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Mutations of the human TRAFFICKING PROTEIN PARTICLE COMPLEX SUBUNIT 9 (TRAPPC9) cause a neurodevelopmental disorder characterised by microcephaly and intellectual disability. Trappc9 constitutes a subunit specific to the intracellular membrane-associated TrappII complex. The TrappII complex interacts with Rab11 and Rab18, the latter being specifically associated with lipid droplets (LDs). Here we used non-invasive imaging to characterise Trappc9 knock-out (KO) mice as a model of the human hereditary disorder. KOs developed postnatal microcephaly with many grey and white matter regions being affected. In vivo magnetic resonance imaging (MRI) identified a disproportionately stronger volume reduction in the hippocampus, which was associated with a significant loss of Sox2-positive neural stem and progenitor cells. Diffusion tensor imaging indicated a reduced organisation or integrity of white matter areas. Trappc9 KOs displayed behavioural abnormalities in several tests related to exploration, learning and memory. Trappc9-deficient primary hippocampal neurons accumulated a larger LD volume per cell following Oleic Acid stimulation, and the coating of LDs by Perilipin-2 was much reduced. Additionally, Trappc9 KOs developed obesity, which was significantly more severe in females than in males. Our findings indicate that, beyond previously reported Rab11-related vesicle transport defects, dysfunctions in LD homeostasis might contribute to the neurobiological symptoms of Trappc9 deficiency.
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Microcefalia , Animales , Femenino , Humanos , Masculino , Ratones , Imagen de Difusión Tensora , Gotas Lipídicas , Ratones Noqueados , Microcefalia/genética , Microcefalia/metabolismo , Neuronas/metabolismoRESUMEN
Changes in glioblastoma (GBM) metabolism was investigated in response to JAS239, a choline kinase inhibitor, using MRS. In addition to the inhibition of phosphocholine synthesis, we investigated changes in other key metabolic pathways associated with GBM progression and treatment response. Three syngeneic rodent models of GBM were used: F98 (N = 12) and 9L (N = 8) models in rats and GL261 (N = 10) in mice. Rodents were intracranially injected with GBM cells in the right cortex and tumor growth was monitored using T2 -weighted images. Animals were treated once daily with intraperitoneal injections of 4 mg/kg JAS239 (F98 rats, n = 6; 9L rats, n = 6; GL261 mice, n = 5) or saline (control group, F98 rats, n = 6; 9L rats, n = 2; GL261 mice, n = 5) for five consecutive days. Single voxel spectra were acquired on Days 0 (T0, baseline) and 6 (T6, end of treatment) from the tumor as well as the contralateral normal brain using a PRESS sequence. Changes in metabolite ratios (tCho/tCr, tCho/NAA, mI/tCr, Glx/tCr and (Lip + Lac)/Cr) were used to assess metabolic pathway alterations in response to JAS239. Tumor growth arrest was noted in all models in response to JAS239 treatment compared with saline-treated animals, with a significant reduction (p < 0.05) in the F98 model. A reduction in tCho/tCr was observed with JAS239 treatment in all GBM models, indicating reduced phospholipid metabolism, with the highest reduction in 9L followed by GL261 and F98 tumors. A significant reduction (p < 0.05) in the tCho/NAA ratio was observed in the 9L model. A significant reduction in mI/tCr (p < 0.05) was found in JAS239-treated F98 tumors compared with the saline-treated animals. A non-significant trend of reduction in Glx/tCr was observed only in F98 and 9L tumors. JAS239-treated F98 tumors also showed a significant increase in Lip + Lac (p < 0.05), indicating increased cell death. This study demonstrated the utility of MRS in assessing metabolic changes in GBM in response to choline kinase inhibition.
