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1.
Ann Neurol ; 94(2): 332-349, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37062836

RESUMEN

OBJECTIVE: Pathogenic variants in KCNT2 are rare causes of developmental epileptic encephalopathy (DEE). We herein describe the phenotypic and genetic features of patients with KCNT2-related DEE, and the in vitro functional and pharmacological properties of KCNT2 channels carrying 14 novel or previously untested variants. METHODS: Twenty-five patients harboring KCNT2 variants were investigated: 12 were identified through an international collaborative network, 13 were retrieved from the literature. Clinical data were collected and included in a standardized phenotyping sheet. Novel variants were detected using exome sequencing and classified using ACMG criteria. Functional and pharmacological studies were performed by whole-cell electrophysiology in HEK-293 and SH-SY5Y cells. RESULTS: The phenotypic spectrum encompassed: (a) intellectual disability/developmental delay (21/22 individuals with available information), ranging from mild to severe/profound; (b) epilepsy (15/25); (c) neurological impairment, with altered muscle tone (14/22); (d) dysmorphisms (13/20). Nineteen pathogenic KCNT2 variants were found (9 new, 10 reported previously): 16 missense, 1 in-frame deletion of a single amino acid, 1 nonsense, and 1 frameshift. Among tested variants, 8 showed gain-of-function (GoF), and 6 loss-of-function (LoF) features when expressed heterologously in vitro. Quinidine and fluoxetine blocked all GoF variants, whereas loxapine and riluzole activated some LoF variants while blocking others. INTERPRETATION: We expanded the phenotypic and genotypic spectrum of KCNT2-related disorders, highlighting novel genotype-phenotype associations. Pathogenic KCNT2 variants cause GoF or LoF in vitro phenotypes, and each shows a unique pharmacological profile, suggesting the need for in vitro functional and pharmacological investigation to enable targeted therapies based on the molecular phenotype. ANN NEUROL 2023;94:332-349.


Asunto(s)
Discapacidad Intelectual , Neuroblastoma , Humanos , Células HEK293 , Fenotipo , Genotipo , Discapacidad Intelectual/tratamiento farmacológico , Discapacidad Intelectual/genética , Canales de potasio activados por Sodio/genética
2.
Genet Med ; 24(12): 2453-2463, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36305856

RESUMEN

PURPOSE: Cerebellar hypoplasia and atrophy (CBHA) in children is an extremely heterogeneous group of disorders, but few comprehensive genetic studies have been reported. Comprehensive genetic analysis of CBHA patients may help differentiating atrophy and hypoplasia and potentially improve their prognostic aspects. METHODS: Patients with CBHA in 176 families were genetically examined using exome sequencing. Patients with disease-causing variants were clinically evaluated. RESULTS: Disease-causing variants were identified in 96 of the 176 families (54.5%). After excluding 6 families, 48 patients from 42 families were categorized as having syndromic associations with CBHA, whereas the remaining 51 patients from 48 families had isolated CBHA. In 51 patients, 26 aberrant genes were identified, of which, 20 (76.9%) caused disease in 1 family each. The most prevalent genes were CACNA1A, ITPR1, and KIF1A. Of the 26 aberrant genes, 21 and 1 were functionally annotated to atrophy and hypoplasia, respectively. CBHA+S was more clinically severe than CBHA-S. Notably, ARG1 and FOLR1 variants were identified in 2 families, leading to medical treatments. CONCLUSION: A wide genetic and clinical diversity of CBHA was revealed through exome sequencing in this cohort, which highlights the importance of comprehensive genetic analyses. Furthermore, molecular-based treatment was available for 2 families.


