RESUMEN
Present coagulation assays fail to detect mild coagulation disorders, while thrombin-generation (TG) assays solve this problem. However, most of them only work with threated blood samples, which makes them labor intensive, time consuming, unreliable, and expensive. We have developed a TG electrophoretic assay that uses a thrombin specific charge-changing fluorescent peptide substrate, electrophoretic separation, and requires a drop of blood. The limit of detection of the assay was 1.97 nM in phosphate buffer saline and 6.82 nM in citrated whole blood. The assay was used to determine the TG in whole blood from healthy volunteers (n = 6, one aspirin user), over 30 min, after the blood was drawn; the TG increased from a baseline level of 2 × 10(6) RFU to 1.2 × 10(13) RFU. The lag time between the blood draw and initial burst of TG was 6 min for the volunteers (n = 5) and 15 min for the aspirin user. Specificity of the assay was evaluated by reacting our substrate with the heparinized blood samples and other proteases. The TG electrophoretic assay was designed and tested in the whole human blood, requiring no sample preparation, 5 µL of blood, 45 min, and it detected differences in coagulation patterns between a volunteer taking aspirin and non-aspirin users.
Asunto(s)
Pruebas de Coagulación Sanguínea/métodos , Electroforesis/métodos , Aspirina/farmacología , Coagulación Sanguínea/efectos de los fármacos , Humanos , Límite de Detección , Sensibilidad y Especificidad , Tiempo de TrombinaRESUMEN
BACKGROUND: Current cell-free DNA assessment of fetal chromosomes does not analyze and report on all chromosomes. Hence, a significant proportion of fetal chromosomal abnormalities are not detectable by current noninvasive methods. Here we report the clinical validation of a novel noninvasive prenatal test (NIPT) designed to detect genomewide gains and losses of chromosomal material ≥7 Mb and losses associated with specific deletions <7 Mb. OBJECTIVE: The objective of this study is to provide a clinical validation of the sensitivity and specificity of a novel NIPT for detection of genomewide abnormalities. STUDY DESIGN: This retrospective, blinded study included maternal plasma collected from 1222 study subjects with pregnancies at increased risk for fetal chromosomal abnormalities that were assessed for trisomy 21 (T21), trisomy 18 (T18), trisomy 13 (T13), sex chromosome aneuploidies (SCAs), fetal sex, genomewide copy number variants (CNVs) ≥7 Mb, and select deletions <7 Mb. Performance was assessed by comparing test results with findings from G-band karyotyping, microarray data, or high coverage sequencing. RESULTS: Clinical sensitivity within this study was determined to be 100% for T21 (95% confidence interval [CI], 94.6-100%), T18 (95% CI, 84.4-100%), T13 (95% CI, 74.7-100%), and SCAs (95% CI, 84-100%), and 97.7% for genomewide CNVs (95% CI, 86.2-99.9%). Clinical specificity within this study was determined to be 100% for T21 (95% CI, 99.6-100%), T18 (95% CI, 99.6-100%), and T13 (95% CI, 99.6-100%), and 99.9% for SCAs and CNVs (95% CI, 99.4-100% for both). Fetal sex classification had an accuracy of 99.6% (95% CI, 98.9-99.8%). CONCLUSION: This study has demonstrated that genomewide NIPT for fetal chromosomal abnormalities can provide high resolution, sensitive, and specific detection of a wide range of subchromosomal and whole chromosomal abnormalities that were previously only detectable by invasive karyotype analysis. In some instances, this NIPT also provided additional clarification about the origin of genetic material that had not been identified by invasive karyotype analysis.