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Neoplasias Encefálicas , Glioblastoma , Ratas , Ratones , Animales , Glioblastoma/diagnóstico por imagen , Glioblastoma/tratamiento farmacológico , Roedores/metabolismo , Colina Quinasa , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Receptores de Antígenos de Linfocitos T , Colina/metabolismoRESUMEN
The immunomodulatory properties of MSCs can be recreated using their extracellular vesicles (EVs). Yet, the true capabilities of the MSC EVs cannot be distinguished from contaminating bovine EVs and protein derived from supplemental foetal bovine serum (FBS). FBS EV depletion protocols can minimise this, but vary in terms of depletion efficiency, which can negatively impact the cell phenotype. We explore the impact of FBS EV depletion strategies, including ultracentrifugation, ultrafiltration, and serum-free, on umbilical cord MSC characteristics. Whilst a greater depletion efficiency, seen in the ultrafiltration and serum-free strategies, did not impact the MSC markers or viability, the MSCs did become more fibroblastic, had slower proliferation, and showed inferior immunomodulatory capabilities. Upon MSC EV enrichment, more particles, with a greater particle/protein ratio, were isolated upon increasing the FBS depletion efficiency, except for serum-free, which showed a decreased particle number. Whilst all conditions showed the presence of EV-associated markers (CD9, CD63, and CD81), serum-free was shown to represent a higher proportion of these markers when normalised by total protein. Thus, we caution MSC EV researchers on the use of highly efficient EV depletion protocols, showing that it can impact the MSC phenotype, including their immunomodulatory properties, and stress the importance of testing in consideration to downstream objectives.
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Vesículas Extracelulares , Células Madre Mesenquimatosas , Albúmina Sérica Bovina/metabolismo , Cordón Umbilical , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , InmunomodulaciónRESUMEN
Resistive pulse sensors have been used to characterise everything from whole cells to small molecules. Their integration into microfluidic devices has simplified sample handling whilst increasing throughput. Typically, these devices measure a limited size range, making them prone to blockages in complex sample matrixes. To prolong their life and facilitate their use, samples are often filtered or prepared to match the sample with the sensor diameter. Here, we advance our tuneable flow resistive pulse sensor which utilises additively manufactured parts. The sensor allows parts to be easily changed, washed and cleaned, its simplicity and versatility allow components from existing nanopore fabrication techniques such as glass pipettes to be integrated into a single device. This creates a multi-nanopore sensor that can simultaneously measure particles from 0.1 to 30 µm in diameter. The orientation and controlled fluid flow in the device allow the sensors to be placed in series, whereby smaller particles can be measured in the presence of larger ones without the risk of being blocked. We illustrate the concept of a multi-pore flow resistive pulse sensor, by combining an additively manufactured tuneable sensor, termed sensor 1, with a fixed nanopore sensor, termed sensor 2. Sensor 1 measures particles as small as 10 µm in diameter, whilst sensor 2 can be used to characterise particles as small as 100 nm, depending upon its dimensions. We illustrate the dual pore sensor by measuring 1 and 10 µm particles simultaneously.
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Técnicas Analíticas Microfluídicas , Nanoporos , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , Microfluídica , Tamaño de la PartículaRESUMEN
Prion diseases are a group of fatal transmissible neurological conditions caused by the change in conformation of intrinsic cellular prion protein (PrPC). We present a rapid assay using aptamers and resistive pulse sensing, RPS, to extract and quantify PrPC from complex sample matrices. We functionalise the surface of superparamagnetic beads, SPBs, with a DNA aptamer. First SPB's termed P-beads, are used to pre-concentrate the analyte from a large sample volume. The PrPC protein is then eluted from the P-beads before aptamer modified sensing beads, S-beads, are added. The velocity of the S-beads through the nanopore reveals the concentration of the PrPC protein. The process is done in under an hour and allows the detection of picomol's of protein.