Asunto(s)
Exoma , Malformaciones del Sistema Nervioso , Niño , Humanos , Exoma/genética , Mutación , Malformaciones del Sistema Nervioso/genética , Atrofia/genética , Receptor 1 de Folato/genética , Cinesinas
3.
Brain ; 141(11): 3098-3114, 2018 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-30256902

RESUMEN

Trio-based whole exome sequencing identified two de novo heterozygous missense mutations [c.1449T > C/p.(Leu500Pro) and c.1436A > T/p.(Asn479Ile)] in PHACTR1, encoding a molecule critical for the regulation of protein phosphatase 1 (PP1) and the actin cytoskeleton, in unrelated Japanese individuals with West syndrome (infantile spasms with intellectual disability). We then examined the role of Phactr1 in the development of mouse cerebral cortex and the pathophysiological significance of these two mutations and others [c.1561C > T/p.(Arg521Cys) and c.1553T > A/p.(Ile518Asn)], which had been reported in undiagnosed patients with intellectual disability. Immunoprecipitation analyses revealed that actin-binding activity of PHACTR1 was impaired by the p.Leu500Pro, p.Asn479Ile and p.Ile518Asn mutations while the p.Arg521Cys mutation exhibited impaired binding to PP1. Acute knockdown of mouse Phactr1 using in utero electroporation caused defects in cortical neuron migration during corticogenesis, which were rescued by an RNAi-resistant PHACTR1 but not by the four mutants. Experiments using knockdown combined with expression mutants, aimed to mimic the effects of the heterozygous mutations under conditions of haploinsufficiency, suggested a dominant negative effect of the mutant allele. As for dendritic development in vivo, only the p.Arg521Cys mutant was determined to have dominant negative effects, because the three other mutants appeared to be degraded with these experimental conditions. Electrophysiological analyses revealed abnormal synaptic properties in Phactr1-deficient excitatory cortical neurons. Our data show that the PHACTR1 mutations may cause morphological and functional defects in cortical neurons during brain development, which is likely to be related to the pathophysiology of West syndrome and other neurodevelopmental disorders.


Asunto(s)
Salud de la Familia , Proteínas de Microfilamentos/genética , Mutación/genética , Espasmos Infantiles/genética , Espasmos Infantiles/fisiopatología , Animales , Células COS , Movimiento Celular/genética , Células Cultivadas , Chlorocebus aethiops , Embrión de Mamíferos , Agonistas de Aminoácidos Excitadores/farmacología , Femenino , Humanos , Lactante , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , N-Metilaspartato/farmacología , Plasticidad Neuronal/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Urea/administración & dosificación , Urea/análogos & derivados
4.
Anal Chem ; 90(5): 3211-3219, 2018 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-29446612

RESUMEN

Understanding nanoconfinement phenomena is necessary to develop nanofluidic technology platforms. One example of nanoconfinement phenomena is shifts in reaction equilibria toward reaction products in nanoconfined systems, which have been predicted theoretically and observed experimentally in DNA hybridization. Here we demonstrate a convection-limited nanofluidic immunoassay that achieves total capture of a target analyte and an apparent shift in the antibody-antigen reaction equilibrium due to nanoconfinement. The system exhibits wavefronts of the target analyte that propagate along the length of the nanochannel at a velocity much slower than that of the carrier fluid. We apply an analytical model describing the propagation of these wavefronts to determine the density of capture antibody binding sites in the enclosed nanochannel for a known concentration of the target analyte. We then use this binding site density to estimate the concentration of solutions with 5× and 10× less analyte. Our analysis suggests that nanoconfinement results in a preference toward binding of the target analyte with the surface-grafted capture antibody, as evidenced by an apparent reduction in the equilibrium dissociation constant. Our findings motivate the advancement of new biomedical and chemical synthesis technologies by leveraging nanoconfinement effects, and demonstrate a useful platform for studying the effect of nanoconfinement on chemical systems.


Asunto(s)
Proteínas Fluorescentes Verdes/química , Inmunoensayo/métodos , Técnicas Analíticas Microfluídicas/métodos , Nanoestructuras/química , Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo , Sitios de Unión , Convección , Proteínas Fluorescentes Verdes/inmunología , Técnicas Analíticas Microfluídicas/instrumentación
5.
Am J Med Genet A ; 176(5): 1195-1199, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29681101

