RESUMEN
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are frequently used for preclinical cardiotoxicity testing and remain an important tool for confirming model-based predictions of drug effects in accordance with the comprehensive in vitro proarrhythmia assay (CiPA). Despite the considerable benefits hiPSC-CMs provide, concerns surrounding experimental reproducibility have emerged. We investigated the effects of temporal changes and experimental parameters on hiPSC-CM electrophysiology. iCell cardiomyocytes2 were cultured and biosignals were acquired using a microelectrode array (MEA) system (2-14 days). Continuous recordings revealed a 22.6% increase in the beating rate and 7.7% decrease in the field potential duration (FPD) during a 20-min equilibration period. Location-specific differences across a multiwell plate were also observed, with iCell cardiomyocytes2 in the outer rows beating 8.8 beats/min faster than the inner rows. Cardiac endpoints were also impacted by cell culture duration; from 2 to 14 days, the beating rate decreased (-12.7 beats/min), FPD lengthened (+257 ms), and spike amplitude increased (+3.3 mV). Cell culture duration (4-10 days) also impacted cardiomyocyte drug responsiveness (E-4031, nifedipine, isoproterenol). qRT-PCR results suggest that daily variations in cardiac metrics may be linked to the continued maturation of hiPSC-CMs in culture (2-30 days). Daily experiments were also repeated using a second cell line (Cor.4U). Collectively, our study highlights multiple sources of variability to consider and address when performing hiPSC-CM MEA studies. To improve reproducibility and data interpretation, MEA-based studies should establish a standardized protocol and report key experimental conditions (e.g., cell line, culture time, equilibration time, electrical stimulation settings, and raw data values).NEW & NOTEWORTHY We demonstrate that iCell cardiomyocytes2 electrophysiology measurements are impacted by deviations in experimental techniques including electrical stimulation protocols, equilibration time, well-to-well variability, and length of hiPSC-CM culture. Furthermore, our results indicate that hiPSC-CM drug responsiveness changes within the first 2 wk following defrost.
Asunto(s)
Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Reproducibilidad de los Resultados , Factores de Tiempo , Potenciales de Acción/efectos de los fármacos , Células Cultivadas , Isoproterenol/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Microelectrodos , Línea Celular , CardiotoxicidadRESUMEN
In silico modeling offers an opportunity to supplement and accelerate cardiac safety testing. With in silico modeling, computational simulation methods are used to predict electrophysiological interactions and pharmacological effects of novel drugs on critical physiological processes. The O'Hara-Rudy's model was developed to predict the response to different ion channel inhibition levels on cardiac action potential duration (APD) which is known to directly correlate with the QT interval. APD data at 30% 60% and 90% inhibition were derived from the model to delineate possible ventricular arrhythmia scenarios and the marginal contribution of each ion channel to the model. Action potential values were calculated for epicardial, myocardial, and endocardial cells, with action potential curve modeling. This study assessed cardiac ion channel inhibition data combinations to consider when undertaking in silico modeling of proarrhythmic effects as stipulated in the Comprehensive in Vitro Proarrhythmia Assay (CiPA). As expected, our data highlight the importance of the delayed rectifier potassium channel (IKr) as the most impactful channel for APD prolongation. The impact of the transient outward potassium channel (Ito) inhibition on APD was minimal while the inward rectifier (IK1) and slow component of the delayed rectifier potassium channel (IKs) also had limited APD effects. In contrast, the contribution of fast sodium channel (INa) and/or L-type calcium channel (ICa) inhibition resulted in substantial APD alterations supporting the pharmacological relevance of in silico modeling using input from a limited number of cardiac ion channels including IKr, INa, and ICa, at least at an early stage of drug development.