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Técnicas Biosensibles/métodos , Proteínas Priónicas/análisis , Proteínas Recombinantes/análisis , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Humanos , Magnetismo , Nanoporos , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
DNAzymes are DNA oligonucleotides that can undergo a specific chemical reaction in the presence of a cofactor. Ribonucleases are a specific form of DNAzymes where a tertiary structure undergoes cleavage at a single ribonuclease site. The cleavage is highly specificity to co-factors, which makes them excellent sensor recognition elements. Monitoring the change in structure upon cleavage has given rise to many sensing strategies; here we present a simple and rapid method of following the reaction using resistive pulse sensors, RPS. To demonstrate this methodology, we present a sensor for Ca2+ ions in solution. A nanoparticle was functionalised with a Ca2+ DNAzyme, and it was possible to follow the cleavage and rearrangement of the DNA as the particles translocate the RPS. The binding of Ca2+ caused a conformation change in the DNAzyme, which was monitored as a change in translocation speed. A 30 min assay produced a linear response for Ca2+ between 1-9 µm, and extending the incubation time to 60 min allowed for a concentration as low as 0.3 µm. We demonstrate that the signal is specific to Ca2+ in the presence of other metal ions, and we can quantify Ca2+ in tap and pond water samples.
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Técnicas Biosensibles , Calcio/análisis , ADN Catalítico , ADN , Iones , Metales , OligonucleótidosRESUMEN
The use of nanocarriers within resistive pulse sensing facilitates the detection and quantification of analytes. To date the field has been dominated by polyionic carriers or nanomaterials. Together they combine the recognition elements of a ligand with a stable support, facilitating the sample handling, analysis times, and multiplex detection. Here we develop the use of peptide-functionalized superparamagnetic nanocarriers to extract and quantify metal ions in solution. The interaction between nickel and the peptide ligand is measured as a change in translocation velocity of the carrier. The magnitude of change is proportional to the concentration of the metal ions in solution. Unlike DNA aptamers where a change in the tertiary structure and the folding of the polyanionic backbone influences the carrier velocity, the peptides here had a lower net charge under the assay conditions. To try and enhance the signal we engineered charged groups within the peptide to explore the effects on the signal. In all cases the metal ion binding dominated the velocity of the carrier. The assay was shown to work across 3 orders of magnitude and can detect Ni2+ in the presence of some other heavy metal ions. We demonstrate this by quantifying Ni2+ in both tap and pond water. The work allows for future multiplexed sensing strategies using both peptides and DNA aptamers in resistive pulse sensors.
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Nanopartículas de Magnetita/química , Níquel/análisis , Péptidos/química , Aptámeros de Nucleótidos/química , LigandosRESUMEN
Resistive pulse sensors (RPSs) provide detailed characterization of materials from the nanoparticle up to large biological cells on a particle-to-particle basis. During the RPS experiment, particles pass through a channel or pore that conducts ions, and the change in the ionic current versus time is monitored. The change in current during each translocation, also known as a "pulse", is dependent on the ratio of the particle and channel dimensions. Here we present a facile and rapid method for producing flow-RPSs that do not require lithographic processes. The additively manufactured sensor has channel dimensions that can be easily controlled. In addition, the fabrication process allows the sensor to be quickly assembled, disassembled, cleaned, and reused. Furthermore, the RPS can be created with a direct interface for fluidic pumps or imaging window for complementary optical microscopy. We present experiments and simulations of the RPS, showing how the pulse shapes are dependent on the channel morphology and how the device can count and size particles across a range of flow rates and ionic strengths. The use of pressure-driven fluid flow through the device allowed a rapid characterization of particles down to concentrations as low as 1 × 10-3 particles per mL, which equated to one event per second.
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Técnicas Analíticas Microfluídicas , Nanopartículas/química , Técnicas Analíticas Microfluídicas/instrumentación , Concentración Osmolar , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation in order to acquire the ability to fertilize. Although the increase in phosphorylation associated with mouse sperm capacitation is well established, the identity of the proteins involved in this signaling cascade remains largely unknown. Tandem mass spectrometry (MS/MS) has been used to identify the exact sites of phosphorylation and to compare the relative extent of phosphorylation at these sites. In the present work, we find that a novel site of phosphorylation on a peptide derived from the radial spoke protein Rsph6a is more phosphorylated in capacitated mouse sperm. The Rsph6a gene has six exons, five of which are conserved during evolution in flagellated cells. The exon containing the capacitation-induced phosphorylation site was found exclusively in eutherian mammals. Transcript analyses revealed at least two different testis-specific splicing variants for Rsph6a.Rsph6a mRNA expression was restricted to spermatocytes. Using antibodies generated against the Rsph6a N-terminal domain, western blotting and immunofluorescence analyses indicated that the protein remains in mature sperm and localizes to the sperm flagellum. Consistent with its role in the axoneme, solubility analyses revealed that Rsph6 is attached to cytoskeletal structures. Based on previous studies in Chlamydomonas reinhardtii, we predict that Rsph6 participates in the interaction between the central pair of microtubules and the surrounding pairs. The findings that Rsph6a is more phosphorylated during capacitation and is predicted to function in axonemal localization make Rsph6a a candidate protein mediating signaling processes in the sperm flagellum.
