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1.
BMC Microbiol ; 22(1): 119, 2022 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-35501697

RESUMEN

BACKGROUND: 16S rDNA-PCR for the identification of a bacterial species is an established method. However, the DNA extraction reagents as well as the PCR reagents may contain residual bacterial DNA, which consequently generates false-positive PCR results. Additionally, previously used methods are frequently time-consuming. Here, we describe the results obtained with a new technology that uses DNA-free reagents for automated DNA extraction and subsequent real time PCR using sterile clinical specimens. RESULTS: In total, we compared 803 clinical specimens using real time PCR and culturing. The clinical specimens were mainly of orthopedic origin received at our diagnostic laboratory. In 595 (74.1%) samples, the results were concordant negative, and in 102 (12.7%) the results were concordant positive. A total of 170 (21.2%) clinical specimens were PCR-positive, of which 62 (36.5% from PCR positive, 7.7% in total) gave an additional benefit to the patient since only the PCR result was positive. Many of these 62 positive specimens were strongly positive based on crossingpoint values (54% < Cp 30), and these 62 positive clinical specimens were diagnosed as medically relevant as well. Thirty-eight (4.2%) clinical specimens were culture-positive (25 of them were only enrichment culture positive) but PCR-negative, mainly for S. epidermidis, S. aureus and C. acnes. The turnaround times for negative specimens were 4 hours (automated DNA extraction and real time PCR) and 1 working day for positive specimens (including Sanger sequencing). Melting-curve analysis of SYBR Green-PCR enables the differentiation of specific and unspecific PCR products. Using Ripseq, even mixed infections of 2 bacterial species could be resolved. CONCLUSIONS: For endocarditis cases, the added benefit of PCR is obvious. The crucial innovations of the technology enable timely reporting of explicit reliable results for adequate treatment of patients. Clinical specimens with truly PCR-positive but culture-negative results represent an additional benefit for patients. Very few results at the detection limit still have to be critically examined.


Asunto(s)
Bacterias , Staphylococcus aureus , Bacterias/genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , ADN Ribosómico/genética , Humanos , Indicadores y Reactivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Staphylococcus aureus/genética
2.
J Am Chem Soc ; 144(16): 7189-7197, 2022 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-35436110

RESUMEN

A multi-component approach to structurally complex organosulfur products is described via the nickel-catalyzed 1,2-carbosulfenylation of unactivated alkenes with organoboron nucleophiles and tailored organosulfur electrophiles. The key to the development of this transformation is the identification of a modular N-alkyl-N-(arylsulfenyl)arenesulfonamide family of sulfur electrophiles. Tuning the electronic and steric properties of the leaving group in these reagents controls pathway selectivity, favoring three-component coupling and suppressing side reactions, as examined via computational studies. The unique syn-stereoselectivity differs from traditional electrophilic sulfenyl transfer processes involving a thiiranium ion intermediate and arises from the directed arylnickel(I) migratory insertion mechanism, as elucidated through reaction kinetics and control experiments. Reactivity and regioselectivity are facilitated by a collection of monodentate, weakly coordinating native directing groups, including sulfonamides, alcohols, amines, amides, and azaheterocycles.


Asunto(s)
Alquenos , Níquel , Catálisis , Indicadores y Reactivos , Azufre
3.
Nat Commun ; 13(1): 1815, 2022 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-35383192

RESUMEN

The ability to detect and target ß cells in vivo can substantially refine how diabetes is studied and treated. However, the lack of specific probes still hampers a precise characterization of human ß cell mass and the delivery of therapeutics in clinical settings. Here, we report the identification of two RNA aptamers that specifically and selectively recognize mouse and human ß cells. The putative targets of the two aptamers are transmembrane p24 trafficking protein 6 (TMED6) and clusterin (CLUS). When given systemically in immune deficient mice, these aptamers recognize the human islet graft producing a fluorescent signal proportional to the number of human islets transplanted. These aptamers cross-react with endogenous mouse ß cells and allow monitoring the rejection of mouse islet allografts. Finally, once conjugated to saRNA specific for X-linked inhibitor of apoptosis (XIAP), they can efficiently transfect non-dissociated human islets, prevent early graft loss, and improve the efficacy of human islet transplantation in immunodeficient in mice.


