RESUMEN

Familial hyperlysinemia is a rare autosomal recessive disorder due to defects of the AASS (α-aminoadipate δ-semialdehyde synthase) gene, which encodes for a bifunctional enzyme. Two types of hyperlysinemia have been identified namely type 1, due to the deficit of the alfa-ketoglutarate activity, and type 2, due to the deficit of the saccharopine dehydrogenase activity. METHODS: To better characterize the phenotypic spectrum of familial hyperlysinemia type 1, we conducted a systematic review of cases in the literature following PRISMA guidelines. We selected 16 articles describing 23 patients with hyperlysinemia type 1, twelve of whom with homozygous or compound heterozygous mutations in AASS gene. We also included a novel patient with a homozygous c.799C>T; p.(Arg267Cys) mutation in AASS gene. We collected genetic, clinical, brain imaging and electroencephalogram (EEG) features when available. RESULTS: The phenotype of this disease is heterogeneous, ranging from more severe forms with spastic tetraparesis, intellectual disability and epilepsy and mild-moderate forms with only intellectual disability or behavioural problem and/or epilepsy to normal clinical conditions. Only our patient has neuropathy unrelated to infectious event. CONCLUSIONS: We described the heterogeneous phenotypic spectrum of familial hyperlysinemia type 1 and we identified a new symptom, axonal neuropathy, never before described in this condition.

Asunto(s)

Hiperlisinemias , Humanos , Hiperlisinemias/genética

RESUMEN

Mutant-specific inhibitors of KRASG12C, such as AMG510 (sotorasib) and MRTX849 (adagrasib), offer the unprecedented opportunity to inhibit KRAS, the most frequently mutated and heretofore undruggable oncoprotein. While clinical data are still limited, on-target mutations in KRASG12C at position 12 and other sites are emerging as major drivers of clinical relapse. We identified additional mutations in KRASG12C that impact inhibitor sensitivity through a saturation mutagenesis screen in the KRASG12C NCI-H358 non­small-cell lung cancer (NSCLC) cell line. We also identified individuals in population genetic databases harboring these resistance mutations in their germline and in tumors, including a subset that co-occur with KRASG12C, indicating that these mutations may preexist in patients treated with KRASG12C inhibitors. Notably, through structural modeling, we found that one such mutation (R68L) interferes with the critical protein­drug interface, conferring resistance to both inhibitors. Finally, we uncovered a mutant (S17E) that demonstrated a strong sensitizing phenotype to both inhibitors. Functional studies suggest that S17E sensitizes KRASG12C cells to KRASG12C inhibition by impacting signaling through PI3K/AKT/mTOR but not the MAPK signaling pathway. Our studies highlight the utility of unbiased mutation profiling to understand the functional consequences of all variants of a disease-causing genetic mutant and predict acquired resistant mutations in the targeted therapeutics.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutagénesis , Mutación , Piperazinas , Proteínas Proto-Oncogénicas p21(ras)/genética , Piridinas , Pirimidinas

RESUMEN

The necroptotic cell death pathway is a key component of human pathogen defense that can become aberrantly derepressed during tissue homeostasis to contribute to multiple types of tissue damage and disease. While formation of the necrosome kinase signaling complex containing RIPK1, RIPK3, and MLKL has been extensively characterized, additional mechanisms of its regulation and effector functions likely remain to be discovered. We screened 19,883 mouse protein-coding genes by CRISPR/Cas9-mediated gene knockout for resistance to cytokine-induced necroptosis and identified 112 regulators and mediators of necroptosis, including 59 new candidate pathway components with minimal or no effect on cell growth in the absence of necroptosis induction. Among these, we further characterized the function of PTBP1, an RNA binding protein whose activity is required to maintain RIPK1 protein abundance by regulating alternative splice-site selection.

Asunto(s)

Empalme Alternativo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Fibroblastos/enzimología , Marcación de Gen/métodos , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Necroptosis , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Fibroblastos/patología , Regulación Enzimológica de la Expresión Génica , Células HEK293 , Células HT29 , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Ratones , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal

