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Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.
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Barrera Hematoencefálica , Modelos Animales de Enfermedad , Accidente Cerebrovascular Isquémico , Liposomas , Nanopartículas , Molécula 1 de Adhesión Celular Vascular , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Animales , Ratones , Molécula 1 de Adhesión Celular Vascular/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Nanopartículas/química , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Lípidos/química , Sistemas de Liberación de Medicamentos/métodos , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , HumanosRESUMEN
Affinity chromatography is a technique that uses a stationary phase based on the supramolecular interactions that occur in biological systems or mimics of these systems. This method has long been a popular tool for the isolation, measurement, and characterization of specific targets in complex samples. This review discusses the basic concepts of this method and examines recent developments in affinity chromatography and related supramolecular separation methods. Topics that are examined include advances that have occurred in the types of supports, approaches to immobilization, and binding agents that are employed in this method. New developments in the applications of affinity chromatography are also summarized, including an overview on the use of this method for biochemical purification, sample preparation or analysis, chiral separations, and biointeraction studies.
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Human serum albumin (HSA) is known to undergo modifications by glucose during diabetes. This process produces glycated HSA that can have altered binding to some drugs. In this study, high-performance affinity microcolumns and competition studies were used to see how glycation affects the binding by two thiazolidinedione-class drugs (i.e., pioglitazone and rosiglitazone) at specific regions of HSA. These regions included Sudlow sites I and II, the tamoxifen and digitoxin sites, and a drug-binding site located in subdomain IB. At Sudlow site II, the association equilibrium constants (or binding constants) for pioglitazone and rosiglitazone with normal HSA were 1.7 × 105 M-1 and 2.0 × 105 M-1 at pH 7.4 and 37 °C, with values that changed by up to 5.7-fold for glycated HSA. Sudlow site I of normal HSA had binding constants for pioglitazone and rosiglitazone of 3.4 × 105 M-1 and 4.6 × 105 M-1, with these values changing by up to 1.5-fold for glycated HSA. Rosiglitazone was found to also bind a second region that had a positive allosteric effect on Sudlow site I for all the tested preparations of HSA (binding affinity, 1.1-3.2 × 105 M-1; coupling constant for Sudlow site I, 1.20-1.34). Both drugs had a strong positive allosteric effect on the tamoxifen site of HSA (coupling constants, 13.7-19.9 for pioglitazone and 3.7-11.5 for rosiglitazone). Rosiglitazone also had weak interactions at a site in subdomain IB, with a binding constant of 1.4 × 103 M-1 for normal HSA and a value that was altered by up to 6.8-fold with glycated HSA. Neither of the tested drugs had any significant binding at the digitoxin site. The results were used to produce affinity maps that described binding by these thiazolidinediones with HSA and the effects of glycation on these interactions during diabetes.
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Diabetes Mellitus , Albúmina Sérica Humana , Humanos , Albúmina Sérica Humana/química , Hipoglucemiantes/química , Reacción de Maillard , Rosiglitazona , Pioglitazona , Unión Proteica , Albúmina Sérica/química , Tamoxifeno , Digitoxina , Cromatografía de Afinidad/métodos , Sitios de UniónRESUMEN
Increased access to cheap and rapid mass spectrometry testing of biofluids is desirable for the analysis of disorders and diseases that may be linked to alterations in metabolite or lipid levels. The objective of this study is to establish an easily customized high-throughput workflow for the analysis of biological samples using desorption electrospray ionization-mass spectrometry (DESI-MS). The guiding principles of this workflow are the use of low-cost, open-source, and readily accessible materials with high-throughput and reproducibility. The design consists of 3 steps: (1) PARAFILM surface customization of size, shape, and depth of features on PARAFILM via 3D printed molds; (2) sample spotting via high-throughput robotics using the relatively inexpensive and open-source Opentrons platform to reduce variability and increase reliability of sample spotting; and (3) an open-source point-and-click graphical user interface (MSI.EAGLE) for data analysis via the R statistical language building on the Cardinal package. Here we describe this workflow and test optimal surface ionization characteristics by comparison of serum extracts spotted on PARAFILM and on PTFE (porous and nonporous). Untargeted analysis across three surfaces suggests that they are all suitable for ionization of a wide range of metabolites and lipids, with 3983 m/z features detected. Differential analysis of polar vs nonpolar serum extracts suggests that â¼80% of ions are desorbed preferentially from different surfaces. PARAFILM is less impacted by the interference of background ions derived from the surface. The developed system allows for a wide range of researchers to access custom surface design workflows and high-throughput analyses in a highly cost-effective manner.
