treatment, which may due to decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and NOX2 expression. Our results showed that LPS-induced BBB dysfunction could be attenuated by DDS, indicated that DDS has a therapeutic potential for treating CNS infection and other BBB related diseases.
Assuntos
Barreira Hematoencefálica , Dapsona/farmacologia , Lipopolissacarídeos/farmacologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismoRESUMO
PIP2;1 is an integral membrane protein that facilitates water transport across plasma membranes. To address the dynamics of Arabidopsis thaliana PIP2;1 at the single-molecule level as well as their role in PIP2;1 regulation, we tracked green fluorescent protein-PIP2;1 molecules by variable-angle evanescent wave microscopy and fluorescence correlation spectroscopy (FCS). Single-particle tracking analysis revealed that PIP2;1 presented four diffusion modes with large dispersion of diffusion coefficients, suggesting that partitioning and dynamics of PIP2;1 are heterogeneous and, more importantly, that PIP2;1 can move into or out of membrane microdomains. In response to salt stress, the diffusion coefficients and percentage of restricted diffusion increased, implying that PIP2;1 internalization was enhanced. This was further supported by the decrease in PIP2;1 density on plasma membranes by FCS. We additionally demonstrated that PIP2;1 internalization involves a combination of two pathways: a tyrphostin A23-sensitive clathrin-dependent pathway and a methyl-ß-cyclodextrin-sensitive, membrane raft-associated pathway. The latter was efficiently stimulated under NaCl conditions. Taken together, our findings demonstrate that PIP2;1 molecules are heterogeneously distributed on the plasma membrane and that clathrin and membrane raft pathways cooperate to mediate the subcellular trafficking of PIP2;1, suggesting that the dynamic partitioning and recycling pathways might be involved in the multiple modes of regulating water permeability.
Assuntos
Aquaporinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Aquaporinas/análise , Aquaporinas/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/genética , Clatrina/metabolismo , Proteínas de Fluorescência Verde , Modelos Biológicos , Permeabilidade , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Água/metabolismo , beta-Ciclodextrinas/metabolismoRESUMO
Human embryonic germ cells (hEGCs) are a valuable and underutilized source of pluripotent stem cells. Unlike embryonic stem cells, which have been extensively studied, little is known about the factors that regulate hEGC derivation and maintenance. This study demonstrates for the first time a central role for selective activation of PDGFR signaling in the derivation and maintenance of pluripotency in hEGCs. In the study, hEGCs were found to express PDGF receptor α at high levels compared to human embryonic stem cells (hESCs). PDGF significantly improved formation of alkaline phosphatase (AP) positive hEGC colonies. We subsequently determined that PDGF activates the phosphatidylinositol-3-kinase (PI3K) pathway as phosphorylation of AKT was up-regulated in response to PDGF. Furthermore, inhibition of PI3K signaling using small molecular inhibitor LY294002 led to significantly decreased AP positive hEGC colony formation whereas inhibition of MAPK pathway using U0126 had a negligible effect. We established a primary mechanism for PDGF mediated derivation and maintenance of hEGCs by demonstrating that OCT4 was upregulated and PTEN was suppressed in a dose dependent manner in response to PDGF.
Assuntos
Células-Tronco Embrionárias/citologia , Células Germinativas/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fosfatase Alcalina/metabolismo , Células Germinativas/metabolismo , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de SinaisRESUMO
Vision plays a crucial role in the survival of animals, and the visual system has particularly selectively evolved in response to the visual environment, ecological niche, and species habitats in vertebrate species. To date, a horizontal streak of retinal ganglion cell (RGC) distribution pattern is observed across mammal species. Here, we report that the giant panda's vertically oriented visual streak, combined with current evidence of the animal's forward-placed eyes, ocular structure, and retinal neural topographic distribution patterns, presents the emergence of a well-adapted binocular visual system. Our results suggest that the giant panda may use a unique way to processing binocular visual information. Results of mathematical simulation are in favor of this hypothesis. The topographic distribution properties of RGCs reported here could be essential for understanding the visual adaptation and evolution of this living fossil.
Assuntos
Células Ganglionares da Retina , Ursidae , Animais , Células Ganglionares da Retina/citologia , Ursidae/anatomia & histologia , Ursidae/fisiologia , Retina/citologia , Retina/anatomia & histologiaRESUMO
BACKGROUND: Cry1Ab has emerged as a bio-insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets. RESULTS: After sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag-based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up-regulated and significantly enriched in the serine protease activity pathway, and up-regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down-regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein-protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt-resistance of S. litura larvae under sublethal Cry1Ab exposure. CONCLUSION: Under sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry.