Asunto(s)
Aberraciones Cromosómicas , Trastornos de los Cromosomas/diagnóstico , Variaciones en el Número de Copia de ADN , Diagnóstico Prenatal/métodos , Adolescente , Adulto , Trastornos de los Cromosomas/diagnóstico por imagen , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Cariotipificación , Edad Materna , Persona de Mediana Edad , Embarazo , Estudios Retrospectivos , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
In real time: thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual-emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage-dependent spatial localization. The most advanced RACPP uses norleucine (Nle)-TPRSFL as a linker that increases sensitivity to thrombin by about 90-fold.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Trombina/metabolismo , Animales , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , Ratones , Ratones TransgénicosRESUMEN
The ability to measure protease activity in the blood is important for the development of future diagnostics and for biomedical research. Presently, protease assays require sample preparation, making them time-consuming, costly, less accurate, and unsuitable for point-of-care (POC) diagnostics. Recently, we demonstrated a unique method for measuring clinically relevant levels of trypsin activity in only a few microliters of whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target protease. Using a simple electrophoretic format, the fragments could be rapidly separated, concentrated, and detected directly from a whole blood sample. We now report on the development of new protease substrates for the measurement of elastase, chymotrypsin, matrix metalloproteinase (MMP)-2, and MMP-9 activity in whole blood. In these studies, detection limits ranging from 1 to 40 pg in 6 µL of 1× phosphate-buffered saline (PBS) (0.2-6 ng/mL) were achieved after a only 1 h reaction of enzyme and substrate. In subsequent experiments measuring spiked protease in whole blood (with endogenous protease present), detection limits ranging from 100 to 200 ng/mL were achieved after a 1 h reaction. Thus, these new substrates demonstrate broad applicability toward clinically relevant detection of important disease-relevant proteases.
Asunto(s)
Quimotripsina/sangre , Pruebas de Enzimas/métodos , Metaloproteinasa 2 de la Matriz/sangre , Metaloproteinasa 9 de la Matriz/sangre , Elastasa Pancreática/sangre , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Péptidos/química , Sistemas de Atención de Punto , Espectrometría de Fluorescencia/métodos , Especificidad por SustratoRESUMEN
The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly-L-glutamic acid-doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 microL (0.3 ng/mL) in whole human blood was achieved after a 1-h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50- to 200-fold better than the estimated reference levels for trypsin (15-60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics.
Asunto(s)
Electroforesis/métodos , Tripsina/sangre , Resinas Acrílicas/química , Electroforesis/economía , Humanos , Límite de Detección , Polielectrolitos , Ácido Poliglutámico/química , Polímeros/químicaRESUMEN
In biomedical research and clinical diagnostics, it is a major challenge to measure disease-related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time-consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge-changing fluorescent peptide substrates. Charge-changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic alpha-chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both alpha-chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross-reactivity with the trypsin-like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin-specific), a detection limit of about 10-20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point-of-care diagnostics.
Asunto(s)
Quimotripsina/sangre , Electroforesis/métodos , Tripsina/sangre , Animales , Humanos , Cinética , Péptidos/metabolismo , Ratas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Obesity is a state of subclinical inflammation resulting in loss of function of insulin receptors and decreased insulin sensitivity. Inhibition of the inflammatory enzymes, matrix metalloproteinases (MMPs), for 6 months in rodent models restores insulin receptor function and insulin sensitivity. METHODS: This 12-week double-blind, randomized, placebo (PL)-controlled proof-of-concept study was performed to determine if the MMP inhibitor (MMPI), doxycycline, decreased global markers of inflammation and enhanced muscle insulin sensitivity in obese people with type 2 diabetes (DM2). The study included non-DM2 controls (n = 15), and DM2 subjects randomized to PL (n = 13) or doxycycline 100 mg twice daily (MMPI; n = 11). All participants were evaluated on Day 1; MMPI and PL groups were also evaluated after 84 days of treatment. RESULTS: There was a significant decrease in inflammatory markers C-reactive protein (P < 0.05) and myeloperoxidase (P = 0.01) in the MMPI but not PL group. The MMPI also significantly increased skeletal muscle activated/total insulin signaling mediators: 3'phosphoinositide kinase-1 (PDK1) (p < 0.03), protein kinase B (PKB/Akt) (p < 0.004), and glycogen synthase kinase 3ß (GSK3ß) (p < 0.03). CONCLUSIONS: This study demonstrated short term treatment of people with diabetes with an MMPI resulted in decreased inflammation and improved insulin sensitivity. Larger, longer studies are warranted to determine if doxycycline can improve glucose control in people with diabetes. TRIAL REGISTRATION: Clinicaltrials.gov NCT01375491.