A high throughput scintillation proximity imaging assay for protein methyltransferases

Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers, it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on lysine residues of biotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and optimized assay conditions to achieve Z' values ranging from 0.48 to 0.91. The robust performance of this SPIA for the four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the four targets. NF279, a suramin analogue, was found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and 1.1 μM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may constitute a new class of anti-proliferative agents for potential therapeutic use.     

Liquid scintillation assay of 3H and 14C-labelled polymers

The assay of a range of ³H and ¹⁴C-labelled polymers by liquid scintillation counting is reported. Methods of sample preparation such as combustion, solution, gelation and suspension techniques are described and compared. The validity of a variety of procedures of counting efficiency determination for the above methods is examined and critically discussed.     

Hetarylethylene derivatives of 2,5-diaryloxazoles and 2,5-diarloxadiazoles and their luminescence and scintillation properties

A number of new organic luminophores were synthesized via the Wittig reaction from 2-(4-bromomethylphenyl)-5-phenyloxazole or 2-(4-bromomethylphenyl)-5-phenyl-1,3,4-oxadiazole and various heterocyclic aldehydes containing a thiophene ring.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 9, pp. 1267–1270, September, 1986.     

Synthesis, evaluation and incorporation into liposomes of 4-functionalised-2,5-diphenyloxazole derivatives for application in scintillation proximity assays

The preparation of a series of 4-functionalised-2,5-diphenyloxazoles is described. The scintillating efficiency of each of these ‘scintilipid’ molecules has been evaluated in the presence of ionising radiation. Each ‘scintilipid’ has been assessed for the ability to assemble, with other lipids, into liposomes, under a variety of preparative conditions. Each liposomal preparation has been monitored for the ability to scintillate in the presence of ionising radiation. The optimal ‘scintilipid’, both in terms of effective liposomal formation and scintillation efficiency, has been determined.     

A serological aptamer-assisted proximity ligation assay for COVID-19 diagnosis and seeking neutralizing aptamers

Rapid and accurate diagnosis of COVID-19 plays an essential role in the current epidemic prevention and control. Despite the promise of nucleic acid and antibody tests, there is still a great challenge to reduce the misdiagnosis, especially for asymptomatic individuals. Here we report a generalizable method for highly specific and ultrasensitive detection of serum COVID-19-associated antigens based on an aptamer-assisted proximity ligation assay. The sensor is based on binding two aptamer probes to the same protein target that brings the ligation DNA region into close proximity, thereby initiating ligation-dependent qPCR amplification. Using this system, serum nucleocapsid protein has been detected quantitatively by converting protein recognition into a detectable qPCR signal using a simple, homogeneous and fast detection workflow in ∼2 hours. In addition, this system has also been transformed into a universal platform for measuring specific interactions between spike S1 and its receptor ACE2, and more importantly demonstrated the feasibility for screening and investigation of potential neutralizing aptamers. Since in vitro selection can obtain aptamers selective for many COVID-19-associated antigens, the method demonstrated here will serve as an important tool for the diagnosis and therapeutics of COVID-19.

A versatile aptamer-assisted proximity ligation system improves diagnosis of COVID-19, and allows the evaluation of potential neutralizing aptamers.     

Comparison of radiometric and non-radiometric methods for uranium determination in groundwater of Punjab,India

Uranium in groundwater samples collected from Punjab state, India was determined using radiometric methods (extractive liquid scintillation and Cerenkov counting). Experimental conditions were optimized by studying the effect of sample pH, quenching and amount of extracting agent added to the scintillation cocktail on recovery of uranium. To ensure the accuracy of results, both radiometric methods were compared with non radiometric method such as adsorptive stripping voltammetry and found to be very good agreement. The distinct advantage of proposed radiometric methods is almost 100 % effective especially extractive liquid scintillation for simultaneous measurement of alpha and beta emitting radionuclides with fast and simple sample preparation.     

Comparison of fluorimetric methods of assay for 7 H-Benz(de)anthracen-7-one in airborne particulates and air pollution source effluents

Summary Various modifications of a thin-layer chromatography fluorimetric method for the determination of 7 H-benz(de)anthracen-7-one (BO) and phenalen-1-one (PO) in complex mixtures have been compared. The best modifications consisted of one-dimensional thin-layer chromatography followed by elution, evaporation, solution in trifluoroacetic acid, followed by either spectrophotofluorimetric or fluorimetric assay. In some cases a quenching agent was necessary to quench the fluorescence of the interferences. Urban atmospheres and air pollution source effluents can be analyzed by these micromethods. These methods with slight modification could be applied to the determination of the aza heterocyclic and the other ring-carbonyl compounds found in urban atmospheres and air pollution source effluents.