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Potenciales de Acción , Simulación por Computador , Canales Iónicos , Miocitos Cardíacos , Potenciales de Acción/efectos de los fármacos , Humanos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Canales Iónicos/fisiología , Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/fisiopatologíaRESUMEN
In recent decades, drug development costs have increased by approximately a hundredfold, and yet about 1 in 7 licensed drugs are withdrawn from the market, often due to cardiotoxicity. This review considers whether technologies using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could complement existing assays to improve discovery and safety while reducing socioeconomic costs and assisting with regulatory guidelines on cardiac safety assessments. We draw on lessons from our own work to suggest a panel of 12 drugs that will be useful in testing the suitability of hiPSC-CM platforms to evaluate contractility. We review issues, including maturity versus complexity, consistency, quality, and cost, while considering a potential need to incorporate auxiliary approaches to compensate for limitations in hiPSC-CM technology. We give examples on how coupling hiPSC-CM technologies with Cas9/CRISPR genome engineering is starting to be used to personalize diagnosis, stratify risk, provide mechanistic insights, and identify new pathogenic variants for cardiovascular disease.
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Cardiotoxicidad/prevención & control , Descubrimiento de Drogas/métodos , Miocitos Cardíacos/efectos de los fármacos , Animales , Sistemas CRISPR-Cas/genética , Desarrollo de Medicamentos/métodos , Ingeniería Genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Medicina de Precisión/métodosRESUMEN
Among various cardiac safety concerns, proarrhythmia risks, including QT prolongation leading to Torsade de Pointes, is one of major cause for drugs being withdrawn (~45% 1975-2007). Preclinical study requires the evaluation of proarrhythmia using in silico, in vitro, and/or animal models. Considering that the primary consumers of prescription drugs are elderly patients, applications of "aging-in-a-dish" models would be appropriate for screening proarrhythmia risks. However, acquiring such models, including cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs), presents extensive challenges. We proposed the hypothesis that CMs differentiated from iPSCs derived from Hutchinson-Gilford progeria syndrome (HGPS, progeria) patients, an ultra-rare premature aging syndrome, can mimic the phenotypes of aging CMs. Our objective, therefore, was to examine this hypothesis by analyzing the response of 11 reference compounds utilized by the Food and Drug Administration (FDA)'s Comprehensive in vitro Proarrhythmia Assay (CiPA) using progeria and control CMs. As a sensitive surrogate marker of modulating cardiac excitation-contraction coupling, we evaluated drug-induced changes in calcium transient (CaT). We observed that the 80% CaT peak duration in the progeria CMs (0.98 ± 0.04 s) was significantly longer than that of control CMs (0.70 ± 0.05 s). Furthermore, when the progeria CMs were subjected to four doses of 11 compounds from low-, intermediate-, and high-risk categories, they demonstrated greater arrhythmia susceptibility than control cells, as shown through six-parameter CaT profile analyses. We also employed the regression analysis established by CiPA to classify the 11 reference compounds and compared proarrhythmia susceptibilities between the progeria and control CMs. This analysis revealed a greater proarrhythmia susceptibility in the progeria CMs compared to the control CMs. Interestingly, in both CMs, the compounds categorized as low risk did not exceed the safety risk threshold of 0.8. In conclusion, our study demonstrates increased proarrhythmia sensitivity in progeria CMs when tested with reference compounds. Future studies are needed to analyze underlying mechanisms and further validate our findings using a larger array of reference compounds.
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Células Madre Pluripotentes Inducidas , Progeria , Animales , Miocitos Cardíacos/fisiología , Preparaciones Farmacéuticas , EnvejecimientoRESUMEN
The goal of the CiPA initiative (Comprehensive in vitro Proarrhythmia Assay) was to assess a more accurate prediction of new drug candidate proarrhythmic severe liabilities such as torsades de pointes, for example. This new CiPA paradigm was partly based on in silico reconstruction of human ventricular cardiomyocyte action potential useful to identify repolarization abnormalities such early afterdepolarization (EAD), for example. Using the ToR-ORd algorithm (Tomek-Rodriguez-O'Hara-Rudy dynamic model), the aim of the present work was (i) to identify intracellular parameters leading to EAD occurrence under healthy and hypertrophic cardiomyopathy (HCM) conditions and (ii) to evaluate the prediction accuracy of compound torsadogenic risk based on EAD occurrence using a large set of 109 torsadogenic and non-torsadogenic compounds under both experimental conditions. In silico results highlighted the crucial involvement of Ca++ handling in the ventricular cardiomyocyte intracellular subspace compartment for the initiation of EAD, demonstrated by a higher amplitude of Ca++ release from junctional sarcoplasmic reticulum to subspace compartments (Jrel) measured at EAD take-off voltage in the presence vs. the absence of EAD initiated either by high IKr inhibition or by high enough concentration of a torsadogenic compound under both experimental conditions. Under healthy or HCM conditions, the prediction accuracy of the torsadogenic risk of compound based on EAD occurrence was observed to be 61 or 92%, respectively. This high accuracy under HCM conditions was discussed regarding its usefulness for cardiac safety pharmacology at least at early drug screening/preclinical stage of the drug development process.
