Dual-labeled Fluorescent Probe Research Articles

Spatiotemporal Quantification of HER2-targeting Antibody-Drug Conjugate Bystander Activity and Enhancement of Solid Tumor Penetration.

Antibody-drug conjugate (ADC) has had a transformative effect on the treatment of many solid tumors, yet it remains unclear how ADCs exert bystander activity in the tumor microenvironment. Here, we directly visualized and spatiotemporally quantified the intratumor biodistribution and pharmacokinetics of different ADC components by developing dual-labeled fluorescent probes. Mechanistically, we found that tumor penetration of ADCs is distinctly affected by their ability to breach the binding site barrier (BSB) in perivascular regions of tumor vasculature, and bystander activity of ADC can only partially breach BSB. Furthermore, bystander activity of ADCs can work in synergy with coadministration of their parental antibodies, leading to fully bypassing BSBs and enhancing tumor penetration via a two-step process. These promising preclinical data allowed us to initiate a phase I/II clinical study of coadministration of RC48 and trastuzumab in patients with malignant stomach cancer to further evaluate this treatment strategy in humans.

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences.

Blocking PCR is a method that inhibits amplification of DNA possessing a nucleotide sequence complementary to that of a blocker; the method can be used to suppress amplification of target wild-type DNA while amplifying mutated DNA. Previously, we demonstrated that an oligoribonucleotide (ORN) functions as a cost-effective and sequence-specific blocker. This blocking PCR system, named ORN interference-PCR (ORNi-PCR), is compatible with DNA polymerases lacking 5'-3' exonuclease activity but not with those possessing the activity (e.g., Taq DNA polymerase), which can remove a hybridized ORN during DNA extension. Here, we demonstrate that under specific experimental conditions, an intact or phosphorothioated ORN strongly suppresses extension of target DNA by Taq DNA polymerases. This method was applied successfully to real-time ORNi-PCR and one-step real-time reverse transcription-ORNi-PCR using a dual-labeled fluorescent probe to detect a single-nucleotide mutation in DNA and RNA in a sequence-specific manner. The results reaffirm the utility of blocking PCR and provide technical hints for its improvement.

A fluorogenic probe for core-fucosylated glycan-preferred ENGase

A fluorescence-quenching-based assay system to determine the hydrolytic activity of endo-β-N-acetylglucosaminidases (ENGases), which act on the innermost N-acetylglucosamine (GlcNAc) residue of the chitobiose segment of core-fucosylated N-glycans, was constructed using a dual-labeled fluorescent probe with a hexasaccharide structure. The fluorogenic probe was evaluated using a variety of ENGases, including Endo-M W251N mutant, Endo-F3, and Endo-S, which recognize core fucosylated N-glycans. The occurrence of a hydrolysis reaction was detected by observing an increased fluorescence intensity, ultimately allowing the ENGase activities to be easily and quantitatively evaluated, with the exception of Endo-S. The obtained results clearly indicated the substrate specificities of the examined ENGases.

A dual-labeled fluorescent probe for visualization of dextranase activity in a simulated food digestion system

Molecular bioimaging of enzyme activity is rapidly emerging as a powerful strategy for accurate disease diagnostics. This work aims to prove that bioimaging of enzyme activity in food digestion with a fluorescent probe is feasible. In this study, a dual-labeled fluorescent probe with dextran-tetramethylrhodamine (TMR)-biotin conjugate (DTB) as the enzyme-cleavable unit, and biotin-(5-fluorescein) conjugate (FB) as the reference unit, was developed. It was immobilized in the agarose gel (the model food matrix) for the fluorescence quantification of dextranase activity. The probe manifested significantly ratiometric fluorescent signals (Igreen/Ired) in response to the enzyme-active reaction. Linear relationships of Igreen/Ired were obtained against the dextranase concentration ratio (C/C0). Igreen/Ired increased more rapidly with a greater dextranase diffusion rate, also supported by the more significant diffusion coefficient of fluorescently labeled dextranase in 0.5 wt% agarose gel (1.87 × 10-6 cm2 s−1). Our work provides more mechanistic evidence for enzyme activity imaging in food digestion.

