RESUMEN

This paper presents an aptameric graphene nanosensor for rapid and sensitive measurement of arginine vasopressin (AVP) toward continuous monitoring of critical care patients. The nanosensor is a field-effect transistor (FET) with monolayer graphene as the conducting channel and is functionalized with a new custom-designed aptamer for specific AVP recognition. Binding between the aptamer and AVP induces a change in the carrier density in the graphene and resulting in measurable changes in FET characteristics for determination of the AVP concentration. The aptamer, based on the natural enantiomer D-deoxyribose, possess optimized kinetic binding properties and is attached at an internal position to the graphene for enhanced sensitivity to low concentrations of AVP. Experimental results show that this aptameric graphene nanosensor is highly sensitive (with a limit of detection of 0.3 pM and a resolution of 0.1 pM) to AVP, and rapidly responsive (within 90 s) to both increasing and decreasing AVP concentration changes. The device is also reversable (within 4%), repeatable (within 4%) and reproducible (within 5%) in AVP measurements.

RESUMEN

BACKGROUND: The dopamine transporter striatal binding ratio (DAT SBR) has been used as an outcome measure in Parkinson's disease (PD) trials of potential disease-modifying therapies; however, both patient characteristics and analysis approach potentially complicate its interpretation. OBJECTIVE: The aim was to explore how well DAT SBR reflects PD motor severity across different striatal subregions and the relationship to disease duration, and side of onset. METHODS: DAT SBR for the anterior and posterior putamen and caudate in both hemispheres was obtained using validated automated quantitative software on baseline scans of 132 patients recruited for the Exenatide PD2 and PD3 trials. Associations between mean and lateralized SBR subregions (posterior and anterior putamen and caudate) and summed and lateralized motor characteristics were explored using regression analysis. Analyses were repeated considering disease duration and limiting analysis to the less-affected hemisphere. RESULTS: Lateralized bradykinesia was most consistently associated with the loss of DAT uptake in the contralateral anterior putamen. There was much higher variance in the posterior putamen, and in all regions in those with longer duration disease, although bradykinesia remained robustly associated with anterior putaminal DAT uptake even in longer-duration patients. Restricting analyses to the less-affected side did not usefully reduce the variance compared to the overall cohort. CONCLUSION: These data suggest that DAT SBR could be a useful biomarker in disease-modifying trials, but a focus on anterior striatal subregions and incorporating disease duration into analyses may improve its utility.

RESUMEN

c-MYC is one of the most important oncogenes, which is overexpressed in many cancers, and is highly related to development, metastasis, and drug resistance of cancers. The G4 structure in the promoter of c-MYC oncogene contributes a lot to the gene transcriptional mechanism. Small-molecule ligands binding to the c-MYC G4 appear to be a new class of anticancer agents. However, selective ligands for the c-MYC G4 over other G4s have been rarely reported. In this study, we reported a novel fluorescent ligand by migrating the benzene group on a carbazole-benzothiazolium scaffold, which was demonstrated to exhibit considerable specificity to the c-MYC G4, which was distinguished from other small-molecule ligands. The further cellular experiments suggested that this ligand may indeed target the promoter G4 and cause apparent transcriptional inhibition of the c-MYC oncogene instead of other G4-mediated oncogenes, which thereby resulted in cancer cell growth inhibition. Collectively, this study provided a good example for developing specific c-MYC G4 ligands, which may further develop into an effective anticancer agent that inhibit the c-MYC expression.

Asunto(s)

Antineoplásicos , Benzotiazoles , Carbazoles , Proliferación Celular , Colorantes Fluorescentes , G-Cuádruplex , Proteínas Proto-Oncogénicas c-myc , Carbazoles/química , Carbazoles/farmacología , G-Cuádruplex/efectos de los fármacos , Humanos , Ligandos , Benzotiazoles/química , Benzotiazoles/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacología , Estructura Molecular , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Proliferación Celular/efectos de los fármacos , Relación Estructura-Actividad , Ensayos de Selección de Medicamentos Antitumorales , Relación Dosis-Respuesta a Droga , Benceno/química , Benceno/farmacología , Línea Celular Tumoral