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Potenciales de Acción/fisiología , Cardiomiopatía Hipertrófica/tratamiento farmacológico , Fármacos Cardiovasculares/farmacología , Miocitos Cardíacos/efectos de los fármacos , Torsades de Pointes/tratamiento farmacológico , Algoritmos , Calcio/metabolismo , Cardiomiopatía Hipertrófica/metabolismo , Simulación por Computador , Evaluación Preclínica de Medicamentos/métodos , Electrocardiografía/efectos de los fármacos , Humanos , Miocitos Cardíacos/fisiología , Medición de Riesgo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Torsades de Pointes/fisiopatologíaRESUMEN
Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal-recessive hereditary neuropathy causing congenital loss of pain sensation, thermoception, and perspiration. CIPA sometimes causes destructive spondyloarthropathy, the so-called Charcot spine, because of insensitivity to pain stimuli. Herein, we report a case of CIPA with severe spinal destruction treated by multiple spinal reconstructive surgeries and over 15 years of follow-up. A 15-year-old male patient who had been diagnosed with CIPA at the age of 17 months presented to his previous spine clinic with gait disturbance due to muscle weakness in his lower extremities. Imaging studies revealed that collapsed L3 and L4 vertebral bodies involved the spinal canal, and it was treated by L3-L4 instrumented posterior fusion. Fourteen years after surgery, the patient became unable to walk again due to spinal canal stenosis at the proximal fusion segment. An L2-L3 posterior interbody fusion alleviated his gait ability for 2 years; however, he became unable to stand again because of the collapsed fusion segment that caused severe lumbar kyphosis. Subsequently, a two-staged posterior and anterior fusion surgery from the lower thoracic spine to the pelvis was performed, and spinal fusion and neurological recovery were achieved 3 years after surgery. A kyphotic deformity in patients with CIPA-associated Charcot spine could be favorably treated by a long spinal fusion in combination with a reconstruction of an anterior spinal column. This case report provides a significant lesson for a treatment of CIPA-associated Charcot spine.
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Neuropatías Hereditarias Sensoriales y Autónomas , Cifosis , Espondiloartropatías , Adolescente , Canalopatías , Estudios de Seguimiento , Humanos , Lactante , Vértebras Lumbares , Masculino , Dolor , Insensibilidad Congénita al DolorRESUMEN
Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disease resulting from mutations in the NTRK1 gene encoding the neurotrophic tyrosine kinase-1 receptor. In this multicenter observational retrospective study, we investigated CIPA patients identified from French laboratories sequencing the NTRK1 gene, and seven patients were identified. Patients originated from France (2), Suriname (2), Mali (1), Kazakhstan (1), and Algeria (1). Mean age of patients was 9.8 years (4-20), four patients were female (57%), infant developmental milestones were delayed in four cases (57%), and four patients had a family history of consanguinity (57%). Mean age at diagnosis was 4.8 months (3-6), and all patients presented with pain insensitivity, anhidrosis, intellectual disability, self-mutilation, febrile episodes, impaired temperature perception, and autonomous nervous system impairment. Patients also showed an assortment of associated findings, including hyperactivity (86%), emotional lability (86%), joint deformities (71%), bone fractures (57%), abnormal sense of touch, vibration and position (50%), skin, hair and nails abnormalities (28%), and hypothermia episodes (28%). Two patients died at age 9 and 12 years from infection. In three cases, nerve conduction studies showed absent lower limbs sensory nerve action potentials. In one case, sensory nerve biopsy showed complete absence of unmyelinated fibers. Nine NTRK1 pathogenic variants were found, including three newly described mutations. This nationwide study confirms that NTRK1 gene-related CIPA is an extremely rare disorder and expands the genotypic spectrum of NTRK1 mutations.
