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Legged robots have received widespread attention in academia and engineering owing to their excellent terrain adaptability. However, most legged robots can only adapt to high-hardness environments instead of flexible environments. Expanding the motion range of legged robots to water is a promising but challenging work. Inspired by basilisk lizards which can run on water surfaces by feet, this paper proposes a bipedal robot for water running by hydrodynamics instead of buoyancy. According to the motion parameters of the basilisk lizard during water running, a single-degree of freedom bipedal mechanism is proposed to reproduce the motion trajectory of the feet of the basilisk lizard. Scale optimization is conducted by a particle swarm optimization algorithm to determine the geometrical parameters of the mechanism. The effects of motion frequency and foot area on mechanism performance are studied and the optimal solutions are determined by the maximum single-cycle lift impulse through numerical calculations. A bipedal water running robot prototype was fabricated, and the experimental results show that the prototype can generate enough support for the robot running on the water by providing a maximum lift of 2.4 times its weight (160 g) and reaching a horizontal forward speed range of 0.3-0.8 m s-1, compared with the basilisk lizard weighs 2-200 g, generates a lift impulse that is 111%-225% of its body weight, and moves at a speed of 1.3 ± 0.1 m s-1.
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Diseño de Equipo , Lagartos , Robótica , Carrera , Animales , Lagartos/fisiología , Robótica/instrumentación , Carrera/fisiología , Agua , Hidrodinámica , Biomimética/instrumentación , Biomimética/métodos , Fenómenos Biomecánicos , Algoritmos , Modelos BiológicosRESUMEN
The complexity and heterogeneity of protein glycosylation present an analytical challenge to the studies of characterization and quantitation. Various LC-MS-based quantitation strategies have emerged in recent decades. Metabolic stable isotope labeling has been developed to enhance the accurate LC/MS-based quantitation between different cell lines. Stable isotope labeling by amino acids in a cell culture (SILAC) is the most widely used metabolic labeling method in proteomic analysis. However, it can only label the peptide backbone and is thus limited in glycomic studies. Here, we present a metabolic isotope labeling strategy, named GlyProSILC (Glycan Protein Stable Isotope Labeling in Cell Culture), that can label both the glycan motif and peptide backbone from the same batch of cells. It was performed by feeding cells with a heavy medium containing amide-15N-glutamine, 13C6-arginine (Arg6), and 13C6-15N2-lysine (Lys8). No significant change of cell line metabolism after GlyProSILC labeling was observed based on transcriptomic, glycomic, and proteomic data. The labeling conditions, labeling efficiency, and quantitation accuracy were investigated. After quantitation correction, we simultaneously quantified 62 N-glycans, 574 proteins, and 344 glycopeptides using the same batch of mixed 231BR/231 cell lines. So far, GlyProSILC provides an accurate and effective quantitation approach for glycomics, proteomics, and glycoproteomics in a cell culture system.
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Glicómica , Proteómica , Marcaje Isotópico/métodos , Glicómica/métodos , Proteómica/métodos , Proteínas , Técnicas de Cultivo de Célula , Glicopéptidos/metabolismo , Polisacáridos/químicaRESUMEN
Narcolepsy type 1 (NT1) is the most common type of narcolepsy known to be caused by the loss of specific neurons responsible for producing peptide neurotransmitters (orexins/hypocretins), resulting in a sleep-wake cycle disorder. It is characterized by its association with cataplexy and abnormalities in rapid eye movement. To date, no cure has been established for this life-threatening condition. Misdiagnosis of NT1 is also quite common, although it is not exceedingly rare. Therefore, successfully identifying candidate serum biomarkers for NT1 would be a head start for accurate diagnosis and development of therapeutics for this disorder. This study aims to identify such potential serum biomarkers. A depletion protocol was employed for 27 human serum samples (16 NT1 and 11 healthy controls), followed by applying LC-MS/MS bottom-up proteomics analysis, then LC-PRM-MS for validation. The comparison of the proteome profiles of the low-abundant proteins in the samples was then investigated based on age, sex, sample groups, and the presence of the Human Leukocyte Antigen (HLA) DQB1*0602 allele. The results were tracked to gene expression studies as well as system biology to identify key proteins and understand their relationship in the pathogenesis of NT1. Our results revealed 36 proteins significantly and differentially expressed. Among the impaired pathways and bioprocesses, the complement activation pathway is impaired by six of the differentially expressed proteins (DEPs). They are coded by the genes C2, CFB, C5, C1R, C1S, and MASP1, while 11 DEPs are involved in Acute Phase Response Signaling (APRS), which are coded by the genes FN1, AMBP, APOH, CFB, CP, ITIH2, C5, C2, F2, C1, and ITIH4. The combined AUCs of the downregulated and upregulated DEPs are 0.95 and 0.76, respectively. Overall, this study reveals potential serum-protein biomarkers of NT1 and explains the possible correlation between the biomarkers and pathophysiological effects, as well as important biochemical pathways involved in NT1.