Early Growth Response-1 (EGR-1) and Nuclear Factor of Activated T Cells (NFAT) Cooperate to Mediate CD40L Expression in Megakaryocytes and Platelets

Increasing evidence implicates circulating platelets as mediators of chronic inflammatory and autoimmune diseases via the expression and release of CD40L, an important modulator of inflammation and adaptive immune responses traditionally associated with activated T cells. Emerging evidence suggests that platelet CD40L is dynamically regulated in several chronic inflammatory and autoimmune diseases and may mediate progression and secondary pathology associated with those disease states. The present study identifies NFATc2 as a key transcriptional modulator of CD40L expression in megakaryocytes and inflammatory activity of platelets. Furthermore, the current data show that EGR-1, a member of the early growth response family of zinc finger transcription factors, modulates NFATc2-dependent regulation of CD40L expression in megakaryocytes. Our novel demonstration that in vivo biochemical or genetic inhibition of NFATc2 activity in megakaryocyte diminishes platelet CD40L implicates the NFATc2/EGR-1 axis as a key regulatory pathway of inflammatory and immunomodulatory activity in platelets and represents a target for the development of therapeutics for the potential treatment of chronic inflammatory and autoimmune diseases.

Specific real-time reverse transcription-polymerase chain reaction for detection and quantitation of turkey coronavirus RNA in tissues and feces from turkeys infected with turkey coronavirus

Turkey coronavirus (TCoV) infection causes acute atrophic enteritis in the turkey poults, leading to significant economic loss in the U.S. turkey industry. Rapid detection, differentiation, and quantitation of TCoV are critical to the diagnosis and control of the disease. A specific one-step real-time reverse transcription-polymerase chain reaction (RRT-PCR) assay for detection and quantitation of TCoV in the turkey tissues was developed using a dual-labeled fluorescent probe. The fluorogenic probe labeled with a reporter dye (FAM, 6-carboxytetramethylrhodamin) and a quencher dye (AbsoluteQuencher™) was designed to bind to a 186 base-pair fragment flanked by the two PCR primers targeting the 3′ end of spike gene of TCoV. The assay was performed on different avian viruses and bacteria to determine the specificity as well as serial dilutions of TCoV for the sensitivity. Three animal trials were conducted to further validate the assay. Ten-day-old turkey poults were inoculated orally with 100 EID 50 of TCoV. Intestinal tissues (duodenum, jejunum, ileum, cecum), feces from the cloacal swabs, or feces from the floor were collected at 12 h, 1, 2, 3, 5, 7, and/or 14 days post-inoculation (DPI). RNA was extracted from each sample and subjected to the RRT-PCR. The designed primers and probe were specific for TCoV. Other non-TCoV avian viruses and bacteria were not amplified by RRT-PCR. The assay was highly sensitive and could quantitate between 10 2 and 10 10 copies/μl of viral genome. The viral RNA in the intestine segments reached the highest level, 6 × 10 15 copies/μl, in the jejunum at 5 DPI. Eighty-four intestine segments assayed by the developed RRT-PCR and immunofluorescence antibody assay (IFA) revealed that there were 6 segments negative for TCoV by both assays, 45 positive for TCoV by IFA, and 77 positive for TCoV by RRT-PCR. Turkey coronavirus was detected in the feces from the cloacal swabs or floor 1–14 DPI; however, the viral RNA load varied among different turkey poults at different intervals from different trials. The highest amount of viral RNA, 2.8 × 10 10 copies/μl, in the feces was the one from the cloacal swab collected at 1 DPI. The average amount of TCoV RNA in the cloacal fecal samples was 10 times higher than that in the fecal droppings on the floor. Taken together, the results indicated that the developed RRT-PCR assay is rapid, sensitive, and specific for detection, differentiation, and quantitation of TCoV in the turkey tissues and should be helpful in monitoring the progression of TCoV induced acute enteritis in the turkey flocks.