Asunto(s)
Hipohidrosis/genética , Mutación/genética , Dolor/genética , Receptor trkA/genética , Niño , Preescolar , Femenino , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Humanos , Discapacidad Intelectual/genética , Masculino , Estudios RetrospectivosRESUMEN
The diverse function of the moonlighting proteins in Acinetobacter baumannii is highly associated with its virulence that had spurred renewed attention in recent years. The existing and newly formed hypothetical moonlighting proteins, evolve without jeopardizing the structural constraints of their original roles. It is yet uncertain and undefined to lucidly describe the functions of the moonlighting proteins in A. baumannii albeit its overwhelming evidences on few proteins. This commentary thus highlights the expression and occurrence of potent moonlighting proteins in A. baumannii, rendering virulence to the strains and the reasons to target the same portraying an active arena of research.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Acinetobacter baumannii/genética , Humanos , Virulencia , Factores de Virulencia/genéticaRESUMEN
In this review, we consider the pharmacology of potassium channels from the perspective of these channels as therapeutic targets. Firstly, we describe the three main families of potassium channels in humans and disease states where they are implicated. Secondly, we describe the existing therapeutic agents which act on potassium channels and outline why these channels represent an under-exploited therapeutic target with potential for future drug development. Thirdly, we consider the evidence desired in order to embark on a drug discovery programme targeting a particular potassium channel. We have chosen two "case studies": activators of the two-pore domain potassium (K2P) channel TREK-2 (K2P10.1), for the treatment of pain and inhibitors of the voltage-gated potassium channel KV1.3, for use in autoimmune diseases such as multiple sclerosis. We describe the evidence base to suggest why these are viable therapeutic targets. Finally, we detail the main technical approaches available to characterise the pharmacology of potassium channels and identify novel regulatory compounds. We draw particular attention to the Comprehensive in vitro Proarrhythmia Assay initiative (CiPA, https://cipaproject.org ) project for cardiac safety, as an example of what might be both desirable and possible in the future, for ion channel regulator discovery projects.
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Canales Iónicos , Canales de Potasio , Corazón , HumanosRESUMEN
Human stem cell-derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and "cellrhythmias" (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches and experimenter, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects, there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CM preparations to define "fit for purpose" applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable "fit for purpose" hSC-CM-based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings.
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Células Madre Adultas/efectos de los fármacos , Arritmias Cardíacas/inducido químicamente , Cardiotoxinas/toxicidad , Miocitos Cardíacos/efectos de los fármacos , Guías de Práctica Clínica como Asunto/normas , Estudios de Validación como Asunto , Células Madre Adultas/patología , Células Madre Adultas/fisiología , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Miocitos Cardíacos/patologíaRESUMEN
INTRODUCTION: The Safety Pharmacology Society (SPS) conducted a membership survey to examine industry practices related mainly to cardiovascular (CV) safety pharmacology (SP). METHODS: Questions addressed nonclinical study design, data analysis methods, drug-induced effects, and conventional and novel CV assays. RESULTS: The most frequent therapeutic area targeted by drugs developed by the companies/institutions that employ survey responders was oncology. The most frequently observed drug-mediated effects included an increased heart rate, increased arterial blood pressure, hERG (IKr) block, decreased arterial blood pressure, decreased heart rate, QTc prolongation, and changes in body temperature. Broadly implemented study practices included Latin square crossover study design with n = 4 for nonrodent CV studies, statistical analysis of data (eg, analysis of variance), use of arrhythmia detection software, and the inclusion of data from all study animals when integrating SP studies into toxicology studies. Most responders frequently used individual animal housing conditions. Responders commonly evaluated drug effects on multiple ion channels, but in silico modeling methods were used much less frequently. Most responders rarely measured the J-Tpeak interval in CV studies. Uncertainties relative to Standard for Exchange of Nonclinical Data applications for data derived from CV SP studies were common. Although available, the use of human induced pluripotent stem cell cardiomyocytes remains rare. The respiratory SP study was rarely involved with identifying drug-induced functional issues. Responders indicated that the study-derived no observed effect level was more frequently determined than the no observed adverse effect level in CV SP studies; however, a large proportion of survey responders used neither.