RESUMEN

In this report, we present the case of a female infant with peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, and Hirschsprung disease (PCWH) associated with a novel frameshift mutation (c.842dupT) in exon 5, the last exon of SOX10. She had severe hypoganglionosis in the small intestine and entire colon, and suffered from frequent enterocolitis. The persistence of ganglion cells made both the diagnosis and treatment difficult in the neonatal period. She also showed hypopigmentation of the irises, hair and skin, bilateral sensorineural deafness with hypoplastic inner year, severe demyelinating neuropathy with hypotonia, and diffuse brain hypomyelination. The p.Ser282GlnfsTer12 mutation presumably escapes from nonsense-mediated decay and may generate a dominant-negative effect. We suggest that hypoganglionosis can be a variant intestinal manifestation associated with PCWH and that hypoganglionosis and aganglionosis may share the same pathoetiological mechanism mediated by SOX10 mutations.


Asunto(s)
Enfermedades Desmielinizantes/genética , Estudios de Asociación Genética , Enfermedad de Hirschsprung/genética , Mutación , Factores de Transcripción SOXE/genética , Síndrome de Waardenburg/genética , Biopsia , Encéfalo/anomalías , Encéfalo/diagnóstico por imagen , Análisis Mutacional de ADN , Enfermedades Desmielinizantes/diagnóstico , Exones , Facies , Femenino , Mutación del Sistema de Lectura , Enfermedad de Hirschsprung/diagnóstico , Humanos , Inmunohistoquímica , Lactante , Intestinos/patología , Imagen por Resonancia Magnética , Fenotipo , Cráneo/anomalías , Cráneo/diagnóstico por imagen , Síndrome de Waardenburg/diagnóstico
6.
Analyst ; 143(4): 943-948, 2018 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-29364290

RESUMEN

Single molecule analysis is desired in many areas that require the analysis of ultra-small volume and/or extremely low concentration samples (e.g., single-cell biology, medicine diagnosis, virus detection, etc.). Due to the ultra-small volume or concentration, the sample contains only single or countable analyte molecules. Thus, specific single molecules should be precisely processed and detected for analysis. However, except nucleic acids, most molecules are difficult to amplify, and a new analytical methodology for specific single molecules is thus essential. For this, efficient chemical processing and detection, which are important analytical elements, should be developed. Here, we report a single-molecule ELISA (enzyme-linked immunosorbent assay) device utilizing micro/nanofluidic technology. Both chemical processing and detection were integrated into an ultra-small space (102 nm in size), and the integration allowed precise processing (∼100% capture) and detection of a specific single molecule (protein) for the first time. This new concept and enabling technology represent a significant innovation in analytical chemistry and will have a large impact on general biology and medicine.


Asunto(s)
Ensayo de Inmunoadsorción Enzimática/instrumentación , Técnicas Analíticas Microfluídicas , Nanotecnología/instrumentación
8.
Am J Med Genet A ; 173(4): 1124-1127, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28328133

RESUMEN

The proteolipid protein 1 gene (PLP1) is located on chromosome Xq22.2 and is related to X-linked recessive leukoencephalopathy (Pelizaeus-Merzbacher disease: PMD). Compared to PLP1 duplications, which are a major contributor to PMD, chromosomal deletions in this region are rare and only a few PMD patients with small deletions have been reported, suggesting that large deletions of this region would cause embryonic lethality. Previously, we have reported female patients, with chromosomal deletions in this region, who showed severe developmental delays and behavioral abnormalities. In this study, we identified the first case of a male patient associated with an Xq22 nullisomy in a region proximal to PLP1. The patient showed severe neurological impairment and was bedridden. Brain magnetic resonance imaging revealed a severely reduced cerebral volume. The chromosomal region proximal to PLP1 was considered to be significantly important for brain development.


Asunto(s)
Secuencia de Bases , Cromosomas Humanos X/química , Enfermedad de Pelizaeus-Merzbacher/diagnóstico por imagen , Enfermedad de Pelizaeus-Merzbacher/genética , Eliminación de Secuencia , Mapeo Cromosómico , Expresión Génica , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Proteína Proteolipídica de la Mielina/genética , Neuroimagen , Enfermedad de Pelizaeus-Merzbacher/patología
9.
Anal Chem ; 88(14): 7123-9, 2016 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-27322525

RESUMEN

Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers.