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Proteínas del Citoesqueleto/metabolismo , Capacitación Espermática/fisiología , Testículo/metabolismo , Animales , Anticuerpos , Clonación Molecular , Proteínas del Citoesqueleto/genética , Regulación de la Expresión Génica/fisiología , Masculino , Ratones , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas RecombinantesRESUMEN
Raman spectroscopy was applied to the measurement of urinary and in vitro endothelium-derived extracellular vesicles (EVs) isolated by hydrostatic filtration dialysis (HFD) method. Raman spectra obtained for urinary EVs (UEVs) showed distinct differences in the fingerprint region. In contrast, average Raman spectra of endothelium-derived EVs samples were almost identical. Cluster Analysis of UEVs significantly discriminated diabetic samples from control, moreover endothelium-derived EVs revealed stronger similarity between long hyperglycemia and normoglycemia samples compared to short hyperglycemia. Results obtained from Partial Least Squares analysis corresponded well with integral intensities of selected bands. Our proof-of-concept approach demonstrates the potential for Raman spectroscopy to be used both for identification of EVs molecular signatures in urine samples from patients with type 2 diabetes mellitus and good glycemic control and unsatisfactory glycemic control as well as for in vitro hyperglycemic model. This noninvasive technique may be useful in identifying new biomarkers of diabetes and renal complications.
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Diabetes Mellitus Tipo 2/diagnóstico , Células Endoteliales/patología , Vesículas Extracelulares/patología , Hiperglucemia/diagnóstico , Diabetes Mellitus Tipo 2/orina , Células Endoteliales/química , Vesículas Extracelulares/química , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hiperglucemia/orina , Masculino , Espectrometría Raman/métodos , Urinálisis/métodos , Orina/químicaRESUMEN
A facile and rapid method for synthesizing single crystal gold spherical or platelet (nonspherical) particles is reported. The reaction takes place at the interface of two immiscible liquids where the reducing agent decamethylferrocene (DmFc) was initially added to hexane and gold chloride (AuCl4-) to an aqueous phase. The reaction is spontaneous at room temperature, leading to the creation of Au nanoparticles (AuNP). A flow focusing microfluidic chip was used to create emulsion droplets, allowing the same reaction to take place within a series of microreactors. The technique allows the number of droplets, their diameter, and even the concentration of reactants in both phases to be controlled. The size and shape of the AuNP are dependent upon the concentration of the reactants and the size of the droplets. By tuning the reaction parameters, the synthesized nanoparticles vary from nanometer to micrometer sized spheres or platelets. The surfactant used to stabilize the emulsion was also shown to influence the particle shape. Finally, the addition of other nanoparticles within the droplet allows for core@shell particles to be readily formed, and we believe this could be a versatile platform for the large scale production of core@shell particles.
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A novel approach for rapid and sensitive detection of matrilysin (MMP-7, a biomarker involved in the degradation of various macromolecules) based on a polypeptide (JR2EC) functionalized reduced graphene oxide (rGO) field effect transistor (FET) is reported. MMP-7 specifically digests negatively charged JR2EC immobilized on rGO, thereby modulating the conductance of rGO-FET. The proposed assay enabled detection of MMP-7 at clinically relevant concentrations with a limit of detection (LOD) of 10 ng/mL (400 pM), attributed to the significant reduction of the net charge of JR2EC upon digestion by MMP-7. Quantitative detection of MMP-7 in human plasma was further demonstrated with a LOD of 40 ng/mL, illustrating the potential for the proposed methodology for tumor detection and carcinoma diagnostic (e.g., lung cancer and salivary gland cancer). Additionally, excellent specificity of the proposed assay was demonstrated using matrix metallopeptidase 1 (MMP-1), a protease of the same family. With appropriate selection and modification of polypeptides, the proposed assay could be extended for detection of other enzymes with polypeptide digestion capability.