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Enfermedades Cardiovasculares/inducido químicamente , Evaluación Preclínica de Medicamentos/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Farmacología/métodos , Animales , Sistema Cardiovascular , Interpretación Estadística de Datos , Industria Farmacéutica , Humanos , Proyectos de Investigación , Encuestas y CuestionariosRESUMEN
The QT interval occupies a pivotal role in drug development as a surface biomarker of ventricular repolarization. The electrophysiologic substrate for QT prolongation coupled with reports of non-cardiac drugs producing lethal arrhythmias captured worldwide attention from government regulators eventuating in a series of guidance documents that require virtually all new chemical compounds to undergo rigorous preclinical and clinical testing to profile their QT liability. While prolongation or shortening of the QT interval may herald the appearance of serious cardiac arrhythmias, the positive predictive value of an abnormal QT measurement for these arrhythmias is modest, especially in the absence of confounding clinical features or a congenital predisposition that increases the risk of syncope and sudden death. Consequently, there has been a paradigm shift to assess a compound's cardiac risk of arrhythmias centered on a mechanistic approach to arrhythmogenesis rather than focusing solely on the QT interval. This entails both robust preclinical and clinical assays along with the emergence of concentration QT modeling as a primary analysis tool to determine whether delayed ventricular repolarization is present. The purpose of this review is to provide a comprehensive understanding of the QT interval and highlight its central role in early drug development.
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Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/fisiopatología , Desarrollo de Medicamentos/métodos , Electrocardiografía/métodos , Síndrome de QT Prolongado/tratamiento farmacológico , Síndrome de QT Prolongado/fisiopatología , Animales , Arritmias Cardíacas/diagnóstico , Corazón/efectos de los fármacos , Corazón/fisiopatología , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/fisiopatología , Humanos , Síndrome de QT Prolongado/diagnósticoRESUMEN
Bacterial cellulases are drawing increased attention as a means to obtain plentiful chemical feedstocks and fuels from renewable lignocellulosic biomass sources. Certain bacteria deploy a large extracellular multi-protein complex, called the cellulosome, to degrade cellulose. Scaffoldin, a key non-catalytic cellulosome component, is a large protein containing a cellulose-specific carbohydrate-binding module and several cohesin modules which bind and organize the hydrolytic enzymes. Despite the importance of the structure and protein/protein interactions of the cohesin module in the cellulosome, its structure in solution has remained unknown to date. Here, we report the backbone 1H, 13C and 15N NMR assignments of the Cohesin module 5 from the highly stable and active cellulosome from Clostridium thermocellum. These data reveal that this module adopts a tightly packed, well folded and rigid structure in solution. Furthermore, since in scaffoldin, the cohesin modules are connected by linkers we have also characterized the conformation of a representative linker segment using NMR spectroscopy. Analysis of its chemical shift values revealed that this linker is rather stiff and tends to adopt extended conformations. This suggests that the scaffoldin linkers act to minimize interactions between cohesin modules. These results pave the way towards solution studies on cohesin/dockerin's fascinating dual-binding mode.
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Proteínas Bacterianas/química , Proteínas de Ciclo Celular/química , Celobiosa/química , Proteínas Cromosómicas no Histona/química , Clostridium thermocellum/química , Complejos Multiproteicos/química , Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/genética , Celobiosa/genética , Proteínas Cromosómicas no Histona/genética , Clostridium thermocellum/genética , Complejos Multiproteicos/genética , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , CohesinasRESUMEN
We analyzed torsadogenic and pharmacokinetic profile of E-4031 using chronic atrioventricular block dogs. E-4031 in intravenous doses of 0.03, 0.1 and 0.3 mg/kg over 10 min prolonged QT/QTc, and increased short-term variability of QT in a dose-related manner (n = 4), resulting in onset of torsade de pointes in 1 animal after the middle dose and 4 animals after the high dose, while it attained peak plasma concentrations of 16.5, 60.5 and 182.5 ng/mL at 10 min after their start of administration, respectively (n = 2). These results bridge the gap of information between in vitro proarrhythmia assay and clinical observation in human subjects.
