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The nearby radio galaxy M87 offers a unique opportunity to explore the connections between the central supermassive black hole and relativistic jets. Previous studies of the inner region of M87 revealed a wide opening angle for the jet originating near the black hole1-4. The Event Horizon Telescope resolved the central radio source and found an asymmetric ring structure consistent with expectations from general relativity5. With a baseline of 17 years of observations, there was a shift in the jet's transverse position, possibly arising from an 8- to 10-year quasi-periodicity3. However, the origin of this sideways shift remains unclear. Here we report an analysis of radio observations over 22 years that suggests a period of about 11 years for the variation in the position angle of the jet. We infer that we are seeing a spinning black hole that induces the Lense-Thirring precession of a misaligned accretion disk. Similar jet precession may commonly occur in other active galactic nuclei but has been challenging to detect owing to the small magnitude and long period of the variation.
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Fusobacterium nucleatum is classified into five subspecies. F. nucleatum ChDC F128 was isolated from a periodontitis lesion and proposed as a new subspecies based on the comparison of the nucleotide sequences of the RNA polymerase beta subunit and zinc protease genes. Here, we report the draft genome sequence of the strain.
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Infecciones por Fusobacterium/microbiología , Fusobacterium nucleatum/genética , Genoma Bacteriano , Periodontitis/microbiología , Fusobacterium nucleatum/clasificación , Humanos , Datos de Secuencia MolecularRESUMEN
Fusobacterium nucleatum, one of the major causative bacteria of periodontitis, is classified into five subspecies (nucleatum, polymorphum, vincentii, animalis, and fusiforme) on the basis of the several phenotypic characteristics and DNA homology. This is the first report of the draft genome sequence of F. nucleatum subsp. fusiforme ATCC 51190(T).
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Fusobacterium nucleatum/clasificación , Fusobacterium nucleatum/genética , Genoma Bacteriano , Datos de Secuencia MolecularRESUMEN
Down syndrome is the most common genetic disorder and is characterized by three copies of chromosome 21. Regulator of calcineurin 1 (RCAN1) is located close to the Down syndrome critical region (distal part of chromosome 21), and its product functions as an endogenous inhibitor of calcineurin signaling. RCAN1 protein stability is regulated by several inflammatory signaling factors, though the underlying mechanisms remain incompletely understood. Here, we report that RCAN1 interacts with the inflammation-linked transcription factor, signal transducer and activator of transcription 2 (STAT2) in mammalian cells. STAT2 overexpression decreased levels of RCAN1 protein. Decreases in RCAN1 were blocked by a proteasome inhibitor, indicating that STAT2 regulates RCAN1 degradation via the ubiquitin-proteasome system. Co-immunoprecipitation/immunoblot analyses showed that STAT2 enhanced RCAN1 ubiquitination through the ubiquitin E3 ligase FBW7. This pathway appeared to be physiologically relevant, as treatment of cells with interferon-α reduced RCAN1 levels through the activation of STAT2 and FBW7. Together, these results suggest that STAT2 influences diverse cellular processes linked to RCAN1 by negatively affecting RCAN1 protein stability.
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Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas Musculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Factor de Transcripción STAT2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Humanos , Mediadores de Inflamación/metabolismo , Interferón-alfa/metabolismo , Proteolisis , Factor de Transcripción STAT2/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Recently, a method of identifying body fluids using DNA methylation has been developed (Frumkin et al., 2011). An existing multiplex assay using 9 CpG markers could differentiate 5 body fluids: semen, blood, saliva, menstrual blood, and vaginal fluid. To validate this technique, we evaluated the previously described body fluid identification method by means of single base extension (SBE). DNA methylation was applied to 22 samples in 18 forensic cases; seven of these were semen, three were blood, eight were saliva, three were vaginal fluid, and one was menstrual blood. Total of 18 samples were tested, the DNA methylation profiles were coincident from preliminary tests (acid phosphatase (AP), leucomalachite green (LMG, Sigma Aldrich, St Louis, MO, USA) and SALIgAE®) except one sample which displayed an all-negative result. After applying the DNA methylation method to forensic samples, we determined that it could be very useful for differentiating vaginal secretions from menstrual blood, for which there is no conventional preliminary testing method.