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Zusammenfassung Verschiedene Modifikationen einer dünnschichtchromatographischen, fluorimetrischen Methode zur Bestimmung von 7 H-Benz(de)-anthracen-7-on (BO) und Phenalen-1-on (PO) in komplexen Gemischen wurden verglichen. Am besten bewährte sich eindimensionale Dünnschichtchromatographie, nachfolgende Elution, Abdampfen, Aufnehmen in Trifluoressigsäure und schließlich Bestimmung durch Spektralfluoreszenz-oder Fluoreszenzmessung. In manchen Fällen war es nötig, die Fluoreszenz störender Substanzen mit einem geeigneten Reagens zu löschen. Stadtatmosphäre oder Quellen der Luftverunreinigung können mit diesen Mikromethoden analysiert werden. Sie eignen sich nach geringfügiger Modifikation auch zur Bestimmung von azaheterocyklischen und anderen Ring-Carbonylverbindungen in der Atmosphäre.

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Résumé On a comparé entre elles différentes modifications d'une méthode de fluorimétrie avec chromatographie en couche mince pour le dosage de la benzo-7 H anthracène-one-7 (BO) et de la phénalène-one-1 (PO) en mélanges complexes. Les modifications les meilleures ont consisté en une Chromatographie en couche mince unidimensionnelle suivie d'élution, évaporation, mise en solution dans l'acide trifluoroacétique, opérations suivies d'une recherche spectrophotofluorimétrique ou fluorimétrique. Dans certains cas, un agent d'extinction s'est montré nécessaire pour éteindre la fluorescence due aux interférences. On peut analyser par ces microméthodes les atmosphères urbaines et les écoulements sources de pollution de l'air. Ces méthodes ont pu être appliquées après légère modification au dosage des composés aza hétérocycliques et autres composés carbonyles cycliques trouvés dans les atmosphères urbaines et dans les écoulements sources de pollution de l'air.

     

Efficient production of ligand-free microscintillators at gram-scale for high-resolution X-ray luminescence imaging

The efficient production of high-quality scintillators with long radioluminescence afterglow is crucial for high-performance X-ray luminescence extension imaging. However, scaling-up the synthesis of ligand-free scintillators to fabricate large-area X-ray imaging screens for industrial applications remains a challenge.In this study, we report an efficient method to synthesize ligand-free, lanthanide-doped microscintillators by a one-pot reaction via the concentrated hydrothermal method. The as-s...     

A novel method for the sensitive detection of mutant proteins using a covalent-bonding tube-based proximity ligation assay

Tumorigenesis is the cumulative result of multiple gene mutations. The mutant proteins that are expressed by mutant genes in cancer cells are secreted into the blood and are useful biomarkers for the early diagnosis of cancer. However, some difficulties exist; for example, the same gene will express different protein mutants in different patients, and early tumors secrete only small amounts of mutant protein. Thus, the presence of mutant proteins in plasma has not previously been exploited for the early diagnosis of cancer. Proximity ligation assay is a protein-detection method that has been developed in recent years and has been widely used because of its high sensitivity. However, this approach still suffers from some shortcomings that should be addressed. In this paper, we develop a covalent-bonding tube-based proximity ligation assay (TB-PLA). The limit of detection of TB-PLA for 0.001 pM, and the method exhibited a broad dynamic range of up to seven orders of magnitude. Furthermore, we coupled the conformation-specific antibody PAb240 of p53 mutants to PCR tubes for TB-PLA. The assay was capable of detecting an approximately 500-fold lower concentration of mutant p53 in serum compared with sandwich ELISA. Thus, we demonstrate TB-PLA to be a highly sensitive and effective approach that is suitable for the early clinical diagnosis of cancer using the conformation-specific antibodies of protein mutants.     

Dual energy converting nano-phosphors: upconversion luminescence and X-ray excited scintillation from a single composition of lanthanide-doped yttrium oxide

We report an upconverting nanomaterial composition, [Y(2)O(3); Yb (2%), Er (1%)], that converts both X-ray and high-fluence NIR irradiation to visible light. This composition is compared to a higher Yb(3+) doped composition, [Y(2)O(3); Yb (10%), Er (1%)], that displays diminished visible X-ray scintillation, but shows enhanced red wavelength centered upconversion emission. These nanocrystals have been characterized by TEM, X-ray diffraction, power-dependent upconversion luminescence, and X-ray scintillation spectroscopy. We further demonstrate that lithium ion doping of the [Y(2)O(3); Yb (2%), Er (1%)] nanoscale composition leads to enhanced X-ray and NIR excited emission intensities through the production of nanoparticles that feature slightly enhanced sizes and increased crystallinity.     