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Narcolepsia , Proteómica , Humanos , Cromatografía Liquida , Espectrometría de Masas en Tándem , Narcolepsia/etiología , Narcolepsia/genética , Biomarcadores , OrexinasRESUMEN
PURPOSE: Chronic kidney disease (CKD) is defined by a reduced renal function, that is, glomerular filtration rate, and the extent of kidney damage is assessed by determining serum creatinine levels and proteins in urine, diagnosed as proteinuria/albuminuria. Albuminuria increases with age and can result from glomerular and/or proximal tubule (PT) alterations. Brush border membranes (BBMs) on PT cells are important in maintaining the stability of PT functions. EXPERIMENTAL DESIGN: An LC-MS/MS bottom-up proteomics analysis of BBMs from four groups of rat models was applied to investigate protein abundance alterations associated with CKD progression. Moreover, systems biology analyses were used to identify key proteins that can provide insight into the different regulated molecular pathways and processes associated with CKD. RESULTS: Our results indicated that 303 proteins showed significantly altered expressions from the severe CKD BBM group when compared to the control. Focusing on renal diseases, several proteins including Ctnnb1, Fah, and Icam1 were annotated to kidney damage and urination disorder. The up-regulation of Ctnnb1 (ß-catenin) could contribute to CKD through the regulation of the WNT signaling pathway. CONCLUSION AND CLINICAL RELEVANCE: Overall, the study of protein abundance changes in BBMs from rat models helps to reveal protein corrections with important pathways and regulator effects involved in CKD. Although this study is focused on rat models, the results provided more information for a deeper insight into possible CKD mechanisms in humans.
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Albuminuria , Insuficiencia Renal Crónica , Humanos , Ratas , Animales , Albuminuria/complicaciones , Albuminuria/diagnóstico , Microvellosidades , Cromatografía Liquida , Proteómica , Espectrometría de Masas en Tándem , Riñón/metabolismoRESUMEN
Kidney disease often manifests with an increase in proteinuria, which can result from both glomerular and/or proximal tubule injury. The proximal tubules are the major site of protein and peptide endocytosis of the glomerular filtrate, and cubilin is the proximal tubule brush border membrane glycoprotein receptor that binds filtered albumin and initiates its processing in proximal tubules. Albumin also undergoes multiple modifications depending upon the physiologic state. We previously documented that carbamylated albumin had reduced cubilin binding, but the effects of cubilin modifications on binding albumin remain unclear. Here, we investigate the cubilin-albumin binding interaction to define the impact of cubilin glycosylation and map the key glycosylation sites while also targeting specific changes in a rat model of proteinuria. We identified a key Asn residue, N1285, that when glycosylated reduced albumin binding. In addition, we found a pH-induced conformation change may contribute to ligand release. To further define the albumin-cubilin binding site, we determined the solution structure of cubilin's albumin-binding domain, CUB7,8, using small-angle X-ray scattering and molecular modeling. We combined this information with mass spectrometry crosslinking experiments of CUB7,8 and albumin that provides a model of the key amino acids required for cubilin-albumin binding. Together, our data supports an important role for glycosylation in regulating the cubilin interaction with albumin, which is altered in proteinuria and provides new insight into the binding interface necessary for the cubilin-albumin interaction.