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Líquidos Corporales , Metilación de ADN , Femenino , Genética Forense , Humanos , Saliva , SemenRESUMEN
Micro/nanofluidics are excellent candidates for biological sample preparation. However, the limited process volume in micro/nanofluidics is the main hurdle limiting their practical applications. To date, most micro/nanofluidics have processed sample volumes of several microliters and have rarely been used to handle large-volume samples. Herein, we propose a microfluidic paper-based large-volume preconcentrator (u-LVP) for enrichment and purification of biomarkers (e.g., miRNA) using ion concentration polarization. A Nafion (ion-selective nanoporous membrane)-functionalized multilayer cellulose paper enables microscale division of milliliter-scale samples, thus electrokinetically separating and preconcentrating the biomarker in different locations within the u-LVP. By inserting collecting discs at optimal positions in the u-LVP, the enriched biomarker is simply recovered with high efficiency. With this approach, as an exemplary biomarker, miRNA-21 in human serum was separated from proteins and preconcentrated with an effective preconcentration factor exceeding 6.63 and a recovery rate above 84%. Thus, our platform offers new opportunities and benefits for biomarker, diagnostic, prognostic, and therapeutic research.
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Técnicas Biosensibles , Técnicas Analíticas Microfluídicas , Biomarcadores , Humanos , Microfluídica , ProteínasRESUMEN
In this paper, we describe the development of a novel method to detect oral bacteria by combining direct polymerase chain reaction (direct PCR) with an immunochromatographic strip (ICS), enabling the identification of saliva in forensic samples. Direct PCR was first used to directly amplify specific oral bacterial sequences (from Streptococcus sanguinis and Streptococcus salivarius) from swab samples, circumventing the need for tedious sample preparation steps such as cell lysis and DNA extraction and purification. The resultant amplicons were then colorimetrically detected on an ICS, a much more convenient, cost-effective, and user-friendly detection method than those currently available, thereby allowing the presence or absence of the target oral bacteria to be determined with the naked eye. Moreover, the entire analysis process was performed rapidly and with ease using this combination of direct PCR amplification from swab samples and ICS-based amplicon detection. This method successfully detected S. sanguinis and S. salivarius in most of the saliva swab samples tested, and returned negative results using blood, semen, urine, and vaginal fluid swab samples. Furthermore, S. sanguinis and S. salivarius were detected in a large number of mock forensic samples using this technique, which suggests that direct PCR and ICS-based detection of oral bacteria is sufficient to demonstrate the presence of saliva. Thus, we believe that the proposed method could be very useful for the identification of saliva in forensic applications.
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Cromatografía de Afinidad , Reacción en Cadena de la Polimerasa , Saliva/microbiología , ADN Bacteriano/genética , Genética Forense , Humanos , Análisis de Secuencia de ADN , Streptococcus salivarius/genética , Streptococcus sanguis/genéticaRESUMEN
OBJECTIVE: These days, young children are exposed to a wide range of smart devices and their usage of smart devices is rapidly increasing worldwide. However, the use of smart devices by young children has not been studied in detail yet because smart device is relatively recent. The purpose of this study was to investigate the exposure status of smart devices among 2-5 years old children in Korea. METHODS: Four hundred parents of 2- to 5-year-old children were invited to enroll. Data on demographic information and the frequency of media use, time of media use, age at first use of media was self-reported. RESULTS: Among 390 toddlers, 39.3% watched TV almost every day, while 12.0% of children used smartphone on a daily basis. During weekdays, 48% of the children watched TV for over an hour. On weekends, 63.1% of the children watched TV for over an hour. On weekends, 23.4% of children use their smartphones for over an hour. Children using smartphones before 24 months of age were 31.3%. CONCLUSION: Research has shown that TV and smartphones are the most popular digital devices used by toddlers. Most toddlers began using smart devices at 12-24 months. This study provides comprehensive information on children's contemporary use of media.