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The post-genomic era has ushered in the extensive application of epigenetic editing tools, allowing for precise alterations of gene expression. The use of reprogrammable editors that carry transcriptional corepressors has significant potential for long-term epigenetic silencing for the treatment of human diseases. The ideal scenario involves precise targeting of a specific genomic location by a DNA-binding domain, ensuring there are no off-target effects and that the process yields no genetic remnants aside from specific epigenetic modifications (i.e., DNA methylation). A notable example is a recent study on the mouse Pcsk9 gene, crucial for cholesterol regulation and expressed in hepatocytes, which identified synthetic zinc-finger (ZF) proteins as the most effective DNA-binding editors for silencing Pcsk9 efficiently, specifically, and persistently. This discussion focuses on enhancing the specificity of ZF-array DNA binding by optimizing interactions between specific amino acids and DNA bases across three promoters containing CpG islands.

RESUMEN

In clinical practice, the determination of unbound drug concentration is very important for dose adjustment and toxicity prediction because only the unbound fraction can achieve a pharmacological effect. A fast, sensitive and accurate analytical method of centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method was developed and applied to allow the quantification of unbound lenvatinib concentration. The application of linear regression analysis was used to examine the effects of centrifugal force, centrifugal time, and protein content on ultrafiltrate volume (Vu). The results indicated that the centrifugal force and centrifugal time have an influence on Vu that is significantly positive (P < 0.05). This developed method with good linearity (r2 = 0.9996), good accuracy (bias % ≤ 2.24 %), good precision (CV % ≤ 7.10 %), and good recovery (95.46 %-106.46 %) was suitable for routine clinical practice and studies. Particularly, the ultrafiltration membrane had no non-specific binding to lenvatinib. The unbound fractions can be separated in just 15 min. This method was applied to quantify clinical samples and to determine the plasma protein binding and unbound fraction of lenvatinib. This study provides a more effective and promising method for determination of unbound lenvatinib. It could be beneficial to measure the unbound concentration of lenvatinib in personalized medicine and therapeutic drug monitoring in routine clinical practice.

Asunto(s)

Compuestos de Fenilurea , Quinolinas , Espectrometría de Masas en Tándem , Ultrafiltración , Humanos , Compuestos de Fenilurea/sangre , Compuestos de Fenilurea/farmacocinética , Compuestos de Fenilurea/química , Compuestos de Fenilurea/análisis , Quinolinas/sangre , Quinolinas/química , Quinolinas/farmacocinética , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión/métodos , Ultrafiltración/métodos , Modelos Lineales , Reproducibilidad de los Resultados , Unión Proteica , Límite de Detección

RESUMEN

The immobilization of proteins on the surface of carriers is challenging due to the loss of protein structure and function in this process. Here, we report the development of the protein immobilization on the surface of the metallated-porphyrin complex in the porphysome nanocarrier. The conjugated Ni-porphyrin to fatty acid (as a tail) has been synthesized and independently placed at the depth of the bilayer center of Dipalmitoylphosphatidylcholine (DPPC) in which the Ni-porphyrin was at the polar region of the membrane and is thus superficial. This porphysome (DPPC: Ni-porphyrin, 4 : 1 mole ratio) was formed by supramolecular self-assembly with a diameter of 173±7 nm and zeta potential -8.5±3.4 mv, which exhibited no significant toxicity at the experimental concentrations and acceptable cellular uptake on MCF-7 cells. The physicochemical properties and specific protein binding sites of the firefly luciferase as a model protein into the porphysome (1 : 2 mole ratio) show the conjugation efficiency about 80 % and the conformation of protein was completely maintained. Furthermore, bioluminescence assay and SDS-PAGE confirmed the preservation of protein function. The stabilized platform of porphyrin-lipid structure can potentially improve the efficacy of protein functionality for a particular display, shifting porphysomes from a simple carrier to a therapeutic agent.