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Antiarrítmicos/efectos adversos , Antiarrítmicos/farmacocinética , Bloqueo Atrioventricular/fisiopatología , Electrocardiografía/efectos de los fármacos , Piperidinas/efectos adversos , Piperidinas/farmacocinética , Piridinas/efectos adversos , Piridinas/farmacocinética , Torsades de Pointes/inducido químicamente , Animales , Antiarrítmicos/administración & dosificación , Bloqueo Atrioventricular/complicaciones , Enfermedad Crónica , Perros , Relación Dosis-Respuesta a Droga , Humanos , Infusiones Intravenosas , Piperidinas/administración & dosificación , Piridinas/administración & dosificación , Torsades de Pointes/etiologíaRESUMEN
Cardiac safety assessment is challenging because a better understanding of torsadogenic mechanisms beyond hERG blockade and QT interval prolongation is necessary for patient safety. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a new human cell-based platform to assess cardiac safety in non-clinical testing during drug development. The multi-electrode array (MEA) platform is a promising electrophysiological technology to assess QT interval prolongation and proarrhythmic potential of drug candidates using hiPSC-CMs. The Japan iPS Cardiac Safety Assessment (JiCSA) has established an MEA protocol to evaluate the applicability of hiPSC-CMs for assessing the torsadogenic potential of compounds and completed a large-scale validation study using 60 compounds. During our study, an international multi-site study of hiPSC-CMs was performed by the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative using 28 compounds. We have comparatively analyzed our JiCSA datasets with those of CiPA using the CiPA logistical and ordinal linear regression model. Regardless of the protocol differences, the evaluation results of the 28 compounds were very similar and highly predictable for torsadogenic risks. Thus, an MEA-based approach using hiPSC-CMs would be a standard testing method to evaluate proarrhythmic potentials. This review paper would provide new insights into the hiPSC-CMs/MEA method required for its regulatory use.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/efectos de los fármacos , Torsades de Pointes/inducido químicamente , Bioensayo , Humanos , Miocitos Cardíacos/fisiología , Medición de RiesgoRESUMEN
Pyrogallol is a valuable phenolic compound and displays various physiological and pharmaceutical functions. Chemical synthesis of pyrogallol suffered from many issues, including environmental pollution, high cost, and low yield. Here, to address the above drawbacks, an artificial pathway for de novo pyrogallol production was established and this pathway only needed two exogenous enzymes (Y385F/T294A PobA and 3,4-dihydroxybenzoic acid decarboxylase (PDC)). Y385F/T294A PobA is a mutant of PobA which is a hydroxylase from Pseudomonas aeruginosa, while PDC is a decarboxylase from Klebsiella pneumoniae subsp. pneumoniae. First, the conversion efficiency of PDC was tested and 1800 ± 100 mg/L pyrogallol was generated from 4 g/L gallic acid (GA). Subsequently, assembly of the whole pathway enabled 33 ± 6 mg/L pyrogallol production from simple carbon sources. After that, based on the assembling property of CipA (a hydrophobic protein) and to enhance the hydroxylation of 3,4-dihydroxybenzoic acid, CipA was employed to organize its fusion (Y385F/T294A PobA) into protein crystalline inclusions (PCIs). Remarkably, the formation of CipA-Y385F/T294A PobA PCIs increased the pyrogallol production to 60 ± 6 mg/L, a 1.8 ± 0.4-fold higher value as compared to the strain without enzyme self-assembly. Additionally, the titer of pyrogallol was enhanced to 80 ± 1 mg/L through yeast extract concentration optimization. This work not only realizes the biosynthesis of pyrogallol from renewable carbon sources but also demonstrates that using CipA-mediating enzyme self-assembly could reinforce the hydroxylation efficiency of Y385F/T294A PobA, resulting in the enhancement of pyrogallol production.