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Lipidómica , Parafina , Reproducibilidad de los Resultados , Lipidómica/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , IonesRESUMEN
Modification of proteins can occur during diabetes due to the formation of advanced glycation end-products (AGEs) with reactive dicarbonyls such as glyoxal (Go) and methylglyoxal (MGo). Human serum albumin (HSA) is a serum protein that binds to many drugs in blood and that is known to be modified by Go and MGo. This study examined the binding of various sulfonylurea drugs with these modified forms of HSA by using high-performance affinity microcolumns prepared by non-covalent protein entrapment. Zonal elution experiments were employed to compare the retention and overall binding constants for the drugs with Go- or MGo-modified HSA vs normal HSA. The results were compared to values from the literature, such as measured or estimated using affinity columns containing covalently immobilized HSA or biospecifically-adsorbed HSA. The entrapment-based approach provided estimates of global affinity constants within 3-5 min for most of the tested drugs and with typical precisions of ±10-23%. Each entrapped protein microcolumn was stable for over at least 60-70 injections and one month of use. The results obtained with normal HSA agreed at the 95% confidence level with global affinity constants that have been reported for the given drugs in the literature. It was found for HSA that had been modified with clinically-relevant levels of either Go or MGo that an increase in the global affinity constant of up to 2.1-fold occurred for some of the tested drugs. The information acquired in this study can be used in the future to adapt this entrapment-based approach to study and evaluate interactions between other types of drugs and normal or modified binding agents for clinical testing and biomedical research.
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Albúmina Sérica Humana , Albúmina Sérica , Humanos , Albúmina Sérica Humana/química , Albúmina Sérica/química , Óxido de Magnesio , Unión Proteica , Cromatografía de Afinidad/métodos , Compuestos de Sulfonilurea/químicaRESUMEN
Lipids may influence cellular penetrance by pathogens and the immune response that they evoke. Here we find a broad based lipidomic storm driven predominantly by secretory (s) phospholipase A 2 (sPLA 2 ) dependent eicosanoid production occurs in patients with sepsis of viral and bacterial origin and relates to disease severity in COVID-19. Elevations in the cyclooxygenase (COX) products of arachidonic acid (AA), PGD 2 and PGI 2 , and the AA lipoxygenase (LOX) product, 12-HETE, and a reduction in the high abundance lipids, ChoE 18:3, LPC-O-16:0 and PC-O-30:0 exhibit relative specificity for COVID-19 amongst such patients, correlate with the inflammatory response and link to disease severity. Linoleic acid (LA) binds directly to SARS-CoV-2 and both LA and its di-HOME products reflect disease severity in COVID-19. AA and LA metabolites and LPC-O-16:0 linked variably to the immune response. These studies yield prognostic biomarkers and therapeutic targets for patients with sepsis, including COVID-19. An interactive purpose built interactive network analysis tool was developed, allowing the community to interrogate connections across these multiomic data and generate novel hypotheses.