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Albúminas , Asparagina , Túbulos Renales Proximales , Receptores de Superficie Celular , Animales , Ratas , Albúminas/metabolismo , Endocitosis/fisiología , Glicosilación , Túbulos Renales Proximales/metabolismo , Proteinuria/metabolismo , Asparagina/genética , Asparagina/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.
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Glicopéptidos/sangre , Glicoproteínas/sangre , Informática/métodos , Proteoma/análisis , Proteómica/métodos , Investigadores/estadística & datos numéricos , Programas Informáticos , Glicosilación , Humanos , Proteoma/metabolismo , Espectrometría de Masas en TándemRESUMEN
Chronic kidney disease (CKD) is defined as a decrease in renal function or glomerular filtration rate (GFR), and proteinuria is often present. Proteinuria increases with age and can be caused by glomerular and/or proximal tubule (PT) alterations. PT cells have an apical brush border membrane (BBM), which is a highly dynamic, organized, and specialized membrane region containing multiple glycoproteins required for its functions including regulating uptake, secretion, and signaling dependent upon the physiologic state. PT disorders contribute to the dysfunction observed in CKD. Many glycoprotein functions have been attributed to their N- and O-glycans, which are highly regulated and complex. In this study, the O-glycans present in rat BBMs from animals with different levels of kidney disease and proteinuria were characterized and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). A principal component analysis (PCA) documented that each group has distinct O-glycan distributions. Higher fucosylation levels were observed in the CKD and diabetic groups, which may contribute to PT dysfunction by altering physiologic glycoprotein interactions. Fucosylated O-glycans such as 1-1-1-0 exhibited higher abundance in the severe proteinuric groups. These glycomic results revealed that differential O-glycan expressions in CKD progressions has the potential to define the mechanism of proteinuria in kidney disease and to identify potential therapeutic interventions.
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Microvellosidades , Animales , Tasa de Filtración Glomerular , Glicosilación , Ratas , Insuficiencia Renal CrónicaRESUMEN
Chronic kidney disease (CKD) is defined by a reduced renal function i.e., glomerular filtration rate (GFR), and the presence of kidney damage is determined by measurement of proteinuria or albuminuria. Albuminuria increases with age and can result from glomerular and/or proximal tubule (PT) alterations. Brush-border membranes (BBMs) on PT cells play an important role in maintaining the stability of PT functions. The PT BBM, a highly dynamic, organized, specialized membrane, contains a variety of glycoproteins required for the functions of PT. Since protein glycosylation regulates many protein functions, the alteration of glycosylation due to the glycan changes has attracted more interests for a variety of disease studies recently. In this work, liquid chromatography-tandem mass spectrometry was utilized to analyze the abundances of permethylated glycans from rats under control to mild CKD, severe CKD, and diabetic conditions. The most significant differences were observed in sialylation level with the highest present in the severe CKD and diabetic groups. Moreover, high mannose N-glycans was enriched in the CKD BBMs. Characterization of all the BBM N-glycan changes supports that these changes are likely to impact the functional properties of the dynamic PT BBM. Further, these changes may lead to the potential discovery of glycan biomarkers for improved CKD diagnosis and new avenues for therapeutic treatments.
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Microvellosidades , Animales , Glicómica , Glicosilación , Riñón , RatasRESUMEN
The mature HIV-1 envelope (Env) glycoprotein is composed of gp120, the exterior subunit, and gp41, the transmembrane subunit assembled as trimer by noncovalent interaction. There is a great body of literature to prove that gp120 binds to CD4 first, then to the coreceptor. Binding experiments and functional assays have demonstrated that CD4 binding induces conformational changes in gp120 that enable or enhance its interaction with a coreceptor. Previous studies provided different glycomic maps for the HIV-1 gp120. Here, we build on previous work to report that the use of LC-MS/MS, in conjunction with hydrophilic interaction liquid chromatography (HILIC) enrichment to glycosylation sites, associated with the assorted neutralizing or binding events of glycosylation targeted antibodies from different clades or strains. In this study, the microheterogeneity of the glycosylation from 4 different clades of gp120s is deeply investigated. Aberrant glycosylation patterns were detected on gp120 that originated from different clades, viral sequences, and host cells. The results of this study may help provide a better understanding of the mechanism of how the glycans participate in the antibody neutralizing process that targets glycosylation sites.