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Grafito/química , Metaloproteinasa 7 de la Matriz/metabolismo , Péptidos/química , Límite de Detección , Microscopía Electrónica de Rastreo , Óxidos/químicaRESUMEN
Resistive pulse sensors, RPS, are allowing the transport mechanism of molecules, proteins and even nanoparticles to be characterized as they traverse pores. Previous work using RPS has shown that the size, concentration and zeta potential of the analyte can be measured. Here we use tunable resistive pulse sensing (TRPS) which utilizes a tunable pore to monitor the translocation times of nanoparticles with DNA modified surfaces. We start by demonstrating that the translocation times of particles can be used to infer the zeta potential of known standards and then apply the method to measure the change in zeta potential of DNA modified particles. By measuring the translocation times of DNA modified nanoparticles as a function of packing density, length, structure, and hybridization time, we observe a clear difference in zeta potential using both mean values and population distributions as a function of the DNA structure. We demonstrate the ability to resolve the signals for ssDNA, dsDNA, small changes in base length for nucleotides between 15 and 40 bases long, and even the discrimination between partial and fully complementary target sequences. Such a method has potential and applications in sensors for the monitoring of nanoparticles in both medical and environmental samples.
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ADN/química , Nanopartículas de Magnetita/química , Poliestirenos/química , Técnicas Electroquímicas , Hibridación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Estreptavidina/químicaRESUMEN
We present the first comparison between assays that use resistive pulses or rectification ratios on a tunable pore platform. We compare their ability to quantify the cancer biomarker Vascular Endothelial Growth Factor (VEGF). The first assay measures the electrophoretic mobility of aptamer modified nanoparticles as they traverse the pore. By controlling the aptamer loading on the particle surface, and measuring the speed of each translocation event we are able to observe a change in velocity as low as 18 pM. A second non-particle assay exploits the current rectification properties of conical pores. We report the first use of Layer-by-Layer (LbL) assembly of polyelectrolytes onto the surface of the polyurethane pore. The current rectification ratios demonstrate the presence of the polymers, producing pH and ionic strength-dependent currents. The LbL assembly allows the facile immobilisation of DNA aptamers onto the pore allowing a specific dose response to VEGF. Monitoring changes to the current rectification allows for a rapid detection of 5 pM VEGF. Each assay format offers advantages in their setup and ease of preparation but comparable sensitivities.
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In this review, recent advances in the development of electronic detection methodologies based on non-antibody recognition elements such as functional liposomes, aptamers and synthetic peptides are discussed. Particularly, we highlight the progress of field effect transistor (FET) sensing platforms where possible as the number of publications on FET-based platforms has increased rapidly. Biosensors involving antibody-antigen interactions have been widely applied in diagnostics and healthcare in virtue of their superior selectivity and sensitivity, which can be attributed to their high binding affinity and extraordinary specificity, respectively. However, antibodies typically suffer from fragile and complicated functional structures, large molecular size and sophisticated preparation approaches (resource-intensive and time-consuming), resulting in limitations such as short shelf-life, insufficient stability and poor reproducibility. Recently, bio-sensing approaches based on synthetic elements have been intensively explored. In contrast to existing reports, this review provides a comprehensive overview of recent advances in the development of biosensors utilizing synthetic recognition elements and a detailed comparison of their assay performances. Therefore, this review would serve as a good summary of the efforts for the development of electronic bio-sensing approaches involving synthetic recognition elements.