Asunto(s)

Porfirinas , Humanos , Sitios de Unión/efectos de los fármacos , Porfirinas/química , Porfirinas/farmacología , Células MCF-7 , Portadores de Fármacos/química , Nanomedicina Teranóstica , Nanopartículas/química , Supervivencia Celular/efectos de los fármacos , 1,2-Dipalmitoilfosfatidilcolina/química , Sistemas de Liberación de Medicamentos , Tamaño de la Partícula

RESUMEN

In the detection of biomolecules, surface plasmon resonance (SPR) sensors require high sensitivity. In this study, we propose a sensitivity-enhanced functionalized plasmonic interface based on Ag-TiO2-Co(OH)2 nanosheets structure. Compared to unmodified SPR sensors, the sensitivity of the sensor decorated with TiO2 and Co(OH)2 nanosheets is increased by 130.84%, reaching 5764.27 nm/RIU. This enhancement is attributed to the high refractive index of the coating, as well as the high specific surface area and abundant active sites provided by the synthesized Co(OH)2 nanosheets with a multi-grooved structure. Additionally, employing a double-antibody sandwich method, the antibody-functionalized plasmonic interface enables specific detection of human serum albumin (HSA). The linear response of this sensor was in the wide range of 0.4-150 µM, and the LOD reached 154.89 nM(KD is approximately 1.73 × 10-6 M). This novel SPR sensor offers a new strategy for biochemical sensing and provides a highly sensitive platform for immunoassays.

Asunto(s)

Técnicas Biosensibles , Humanos , Resonancia por Plasmón de Superficie , Refractometría , Anticuerpos , Alimentos

RESUMEN

Immunoassays (IAs) with fluorescence-based detection are already well-established commercialized biosensing methods, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA). Immunoassays with surface-enhanced Raman spectroscopy (SERS) detection have received significant attention from the research community for at least two decades, but so far they still lack a wide clinical commercial application. This review, unlike any other review that we have seen, performs a three-dimensional performance comparison of SERS IAs vs. fluorescence IAs. First, we compared the limit of detection (LOD) as a key performance parameter for 30 fluorescence and 30 SERS-based immunoassays reported in the literature. We also compared the clinical performances of a smaller number of available reports for SERS vs. fluorescence immunoassays (FIAs). We found that the median and geometric average LODs are about 1.5-2 orders of magnitude lower for SERS-based immunoassays in comparison to fluorescence-based immunoassays. For instance, the median LOD for SERS IA is 4.3 × 10-13 M, whereas for FIA, it is 1.5 × 10-11 M. However, there is no significant difference in average relative standard deviation (RSD)-both are about 5-6%. The analysis of sensitivity, selectivity, and accuracy reported for a limited number of the published clinical studies with SERS IA and FIA demonstrates an advantage of SERS IA over FIA, at least in terms of the median value for all three of those parameters. We discussed common and specific challenges to the performances of both SERS IA and FIA, while proposing some solutions to mitigate those challenges for both techniques. These challenges include non-specific protein binding, non-specific interactions in the immunoassays, sometimes insufficient reproducibility, relatively long assay times, photobleaching, etc. Overall, this review may be useful for a large number of researchers who would like to use immunoassays, but particularly for those who would like to make improvements and move forward in both SERS-based IAs and fluorescence-based IAs.

Asunto(s)

Nanopartículas del Metal , Espectrometría Raman , Reproducibilidad de los Resultados , Espectrometría Raman/métodos , Inmunoensayo/métodos , Colorantes , Ensayo de Inmunoadsorción Enzimática , Oro/química , Nanopartículas del Metal/química