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Carboxiliasas/metabolismo , Escherichia coli/metabolismo , Oxigenasas de Función Mixta/metabolismo , Pirogalol/metabolismo , Carbono/metabolismo , Medios de Cultivo/química , ADN Bacteriano/genética , Escherichia coli/genética , Ácido Gálico/metabolismo , Hidroxibenzoatos/metabolismo , Cuerpos de Inclusión/metabolismo , Indoles/metabolismo , Microbiología Industrial , Klebsiella pneumoniae/enzimología , Propionatos/metabolismo , Pseudomonas aeruginosa/enzimologíaRESUMEN
Hereditary sensory and autonomic neuropathy type IV (HSAN IV) is an autosomal recessive disorder characterized by a complete lack of pain perception and anhidrosis. Here, we studied a cohort of seven patients with HSAN IV and describe a comprehensive functional analysis of seven novel NTRK1 missense mutations, c.1550G >A, c.1565G >A, c.1970T >C, c.2096T >C, c.2254T >A, c.2288G >C, and c.2311C >T, corresponding to p.G517E, p.G522E, p.L657P, p.I699T, p.C752S, p.C763S, and p.R771C, all of which were predicted pathogenic by in silico analysis. The results allowed us to assess the pathogenicity of each mutation and to gain novel insights into tropomyosin receptor kinase A (TRKA) downstream signaling. Each mutation was systematically analyzed for TRKA glycosylation states, intracellular and cell membrane expression patterns, nerve growth factor stimulated TRKA autophosphorylation, TRKA-Y496 phosphorylation, PLCγ activity, and neurite outgrowth. We showed a diverse range of functional effects: one mutation appeared fully functional, another had partial activity in all assays, one mutation affected only the PLCγ pathway and four mutations were proved null in all assays. Thus, we conclude that complete abolition of TRKA kinase activity is not the only pathogenic mechanism underlying HSAN IV. By corollary, the assessment of the clinical pathogenicity of HSAN IV mutations is more complex than initially predicted and requires a multifaceted approach.
Asunto(s)
Neuropatías Hereditarias Sensoriales y Autónomas/genética , Neuropatías Hereditarias Sensoriales y Autónomas/metabolismo , Mutación Missense , Receptor trkA/genética , Receptor trkA/metabolismo , Alelos , Línea Celular , Biología Computacional/métodos , Análisis Mutacional de ADN , Orden Génico , Estudios de Asociación Genética , Sitios Genéticos , Predisposición Genética a la Enfermedad , Genotipo , Glicosilación , Neuropatías Hereditarias Sensoriales y Autónomas/diagnóstico , Humanos , Imagen Molecular , Neuritas/metabolismo , Fosfolipasa C gamma/metabolismo , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptor trkA/química , Proteínas Recombinantes de Fusión , Análisis de Secuencia de ADN , Transducción de SeñalRESUMEN
Cellulosomes are considered to be one of the most efficient systems for the degradation of plant cell wall polysaccharides. The central cellulosome component comprises a large, noncatalytic protein subunit called scaffoldin. Multiple saccharolytic enzymes are incorporated into the scaffoldins via specific high-affinity cohesin-dockerin interactions. Recently, the regulation of genes encoding certain cellulosomal components by multiple RNA polymerase alternative σI factors has been demonstrated in Clostridium (Ruminiclostridium) thermocellum In the present report, we provide experimental evidence demonstrating that the C. thermocellum cipA gene, which encodes the primary cellulosomal scaffoldin, is regulated by several alternative σI factors and by the vegetative σA factor. Furthermore, we show that previously suggested transcriptional start sites (TSSs) of C. thermocellum cipA are actually posttranscriptional processed sites. By using comparative bioinformatic analysis, we have also identified highly conserved σI- and σA-dependent promoters upstream of the primary scaffoldin-encoding genes of other clostridia, namely, Clostridium straminisolvens, Clostridium clariflavum, Acetivibrio cellulolyticus, and Clostridium sp. strain Bc-iso-3. Interestingly, a previously identified TSS of the primary scaffoldin CbpA gene of Clostridium cellulovorans matches the predicted σI-dependent promoter identified in the present work rather than the previously proposed σA promoter. With the exception of C. cellulovorans, both σI and σA promoters of primary scaffoldin genes are located more than 600 nucleotides upstream of the start codon, yielding long 5'-untranslated regions (5'-UTRs). Furthermore, these 5'-UTRs have highly conserved stem-loop structures located near the start codon. We propose that these large 5'-UTRs may be involved