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Técnicas Biosensibles/métodos , Animales , Aptámeros de Nucleótidos/metabolismo , Humanos , Liposomas/química , Péptidos/metabolismoRESUMEN
The zeta potential of the protein corona around carboxyl particles has been measured using tunable resistive pulse sensing (TRPS). A simple and rapid assay for characterising zeta potentials within buffer, serum and plasma is presented monitoring the change, magnitude and distribution of proteins on the particle surface. First, we measure the change in zeta potential of carboxyl-functionalised nanoparticles in solutions that contain biologically relevant concentrations of individual proteins, typically constituted in plasma and serum, and observe a significant difference in distributions and zeta values between room temperature and 37 °C assays. The effect is protein dependent, and the largest difference between the two temperatures is recorded for the γ-globulin protein where the mean zeta potential changes from -16.7 to -9.0 mV for 25 and 37 °C, respectively. This method is further applied to monitor particles placed into serum and/or plasma. A temperature-dependent change is again observed with serum showing a 4.9 mV difference in zeta potential between samples incubated at 25 and 37 °C; this shift was larger than that observed for samples in plasma (0.4 mV). Finally, we monitor the kinetics of the corona reorientation for particles initially placed into serum and then adding 5 % (V/V) plasma. The technology presented offers an interesting insight into protein corona structure and kinetics of formation measured in biologically relevant solutions, i.e. high protein, high salt levels, and its particle-by-particle analysis gives a measure of the distribution of particle zeta potential that may offer a better understanding of the behaviour of nanoparticles in solution. Graphical Abstract The relative velocity of a nanoparticle as it traverses a nanopore can be used to determine its zeta potential. Monitoring the changes in translocation speeds can therefore be used to follow changes to the surface chemistry/composition of 210 nm particles that were placed into protein rich solutions, serum and plasma. The particle-by-particle measurements allow the zeta potential and distribution of the particles to be characterised, illustrating the effects of protein concentration and temperature on the protein corona. When placed into a solution containing a mixture of proteins, the affinity of the protein to the particle's surface determines the corona structure, and is not dependent on the protein concentration.
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Ácidos Carboxílicos/química , Fibrinógeno/química , Corona de Proteínas/química , Albúmina Sérica/química , gammaglobulinas/química , Técnicas Electroquímicas/métodos , Humanos , Nanopartículas/química , Tamaño de la Partícula , Electricidad Estática , Propiedades de SuperficieRESUMEN
A novel approach for enzymatic assay using reporter-encapsulated liposomes on graphene field effect transistors (FET) is proposed. This approach involves real time monitoring of drain current (Id) of reduced graphene oxide (rGO) upon rupture of reporter-encapsulated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes triggered by enzymes. For validation of the proposed approach, 2,4,6-trinitrophenol (TNP) is used as the reporter for specific detection of phospholipase A2 (PLA2), a key enzyme in various membrane related physiological processes. Experimental results revealed that Id increased with PLA2 concentration, which is attributed to the interaction between released TNP and rGO. The limit of detection (LOD) achieved by the proposed approach was 80 pM, which is superior to most assays reported previously and much lower than the cut-off level of circulating secretory PLA2 (2.07 nM). Besides the high accuracy of the electronic detection methodology, the signal enhancement effect realized by the excess concentration of TNP (approximately 1 mM) in liposomes is believed to be the main reason for the significantly enhanced sensitivity of the proposed assay, indicating great potential for further improvement in the sensitivity by increasing the concentration of TNP. In addition, the proposed approach is rapid (incubation time ≤ 10 min) and label-free, thus showing great potential for practical applications in the future.