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BACKGROUND: Lipids may influence cellular penetrance by viral pathogens and the immune response that they evoke. We deeply phenotyped the lipidomic response to SARs-CoV-2 and compared that with infection with other pathogens in patients admitted with acute respiratory distress syndrome to an intensive care unit (ICU). METHODS: Mass spectrometry was used to characterise lipids and relate them to proteins, peripheral cell immunotypes and disease severity. RESULTS: Circulating phospholipases (sPLA2, cPLA2 (PLA2G4A) and PLA2G2D) were elevated on admission in all ICU groups. Cyclooxygenase, lipoxygenase and epoxygenase products of arachidonic acid (AA) were elevated in all ICU groups compared with controls. sPLA2 predicted severity in COVID-19 and correlated with TxA2, LTE4 and the isoprostane, iPF2α-III, while PLA2G2D correlated with LTE4. The elevation in PGD2, like PGI2 and 12-HETE, exhibited relative specificity for COVID-19 and correlated with sPLA2 and the interleukin-13 receptor to drive lymphopenia, a marker of disease severity. Pro-inflammatory eicosanoids remained correlated with severity in COVID-19 28 days after admission. Amongst non-COVID ICU patients, elevations in 5- and 15-HETE and 9- and 13-HODE reflected viral rather than bacterial disease. Linoleic acid (LA) binds directly to SARS-CoV-2 and both LA and its di-HOME products reflected disease severity in COVID-19. In healthy marines, these lipids rose with seroconversion. Eicosanoids linked variably to the peripheral cellular immune response. PGE2, TxA2 and LTE4 correlated with T cell activation, as did PGD2 with non-B non-T cell activation. In COVID-19, LPS stimulated peripheral blood mononuclear cell PGF2α correlated with memory T cells, dendritic and NK cells while LA and DiHOMEs correlated with exhausted T cells. Three high abundance lipids - ChoE 18:3, LPC-O-16:0 and PC-O-30:0 - were altered specifically in COVID. LPC-O-16:0 was strongly correlated with T helper follicular cell activation and all three negatively correlated with multi-omic inflammatory pathways and disease severity. CONCLUSIONS: A broad based lipidomic storm is a predictor of poor prognosis in ARDS. Alterations in sPLA2, PGD2 and 12-HETE and the high abundance lipids, ChoE 18:3, LPC-O-16:0 and PC-O-30:0 exhibit relative specificity for COVID-19 amongst such patients and correlate with the inflammatory response to link to disease severity.
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COVID-19 , Fosfolipasas A2 Secretoras , Sepsis , Humanos , SARS-CoV-2 , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Lipidómica , Leucocitos Mononucleares , Leucotrieno E4 , Prostaglandina D2 , Ciclooxigenasa 2 , EicosanoidesRESUMEN
Activities of dendritic cells (DCs) that present tumor antigens are often suppressed in tumors. Here we report that this suppression is induced by tumor microenvironment-derived factors, which activate the activating transcription factor-3 (ATF3) transcription factor and downregulate cholesterol 25-hydroxylase (CH25H). Loss of CH25H in antigen presenting cells isolated from human lung tumors is associated with tumor growth and lung cancer progression. Accordingly, mice lacking CH25H in DCs exhibit an accelerated tumor growth, decreased infiltration and impaired activation of intratumoral CD8+ T cells. These mice do not establish measurable long-term immunity against malignant cells that undergo chemotherapy-induced immunogenic cell death. Mechanistically, downregulation of CH25H stimulates membrane fusion between endo-phagosomes and lysosomes, accelerates lysosomal degradation and restricts cross-presentation of tumor antigens in the intratumoral DCs. Administration of STING agonist MSA-2 reduces the lysosomal activity in DCs, restores antigen cross presentation, and increases therapeutic efficacy of PD-1 blockade against tumour challenge in a CH25H-dependent manner. These studies highlight the importance of downregulation of CH25H in DCs for tumor immune evasion and resistance to therapy.
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Reactividad Cruzada , Neoplasias Pulmonares , Ratones , Humanos , Animales , Antígenos de Neoplasias , Linfocitos T CD8-positivos , Células Dendríticas , Neoplasias Pulmonares/metabolismo , Lisosomas , Ratones Endogámicos C57BL , Microambiente TumoralRESUMEN
2-Imidazoline drugs are used in a variety of applications, such as the treatment of hypertension and opioid withdrawal. It is known these drugs bind to serum proteins and have significant variations within this class of compounds in the overall level of this binding. However, little specific information is available on the interactions of these compounds with the two major transport proteins for many drugs, human serum albumin (HSA) and alpha1-acid glycoprotein (AGP). This study examined binding by 2-imidazolines to these proteins by using 25â¯mmâ¯×â¯2.1â¯mm i.d. high-performance affinity microcolumns that contained HSA or AGP. The drugs that were examined were antazoline, clonidine, dexmedetomidine, lofexidine, moxonidine, phentolamine, and tizanidine, which represented a wide range of structures and pharmaceutical applications. The major metabolite of lofexidine, N-(2-aminoethyl)-2-(2,6-dichlorophenoxy) propenamide (LADP), was also examined. All these 2-imidazolines were found to have weak-to-moderate binding to HSA, with global affinities that ranged from 1.62â¯×â¯102 to 1.07â¯×â¯104 M-1 at pH 7.4 and 37⯰C. These compounds had stronger binding with AGP, with global affinities constants ranging from 3.80â¯×â¯102 to 1.85â¯×â¯104 M-1. No stereoselectivity was observed by HSA for the enantiomers of dexmedetomidine, lofexidine, or LADP. However, AGP did show some stereoselectivity for lofexidine and LADP but not for dexmedetomidine. These results provide a better understanding of interactions of 2-imidazoline with HSA vs AGP in the circulation and of how this binding can change between drugs within this class of compounds.