RESUMEN

The clinical assessment of multiple organ dysfunctions at early stages is recognized to be an important factor in prompting definitive treatment decisions that prevent irreversible organ damage. In this article, we propose a real-time, label-free, and multiplex nanoenhanced SPRi platform to quantitatively assess two biomarkers, kidney injury molecule (KIM-1) and high mobility group box-1 (HMGB-1) simultaneously in buffer. Our work involves three major contributions in the design of the immunosensor: (1) we applied site-specific immobilization of antibodies to the solid surface that avoids loss of biological activity caused by covalent attachment; (2) we constructed a well-blocked sensor surface that exhibits minimal non-specific adsorption for singleplex measurements of each biomarker in buffer; and (3) we adopted a sandwich assay that implements functionalized quantum dots (NanoEnhancers) as signal amplifiers to achieve a sensitivity level of 5 pg/mL for KIM-1 and HMGB-1 in buffer. We foresee great potential and success in extending this multiplex and ultra-sensitive platform to assess a variety of other emerging clinical biomarkers at low concentrations and in complex matrices.

Asunto(s)

Técnicas Biosensibles/métodos , Proteína HMGB1/metabolismo , Receptor Celular 1 del Virus de la Hepatitis A/metabolismo , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/metabolismo , Anticuerpos/metabolismo , Biomarcadores/metabolismo , Humanos , Fallo Hepático Agudo/diagnóstico , Fallo Hepático Agudo/metabolismo , Análisis por Matrices de Proteínas , Puntos Cuánticos

RESUMEN

Progesterone is a steroid hormone that plays a central role in the female reproductive processes such as ovulation and pregnancy with possible effects on other organs as well. The measurement of progesterone levels in bodily fluids can assist in early pregnancy diagnosis and can provide insight for other reproductive functions. In this work, the detection of progesterone was examined by integrating novel aptamer development with a nanoEnhanced surface plasmon resonance imaging sensor. First, we developed X-aptamers and selected them for binding to progesterone. Then, we took advantage of the multi-array feature of SPRi to develop an optimized biosensor capable of simultaneously screening the 9 X-aptamers developed to determine the binding capabilities of each aptamer. The sensor surface design conditions were further optimized for the sandwich assay, which employed nanoEnhancers (NIR-streptavidin coated quantum dots) for ultrasensitive detection of progesterone molecules. The assay designed was examined over a concentration range of 1.575 ng/mL to 126 µg/mL resulting in a limit of detection (LOD) of 1.575 ng/mL (5 nM) in phosphate buffer.

Asunto(s)

Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Progesterona/análisis , Tampones (Química) , Sensibilidad y Especificidad

RESUMEN

CONTEXT: Vitamin B12 deficiency may occur with metformin treatment, but few studies have assessed risk with long-term use. OBJECTIVE: To assess the risk of B12 deficiency with metformin use in the Diabetes Prevention Program (DPP)/DPP Outcomes Study (DPPOS). DESIGN: Secondary analysis from the DPP/DPPOS. Participants were assigned to the placebo group (PLA) (n = 1082) or the metformin group (MET) (n = 1073) for 3.2 years; subjects in the metformin group received open-label metformin for an additional 9 years. SETTING: Twenty-seven study centers in the United States. PATIENTS: DPP eligibility criteria were: elevated fasting glucose, impaired glucose tolerance, and overweight/obesity. The analytic population comprised participants with available stored samples. B12 levels were assessed at 5 years (n = 857, n = 858) and 13 years (n = 756, n = 764) in PLA and MET, respectively. INTERVENTION: Metformin 850 mg twice daily vs placebo (DPP), and open-label metformin in the metformin group (DPPOS). MAIN OUTCOME MEASURES: B12 deficiency, anemia, and peripheral neuropathy. RESULTS: Low B12 (≤ 203 pg/mL) occurred more often in MET than PLA at 5 years (4.3 vs 2.3%; P = .02) but not at 13 years (7.4 vs 5.4%; P = .12). Combined low and borderline-low B12 (≤ 298 pg/mL) was more common in MET at 5 years (19.1 vs 9.5%; P < .01) and 13 years (20.3 vs 15.6%; P = .02). Years of metformin use were associated with increased risk of B12 deficiency (odds ratio, B12 deficiency/year metformin use, 1.13; 95% confidence interval, 1.06­1.20). Anemia prevalence was higher in MET, but did not differ by B12 status. Neuropathy prevalence was higher in MET with low B12 levels. CONCLUSIONS: Long-term use of metformin in DPPOS was associated with biochemical B12 deficiency and anemia. Routine testing of vitamin B12 levels in metformin-treated patients should be considered.