Direct Measurement of Free Chloride Concentrations in the Phagolysosomes of Human Neutrophils

Human neutrophils synthesize hypochlorous acid, a nonradical oxidant, as one of the antimicrobial agents to kill phagocytosed pathogens within phagolysosomes. The production of HOCl is catalyzed by myeloperoxidase using chloride anions (Cl-). Even though various approaches have been documented to measure cytosolic Cl- concentrations, direct detection of phagolysosomal free Cl- in a real-time fashion is not available. Here we report the development of a new dual-labeled fluorescent probe by conjugation of the chloride-sensitive 6-methoxyquinoline and chloride-insensitive tetramethylrhodamine to the zymosan particles. On the surface of the zymosan particles, the chloride-sensitive fluorophore responded linearly to chloride concentrations when plotted according to the Stern-Volmer collisional quenching equation and the chloride-insensitive fluorophore served as an internal reference. After phagocytosis into neutrophils, the probe was able to monitor intraphagosomal chloride levels authentically. Human neutrophils in the medium containing 122 mM chloride had a phagolysosomal chloride level of 73.3 +/- 12.2 mM (N = 5). In contrast, the neutrophils in a chloride-free isoosmotic medium had a chloride level of 6.6 +/- 1.9 mM (N = 5) in the phagolysosomal compartment. These data clearly demonstrated that the extracellular chloride levels affect intraphagolysosomal chloride levels in human neutrophils. Moreover, when the extracellular chloride concentration was switched from high level (122 mM) to low level (0 mM) or vice versa, the probe demonstrated complete reversibility. The data indicate that the new zymosan-conjugated chloride probe described here can be used as an indicator for measurement of the free chloride level within the phagolysosomes of neutrophils and other phagocytic cell types.

Intrahepatic Hepatitis B Virus Covalently Closed Circular DNA Can Be a Predictor of Sustained Response to Therapy

This study aimed to determine whether intrahepatic hepatitis B virus (HBV) covalently closed circular (ccc) DNA and total HBV DNA levels at the end of therapy would predict sustained response to therapy. Hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients receiving either lamivudine monotherapy or combination of peginterferon and lamivudine had liver biopsy at the end of 1 year therapy and were followed for 52 more weeks after cessation of therapy. Serum HBV DNA, intrahepatic HBV ccc DNA, and total HBV DNA levels were determined. Forty-seven patients, including 34 males and 13 females, were studied. Twenty-seven patients received combination therapy, and 20 patients received lamivudine monotherapy. Twenty-nine patients had end-of-treatment virologic response, and 15 patients had sustained response 52 weeks after therapy. At the end of treatment, log serum HBV DNA levels correlated well with log intrahepatic HBV cccDNA and log intrahepatic total HBV DNA levels. Log intrahepatic cccDNA and log intrahepatic total DNA levels were significantly lower among patients with sustained virologic response. The adjusted odds ratio for log cccDNA was 5.3 (95% CI: 1.5-18.2, P = .009) and, for log intrahepatic HBV DNA, was 4.4 (95% CI: 1.3-14.7, P = .015) to predict sustained virologic response. Using log cccDNA at -0.80 copies/genome equivalent as cutoff, the sensitivity, specificity, and positive and negative predictive values and accuracy of predicting sustained virologic response were 73%, 78%, 56%, 86%, and 77% respectively. Intrahepatic HBV cccDNA and intrahepatic total HBV DNA levels at the end of therapy are superior to serum HBV DNA as surrogates of sustained virologic response.

Real-time RT-PCR differentiation and quantitation of infectious bursal disease virus strains using dual-labeled fluorescent probes

A real-time RT-PCR assay was developed utilizing dual-labeled fluorescent probes binding to VP4 sequence that are specific to the classical (Cl), variant (V) and very virulent (vv) strains of infectious bursal disease virus (IBDV). The assay was highly sensitive and could detect as little as 3 × 10 2 to 3 × 10 3 copies of viral template. Viral genomic copy number could be accurately assayed over a broad range of 7–8 logs of viral genome. The variant sequence-specific probe was found to be highly specific in detecting isolates classified as variant A, D, E, G and GLS-5, and did not react with classical strains. A total of 130 field and experimental variant strain isolates were tested using this assay. The classical sequence-specific probe also demonstrated high sensitivity and specificity, and positively detected a total of 87 STC isolates, both field and experimental isolates, while differentiating between isolates that were variant and classical strains. The very virulent sequence-specific probe detected positively the Holland vvIBDV isolate and did not react with classical or variant strains. Rapid identification of viral strain is a primary concern to poultry flock health programs to ensure administered vaccines will protect against current strains of virus circulating in the flock. The ability to quantify virus concurrently is also of assistance in identifying the progression of disease outbreaks within the flock.