Asunto(s)
Biotina/análogos & derivados , Ensayo de Inmunoadsorción Enzimática/métodos , Fenoles/química , Tiramina/análogos & derivados , Biotina/química , Colorantes Fluorescentes/química , Antígenos de Superficie de la Hepatitis B/análisis , Peroxidasa de Rábano Silvestre/química , Peróxido de Hidrógeno/química , Límite de Detección , Microesferas , Tiramina/química
10.
Small ; 10(8): 1514-22, 2014 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-24339226

RESUMEN

The growing need to optimize immunoassay performance driven by interest in analyzing individual cells has resulted in a decrease in the amount of sample required. Miniaturized immunoassays that use ultra-small femtoliter to attoliter sample volumes, a range known as the extended nanospace, can satisfy this analytical need; however, capturing every targeted molecule without loss in extended nanochannels for subsequent detection remains challenging. This is the first report of a successful extended nanofluidics-based quantitative immunochemical reaction capable of high capture efficiency using a femtoliter-scale sample volume. A novel patterning method using a photolithographic technique with vacuum ultraviolet light and low-temperature (100 °C) bonding enables patterning of functional groups for antibody immobilization before bonding, resulting in an immunochemical reaction space of only 86 fL. Reaction rate analyses indicate a decrease in the required sample volume to 810 fL and improvement in the limit of detection to 3 zmol, 5-6 orders of magnitude better than possible with the microfluidic immunoassay format. Highly efficient (near 100%) immunochemical reactions on a seconds time scale are possible due to the nm-scale diffusion length, which should be advantageous for the analysis of ultra-low-volume samples.


Asunto(s)
Inmunoquímica/métodos , Técnicas Analíticas Microfluídicas/métodos , Animales , Anticuerpos Inmovilizados , Bovinos , Límite de Detección , Ratones , Nanotecnología , Impresión , Análisis de la Célula Individual/métodos
11.
Pediatr Int ; 56(2): 240-3, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24476552

RESUMEN

BACKGROUND: Acute pancreatitis in patients with severe motor and intellectual disability (SMID) is a rare but life-threatening condition. Possible causes of acute pancreatitis in these patients including valproic acid therapy, hypothermia and nasoduodenal tube feeding, have not yet been investigated in detail. The aim of this study was therefore to investigate the risk factors for acute pancreatitis in patients with SMID. METHODS: Five SMID patients with acute pancreatitis and 15 SMID patients without acute pancreatitis were reviewed. Age; serum total cholesterol, triglyceride, total protein, and albumin; height; bodyweight; body surface area; body mass index; daily calorie intake; daily calorie intake per unit of body mass surface area; daily calorie intake per kilogram bodyweight; and valproic acid usage were examined. RESULTS: A statistically significant difference was observed in serum albumin level between the two groups (P = 0.026). CONCLUSION: The mechanism of acute pancreatitis in these patients was considered as pancreatic morphological change, acinar damage, and elevated serum trypsinogen level caused by malnutrition. It is likely that acute pancreatitis in patients with SMID occurs due to the same mechanism as in anorexia nervosa and malnourished patients. To prevent acute pancreatitis in these patients, it is important to maintain adequate nutritional status.


Asunto(s)
Discapacidad Intelectual/complicaciones , Trastornos de la Destreza Motora/complicaciones , Pancreatitis/complicaciones , Enfermedad Aguda , Adolescente , Niño , Preescolar , Femenino , Humanos , Masculino , Estudios Retrospectivos , Factores de Riesgo , Índice de Severidad de la Enfermedad , Adulto Joven
12.
Acta Neuropathol Commun ; 11(1): 33, 2023 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-36864519