Asunto(s)

Anemia/inducido químicamente , Diabetes Mellitus Tipo 2/prevención & control , Hipoglucemiantes/efectos adversos , Metformina/efectos adversos , Deficiencia de Vitamina B 12/inducido químicamente , Adulto , Anciano , Femenino , Homocisteína/sangre , Humanos , Hipoglucemiantes/uso terapéutico , Masculino , Metformina/uso terapéutico , Persona de Mediana Edad , Vitamina B 12/sangre

RESUMEN

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml(-) (1) and minimum levels of 0.03 ng ml(-) (1)) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

Asunto(s)

Ensayo de Inmunoadsorción Enzimática/métodos , Hormona de Crecimiento Humana/sangre , Resonancia por Plasmón de Superficie/métodos , Humanos , Nanotecnología/métodos , Puntos Cuánticos , Proteínas Recombinantes/sangre

RESUMEN

Surface plasmon resonance (SPR) is a popular technique that allows for sensitive, specific, label-free and real-time assessment of biomolecular interactions. SPR is a nondestructive, modular and flexible tool for various applications in biomedical sciences ranging from cell sorting, cell surface characterization and drug discovery. In this review, we will discuss more specifically how SPR is used to monitor the dynamics of various types of cellular binding events and morphological adherence changes in response to external stimuli.

Asunto(s)

Rastreo Celular , Sistemas de Liberación de Medicamentos , Imagen Molecular , Resonancia por Plasmón de Superficie , Animales , Biopelículas/crecimiento & desarrollo , Fenómenos Biofísicos , Escherichia coli/crecimiento & desarrollo , Células HEK293 , Humanos

RESUMEN

The fabrication of 4-mercaptobenzoic acid (4-MBA) antibody-functionalized gold nanoparticles via microwave technology for surface-enhanced Raman scattering (SERS)-based cellular nanosensing is reported. Nanoprobes were characterized by UV-vis absorbance, Raman scattering properties, and observed by TEM imaging. Results showed that microwave irradiation rapidly yielded nanoprobes with significant Raman scattering intensity and suitable stability to support antibody conjugation in under 10min. Functionalized nanoprobes demonstrated the ability to map the expression of vascular adhesion molecule-1 (VCAM-1) in human coronary artery endothelial (HCAE) cells, indicating that microwave fabrication presents a viable and rapid approach to SERS nanoprobe construction. The successful application of SERS nanoprobes to localize biomarker expression in vitro may ultimately be used for early diagnostic and preventative functions in medicine.

Asunto(s)

Técnicas Biosensibles , Microondas , Nanoestructuras , Espectrometría Raman/métodos , Línea Celular , Humanos , Microscopía Electrónica de Transmisión , Espectrofotometría Ultravioleta , Propiedades de Superficie , Molécula 1 de Adhesión Celular Vascular/análisis

RESUMEN

OBJECTIVE: We evaluate whether lifestyle and metformin interventions used to prevent diabetes have durable effects on markers of inflammation and coagulation and whether the effects are influenced by the development of diabetes. RESEARCH DESIGN AND METHODS: The Diabetes Prevention Program was a controlled clinical trial of 3,234 subjects at high risk for diabetes who were randomized to lifestyle, metformin, or placebo interventions for 3.4 years. Diabetes was diagnosed semiannually by fasting glucose and annually by oral glucose tolerance testing. In addition to baseline testing, anthropometry was performed every 6 months; fasting insulin yearly; and hs-CRP, tissue plasminogen activator (tPA), and fibrinogen at 1 year and end of study (EOS). RESULTS: CRP and tPA levels were unchanged in the placebo group but fell in the lifestyle and metformin groups at 1 year and remained lower at EOS. These reductions were not seen in those who developed diabetes over the course of the study despite intervention. Fibrinogen was lower at 1 year in the lifestyle group. Differences in weight and weight change explained most of the influence of diabetes on the CRP response in the lifestyle group, but only partly in the placebo and metformin groups. Weight, insulin sensitivity, and hyperglycemia differences each accounted for the influence of diabetes on the tPA response. CONCLUSIONS: Lifestyle and metformin interventions have durable effects to lower hs-CRP and tPA. Incident diabetes prevented these improvements, and this was accounted for by differences in weight, insulin resistance, and glucose levels.