RESUMEN

AIMS/HYPOTHESIS: Compromised pancreatic sympathetic innervation has been suggested as a factor involved in both immune-mediated beta cell destruction and endocrine dysregulation of pancreatic islets. To further explore these intriguing findings, new techniques for in vivo assessment of pancreatic innervation are required. This is a retrospective study that aimed to investigate whether the noradrenaline (norepinephrine) analogue 11C-hydroxy ephedrine (11C-HED) could be used for quantitative positron emission tomography (PET) imaging of the sympathetic innervation of the human pancreas. METHODS: In 25 individuals with type 2 diabetes and 64 individuals without diabetes, all of whom had previously undergone 11C-HED-PET/CT because of pheochromocytoma or paraganglioma (or suspicion thereof), the 11C-HED standardised uptake value (SUVmean), 11C-HED specific binding index (SBI), pancreatic functional volume (FV, in ml), functional neuronal volume (FNV, calculated as SUVmean × FV), specific binding index with functional volume (SBI FV, calculated as SBI × FV) and attenuation on CT (HU) were investigated in the entire pancreas, and additionally in six separate anatomical pancreatic regions. RESULTS: Generally, 11C-HED uptake in the pancreas was high, with marked individual variation, suggesting variability in sympathetic innervation. Moreover, pancreatic CT attenuation (HU) (p<0.001), 11C-HED SBI (p=0.0049) and SBI FV (p=0.0142) were lower in individuals with type 2 diabetes than in individuals without diabetes, whereas 11C-HED SUVmean (p=0.15), FV (p=0.73) and FNV (p=0.30) were similar. CONCLUSIONS/INTERPRETATION: We demonstrate the feasibility of using 11C-HED-PET for non-invasive assessment of pancreatic sympathetic innervation in humans. These findings warrant further prospective evaluation, especially in individuals with theoretical defects in pancreatic sympathetic innervation, such as those with type 1 diabetes.

Asunto(s)

Diabetes Mellitus Tipo 2 , Humanos , Estudios Retrospectivos , Tomografía Computarizada por Tomografía de Emisión de Positrones , Sistema Nervioso Simpático , Tomografía de Emisión de Positrones/métodos , Páncreas/diagnóstico por imagen , Efedrina , Corazón

RESUMEN

BACKGROUND: Allele-specific binding (ASB) events occur when transcription factors (TFs) bind more favorably to one of the two parental alleles at heterozygous single nucleotide polymorphisms (SNPs). Evidence suggests that ASB events could reveal the impact of sequence variations on TF binding and may have implications for the risk of diseases. RESULTS: Here we present ASB-analyzer, a software platform that enables the users to quickly and efficiently input raw sequencing data to generate individual reports containing the cytogenetic map of ASB SNPs and their associated phenotypes. This interactive tool thereby combines ASB SNP identification, biological annotation, motif analysis, phenotype associations and report summary in one pipeline. With this pipeline, we identified 3772 ASB SNPs from thirty GM12878 ChIP-seq datasets and demonstrated that the ASB SNPs were more likely to be enriched at important sites in TF-binding domains. CONCLUSIONS: ASB-analyzer is a user-friendly tool that enables the detection, characterization and visualization of ASB SNPs. It is implemented in Python, R and bash shell and packaged in the Conda environment. It is available as an open-source tool on GitHub at https://github.com/Liying1996/ASBanalyzer .

Asunto(s)

Polimorfismo de Nucleótido Simple , Factores de Transcripción , Alelos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Programas Informáticos , Unión Proteica , Sitios de Unión

RESUMEN

BACKGROUND: Intravitreal injection (IVI) of antibody biologics is a key treatment approach in ophthalmology. Pharmaceutical compounding and storage of prefilled syringes for IVI must take place without impairing the structure and function of the biologics. This study investigated the effect of withdrawing and storing the therapeutic antibody faricimab (Vabysmo, Roche, Basel, Switzerland) in the Zero Residual silicone oil-free, 0.2-mL syringe (SJJ Solutions, The Hague, the Netherlands). METHODS: To assess the effect of syringe withdrawal on faricimab, we compared samples from syringes prepared at day 0 with samples taken directly from faricimab vials. To assess the effect of syringe storage on faricimab, we kept prefilled syringes in the dark at 4 oC for 7, 14, or 37 days and compared samples from these syringes with day 0. We measured protein concentration (with spectrophotometry), stability and integrity (with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), size-exclusion chromatography (SEC), and melting temperature (Tm)), as well as binding of faricimab to its cognate antigens: vascular endothelial growth factor A (VEGF-A) and angiopoietin-2 (Ang-2) (with enzyme-linked immunosorbent assay (ELISA)). RESULTS: Faricimab migrated in line with its expected molecular mass under both reducing and non-reducing conditions for all time points when analyzed with SDS-PAGE, without any sign of degradation products or aggregation. The SEC elution profiles were identical for all time points. There were slight variations in Tm for different time points compared to day 0 but without consistent relationship with storage time. ELISA did not detect differences in VEGF-A or Ang-2 binding between time points, and faricimab did not bind the neonatal Fc receptor. CONCLUSIONS: Withdrawal and storage of faricimab in syringes for up to day 37 did not impair the structure and bi-specific binding properties of the therapeutic antibody.