Asunto(s)
Aptámeros de Nucleótidos , Clusterina , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Proteínas de Transporte Vesicular , Animales , Aptámeros de Nucleótidos/genética , Clusterina/genética , Rechazo de Injerto , Humanos , Indicadores y Reactivos , Islotes Pancreáticos/metabolismo , Ratones , ARN/metabolismo , Proteínas de Transporte Vesicular/genética
4.
Sensors (Basel) ; 22(8)2022 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-35458878

RESUMEN

Microfluidic paper-based analytical devices (µPADs) represent one of the promising green analytical strategies for low-cost and simple determination of various analytes. The actual task is the development of such devices for quantitation of antioxidants, e.g., flavonoids. In this paper, possibilities of a novel three-reagent µPAD including silver nitrate, 4-nitrophenyldiazonium tetrafluoroborate, and iron(III) chloride as reagents are assessed with respect to the determination of dihydroquercetin. It is shown that all the three reagents produce different colorimetric responses that can be detected by a mini-spectrophotometer-monitor calibrator or by a smartphone. The method is applicable to direct measuring high contents of dihydroquercetin (the linearity range is 0.026-1 mg mL-1, and the limit of detection is 7.7 µg mL-1), which is favorable for many dietary supplements. The analysis of a food supplement was possible with the relative standard deviations of 9-26%, which is satisfactory for quantitative and semiquantitative determinations. It was found that plotting a calibration graph in 3D space of the three reagents' responses allows us to distinguish dihydroquercetin from its close structural analogue, quercetin.


Asunto(s)
Colorimetría , Técnicas Analíticas Microfluídicas , Colorimetría/métodos , Compuestos Férricos , Indicadores y Reactivos , Papel , Quercetina/análogos & derivados
5.
Sensors (Basel) ; 22(8)2022 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-35458994

RESUMEN

The maintenance of uric acid levels is crucial for the human body. In this study, the feasibility of using portable ultraviolet (UV) spectrophotometry to measure the uric acid of spot urine without the need to add reagents has been demonstrated for the first time. UV spectral analysis has been used to inspect the uric acid concentration in urine. It is found that the absorption spectrum of urine has a high correlation with the concentration of uric acid at a wavelength of around 290-300 nm. Uric acid levels measured with a spectral analyzer compared to uric acid concentrations measured with a traditional biochemical analysis showed good agreement. The portable prototype is label-free and capable of displaying the inspection result of each measurement within 10 s. In the long run, this device can assist people in checking uric acid levels of spot urine with higher frequency and can adjust diet or medication in real time for more efficient health management.


Asunto(s)
Dieta , Ácido Úrico , Humanos , Indicadores y Reactivos , Espectrofotometría Ultravioleta , Ácido Úrico/orina
6.
Methods Enzymol ; 665: 105-133, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35379431

RESUMEN

Peptide natural products produced by microorganisms have attracted considerable attention as ideal drug leads owing to their low toxicity and high specificity toward target proteins compared with small-sized molecules. These peptide drug leads possess unusual structural features that endow them with unique biological activities and ideal physicochemical properties. In particular, these peptides often have d-amino acids, and therefore the absolute configuration of the component amino acids must be determined during the structural elucidation of newly isolated peptide drug leads. Recently, we developed highly sensitive labeling reagents FDVDA and FDLDA for the structural determination of the component amino acids in peptides. In an LC-MS-based structural study of peptides, these reagents enabled us to detect infinitesimal amounts of amino acids derived from mild degradation of the samples; we named this method the highly sensitive-advanced Marfey's method (HS-advanced Marfey's method). Herein, we first report the synthesis of these reagents and the LC-MS protocols for highly sensitive analyses of amino acids. Second, we discuss applications of the design concept. Specifically, two other labeling reagents were synthesized and their performance in terms of detection sensitivity was evaluated. These investigations provide insights on the structure-property relationship of these labeling reagents and therefore facilitate future on-demand structural modifications of the reagents to enhance their hydrophobicity, stability, and affinity for use with specialized HPLC columns. Finally, we demonstrated the effectiveness of our highly sensitive labeling reagents by using them to detect component amino acids in peptide natural products.