Gold nanocluster-based fluorescent assay for label-free detection of protein kinase and its inhibitors

Microchimica Acta - A sensitive fluorometric assay is reported for label-free detection of the activity of protein kinase (PKA) and of its inhibitors. It is based on the europium(III)-modulated...     

A ratiometric and non-enzymatic luminescence assay for uric acid: differential quenching of lanthanide excited states by anti-oxidants

The excited states of Tb and Eu complexes of a common macrocyclic ligand are quenched preferentially by electron transfer from the urate anion, allowing the creation of a new assay to measure uric acid in biological fluids.     

Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay

Herein, we describe an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced indiscrimitive single-stranded DNase activity of Cas12a for the quantitative profiling of gene expression at the mRNA level and detection of proteins with high sensitivity and specificity. The target recognition is achieved through proximity binding rather than recognition by CRISPR RNA (crRNA), which allows for flexible assay design. A binding-induced primer extension reaction is used to generate a predesigned CRISPR-targetable sequence as a barcode for further signal amplification. Through this dual amplification protocol, we were able to detect as low as 1 fM target nucleic acid and 100 fM target protein isothermally. The practical applicability of this assay was successfully demonstrated for the temporal profiling of interleukin-6 gene expression during allergen-mediated mast cell activation.

Herein, we develop an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced collateral cleavage activity of Cas12a for the quantitative profiling of gene expression and detection of proteins with high sensitivity and specificity.     

Improved method for the determination of the cortisol production rate using high-performance liquid chromatography and liquid scintillation counting

Two new methods for the determination of the cortisol production rate using reversed-phase high-performance liquid chromatography are described. One uses ultraviolet detection at 205 nm, the other on-line post-column derivatization with benzamidine, followed by fluorimetric detection. The specific activity of tetrahydrocortisol and tetrahydrocortisone in urine from patients who had received tritium-labelled cortisol was determined by the indicated methods, followed by fraction collection and liquid scintillation counting. The post-column reaction detection procedure was superior to ultraviolet detection, both in selectivity and analysis time. Intra- and inter-assay variance of the post-column reaction detection procedure were 3.7 and 4.7%, respectively. A good correlation (r = 0.99) was obtained between values determined by this procedure and by a thin-layer chromatographic procedure.     

Upconversion luminescence assay for the detection of the vascular endothelial growth factor,a biomarker for breast cancer

Upconversion nanoparticles (UCNPs) of the type α-NaYF₄:Yb³⁺, Er³⁺ and a typical diameter of 6–7 nm were synthesized by thermal decomposition of the respective rare-earth stearate. The oleic acid on the surface of the UCNPs was then replaced by aptamer DNA. The assay was performed in a microplate format with a capture probe immobilized in the wells. Following binding of the vascular endothelial growth factor (VEGF), an auxiliary probe DNA is added that is labeled with UCNPs and binds to the VEGF-loaded capture probe. The method enables highly sensitive and highly specific detection of the VEGF which is a marker for breast cancer. Under the optimum conditions, the intensity of the upconversion luminescence (at excitation/emission wavelengths of 980/541 nm) is linearly proportional to the VEGF concentration in the 50 pM to 2000 pM concentration range, with a 6 pM detection limit. The method was applied to the determination of VEGF in spiked serum, typically at a 500 pM level, and gave recoveries that ranged from 98 to 113 %, with RSDs between 2.9 and 3.6 %. This makes it a viable tool for early diagnosis of breast cancer.

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Graphical abstract An upconversion luminescence based assay is presented for trace analysis of vascular endothelial growth factor. It is based on a sequence fragment-linked technique of target-induced aptamer (AP) and upconversion nanoparticles (UCNPs) and has potential application in the early diagnosis of breast cancer.

     

Chromatographic and luminescence properties of a 7-aminocoumarin derivative with peroxyoxalate chemiexcitation

A new derivatizing reagent for amines, luminarine 1, is described, and tested as its pentylamine derivative. Chromatographic separation in both normal- and reversed-phase systems was achieved. With fluorescence detection, the limit of detection was in the subpicomole range. With the optimization procedure for chemiluminescence detection, the limit of detection was 1 fmol in the normal-phase system (using bis (2,4-dinitrophenyl) oxalate and hydrogen peroxide), and 6 fmol in the reversed-phase system (using bis (2,4,6-trichlorophenyl) oxalate and hydrogen peroxide ), but the coefficient of variation was lower for the latter system (3.4% vs. 5.8%).