RESUMEN

Focal cortical dysplasia is the most common malformation during cortical development, sometimes excised by epilepsy surgery and often caused by somatic variants of the mTOR pathway genes. In this study, we performed a genetic analysis of epileptogenic brain malformed lesions from 64 patients with focal cortical dysplasia, hemimegalencephy, brain tumors, or hippocampal sclerosis. Targeted sequencing, whole-exome sequencing, and single nucleotide polymorphism microarray detected four germline and 35 somatic variants, comprising three copy number variants and 36 single nucleotide variants and indels in 37 patients. One of the somatic variants in focal cortical dysplasia type IIB was an in-frame deletion in MTOR, in which only gain-of-function missense variants have been reported. In focal cortical dysplasia type I, somatic variants of MAP2K1 and PTPN11 involved in the RAS/MAPK pathway were detected. The in-frame deletions of MTOR and MAP2K1 in this study resulted in the activation of the mTOR pathway in transiently transfected cells. In addition, the PTPN11 missense variant tended to elongate activation of the mTOR or RAS/MAPK pathway, depending on culture conditions. We demonstrate that epileptogenic brain malformed lesions except for focal cortical dysplasia type II arose from somatic variants of diverse genes but were eventually linked to the mTOR pathway.


Asunto(s)
Neoplasias Encefálicas , Displasia Cortical Focal , Malformaciones del Desarrollo Cortical de Grupo I , Malformaciones del Sistema Nervioso , Humanos , Malformaciones del Desarrollo Cortical de Grupo I/genética , Encéfalo
13.
Pediatr Int ; 54(5): 701-3, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23005901

RESUMEN

We report an immunocompetent infantile case wherein group B streptococcus bacteremia recurred two times. The isolated bacteria which colonized in his nasopharyngeal cavity might be a source of repetitive infection. Although the best action has not been established yet, penicillin G as the prophylaxis seemed to be effective in this case.


Asunto(s)
Bacteriemia/inmunología , Inmunocompetencia , Infecciones Estreptocócicas/inmunología , Streptococcus agalactiae/aislamiento & purificación , Bacteriemia/tratamiento farmacológico , Humanos , Lactante , Recién Nacido , Masculino , Nasofaringe/microbiología , Penicilina G/uso terapéutico , Recurrencia , Infecciones Estreptocócicas/tratamiento farmacológico
14.
Lab Chip ; 22(22): 4418-4429, 2022 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-36305222

RESUMEN

Drug selection and treatment monitoring via minimally invasive liquid biopsy using circulating tumor cells (CTCs) are expected to be realized in the near future. For clinical applications of CTCs, simple, high-throughput, single-step CTC isolation from whole blood without red blood cell (RBC) lysis and centrifugation remains a crucial challenge. In this study, we developed a novel cancer cell separation chip, "hybrid double-spiral chip", that involves the serial combination of two different Dean flow fractionation (DFF) separation modes of half and full Dean cycles, which is the hybrid DFF separation mode for ultra-high-throughput blood processing at high precision and size-resolution separation. The chip allows fast processing of 5 mL whole blood within 30 min without RBC lysis and centrifugation. RBC and white blood cell (WBC) depletion rates of over 99.9% and 99%, respectively, were achieved. The average recovery rate of spiked A549 cancer cells was 87% with as low as 200 cells in 5 mL blood. The device can achieve serial reduction in the number of cells from approximately 1010 cells of whole blood to 108 cells, and subsequently to an order of 106 cells. The developed method can be combined with measurements of all recovered cells using imaging flow cytometry. As proof of concept, CTCs were successfully enriched and enumerated from the blood of metastatic breast cancer patients (N = 10, 1-69 CTCs per 5 mL) and metastatic prostate cancer patients (N = 10, 1-39 CTCs per 5 mL). We believe that the developed method will be beneficial for automated clinical analysis of rare CTCs from whole blood.