Asunto(s)
Aminas , Aminoácidos , Aminas/química , Aminoácidos/química , Cromatografía Liquida , Indicadores y Reactivos , Estereoisomerismo
7.
Methods Enzymol ; 665: 49-71, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35379443

RESUMEN

Converting discrete microbial metabolites into chemical probes for chemical biology and medicinal chemistry studies is typically preceded by lengthy purification and chemical derivatization processes. Standard practice involves purifying the target microbial metabolite from culture, followed by derivatization and/or conjugation chemistry to convert the pure metabolite into a tagged species. This multistep approach can pose difficulties in generating useful yields of chemical probes, particularly in the case of low-abundant metabolites, as common in metabolomes. This chapter describes a methodological approach to simplify the steps towards generating chemical probes from complex mixtures, that combines: (a) tailored purification processes; (b) compound identification using state-of-the-art tandem mass spectrometry and data-dependent fragmentation; and (c) in situ bioorthogonal bioconjugation chemistries. The combination of these methods, as illustrated by the conversion of a set of amine-bearing metabolites to the cognate azide analogs suitable for biotinylation through azide-alkyne cycloaddition, describes a powerful approach to access new chemical probes of low-abundant metabolites that might otherwise be inaccessible using traditional methods.


Asunto(s)
Azidas , Química Clic , Alquinos/química , Azidas/química , Química Clic/métodos , Reacción de Cicloadición , Indicadores y Reactivos
8.
Chem Commun (Camb) ; 58(35): 5387-5390, 2022 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-35416220

RESUMEN

Sulfonimidoyl halides have previously shown poor stability and selectivity in reaction with organometallic reagents. Here we report the preparation of enantioenriched sulfonimidoyl fluorides and their stereospecific reaction at sulfur with Grignard reagents. Notably the first enantioenriched alkyl sulfonimidoyl fluorides are prepared, including methyl. The nature of the N-group is important to the success of the stereocontrolled sequence to sulfoximines.


Asunto(s)
Fluoruros , Azufre , Indicadores y Reactivos
9.
J Am Chem Soc ; 144(15): 6928-6935, 2022 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-35380808

RESUMEN

We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The indicators can be specifically localized to different cellular compartments and are compatible with both fluorescence and bioluminescence readouts through conjugation to HaloTag fusion proteins. Importantly, their increase in fluorescence upon localization enables no-wash live-cell imaging, which greatly facilitates their use in biological assays. Applications as fluorescent indicators in rat hippocampal neurons include the detection of single action potentials and of calcium fluxes in the endoplasmic reticulum. Applications as bioluminescent indicators include the recording of the pharmacological modulation of nuclear calcium in high-throughput compatible assays. The versatility and remarkable ease of use of these indicators make them powerful tools for bioimaging and bioassays.


Asunto(s)
Calcio , Colorantes , Animales , Calcio/metabolismo , Color , Colorantes Fluorescentes , Indicadores y Reactivos , Microscopía Fluorescente/métodos , Ratas , Rodaminas
10.
Zhongguo Yi Liao Qi Xie Za Zhi ; 46(2): 230-232, 2022 Mar 30.
Artículo en Chino | MEDLINE | ID: mdl-35411757

RESUMEN

The management of in vitro diagnostic reagents has always been a concern. This paper describes the application of SPD medical consumables fine management system in our hospital. Relying on the brand-new management mode, the whole process from supplier qualification certificate management, in vitro diagnostic reagent procurement management, secondary warehouse management, and then to the use process traceability was realized. The monthly cost of in vitro diagnostic reagents can be accurately counted, which effectively controls the cost of in vitro diagnostic reagents.