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VIH-1 , Anticuerpos Neutralizantes/metabolismo , Cromatografía Liquida , Glicosilación , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Espectrometría de Masas en TándemRESUMEN
PURPOSE: We performed comparative proteomic analyses of blood of patients with RLS and healthy individuals aiming to identify potential biomarker and therapeutic target candidate for RLS. PATIENTS AND METHODS: Blood serum samples from 12 patients with a clinical diagnosis of RLS (8 females and 4 males, with a mean age of 68.52 years) and 10 healthy controls (5 females and 5 males, with a mean age of 67.61 years) underwent proteomic profiling by liquid chromatography coupled with tandem mass spectrometry. Pathway analysis incorporating protein-protein interaction networks was carried out to identify pathological processes linked to the differentially expressed proteins. RESULTS: We quantified 272 proteins in patients with RLS and healthy controls, of which 243 were shared. Five proteins - apolipoprotein C-II, leucine-rich alpha-2-glycoprotein 1, FLJ92374, extracellular matrix protein 1, and FLJ93143 - were substantially increased in RLS patients, whereas nine proteins - vitamin D-binding protein, FLJ78071, alpha-1-antitrypsin, CD5 antigen-like, haptoglobin, fibrinogen alpha chain, complement factor H-related protein 1, platelet factor 4, and plasma protease C1 inhibitor - were decreased. Bioinformatics analyses revealed that these proteins were linked to 1) inflammatory and immune response, and complement activation, 2) brain-related development, cell aging, and memory disorders, 3) pregnancy and associated complications, 4) myocardial infarction, and 5) reactive oxygen species generation and subsequent diabetes mellitus. CONCLUSION: Our findings shed light on the multifactorial nature of RLS and identified a set of circulating proteins that may have clinical importance as biomarkers and therapeutic targets.
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The existence of glycans in isomeric forms is responsible for the multifariousness of their properties and biological functions. Their altered expression has been associated with various diseases and cancers. Analysis of native glycans is not very sensitive due to the low ionization efficiency of glycans. These facts necessitate their comprehensive structural studies and establishes a high demand for sensitive and reliable techniques. In this chapter, we discuss the strategies for effective separation and identification of permethylated isomeric glycans. The sample preparation for permethylated glycans derived from model glycoproteins and complex biological samples, analyzed using LC-MS/MS, is delineated. We introduce protein extraction and release of glycans, followed by strategies to purify the released glycans, which are reduced and permethylated to improve ionization efficiency and stabilize sialic acid residues. High-temperature LC-based separation on PGC (porous graphitized carbon) column is conducive to isomeric separation of glycans and allows their sensitive identification and quantification using MS/MS.
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Cromatografía Liquida , Glicómica , Glicoproteínas/análisis , Polisacáridos/análisis , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Conformación de Carbohidratos , Glicósido Hidrolasas/metabolismo , Glicosilación , Grafito/química , Isomerismo , Metilación , Porosidad , Proyectos de Investigación , Flujo de TrabajoRESUMEN
Protein-based therapeutics such as mAbs have become emerging drugs in modern medicine. Most of the approved therapeutic proteins are glycoproteins. Glycosylation is an essential critical quality attribute (CQA) due to the influence that glycoforms have on the safety, efficacy, and pharmacokinetics/pharmacodynamics (PK/PD) of biotherapeutics. Here, we applied an LC-MS/MS-based glycoproteomics approach to characterize Fc glycans of an NISTmAb reference material (RM) 8671 (sample B) and a ß-1,4-galactosidase-treated NISTmAb (sample A). Overall, 48 glycan compositions were identified and quantified. The glycan structure with the highest abundance was FA2, with a relative abundance of 52% in sample A and 38% in sample B. Over 50% of the identified glycans presented at levels smaller than 0.1%. Important glycan attributes were further derived using the quantitative results. The galactosylation level of modified NISTmAb was found to decrease by â¼10% when compared to the galactosylation level of NISTmAb. There was no significant difference between the two samples in the levels of sialylation, fucosylation, and high mannose. Moreover, unglycosylated peptides were also observed at a level of 1-2%.