RESUMEN

AIM: [123I]Ioflupane (DaTSCAN) has a high binding affinity to the dopamine (DA) transporter (DaT) and tenfold less affinity to serotonin (5-HT) transporter (SERT). Both neurotransmitters are considered to contribute to body weight regulation. This study assesses the association between body mass index (BMI) and DaTSCAN availability in brain. METHOD: Scans from 74 consecutive patients who had undergone DaTSCAN single-photon emission computed tomography-computed tomography (SPECT-CT) were used to obtain semi- and absolute quantitative data in several volumes of interest (VOIs). Relative semi-quantitative specific binding ratios (SBRs) from Chang attenuated SPECT were obtained from GE DaTQUANT. Absolute normalised concentration (NC) was calculated from attenuation/scatter corrected SPECT-CT images, using an adapted version of the EARL Ltd (European Association of Nuclear Medicine (EANM) Research 4 Life) template. Scans were subdivided into either degenerative parkinsonism (abnormal = 49), borderline (n = 14) or scan without evidence of dopaminergic deficit (SWEDD = 11) using visual assessment and SBR values by two nuclear medicine consultants. RESULTS: SBRs did not correlate with BMI. However, NC values correlated negatively in the entire cohort, with the strongest correlation in the frontal (r = - 0.649. p = 0.000), occipital (r = - 0.555, p = 0.000) regions and pons (r = - 0.555, p = 0.000). In the abnormal (n = 49) and SWEDD group (n = 11), NC of the frontal region was the most correlated with BMI (r = - 0.570, p = 0.000; r = - 0.813, p = 0.002, respectively). In the borderline group (n = 14), the left posterior putamen displayed the strongest correlation (r = - 0.765, p = 0.001). CONCLUSION: Absolute NC values demonstrate a strong inverse correlation with BMI, strongest in the extrastriatal regions. Due to the predominately non-overlapping distribution of DaT and SERT, this study suggests greater involvement of SERT in obesity with possible interplay with DA transmission.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Quinolinas , Barrera Hematoencefálica/metabolismo

RESUMEN

A cost-effective approach has been developed to synthesize Cu nanoparticles encapsulated into B and N double-doped carbon nanotubes (Cu@BCNNTs) by one-step pyrolysis. According to the specific binding of Cu-Cl and Cu-glutathione (GSH), we employed Cu@BCNNTs to build an electrochemical sensing platform to detect GSH. The unique space-confined structure can prevent Cu nanoparticles from agglomeration. In addition, B and N co-doped porous hollow tubes can improve the electrochemical conductivity, expand the number of active sites, enhance surface adsorption, and shorten the transport path. These favorable characteristics of Cu@BCNNTs make them have excellent electrocatalytic properties. These results display that the prepared sensor can detect GSH from 0.5 to 120 µM with a detection limit of 0.024 µM. The obtained sensors can be successfully applied in the human serum with recovery of GSH ranging from 100.2 to 103.9%. This work provides a new vision to synthesize nanoparticles confined in a hollow tube for the applications in biosensing and medical diagnostics.