Asunto(s)
Hospitales , Indicadores y Reactivos
11.
Org Lett ; 24(15): 2815-2820, 2022 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-35412324

RESUMEN

Herein, we described an efficient method for the construction of highly functionalized diazirines from the carbohydrazide and diazo-substituted hypervalent iodine reagents. Unambiguous transformation has been designed with user applicable and easy practicable conditions. Remarkably, d-glucose, menthol, aspirin, proline, and lithocholic acid were efficiently diazirinated. Furthermore, the method is mild, robust, and highly selective, which successfully converted a variety of aryl, alkyl, benzyl, and heterocyclic hydrazides into the corresponding diazirine derivatives.


Asunto(s)
Yodo , Diazometano , Indicadores y Reactivos , Yoduros
12.
J Pharm Biomed Anal ; 214: 114738, 2022 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-35395607

RESUMEN

The determination of α-keto acids derived from amino acids is currently the most reliable approach for the diagnosis of some congenital metabolic diseases. An HPLC method for the simultaneous measurement of selected α-keto acids in dried blood samples has been developed and evaluated. Blood spot samples from a group of healthy blood donors were collected onto #903 Specimen Collection Paper. Prior the separation, the α-keto acids were derivatized with 1,2-diamino-4,5-dimethoxybenzene to the corresponding 3-substituted-6,7-dimethoxy-2(1 H)-quinoxalinol derivatives. For the separation, a reverse-phase column LichroCart 125-4, Purospher RP-18e, 5 µm, was used. The mixture of 25% ACN in deionized water (mobile phase A) and 100% ACN (mobile phase B) were used for a gradient elution of α-keto acids derivatives. Analytical performance of this method is satisfactory for all α-keto acids. The intra-assay and inter-assay coefficients were below 10% and recoveries were close to 100%. We have developed relatively simple, rapid, selective and sufficiently sensitive HPLC method with fluorescence detection for the determination of selected α-keto acids in dried blood samples. The presented method is suitable for clinical testing purposes.


Asunto(s)
Aminoácidos , Cetoácidos , Cromatografía Líquida de Alta Presión/métodos , Indicadores y Reactivos
13.
J Org Chem ; 87(8): 5451-5455, 2022 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-35364809

RESUMEN

The accessibility of bromonitromethane has declined in recent years, limiting its viability as a reagent for chemical synthesis. The reinvestigation and optimization of a variety of preparations, and the development of safe operating principles, are described. The reproducible protocol described here leverages the effectiveness of hydroxide for nitromethane bromination while respecting its incompatibility with the product it forms. This careful balance was achieved at scales up to 56 g, resulting in a reproducible procedure that provides straightforward, sustainable, and affordable access to this critical reagent.


Asunto(s)
Etano , Nitrocompuestos , Etano/análogos & derivados , Halogenación , Indicadores y Reactivos
14.
J Org Chem ; 87(8): 5437-5441, 2022 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-35377641

RESUMEN

A new group of nido-carboranyl derivatives of natural (S)-amino acids containing from 9 to 18 boron atoms was obtained in good yields as a result of acylation of 3-amino-1,2-dicarba-closo-dodecaborane followed by deboronation. The proposed approach is convenient and based on the use of readily available reagents and is suitable for the synthesis of enantiopure nido-carboranyl derivatives of amino acids with various side chains, including water-soluble boron-containing amino acids (17 examples).


Asunto(s)
Aminoácidos , Boro , Compuestos de Boro/química , Indicadores y Reactivos
15.
J Hazard Mater ; 433: 128724, 2022 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-35398794

RESUMEN

Electrokinetic in-situ chemical oxidation (EK-ISCO) has attracted much attention during remediation of organic contaminated soil. Oxidants in EK-ISCO brings high cost and negative effects on soil physicochemical properties. In this study, a novel approach of combined electrokinetic treatment and anode oxidation was investigated to remediate phenanthrene polluted soils without adding oxidants. The fabricated Ti4O7 acted as anode, and could generate •OH at the rate of 9.31 × 10-7 mol h-1 at current 5.10 mA cm-2 through direct H2O electrolysis. Electro-osmotic flow (EOF) was used to transport phenanthrene to anode for the subsequent degradation. Sandy soil, fluvo-aquic soil and red soil were selected as typical soil samples, because pH and buffer capacity were two important factors affecting the direction of EOF. Strategies were developed to regulate the direction of EOF, including adding CEM membrane, maintaining soil pH at 3.5-4.0 and mixing solution from anode and cathode chambers. After treatment, more than 81.9% of phenanthrene was removed without adding any oxidants, and the remediated soil had low toxicity for Lolium perenne growth based on 3-d cultivation results. The results indicated that EK-AO had the advantage of less energy consumption and superior environmental friendliness than traditional EK-ISCO.