Asunto(s)

Proteína C-Reactiva/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/prevención & control , Hipoglucemiantes/uso terapéutico , Estilo de Vida , Metformina/uso terapéutico , Activador de Tejido Plasminógeno/sangre , Adulto , Anciano , Anciano de 80 o más Años , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Diabetes Mellitus Tipo 2/epidemiología , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/sangre , Resistencia a la Insulina , Masculino , Persona de Mediana Edad

RESUMEN

Diagnostic biomarkers (i.e. proteins) are often in low abundance in bodily fluids presenting many challenges for their detection. In order to extend the application of SPRi systems in detecting biomarkers at ultralow levels, we combine the advantage of aptamer technology with nanomaterials and microwave-assisted surface functionalization. By implementing a sandwich assay through the introduction of aptamer-modified quantum dots (QDs), it was possible to measure 7 zeptomole (at 5 fg/mL) of C-reactive protein (CRP) selectively in spiked human serum. It is expected that the proposed platform will provide new direction in designing ultrasensitive SPRi biosensors with multiplexing capabilities.

Asunto(s)

Aptámeros de Nucleótidos/química , Técnicas Biosensibles/instrumentación , Proteína C-Reactiva/análisis , Nanopartículas del Metal/química , Puntos Cuánticos , Resonancia por Plasmón de Superficie/instrumentación , Aptámeros de Nucleótidos/genética , Análisis Químico de la Sangre/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Oro/química , Nanopartículas del Metal/ultraestructura , Microquímica/instrumentación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad

RESUMEN

BACKGROUND: The hallmark of atherosclerosis is the accumulation of plaque in vessel walls. This process is initiated when monocytic cells differentiate into macrophage foam cells under conditions with high levels of atherogenic lipoproteins. Vulnerable plaque can dislodge, enter the blood stream, and result in acute myocardial infarction and stroke. Imaging techniques such as cardiovascular magnetic resonance (CMR) provides one strategy to identify patients with plaque accumulation. METHODS: We synthesized an atherosclerotic-targeting contrast agent (ATCA) in which gadolinium (Gd)-containing endohedrals were functionalized and formulated into liposomes with CD36 ligands intercalated into the lipid bilayer. In vitro assays were used to assess the specificity of the ATCA for foam cells. The ability of ATCA to detect atherosclerotic plaque lesions in vivo was assessed using CMR. RESULTS: The ATCA was able to detect scavenger receptor (CD36)-expressing foam cells in vitro and were specifically internalized via the CD36 receptor as determined by focused ion beam/scanning electron microscopy (FIB-SEM) and Western blotting analysis of CD36 receptor-specific signaling pathways. The ATCA exhibited time-dependent accumulation in atherosclerotic plaque lesions of ApoE -/- mice as determined using CMR. No ATCA accumulation was observed in vessels of wild type (C57/b6) controls. Non-targeted control compounds, without the plaque-targeting moieties, were not taken up by foam cells in vitro and did not bind plaque in vivo. Importantly, the ATCA injection was well tolerated, did not demonstrate toxicity in vitro or in vivo, and no accumulation was observed in the major organs. CONCLUSIONS: The ATCA is specifically internalized by CD36 receptors on atherosclerotic plaque providing enhanced visualization of lesions under physiological conditions. These ATCA may provide new tools for physicians to non-invasively detect atherosclerotic disease.

Asunto(s)

Aorta/patología , Enfermedades de la Aorta/diagnóstico , Aterosclerosis/diagnóstico , Medios de Contraste , Fulerenos , Imagen por Resonancia Magnética , Compuestos Organometálicos , Placa Aterosclerótica , Animales , Aorta/metabolismo , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Western Blotting , Antígenos CD36/metabolismo , Medios de Contraste/metabolismo , Medios de Contraste/toxicidad , Modelos Animales de Enfermedad , Células Espumosas/metabolismo , Células Espumosas/patología , Fulerenos/metabolismo , Fulerenos/toxicidad , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Rastreo , Compuestos Organometálicos/metabolismo , Compuestos Organometálicos/toxicidad , Valor Predictivo de las Pruebas , Factores de Tiempo , Células U937

RESUMEN

AIM: To enhance the stability of siRNA while improving their therapeutic properties and visualization at the target site, a novel nanoplex system was developed. MATERIALS & METHODS: The designed nanoplex system involved functionalizing siRNA with near-infrared quantum dots and loading them into histidylated glycol chitosan (GC-His). RESULTS: Colocalization studies revealed a twofold increase in siRNA uptake after encapsulation with GC-His and nanoparticles were localized in cytoplasm, suggesting that histidine promoted their dissociation from the endosomal membranes. Furthermore, as opposed to siRNAs treated with commercial transfection reagent, siRNAs loaded within GC-His showed a marked reduction (64%) of MDM2 protein expression 24 h after transfection. CONCLUSION: These findings concur that GC-His/siRNA-quantum dot nanoplexes are promising multifunctional vehicles for gene inhibitory therapy.