Asunto(s)
Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes , Humanos , Microfluídica , Línea Celular Tumoral , Células Neoplásicas Circulantes/patología , Separación Celular , Eritrocitos/patología
15.
Lab Chip ; 21(16): 3112-3127, 2021 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-34286793

RESUMEN

There is an increasing need for the enrichment of rare cells in the clinical environments of precision medicine, personalized medicine, and regenerative medicine. With the possibility of becoming the next-generation cell sorters, microfluidic fluorescence-activated cell sorting (µ-FACS) devices have been developed to avoid cross-contamination, minimize device footprint, and eliminate bio-aerosols. However, due to highly precise flow control, the achievable throughput of the µ-FACS system is generally lower than the throughput of conventional FACS devices. Here, we report a fully integrated high-throughput microfluidic circulatory fluorescence-activated cell sorting (µ-CFACS) system for the enrichment of clinical rare cells. A microfluidic sorting cartridge has been developed for enriching samples through a sequential sorting process, which was further realized by the integration of both fast amplified piezoelectrically actuated on-chip valves and compact pneumatic cylinders actuated on-chip valves. At an equivalent throughput of ∼8000 events per second (eps), the purity of rare fluorescent microparticles has been significantly increased from ∼0.01% to ∼27.97%. An enrichment of ∼9400-fold from 0.009% to 81.86% has also been demonstrated for isolating fluorescently labelled MCF-7 breast cancer cells from Jurkat cells at an equivalent sorting throughput of ∼6400 eps. With the advantages of high throughput and contamination-free design, the proposed integrated µ-CFACS system provides a new option for the enrichment of clinical rare cells.


Asunto(s)
Técnicas Analíticas Microfluídicas , Microfluídica , Separación Celular , Citometría de Flujo , Humanos , Células Jurkat , Células MCF-7
16.
J Infect Chemother ; 16(6): 436-8, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20454915

RESUMEN

We experienced a case of urinary tract infection in a 3-month-old child caused by Kluyvera ascorbata. The authors report the case and review the literature regarding Kluyvera urinary tract infection exclusively in children. Kluyvera infection, which had been extremely rare, has increasingly been reported, including urinary tract infection. A prompt identification of Kluyvera species in clinical infections is important. Recognition of its disease-producing potential and the subsequent initiation of effective antimicrobial coverage are essential for appropriate management in the pediatric population.


Asunto(s)
Infecciones por Enterobacteriaceae/microbiología , Kluyvera/aislamiento & purificación , Infecciones Urinarias/microbiología , Antibacterianos/uso terapéutico , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Humanos , Lactante , Kluyvera/clasificación , Kluyvera/efectos de los fármacos , Masculino , Pruebas de Sensibilidad Microbiana , Infecciones Urinarias/tratamiento farmacológico
17.
PLoS One ; 15(8): e0237506, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32790768

RESUMEN

Circulating tumor cells (CTCs) invade blood vessels in solid tumors and promote metastases by circulating in the blood. CTCs are thus recognized as targets for liquid biopsy and can provide useful information for design of treatments. This diagnostic approach must consider not only the number of CTCs but also their molecular and genetic characteristics. For this purpose, use of devices that enrich CTCs independent of these characteristics and detectors that recognize various CTC characteristics is essential. In the present study, we developed a CTC detection system comprising ClearCell FX and ImageStream Mark II. We clarified the analytical performance of this system by evaluating recovery rate, lower limits of detection, and linearity. These parameters are critical for detecting rare cells, such as CTCs. We tested these parameters using three cell lines with different expression levels of the epithelial marker-epithelial cell adhesion molecule (EpCAM) and spiked these cells into whole-blood samples from healthy donors. The average recovery rate and lower limit of detection were approximately 40% and five cells/7.5 mL of whole blood, respectively. High linearity was observed for all evaluated samples. We also evaluated the ability of the system to distinguish between normal and abnormal cells based on protein expression levels and gene amplification and found that the system can identify abnormal cells using these characteristics. The CTC detection system thus displays the ability to distinguish specific characteristics of CTC, thereby providing valuable information for cancer treatment.


Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias/sangre , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/patología , Molécula de Adhesión Celular Epitelial/metabolismo , Humanos , Células Neoplásicas Circulantes/metabolismo , Células Tumorales Cultivadas
18.
Brain Dev ; 42(8): 607-611, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32505479

RESUMEN

BACKGROUND: Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the early-onset epileptic encephalopathies resistant to antiepileptic drugs, therefore carrying an extremely poor neurodevelopmental outcome. KCNT1, encoding for a sodium-activated potassium channel (KCa4.1 channel), has recently been reported as the major gene responsible for EIMFS. Since gain of function is the only type of mutation identified in patients with EIMFS, quinidine, a partial antagonist of KCa4.1 channel, is considered as a potential candidate for targeted treatment of EIMFS. However, treatment results reported so far vary from seizure-free state to no response, and cardiac side effect remains a challenge for dose titration and long-term treatment. CASE REPORT: Our case was an infant diagnosed with EIMFS with confirmed mutation in KCNT1 gene. Quinidine therapy was started as early as 9 months old. Within the first month of treatment, the number of seizures reduced to about one third. However, seizure-free state was not obtained and his neuropsychological development remained severely delayed. After 16 months of treatment, quinidine had to be discontinued because of cardiac side effects. At 27 months of age, however, his seizures suddenly stopped and he remained seizure-free for five days. This coincided with the prescription of tipepidine, a commonly used antitussive, administered for his persistent cough. Reduction in seizure frequency was also observed with dextromethorphan, another conventional antitussive drug. Although the relation between these treatments and his symptom improvement is a matter of elucidation, there is a possibility that these nonnarcotic antitussive drugs might play a role in the treatment of EIFMS.


Asunto(s)
Antitusígenos/uso terapéutico , Epilepsia/tratamiento farmacológico , Proteínas del Tejido Nervioso/genética , Canales de potasio activados por Sodio/genética , Convulsiones/tratamiento farmacológico , Dextrometorfano/farmacología , Electroencefalografía , Epilepsia/genética , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Mutación , Piperidinas/farmacología , Quinidina/uso terapéutico , Convulsiones/genética , Insuficiencia del Tratamiento , Resultado del Tratamiento
19.
Electrophoresis ; 30(24): 4251-5, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20013908

RESUMEN

Fused silica glass microchips have several attractive features for lab-on-a-chip applications; they can be machined with excellent precision down to nanospace; are stable; transparent and can be modified with a range of silanization agents to change channel surface properties. For immobilization, however, ligands must be added after bonding, since the harsh bonding conditions using heat or hydrofluoric acid would remove all prior immobilized ligands. For spatial control over immobilization, UV-mediated immobilization offers several advantages; spots can be created in parallel, the feature size can be made small, and spatial control over patterns and positions is excellent. However, UV sensitive groups are often based on hydrophobic chemical moieties, which unfortunately result in greater non-specific binding of biomolecules, especially proteins. Here, we present techniques in which any -CH(x) (x=1,2,3) containing surface coating can be used as foundation for grafting a hydrophilic linker with a chemical anchor, a carboxyl group, to which proteins and amine containing molecules can be covalently coupled. Hence, the attractive features of many well-known protein and biomolecule repelling polymer coatings can be utilized while achieving site-specific immobilization only to pre-determined areas within the bonded microchips.


Asunto(s)
Dispositivos Laboratorio en un Chip , Proteínas/química , Hidrogeles , Rayos Ultravioleta
20.
No To Hattatsu ; 41(6): 447-51, 2009 Nov.
Artículo en Japonés | MEDLINE | ID: mdl-19928544

RESUMEN

We report a 4-year-old boy with fulminating meningitis caused by Haemophilus influenzae (Hib). He suddenly developed fever, vomiting and then somnolence. As bacterial meningitis was suspected, treatment with antibiotics was started at 12 hours after the onset. However, there was a rapid progression of severe brain edema and brain hernia, leading to clinical brain death. His clinical course and neuroradiological findings mimicked those in patients with acute encephalopathy, with cytokine profiles in cerebrospinal fluid demonstrating a marked increase of inflammatory cytokines. From a review of the literature, fulminating Hib meningitis may be classified into two disease types: DIC plus multiple organ failure and acute brain swelling types. The present case belongs to the latter type, in which cytokine storm seems to play an important pathogenic role.


Asunto(s)
Haemophilus influenzae , Meningitis por Haemophilus/diagnóstico , Edema Encefálico/etiología , Preescolar , Citocinas/análisis , Progresión de la Enfermedad , Humanos , Masculino
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