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Imidazolinas , Orosomucoide , Cromatografía de Afinidad , Humanos , Imidazoles , Orosomucoide/metabolismo , Unión Proteica , Albúmina Sérica Humana/metabolismoRESUMEN
Diabetes is characterized by elevated levels of blood glucose, which can result in the modification of serum proteins. The modification of a protein by glucose, or glycation, can also lead to the formation of advanced glycated end-products (AGEs). One protein that can be modified through glycation and AGE formation is human serum albumin (HSA). In this study, immunoextraction based on polyclonal anti-HSA antibodies was used with high-performance affinity microcolumns to see how AGE-related modifications produced by glyoxal (Go) and methylglyoxal (MGo) affected the binding of HSA to several first- and second-generation sulfonylureas, a class of drugs used to treat type II diabetes and known to bind to HSA. With this approach, it was possible to use a single platform to examine drug interactions with several preparations of HSA. Each applied protein sample could be used over 20-50 experiments, and global affinity constants for most of the examined drugs could be obtained in less than 7.5 min. The binding constants measured for these drugs with normal HSA gave good agreement with global affinities based on the literature. Both Go- and MGo-related modifications at clinically relevant levels were found by this method to create significant changes in the binding by some sulfonylureas with HSA. The global affinities for many of the drugs increased by 1.4-fold or more; gliclazide and tolazamide had no significant change with some preparations of modified HSA, and a small-to-moderate decrease in binding strength was noted for glibenclamide and gliclazide with Go-modified HSA. This approach can be adapted for the study of other drug-protein interactions and alternative modified proteins by altering the antibodies that are employed for immunoextraction and within the affinity microcolumn.
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Anticuerpos/aislamiento & purificación , Cromatografía de Afinidad/métodos , Glioxal/química , Piruvaldehído/química , Albúmina Sérica Humana/metabolismo , Compuestos de Sulfonilurea/química , Adsorción , Interacciones Farmacológicas , Gliclazida/química , Gliburida , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Humanos , Cinética , Unión Proteica , Estabilidad Proteica , Albúmina Sérica Humana/química , Warfarina/químicaRESUMEN
Widely accessible food phytochemicals such as curcumin have been reported to have anti-inflammatory and anticarcinogenic properties. However, curcumin has poor absorption in the gut, and piperine has been of interest as a dietary compound that can enhance curcumin bioavailability. The aim of this study was to develop and optimize a technique using reversed-phase chromatography with multi-wavelength detection for the simultaneous measurement of curcumin and piperine in various biological matrices. Emodin was used as an internal standard. Protein precipitation and liquid-liquid extraction based on acetonitrile provided good recovery of these analytes. A 150 mm × 4.6 mm I.D. Luna C18 column was used under isocratic conditions to separate curcumin, piperine, and emodin with baseline resolution, and with good separation from other sample components, in as little as 4 min. The detection limits for curcumin and piperine were 3 and 7 ng/mL, respectively. This method has been used to quantitate these compounds in samples such as human intestinal epithelial cell lysates and mouse plasma or GI tissues in research aimed at examining the bioavailability of curcumin in the presence of piperine.