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Grafito/química , Liposomas/química , Fosfolipasas A2/análisis , Espectrofotometría , Transistores Electrónicos , Cinética , Liposomas/metabolismo , Microscopía Electrónica de Rastreo , Óxidos/química , Fosfatidilcolinas/química , Picratos/química , Dióxido de Silicio/química , Especificidad por SustratoRESUMEN
Aptamers are short single-stranded pieces of DNA or RNA capable of binding to analytes with specificity and high affinity. Due to their comparable selectivity, stability, and cost, over the last two decades, aptamers have started to challenge antibodies in their use on many technology platforms. The binding event often leads to changes in the aptamer's secondary and tertiary structure; monitoring such changes has led to the creation of many new analytical sensors. Here, we demonstrate the use of a tunable resistive pulse sensing (TRPS) technology to monitor the interaction between several DNA aptamers and their target, thrombin. We immobilized the aptamers onto the surface of superparamagnetic beads, prior to their incubation with the thrombin protein. The protein binding to the aptamer caused a conformational change resulting in the shielding of the polyanion backbone; this was monitored by a change in the translocation time and pulse frequency of the particles traversing the pore. This signal was sensitive enough to allow the tagless detection of thrombin down to nanomolar levels. We further demonstrate the power of TRPS by performing real time detection and characterization of the aptamer-target interaction and measuring the association rates of the thrombin protein to the aptamer sequences.
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Aptámeros de Nucleótidos/química , Técnicas Biosensibles , Nanopartículas/química , Trombina/análisis , Aptámeros de Nucleótidos/síntesis química , Técnicas Electroquímicas , Humanos , Cinética , Imanes , Unión Proteica , Conformación Proteica , Sensibilidad y Especificidad , SolucionesRESUMEN
PURPOSE: This study aimed to quantify the relationship between venous and capillary blood sampling methods for the measurement of plasma interleukin-6 (IL-6). A parallel study was conducted to determine the possibility of measuring IL-6 in sweat using an enzyme-linked immunosorbent assay (ELISA) and investigate the relationship between plasma- and sweat-derived measures of IL-6. METHODS: Twelve male participants were recruited for the measurement of IL-6 at rest and during exercise (study 1). An additional group of five female participants was recruited for the measurement of IL-6 in venous blood versus sweat at rest and following exercise (study 2). In study 1, venous and capillary blood samples were collected at rest and in response to exercise. In study 2, venous and sweat samples were collected following exercise. RESULTS: Mean plasma IL-6 concentration was not different between venous and capillary blood sampling methods either at rest (4.27 ± 5.40 vs. 4.14 ± 4.45 pg ml(-1)), during (5.40 ± 5.17 vs. 5.58 ± 6.34 pg ml(-1)), or in response to exercise (6.95 ± 6.37 vs. 6.99 ± 6.74 pg ml(-1)). There was no IL-6 detectable in sweat either at rest or following exercise. CONCLUSION: There are no differences in the measurement of plasma IL-6 using either venous or capillary blood sampling methods. Capillary measurement represents a minimally invasive way of measuring IL-6 and detecting changes in IL-6, which are linked to fatigue and overtraining.
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Recolección de Muestras de Sangre/métodos , Ejercicio Físico , Interleucina-6/sangre , Adulto , Análisis Químico de la Sangre/métodos , Capilares , Femenino , Humanos , Interleucina-6/análisis , Masculino , Descanso , Sudor/química , VenasRESUMEN
Down syndrome (DS) is characterized by skeletal and brain structural malformations, cognitive impairment, altered hippocampal metabolite concentration and gene expression imbalance. These alterations were usually investigated separately, and the potential rescuing effects of green tea extracts enriched in epigallocatechin-3-gallate (GTE-EGCG) provided disparate results due to different experimental conditions. We overcame these limitations by conducting the first longitudinal controlled experiment evaluating genotype and GTE-EGCG prenatal chronic treatment effects before and after treatment discontinuation. Our findings revealed that the Ts65Dn mouse model reflected the pleiotropic nature of DS, exhibiting brachycephalic skull, ventriculomegaly, neurodevelopmental delay, hyperactivity, and impaired memory robustness with altered hippocampal metabolite concentration and gene expression. GTE-EGCG treatment modulated most systems simultaneously but did not rescue DS phenotypes. On the contrary, the treatment exacerbated trisomic phenotypes including body weight, tibia microarchitecture, neurodevelopment, adult cognition, and metabolite concentration, not supporting the therapeutic use of GTE-EGCG as a prenatal chronic treatment. Our results highlight the importance of longitudinal experiments assessing the co-modulation of multiple systems throughout development when characterizing preclinical models in complex disorders and evaluating the pleiotropic effects and general safety of pharmacological treatments.