Asunto(s)

Técnicas Biosensibles , Nanopartículas , Nanotubos de Carbono , Humanos , Nanotubos de Carbono/química , Porosidad , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Electrodos , Nanopartículas/química , Glutatión , Nanotecnología

RESUMEN

This study aimed to develop a new convolutional neural network (CNN) method for estimating the specific binding ratio (SBR) from only frontal projection images in single-photon emission-computed tomography using [123I]ioflupane. We created five datasets to train two CNNs, LeNet and AlexNet: (1) 128FOV used a 0° projection image without preprocessing, (2) 40FOV used 0° projection images cropped to 40 × 40 pixels centered on the striatum, (3) 40FOV training data doubled by data augmentation (40FOV_DA, left-right reversal only), (4) 40FOVhalf, and (5) 40FOV_DAhalf, split into left and right (20 × 40) images of 40FOV and 40FOV_DA to separately evaluate the left and right SBR. The accuracy of the SBR estimation was assessed using the mean absolute error, root mean squared error, correlation coefficient, and slope. The 128FOV dataset had significantly larger absolute errors compared to all other datasets (p < 0. 05). The best correlation coefficient between the SBRs using SPECT images and those estimated from frontal projection images alone was 0.87. Clinical use of the new CNN method in this study was feasible for estimating the SBR with a small error rate using only the frontal projection images collected in a short time.

RESUMEN

The confidence in fraction unbound (ƒu) using equilibrium dialysis (ED) is often questioned (e.g., highly bound, labile compounds) due to uncertainty in whether true equilibrium is achieved. Different methods have been developed to increase confidence in ƒu measurements, such as the presaturation, dilution, and bi-directional ED methods. However, confidence in ƒu measurement can still suffer due to non-specific binding and inter-run variations introduced during equilibrium and analysis. To address this concern, we introduce an orthogonal approach called counter equilibrium dialysis (CED) in which non-labeled and isotope-labeled compounds are dosed counter-directionally in rapid equilibrium dialysis (RED). ƒu values of both non-labeled and labeled compounds are measured simultaneously in the same run. These tactics not only minimize non-specific binding and inter-run variability but also enable the confirmation of true equilibrium. If equilibrium is reached in both dialysis directions, the ƒu for the non-labeled compound and the labeled compound will converge. The refined methodology was extensively tested with various compounds of diverse physicochemical properties and plasma binding characteristics. Our results demonstrated that, by using the CED method, ƒu values for a wide range of compounds could be accurately determined with significantly improved confidence, including the challenging highly bound and labile compounds.

Asunto(s)

Proteínas Sanguíneas , Diálisis Renal , Proteínas Sanguíneas/metabolismo , Unión Proteica , Plasma/metabolismo , Diálisis/métodos

RESUMEN

HYPOTHESIS: Modification of polyallylamine hydrochloride (PAH) with heterobifunctional low molecular weight polyethylene glycol (PEG) (600 and 1395 Da), and subsequent attachment of mannose, glucose, or lactose sugars to PEG, can lead to formation of polyamine phosphate nanoparticles (PANs) with lectin binding affinity and narrow size distribution. EXPERIMENTS: Size, polydispersity, and internal structure of glycosylated PEGylated PANs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). Fluorescence correlation spectroscopy (FCS) was used to study the association of labelled glycol-PEGylated PANs. The number of polymer chains forming the nanoparticles was determined from the changes in amplitude of the cross-correlation function of the polymers after formation of the nanoparticles. SAXS and fluorescence cross-correlation spectroscopy were used to investigate the interaction of PANs with lectins: concanavalin A with mannose modified PANs, and jacalin with lactose modified ones. FINDINGS: Glyco-PEGylated PANs are highly monodispersed, with diameters of a few tens of nanometers and low charge, and a structure corresponding to spheres with Gaussian chains. FCS shows that the PANs are single chain nanoparticles or formed by two polymer chains. Concanavalin A and jacalin show specific interactions for the glyco-PEGylated PANs with higher affinity than bovine serum albumin.

Asunto(s)

Nanopartículas , Fosfatos , Concanavalina A , Lactosa , Manosa , Dispersión del Ángulo Pequeño , Rayos X , Polietilenglicoles/química , Difracción de Rayos X , Nanopartículas/química , Poliaminas , Lectinas/química , Polímeros , Análisis Espectral