Asunto(s)
Restauración y Remediación Ambiental , Contaminantes del Suelo , Electrodos , Indicadores y Reactivos , Oxidantes , Fenantrenos , Suelo , Contaminantes del Suelo/análisis
16.
J Am Chem Soc ; 144(17): 7517-7530, 2022 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-35471019

RESUMEN

Oligophosphates play essential roles in biochemistry, and considerable research has been directed toward the synthesis of both naturally occurring oligophosphates and their synthetic analogues. Greater attention has been given to mono-, di-, and triphosphates, as these are present in higher concentrations biologically and easier to synthesize. However, extended oligophosphates have potent biochemical roles, ranging from blood coagulation to HIV drug resistance. Sporadic reports have slowly built a niche body of literature related to the synthesis and study of extended oligophosphates, but newfound interests and developments have the potential to rapidly expand this field. Here we report on current methods to synthesize oligophosphates longer than triphosphates and comment on the most important future directions for this area of research. The state of the art has provided fairly robust methods for synthesizing nucleoside 5'-tetra- and pentaphosphates as well as dinucleoside 5',5'-oligophosphates. Future research should endeavor to push such syntheses to longer oligophosphates while developing synthetic methodologies for rarer morphologies such as 3'-nucleoside oligophosphates, polyphosphates, and phosphonate/thiophosphate analogues of these species.


Asunto(s)
Nucleósidos , Organofosfonatos , Indicadores y Reactivos , Polifosfatos
17.
Spectrochim Acta A Mol Biomol Spectrosc ; 276: 121188, 2022 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-35395463

RESUMEN

Remdesivir was approved by the Food and Drug Administration for the treatment of COVID -19 in hospitalized adult and pediatric patients. Application of computational calculations for choosing the sensitive reagent in spectrophotometric quantitative analysis is very limited. Computational and theoretical studies were used for choosing the best acid dye for selective visible spectrophotometric quantitative analysis of remdesivir. The calculations were performed using Gaussian 03 software with the density functional theory method using B3LYP/6-31G(d) basis set. The theoretical studies revealed that bromophenol blue is a better match for remdesivir than other acid dyes due to the higher calculated interaction energy. The proposed method was based on the reaction of remdesivir with the computationally selected acid dye bromophenol blue to form a yellow ion-pair complex. The spectra showed absorption peaks at 418 nm. Various factors affecting the reaction were optimized. The method was successfully applied for the determination of remdesivir in the pharmaceutical preparation with good accuracy and precision. Beer's law was observed in the concentration range of 2-12 µg/mL of remdesivir. The proposed reaction was used as a basis for the spectrophotometric determination of remdesivir in pure form and in the pharmaceutical preparation.


Asunto(s)
COVID-19 , Colorantes , Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Azul de Bromofenol/análisis , COVID-19/tratamiento farmacológico , Niño , Humanos , Indicadores y Reactivos , Preparaciones Farmacéuticas/análisis , Estados Unidos
18.
Sci Rep ; 12(1): 6201, 2022 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-35418664

RESUMEN

The Drosophila model has become a leading platform for investigating mechanisms that drive feeding behavior and the effect of diet on physiological outputs. Several methods for tracking feeding behavior in flies have been developed. One method, consumption-excretion or Con-Ex, provides flies with media labeled with dye and then quantifies the amount of dye excreted into the vial as a measure of consumption. We previously found that Blue 1 and Orange 4 work well in Con-Ex and can be used as a dye pair in food preference studies. We have expanded our development of Con-Ex by identifying two additional dyes, Orange G and Yellow 10, that detect the anticipated effects of mating status, strain, starvation and nutrient concentration. Additionally, Orange G and Yellow 10 accumulate linearly in excretion products out to 48 h and the excreted volumes of these two dyes reflect the volumes consumed. Orange G also works with Blue 1 as a dye pair in food preference studies. Finally, consumption of Blue 1, Orange 4, Orange G or Yellow 10 does not affect ethanol sedation or rapid tolerance to ethanol. Our findings establish that Orange G and Yellow 10, like Blue 1 and Orange 4, are suitable for use in Con-Ex.