Asunto(s)

Terapia Genética/métodos , Nanopartículas/química , Western Blotting , Línea Celular Tumoral , Quitosano/química , Ensayo de Cambio de Movilidad Electroforética , Humanos , Espectroscopía de Resonancia Magnética , Microscopía Confocal , Estructura Molecular , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Puntos Cuánticos

RESUMEN

Canonical Wnt signaling is controlled intracellularly by the level of ß-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates ß-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling.

Asunto(s)

Proteína Axina/metabolismo , Transducción de Señal , Tanquirasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Wnt/metabolismo , Centrosoma/metabolismo , Células HEK293 , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Transporte de Proteínas , Proteolisis , Ubiquitinación

RESUMEN

The surface plasmon resonance imaging chip biointerface is fully designed using near-infrared (NIR) quantum dots (QDs) for the enhancement of surface plasmon resonance imaging (SPRi) signals in order to extend their application for medical diagnostics. The measured SPRi detection signal following the QD binding to the surface was amplified 25-fold for a 1 nM concentration of single-stranded DNA (ssDNA) and 50-fold for a 1 µg/mL concentration of prostate-specific antigen (PSA), a cancer biomarker, thus substantiating their wide potential to study interactions of a diverse set of small biomolecules. This significant enhancement is attributed to the QD's mass-loading effect and spontaneous emission coupling with propagating surface plasmons, which allowed the SPRi limit of detection to be reduced to 100 fM and 100 pg/mL for ssDNA and PSA, respectively. Furthermore, this study illustrates the potential of SPRi to be easily integrated with fluorescent imaging for advanced correlative surface-interaction analysis.

Asunto(s)

Técnicas Biosensibles , Puntos Cuánticos , Espectroscopía Infrarroja Corta/métodos , Resonancia por Plasmón de Superficie/métodos , Secuencia de Bases , Cartilla de ADN , ADN de Cadena Simple/análisis , Límite de Detección , Antígeno Prostático Específico/análisis

RESUMEN

AIM: To assess the effects of oleic acid treatment on subcellular distribution of indium gallium phosphide-zinc sulfide (InGaP/ZnS) nanoparticles in microglia and astrocytes. MATERIALS & METHODS: The extent of colocalization between the nanoparticles and organelles was assessed by confocal microscopy, spectrofluorometry and cell sorting. RESULTS: Cell treatment with a common fatty acid (oleic acid) within the range of physiological concentrations markedly enhanced the InGaP/ZnS uptake by microglia and afforded their colocalization within lipid droplets/lysosomes but not with mitochondria. CONCLUSION: These results suggest that the availability of mono-unsaturated fatty acids, such as oleic acid, in different cells could significantly alter nanoparticle uptake and localization, which can in turn affect the functions of cells and tissues coexposed to nanoparticles.

Asunto(s)

Galio/metabolismo , Indio/metabolismo , Nanopartículas/química , Nanotecnología/métodos , Neuroglía/metabolismo , Fosfinas/metabolismo , Sulfuros/metabolismo , Compuestos de Zinc/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Galio/química , Indio/química , Ratones , Microscopía Confocal , Neuroglía/efectos de los fármacos , Ácido Oléico/farmacología , Proteínas Asociadas a Pancreatitis , Fosfinas/química , Sulfuros/química , Compuestos de Zinc/química

RESUMEN

Prodrugs are biologically inactive derivatives of an active drug intended to solve certain problems of the parent drug such as toxicity, instability, minimal solubility and non-targeting capabilities. The majority of drugs for cardiovascular diseases undergo first-pass metabolism, resulting in drug inactivation and generation of toxic metabolites, which makes them appealing targets for prodrug design. Since prodrugs undergo a chemical reaction to form the parent drug once inside the body, this makes them very effective in controlling the release of a variety of compounds to the targeted site. This review will provide the reader with an insight on the latest developments of prodrugs that are available for treating a variety of cardiovascular diseases. In addition, we will focus on several drug delivery methodologies that have merged with the prodrug approach to provide enhanced target specificity and controlled drug release with minimal side effects.