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Anticuerpos Monoclonales , Espectrometría de Masas en Tándem , Anticuerpos Monoclonales/metabolismo , Cromatografía Liquida , Glicoproteínas/metabolismo , Glicosilación , PolisacáridosRESUMEN
Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin's reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.
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Antígenos de Histocompatibilidad Clase I/metabolismo , Túbulos Renales Proximales/metabolismo , Hígado/metabolismo , Receptores Fc/metabolismo , Insuficiencia Renal Crónica/metabolismo , Albúmina Sérica/metabolismo , Animales , Cromatografía Liquida , Modelos Animales de Enfermedad , Tasa de Filtración Glomerular , Túbulos Renales Proximales/fisiopatología , Lisina , Masculino , Microscopía de Fluorescencia por Excitación Multifotónica , Unión Proteica , Carbamilación de Proteína , Ratas Endogámicas , Ratas Sprague-Dawley , Receptores de Superficie Celular/metabolismo , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/fisiopatología , Dispersión del Ángulo Pequeño , Espectrometría de Masas en Tándem , Factores de Tiempo , Difracción de Rayos XRESUMEN
Among the primary contributors to cardiovascular diseases are inflammation and oxidative imbalance within the vessel walls as well as the fibrosis of rat aortic smooth muscle cell (RASMC). Bradykinin (BK) and leptin are inflammatory modulators that are linked to vascular injury. In this study, we employed tandem LC-MS/MS to identify protein signatures that encompass protein abundance in RASMC treated with BK or leptin followed by systems biology analyses to gain insight into the biological pathways and processes linked to vascular remodeling. In the study, 1837 proteins were identified in control untreated RASMC. BK altered the expression of 72 (4%) and 120 (6.5%) proteins, whereas leptin altered the expression of 189 (10.2%) and 127 (6.5%) proteins after 24 and 48 h, respectively, compared to control RASMC. BK increased the protein abundance of leptin receptor, transforming growth factor-ß. On the other hand, leptin increased the protein abundance of plasminogen activator inhibitor 1 but decreased the protein abundance of cofilin. BK and leptin induced the expression of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) and pathway analysis revealed the activation of mitogen-activated protein kinases (MAPKs) and AKT pathways. The proteome profile in response to BK and leptin revealed mechanistic interplay of multiple processes that modulate inflammation and oxidative stress signals in the vasculature.
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Rhabdomyosarcoma (RMS) is a highly malignant soft tissue sarcoma classified into two major histologic subtypes: embryonal (ERMS) and alveolar (ARMS). ARMS subtype is clinically more aggressive, and characterized by an oncogenic fusion protein PAX3-FOXO1 (P3F) that drives oncogenic cellular properties. To understand the role of the fusion oncoprotein in paracrine signaling, we focused on secreted exosomes, which have been demonstrated to contribute to metastasis in multiple tumor types. Advanced Proteomics-bioinformatics analysis of the protein cargo of exosomes isolated from C2C12 myoblasts transduced with P3F fusion gene revealed 52 deregulated proteins compared to control cells, with 26 enriched and 26 depleted proteins. Using both PANTHER gene classification and Ingenuity Pathway Analysis (IPA) software, we found that the main biological processes in which the 52 deregulated proteins are involved, include "catalytic activity," "binding," "metabolic process," and "cellular process." The pathways engaging the 26 enriched proteins include the "14-3-3 mediated signaling," "cell cycle," and "ERK5, VEGF, IGF1,and p70S6K signaling." Furthermore, the main nodes in which deregulated exosome proteins and miRNAs intersected revealed pathways conferring protection from stress and promoting plasticity. Based on the bioinformatics analysis and the altered exosome proteome profile, we performed biochemical functional analysis to study the diverse properties of these exosomes where angiogenesis, stemness, and anti-oxidative stress properties were validated using different platforms. P3F-modulated exosomes activated ERK, 4-EBP1, and MMP-2 in recipient cells, and enhanced angiogenesis and stemness. In addition, P3F led to lower cellular reactive oxygen species levels and enhanced resistance against oxidative stress; and treatment of stromal cells with P3F-modulated exosomes also conferred protection against exogenous oxidative stress. Our findings highlight the role of P3F fusion protein in modulating exosome cargo to confer a protective effect on recipient cells against oxidative stress and to promote plasticity and survival, potentially contributing to the known aggressive phenotype of the fusion gene-positive subtype of RMS.