Assuntos
Toxinas de Bacillus thuringiensis , Endotoxinas , Proteínas Hemolisinas , Larva , Túbulos de Malpighi , Spodoptera , Animais , Spodoptera/efeitos dos fármacos , Spodoptera/genética , Spodoptera/metabolismo , Spodoptera/crescimento & desenvolvimento , Túbulos de Malpighi/efeitos dos fármacos , Túbulos de Malpighi/metabolismo , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Transcriptoma , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Inseticidas/toxicidade , Proteoma , Proteômica , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/metabolismoRESUMO
Introduction: Angong Niuhuang Wan (AGNHW), developed during the Qing dynasty (18th century) for the treatment of consciousness disturbances caused by severe infections, has been used to treat brain edema caused by ischemiaâreperfusion. However, it remains unclear whether AGNHW can ameliorate vascular-origin brain edema caused by lipopolysaccharides (LPS). This study explored the ameliorative effects of AGNHW on LPS-induced cerebrovascular edema in mice, as well as the potential underlying mechanisms. Methods: A cerebrovascular edema model was established in male C57BL/6N mice by two intraperitoneal injections of LPS (15 mg/kg), at 0 and 24 h. AGNHW was administered by gavage at doses of 0.2275 g/kg, 0.455 g/kg, and 0.91 g/kg, 2 h after LPS administration. In control mice, normal saline (NS) or AGNHW (0.455 g/kg) was administered by gavage 2 h after intraperitoneal injection of NS. The survival rate, cerebral water content, cerebral venous FITC-dextran leakage, Evans blue extravasation, and expression of vascular endothelial cadherin (VE-cadherin), zonula occludens-1 (ZO-1), claudin-5, phosphorylated caveolin-1 (CAV-1), and cytomembrane and cytoplasmic aquaporin 1 (AQP1) and aquaporin 4 (AQP4) were evaluated. The cerebral tissue phosphoproteome, blood levels of AGNHW metabolites, and the relationships between these blood metabolites and differentially phosphorylated proteins were analyzed. Results: AGNHW inhibited the LPS-induced decrease in survival rate, increase in cerebral water content, decrease in VE-Cadherin expression and increase in phosphorylated CAV-1 (P-CAV-1). AGNHW treatment increased the expression of AQP4 on astrocyte membrane after LPS injection. AGNHW also inhibited the LPS-induced increases in the phosphorylation of 21 proteins, including protein kinase C-α (PKC-α) and mitogen-activated protein kinase 1 (MAPK1), in the cerebral tissue. Eleven AGNHW metabolites were detected in the blood. These metabolites might exert therapeutic effects by regulating PKC-α and MAPK1. Conclusion: AGNHW can ameliorate cerebrovascular edema caused by LPS. This effect is associated with the inhibition of VE-Cadherin reduction and CAV-1 phosphorylation, as well as the upregulation of AQP4 expression on the astrocyte membrane, following LPS injection.
RESUMO
BACKGROUND: Tertiary lymphoid structure (TLS) is an organized infiltration of immune cells, showing features of germinal center (GC) commonly seen in secondary lymphoid organs. However, its relationship with tumor-draining lymph nodes (TDLNs) has not been studied and we hypothesized that TDLN may influence maturation of intratumoral TLS in non-small cell lung cancer (NSCLC). METHODS: Tissue slides of 616 patients that had undergone surgeries were examined. Cox proportional hazard regression model was used to assess risk factors of patients' survival, and logistic regression model was used for their relationship with TLS. Single-cell RNA-sequencing (scRNA-seq) was employed to explore transcriptomic features of TDLNs. Immunohistochemistry, multiplex immunofluorescence and flow cytometry were performed to analyze cellular composition. Cellular components of NSCLC samples from The Cancer Genome Atlas database were inferred with Microenvironment Cell Populations-counter (MCP-counter) method. Murine NSCLC models were used to dissect underlying mechanisms for relationship between TDLN and TLS maturation. RESULTS: While GC+ TLS was associated with better prognosis, GC- TLS was not. TDLN metastasis reduced the prognostic relevance of TLS, and was associated with less GC formation. Primary tumor sites showed reduced B cell infiltration in TDLN-positive patients, and scRNA-seq revealed diminished memory B cell formation in tumor-invaded TDLNs, together with an emphasis on weakened interferon (IFN)-γ response. Murine NSCLC models revealed that IFN-γ signaling is involved in memory B cell differentiation in TDLNs and GC formation in primary tumors. CONCLUSIONS: Our research emphasizes the influence of TDLN on intratumoral TLS maturation and suggests a role of memory B cells and IFN-γ signaling in this communication.