Real-time PCR assay for detection and genotype differentiation of Giardia lamblia in stool specimens.

Real-time PCR, using dual-labeled fluorescent probes targeting the beta-giardin gene, was used to detect Giardia lamblia in human stool specimens and to discriminate between isolates from the two major genetic assemblages of G. lamblia infective to humans, assemblages A and B.

CARBOHYDRATE INGESTION INFLUENCES SKELETAL MUSCLE CYTOKINE mRNA AND PLASMA CYTOKINE LEVELS AFTER A 3-H RUN

PURPOSE To determine the influence of carbohydrate ingestion on skeletal muscle cytokine mRNA content and plasma cytokine levels after a 3-h run. METHODS Sixteen experienced marathoners ran on treadmills for 3 h at ∼70% VO2max on two occasions while receiving 1 l/h carbohydrate (CHO) or placebo (PLA) beverages. Blood and vastus lateralis muscle biopsy samples were collected before and after exercise. Plasma was analyzed for IL-6, IL-10, IL-1ra, IL-8, cortisol, glucose, and insulin. Muscle was analyzed for glycogen content and relative gene expression of 13 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-γ, TNF-α) using real-time quantitative RT-PCR and the ΔΔCt method that monitors the degradation of a dual-labeled fluorescent probe (TaqMan) in real time concomitant with PCR amplification. The data were analyzed using a 2 × 2 repeated measures ANOVA. RESULTS Plasma glucose and insulin were higher and cortisol, IL-6, IL-10, and IL-1ra, but not IL-8, were significantly lower post-exercise in CHO vs PLA. Change in muscle glycogen content did not differ between CHO and PLA (P=0.246). Muscle cytokine mRNA was detected for IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p35, IL-15, IFN-γ, and TNF-α, but only IL-1β, IL-6, and IL-8 were significantly increased with exercise (P < 0.05). The increase in mRNA (fold difference from pre-exercise) was attenuated in CHO (15.9-fold) compared to PLA (35.2-fold) for IL-6 (P = 0.071) and IL-8 (CHO, 7.8-fold; PLA, 23.3-fold; P = 0.063). CONCLUSION CHO compared to PLA beverage ingestion attenuates the increase in plasma IL-6, IL-10, and IL-1ra, and gene expression for IL-6 and IL-8 in athletes running 3 h at 70% VO2max despite no differences in muscle glycogen content. Supported by a grant from the Gatorade Sports Science Institute.

Fluorescent multicolor multiplex homogeneous assay for the simultaneous analysis of the two most common hemochromatosis mutations

We report the development of a qualitative fluorescent multiplex homogeneous assay designed for the detection of the two most common hemochromatosis mutations using dual-labeled fluorescent probes. The assay is able to detect four allelic variants in a single closed tube using a single thermocycling protocol. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific oligonucleotide hybridization using the 5 ′ nuclease assay format, and the higher throughput of a multicolor fluorescence detection procedure. Genomic DNA was prepared from whole blood specimens using standard procedures. Following DNA sample preparation, two regions of the hemochromatosis gene (HFE) including the H63D and C282Y mutations were coamplified and detected in real-time by four different fluorescently labeled allele-specific oligonucleotide probes. Assay specificity was demonstrated by a blind methods comparison study that included 37 DNA samples from individuals with a known HFE genotype. Results from the study showed that the multicolor multiplex HFE assay unambiguously classified all possible genotypes for the HFE gene C282Y and H63D mutations 1 See, e.g., U.S. Patents 5,712,098 and 6,025,130. 1 . This technique will be useful for research and molecular diagnostic laboratories and can be easily adapted for the detection of other single nucleotide polymorphisms.

Development and Validation of Real-Time Quantitative Reverse Transcriptase–Polymerase Chain Reaction for Monitoring Gene Expression in Cardiac Myocytesin Vitro

In this article we present validation of a real-time RT–PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in anin vitromodel of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT–PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the ΔΔCtmethod. Real-time quantitative RT–PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.