RESUMEN

The functional properties of G protein-coupled receptors (GPCRs) are intimately associated with the different components in their cellular environment. Among them, sodium ions have been proposed to play a substantial role as endogenous allosteric modulators of GPCR-mediated signaling. However, this sodium effect and the underlying mechanisms are still unclear for most GPCRs. Here, we identified sodium as a negative allosteric modulator of the ghrelin receptor GHSR (growth hormone secretagogue receptor). Combining 23Na-nuclear magnetic resonance (NMR), molecular dynamics, and mutagenesis, we provide evidence that, in GHSR, sodium binds to the allosteric site conserved in class A GPCRs. We further leveraged spectroscopic and functional assays to show that sodium binding shifts the conformational equilibrium toward the GHSR-inactive ensemble, thereby decreasing basal and agonist-induced receptor-catalyzed G protein activation. All together, these data point to sodium as an allosteric modulator of GHSR, making this ion an integral component of the ghrelin signaling machinery.

Asunto(s)

Receptores de Ghrelina , Sodio , Regulación Alostérica , Sitio Alostérico , Ghrelina/metabolismo , Iones , Receptores de Ghrelina/metabolismo , Transducción de Señal , Sodio/metabolismo

RESUMEN

BACKGROUND: The specific binding ratio (SBR) of 123I-FP-CIT in the putamen is widely used to support the interpretation of dopamine transporter (DAT) SPECT. Automatic methods for computation of the putamen SBR often include stereotactical normalization of the individual DAT-SPECT image to an anatomical standard space. This study compared using a single 123I-FP-CIT template image as target for stereotactical normalization versus multiple templates representative of normal and different levels of Parkinson-typical reduction of striatal 123I-FP-CIT uptake. METHODS: 1702 clinical 123I-FP-CIT SPECT images were stereotactically normalized (affine) to the anatomical space of the Montreal Neurological Institute (MNI) with SPM12 either using a single custom-made 123I-FP-CIT template representative of normal striatal uptake or using eight different templates representative of normal and different levels of Parkinson-typical reduction of striatal FP-CIT uptake with and without attenuation and scatter correction. In the latter case, SPM finds the linear combination of the multiple templates that best matches the patient's image. The putamen SBR was obtained using hottest voxels analysis in large unilateral regions-of-interest predefined in MNI space. The histogram of the putamen SBR in the whole sample was fitted by the sum of two Gaussians. The power to differentiate between reduced and normal SBR was estimated by the effect size of the distance between the two Gaussians computed as the differences between their mean values scaled to their pooled standard deviation. RESULTS: The effect size of the distance between the two Gaussians was 3.83 with the single template versus 3.96 with multiple templates for stereotactical normalization. CONCLUSIONS: Multiple templates representative of normal and different levels of Parkinson-typical reduction for stereotactical normalization of DAT-SPECT might provide improved separation between normal and reduced putamen SBR that could result in slightly improved power for the detection of nigrostriatal degeneration.

RESUMEN

Human norovirus (HuNoV) is the leading foodborne pathogen causing nonbacterial gastroenteritis worldwide. The oyster is an important vehicle for HuNoV transmission, especially the GI.1 HuNoV. In our previous study, oyster heat shock protein 70 (oHSP 70) was identified as the first proteinaceous ligand of GII.4 HuNoV in Pacific oysters besides the commonly accepted carbohydrate ligands, a histo-blood group antigens (HBGAs)-like substance. However the mismatch of the distribution pattern between discovered ligands and GI.1 HuNoV suggests that other ligands may exist. In our study, proteinaceous ligands for the specific binding of GI.1 HuNoV were mined from oyster tissues using a bacterial cell surface display system. Fifty-five candidate ligands were identified and selected through mass spectrometry identification and bioinformatics analysis. Among them, the oyster tumor necrosis factor (oTNF) and oyster intraflagellar transport protein (oIFT) showed strong binding abilities with the P protein of GI.1 HuNoV. In addition, the highest mRNA level of these two proteins was found in the digestive glands, which is consistent with GI.1 HuNoV distribution. Overall the findings suggested that oTNF and oIFT may play important roles in the bioaccumulation of GI.1 HuNoV.

Asunto(s)

Antígenos de Grupos Sanguíneos , Norovirus , Ostreidae , Animales , Humanos , Ligandos , Norovirus/genética , Norovirus/metabolismo , Carbohidratos , Antígenos de Grupos Sanguíneos/metabolismo