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Cannabinoids have long been used for their psychotropic and possible medical properties of symptom relief. In the past few years, a vast literature shows that cannabinoids are neuroprotective under different pathological situations. Most of the effects of cannabinoids are mediated by the well-characterized cannabinoid receptors, the cannabinoid type 1 receptor (CB1R) and cannabinoid type 2 receptor (CB2R). Even though CB1Rs are highly expressed in the central nervous system (CNS), the adverse central side effects and the development of tolerance resulting from CB1R activation may ultimately limit the clinical utility of CB1R agonists. In contrast to the ubiquitous presence of CB1Rs, CB2Rs are less commonly expressed in the healthy CNS but highly upregulated in glial cells under neuropathological conditions. Experimental studies have provided robust evidence that CB2Rs seem to be involved in the modulation of different neurological disorders. In this paper, we summarize the current knowledge regarding the protective effects of CB2R activation against the development of neurological diseases and provide a perspective on the future of this field. A better understanding of the fundamental pharmacology of CB2R activation is essential for the development of clinical applications and the design of novel therapeutic strategies.
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Agonistas de Receptores de Cannabinoides/uso terapéutico , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/metabolismo , Neuroprotección/fisiología , Receptor Cannabinoide CB2/metabolismo , Animales , Encéfalo/metabolismo , Humanos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismoRESUMEN
Podocyte damage is one of the hallmarks of diabetic nephropathy leading to proteinuria and kidney damage. The underlying mechanisms of podocyte injury are not well defined. Bradykinin (BK) was shown to contribute to diabetic kidney disease. Here, we evaluated the temporal changes in proteome profile and inflammatory signals of podocytes in response to BK (10-7M). Protein profile was evaluated by liquid chromatography mass Spectrometry (LC-MS/MS) analysis. Proteome profile analysis of podocytes treated with BK (10-7M) for 3 and 6 h, revealed 61 proteins that were differentially altered compared to unstimulated control podocytes. Pathway enrichment analysis suggested inhibition of cell death pathways, engagement of cytoskeletal elements and activation of inflammatory pathways. One of the inflammatory proteins that was identified to be induced by BK treatment is Prostaglandin (PG) H Synthase-2 (Cyclooxygenase-2, COX-2). In addition, BK significantly induced the production and release of PGE2 and this effect was inhibited by both COX-2 and MEK Kinase inhibitors, demonstrating that the production of PGE2 by BK is mediated via COX-2 and MAPK-dependent mechanisms. These findings provide a global understanding of the effector modulated proteome in response to BK and also reveal BK as an important modulator of inflammation and a potential player in podocyte injury.
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Protein glycosylation is involved in many biological processes and physiological functions. Despite the recent advances in LC-MS/MS methodologies, the profiling of site-specific glycosylation is one of the major analytical challenges of glycoprotein analysis. Herein, we report that the separation of glycopeptide isomers on porous graphitic carbon (PGC)-LC was significantly improved by elevating the separation temperature under basic mobile phases. These findings permitted the isomeric separation of glycopeptides resulting from highly specific enzymatic digestions. The selectivity for different glycan types was studied using bovine fetuin, asialofetuin, IgG, ribonuclease B, and alpha-1 acid glycoprotein (AGP) by PGC-LC-MS. Comprehensive structural isomeric separation of glycopeptides was observed by high-resolution MS and confirmed by MS/MS. The specific structures of the glycopeptide isomers were identified and confirmed through exoglycosidase digestions. Glycosylation analysis of human AGP revealed the potential use of PGC-LC-MS for extensive glycoprotein analysis for biomarker discovery. This newly developed separation technique was shown as a reproducible and useful analytical method to study site-specific isomeric glycosylation.