in the regulation of both the primary scaffoldin and other cellulosomal components.IMPORTANCE Cellulosome-producing bacteria are among the most effective cellulolytic microorganisms known. This group of bacteria has biotechnological potential for the production of second-generation biofuels and other biocommodities from cellulosic wastes. The efficiency of cellulose hydrolysis is due to their cellulosomes, which arrange enzymes in close proximity on the cellulosic substrate, thereby increasing synergism among the catalytic domains. The backbone of these multienzyme nanomachines is the scaffoldin subunit, which has been the subject of study for many years. However, its genetic regulation is poorly understood. Hence, from basic and applied points of view, it is imperative to unravel the regulatory mechanisms of the scaffoldin genes. The understanding of these regulatory mechanisms can help to improve the performance of the industrially relevant strains of C. thermocellum and related cellulosome-producing bacteria en route to the consolidated bioprocessing of biomass.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Celulosa/metabolismo , Celulosomas/metabolismo , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones no Traducidas 5' , Hidrólisis , Regiones Promotoras Genéticas , Factor sigma/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Hereditary sensory autonomic neuropathy type IV (HSAN-IV) is a rare autosomal recessive disorder that usually begins in infancy and is characterized by anhidrosis, insensitivity to noxious stimuli leading to self-mutilating behavior, and intellectual disability. HSAN-IV is caused by mutations in the neurotrophic tyrosine kinase receptor type 1 gene, NTRK1, encoding the high-affinity receptor of nerve growth factor (NGF) which maps to chromosome 1q21-q22. Patients with HSAN-IV lack all NGF-dependent neurons, the primary afferents and sympathetic postganglionic neurons leading to lack of pain sensation and the presence of anhidrosis, respectively. Herein, we report nine patients from nine unrelated families with HSAN-IV due to various mutations in NTRK1, five of which are novel. These are three missense and two nonsense mutations distributed in various domains of NTRK1 involved in binding of NGF. The affected patients had variable intellectual deficits, and some had delayed diagnosis of HSAN-IV. In addition to being the first report of HSAN-IV from the Arabian Peninsula, this report expands the mutational spectrum of patients with NTRK1 mutations and provides further insights for molecular and clinical diagnosis.
Asunto(s)
Codón sin Sentido , Exoma , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Mutación Missense , Neuronas/metabolismo , Receptor trkA/genética , Adolescente , Secuencia de Bases , Niño , Preescolar , Cromosomas Humanos Par 1 , Consanguinidad , Femenino , Expresión Génica , Genes Recesivos , Neuropatías Hereditarias Sensoriales y Autónomas/diagnóstico , Neuropatías Hereditarias Sensoriales y Autónomas/fisiopatología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hipohidrosis/fisiopatología , Discapacidad Intelectual/fisiopatología , Masculino , Modelos Moleculares , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Neuronas/patología , Fenotipo , Unión Proteica , Estructura Secundaria de Proteína , Receptor trkA/química , Receptor trkA/metabolismo , Arabia Saudita , Conducta Autodestructiva/fisiopatología , Índice de Severidad de la EnfermedadRESUMEN
Congenital insensitivity to pain with anhidrosis (CIPA), also known as hereditary sensory and autonomic neuropathy type IV, features loss of pain sensation, decreased or absent sweating (anhidrosis), recurrent episodes of unexplained fever, self-mutilating behavior, and variable mental retardation. Mutations in neurotrophic receptor tyrosine kinase 1 (NTRK1) have been reported to be associated with CIPA. We identified four novel NTRK1 mutations in six Korean patients from four unrelated families. Of the four mutations, we demonstrated using a splicing assay that IVS14+3A>T causes aberrant splicing of NTRK1 mRNA, leading to introduction of a premature termination codon. An NTRK1 autophosphorylation assay showed that c.1786G>A (p.Asp596Asn) abolished autophosphorylation of NTRK1. In addition, Western blotting showed that c.704C>G (p.Ser235*) and c.2350_2363del (p.Leu784Serfs*79) blunted NTRK1 expression to undetectable levels. The four novel NTRK1 mutations we report here will expand the repertoire of NTRK1 mutations in CIPA patients, and further our understanding of CIPA pathogenesis.