Asunto(s)

Enfermedades Cardiovasculares/tratamiento farmacológico , Profármacos/uso terapéutico , Sistemas de Liberación de Medicamentos , Humanos , Profármacos/administración & dosificación , Profármacos/química

Asunto(s)

Metalotioneína/fisiología , Nanopartículas/química , Nanotecnología/instrumentación , Nanotecnología/métodos , Semiconductores , Compuestos de Cadmio , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Espectrometría de Masas/métodos , Metalotioneína/química , Nanoestructuras/química , Óxido Nítrico/química , Péptidos/química , Proteínas/química , Especies Reactivas de Oxígeno , Compuestos de Selenio , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sulfuros/química , Compuestos de Zinc/química

RESUMEN

Reagentless and reversible maltose biosensors are demonstrated using ZnS coated CdSe (CdSe@ZnS) nanoparticle emission intensities. This method is based on electron transfer quenching of unimolecular protein-CdSe@ZnS nanoparticle assemblies, which is provided by a protein-attached Ru(II) complex. This Ru(II) complex is presumed to reduce a valence band hole of the CdSe@ZnS excited state by tunneling through the ZnS overcoating. The Ru(II) complex mediated quenching of CdSe@ZnS nanoparticle emission was only decreased 1.2-fold relative to the CdSe nanoparticle systems. While four different Ru(II) complex attachment sites provided different amounts of nanoparticle emission quenching (1.20 to 1.75-fold decrease), all of these attachment sites yielded maltose-dependent intensity changes (1.1 to 1.4-fold increase upon maltose addition). Maltose dissociation constants for these four biosensing systems range from 250 nM to 1.0 microM, which are similar to the maltose-maltose binding protein dissociation constant that these sensors are based on. The increased fluorescence intensity was found to only occur in the presence of maltose. Furthermore, the ability of these reagentless protein-nanoparticle assemblies to perform maltose biosensing reversibly is demonstrated with the addition of alpha-glucosidase. Three 50 microM maltose additions after alpha-glucosidase addition showed increases of 2.2 microM, 600 nM, and 150 nM maltose. This result demonstrates a fluorometric method for examining alpha-glucosidase activity. Using maltose binding protein to control Ru(II) complex interactions with CdSe@ZnS nanoparticle surfaces provide a novel class of highly fluorescent, photostable biosensors that are selective for maltose.

Asunto(s)

Técnicas Biosensibles/métodos , Maltosa/análisis , Técnicas Biosensibles/instrumentación , Proteínas Portadoras , Proteínas de Unión a Maltosa , Nanotecnología , Nanotubos , Semiconductores , Espectrometría de Fluorescencia/métodos

RESUMEN

Metalloprotein tethered CdSe nanoparticles have been generated to provide selective and reagentless maltose biosensing. As opposed to cell or protein detection by semiconducting nanoparticle bioconjugates, a modular method for small-molecule detection using semiconducting nanoparticle bioconjugates has been difficult. Here we report a method for reagentless protein-based semiconducting nanoparticle biosensors. This method uses Ru(II) complex-CdSe nanoparticle interactions and the maltose-induced conformation changes of maltose binding protein to alter the CdSe nanoparticle fluorescence emission intensity. In this proof-of-principle system, the maltose-induced protein conformation changes alter the Ru(II) complex-CdSe nanoparticle interaction, which increases the CdSe emission intensity. Altered CdSe emission intensity effects are best described as electron transfer from the Ru(II) complex to the CdSe excited state forming the nonfluorescent CdSe anion. Four surface-cysteine, Ru(II) complex-attached maltose-binding proteins have been studied for maltose dependent alteration of CdSe emission intensities. With 3.0-3.5 nm diameter CdSe nanoparticles, all ruthenated maltose-binding proteins display similar maltose-dependent increases (1.4-fold) in CdSe emission intensity and maltose binding affinities (KA = 3 x 106 M-1). For these four systems, the only difference was the sample-to-sample variation in maltose-dependent responses. Thus, very few surface cysteine mutations need to be examined to find a successful biosensor, as opposed to analogous systems using organic fluorophores. This strategy generates a unimolecular, or reagentless, semiconducting nanoparticle biosensor for maltose, which could be applied to other proteins with ligand-dependent conformation changes.

Asunto(s)

Técnicas Biosensibles , Nanoestructuras/química , Compuestos de Cadmio/química , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Maltosa/química , Maltosa/metabolismo , Proteínas de Unión a Maltosa , Tamaño de la Partícula , Reproducibilidad de los Resultados , Compuestos de Selenio/química , Espectrometría de Fluorescencia