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Alcaloides/sangre , Benzodioxoles/sangre , Cromatografía de Fase Inversa/métodos , Curcumina/análisis , Piperidinas/sangre , Alcamidas Poliinsaturadas/sangre , Alcaloides/química , Alcaloides/farmacocinética , Animales , Benzodioxoles/química , Benzodioxoles/farmacocinética , Disponibilidad Biológica , Cromatografía Líquida de Alta Presión , Curcumina/química , Curcumina/farmacocinética , Emodina , Humanos , Límite de Detección , Modelos Lineales , Masculino , Ratones , Piperidinas/química , Piperidinas/farmacocinética , Alcamidas Poliinsaturadas/química , Alcamidas Poliinsaturadas/farmacocinética , Reproducibilidad de los ResultadosRESUMEN
Affinity capillary electrophoresis (ACE) is a separation technique that combines a biologically-related binding agent with the separating power and efficiency of capillary electrophoresis. This review will examine several classes of binding agents that have been used in ACE and applications that have been described for the resulting methods in clinical or pharmaceutical analysis. Binding agents that will be considered are antibodies, aptamers, lectins, serum proteins, carbohydrates, and enzymes. This review will also describe the various formats in which each type of binding agent has been used in CE, including both homogeneous and heterogeneous methods. Specific areas of applications that will be considered are CE-based immunoassays, glycoprotein/glycan separations, chiral separations, and biointeraction studies. The general principles and formats of ACE for each of these applications will be examined, along with the potential advantages or limitations of these methods.
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Química Farmacéutica/métodos , Electroforesis Capilar/métodos , Inmunoensayo/métodos , Humanos , LigandosRESUMEN
The field of affinity chromatography, which employs a biologically-related agent as the stationary phase, has seen significant growth since the modern era of this method began in 1968. This review examines the major developments and trends that have occurred in this technique over the past five decades. The basic principles and history of this area are first discussed. This is followed by an overview of the various supports, immobilization strategies, and types of binding agents that have been used in this field. The general types of applications and fields of use that have appeared for affinity chromatography are also considered. A survey of the literature is used to identify major trends in these topics and important areas of use for affinity chromatography in the separation, analysis, or characterization of chemicals and biochemicals.
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Cromatografía de Afinidad , Bioquímica , Investigación Biomédica , Biotecnología , Cromatografía de Afinidad/historia , Cromatografía de Afinidad/métodos , Cromatografía de Afinidad/tendencias , Historia del Siglo XX , Historia del Siglo XXI , HumanosRESUMEN
Sulfonylurea drugs are antidiabetic drugs that are utilized in the treatment of type II diabetes and often have significant binding with human serum albumin (HSA). Immobilized samples of normal or glycated HSA in affinity microcolumns were used to investigate interactions of these proteins with the sulfonylurea drug tolazamide. HPLC and frontal analysis were used to first examine the overall binding of this drug with these samples of HSA. It was found that tolazamide had two general classes of binding sites (i.e., high and low affinity) for normal and glycated HSA. The higher affinity sites had binding constants of around 4.3-6.0 × 104 M-1 for these interactions at pH 7.4 and 37 °C, while the lower affinity sites had binding strengths of 4.9-9.1 × 103 M-1. Zonal competition studies between tolazamide and probes for Sudlow sites I and II on HSA were also performed and used to provide site-specific affinities for tolazamide at these sites. A decrease of 22% in affinity was observed for tolazamide at Sudlow site I and an increase up to 58% was seen at Sudlow site II when comparing glycated HSA with normal HSA. These observed changes were compared to those of other first-generation sulfonylurea drugs, providing information on how glycation can alter the total and local binding strength of tolazamide and related compounds with HSA under levels of glycation seen in patients with diabetes.
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Hipoglucemiantes/química , Albúmina Sérica Humana/química , Albúmina Sérica/química , Tolazamida/química , Sitios de Unión , Cromatografía Líquida de Alta Presión , Productos Finales de Glicación Avanzada , Glicosilación , Humanos , Unión Proteica , Albúmina Sérica GlicadaRESUMEN
Serum albumin is the most abundant protein in mammalian blood plasma and is responsible for the transport of metals, drugs, and various metabolites, including hormones. We report the first albumin structure in complex with testosterone, the primary male sex hormone. Testosterone is bound in two sites, neither of which overlaps with the previously suggested Sudlow site I. We determined the binding constant of testosterone to equine and human albumins by two different methods: tryptophan fluorescence quenching and ultrafast affinity extraction. The binding studies and similarities between residues comprising the binding sites on serum albumins suggest that testosterone binds to the same sites on both proteins. Our comparative analysis of albumin complexes with hormones, drugs, and other biologically relevant compounds strongly suggests interference between a number of compounds present in blood and testosterone transport by serum albumin. We discuss a possible link between our findings and some phenomena observed in human patients, such as low testosterone levels in diabetic patients.