Asunto(s)
Colorantes , Preferencias Alimentarias , Animales , Drosophila , Ingestión de Alimentos , Etanol , Indicadores y Reactivos
19.
Acc Chem Res ; 55(9): 1324-1336, 2022 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-35435655

RESUMEN

ConspectusReagent instability reduces the efficiency of chemical processes, and while much effort is devoted to reaction optimization, less attention is paid to the mechanistic causes of reagent decomposition. Indeed, the response is often to simply use an excess of the reagent. Two reaction classes with ubiquitous examples of this are the Suzuki-Miyaura cross-coupling of boronic acids/esters and the transfer of CF3 or CF2 from the Ruppert-Prakash reagent, TMSCF3. This Account describes some of the overarching features of our mechanistic investigations into their decomposition. In the first section we summarize how specific examples of (hetero)arylboronic acids can decompose via aqueous protodeboronation processes: Ar-B(OH)2 + H2O → ArH + B(OH)3. Key to the analysis was the development of a kinetic model in which pH controls boron speciation and heterocycle protonation states. This method revealed six different protodeboronation pathways, including self-catalysis when the pH is close to the pKa of the boronic acid, and protodeboronation via a transient aryl anionoid pathway for highly electron-deficient arenes. The degree of "protection" of boronic acids by diol-esterification is shown to be very dependent on the diol identity, with six-membered ring esters resulting in faster protodeboronation than the parent boronic acid. In the second section of the Account we describe 19F NMR spectroscopic analysis of the kinetics of the reaction of TMSCF3 with ketones, fluoroarenes, and alkenes. Processes initiated by substoichiometric "TBAT" ([Ph3SiF2][Bu4N]) involve anionic chain reactions in which low concentrations of [CF3]- are rapidly and reversibly liberated from a siliconate reservoir, [TMS(CF3)2][Bu4N]. Increased TMSCF3 concentrations reduce the [CF3]- concentration and thus inhibit the rates of CF3 transfer. Computation and kinetics reveal that the TMSCF3 intermolecularly abstracts fluoride from [CF3]- to generate the CF2, in what would otherwise be an endergonic α-fluoride elimination. Starting from [CF3]- and CF2, a cascade involving perfluoroalkene homologation results in the generation of a hindered perfluorocarbanion, [C11F23]-, and inhibition. The generation of CF2 from TMSCF3 is much more efficiently mediated by NaI, and in contrast to TBAT, the process undergoes autoacceleration. The process involves NaI-mediated α-fluoride elimination from [CF3][Na] to generate CF2 and a [NaI·NaF] chain carrier. Chain-branching, by [(CF2)3I][Na] generated in situ (CF2 + TFE + NaI), causes autoacceleration. Alkenes that efficiently capture CF2 attenuate the chain-branching, suppress autoacceleration, and lead to less rapid difluorocyclopropanation. The Account also highlights how a collaborative approach to experiment and computation enables mechanistic insight for control of processes.


Asunto(s)
Ésteres , Fluoruros , Alquenos/química , Ácidos Borónicos/química , Ésteres/química , Indicadores y Reactivos , Cinética
20.
J Vis Exp ; (181)2022 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-35435901

RESUMEN

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.


Asunto(s)
Calcio , Células Madre Pluripotentes Inducidas , Calcio/análisis , Evaluación Preclínica de Medicamentos , Humanos , Indicadores y Reactivos , Células Madre Pluripotentes Inducidas/química , Miocitos Cardíacos/química , Optogenética
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