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Previously, an age-predictive method based on DNA-methylation patterns in semen was developed, using three CpG sites (cg06304190 in the TTC7B gene, cg12837463, and cg06979108 in the NOX4 gene). Before considering the routine use of a new method in forensics, validation studies such as concordance and sensitivity tests are essential for obtaining expanded and more reliable forensic information. Here, we evaluated a previously described age-predictive method for semen for routine forensic use. Concordance testing showed a high correlation between the predicted and chronological age, with a mean absolute deviation from the chronological age of 4.8â¯years. Sensitivity testing suggested that age prediction with reliable accuracy and consistency was possible with >5â¯ng of bisulfite-converted DNA. We also confirmed the applicability of the age-predictive method in forensic casework, using forensic samples. Thus, the proposed method could serve as a very valuable forensics tool for accurate age prediction with semen samples.
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Metilación de ADN , Genética Forense/métodos , Semen , Adulto , Factores de Edad , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Valor Predictivo de las Pruebas , Adulto JovenRESUMEN
This study developed a new method for forensic saliva identification using three oral bacteria, Streptococcus salivarius, Streptococcus sanguinis, and Neisseria subflava, combined with a real-time polymerase chain reaction (RT-PCR) system we called OB mRT-PCR. Analytical sensitivity results showed that the target bacteria were amplified at 102-107 copies/reaction, and analytical specificity was assessed using 24 other viruses, bacteria, and protozoa. To evaluate the OB mRT-PCR kit for forensic applications, saliva from 140 Korean individuals was tested, and at least two target bacteria were detected in all the samples. Additional studies on non-saliva samples demonstrated the specificity of the kit. Comparison of the kit with two conventional saliva test methods, the SALIgAE and RSID-Saliva assays, indicated that it was more sensitive and applicable to saliva samples in long-term storage (up to 14 weeks). Additionally, through amplification of mock forensic items and old DNA samples (isolated without lysis of the bacterial cells, regardless of their Gram-positivity), we found that the kit was applicable to not only saliva swabs, but also DNA samples. We suggest that this simple RT-PCR-based experimental method is feasible for rapid on-site analysis, and we expect this kit to be useful for saliva detection in old forensic DNA samples.
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Bacterias/genética , Infecciones Bacterianas/diagnóstico , ADN Bacteriano/análisis , Medicina Legal , Boca/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Saliva/microbiología , Bacterias/clasificación , Bacterias/aislamiento & purificación , Infecciones Bacterianas/genética , Infecciones Bacterianas/microbiología , ADN Bacteriano/genética , Femenino , Humanos , MasculinoRESUMEN
DNA methylation has important biological roles, such as gene expression regulation, as well as practical applications in forensics, such as in body fluid identification and age estimation. DNA methylation often occurs in the CpG site, and methylation within the CpG islands affects various cellular functions and is related to tissue-specific identification. Several programs have been developed to identify CpG islands; however, the size, location, and number of predicted CpG islands are not identical due to different search algorithms. In addition, they only provide structural information for predicted CpG islands without experimental information, such as primer design. We developed an analysis pipeline package, CpGPNP, to integrate CpG island prediction and primer design. CpGPNP predicts CpG islands more accurately and sensitively than other programs, and designs primers easily based on the predicted CpG island locations. The primer design function included standard, bisulfite, and methylation-specific PCR to identify the methylation of particular CpG sites. In this study, we performed CpG island prediction on all chromosomes and compared CpG island search performance of CpGPNP with other CpG island prediction programs. In addition, we compared the position of primers designed for a specific region within the predicted CpG island using other bisulfite PCR primer programs. The primers designed by CpGPNP were used to experimentally verify the amplification of the target region of markers for body fluid identification and age estimation. CpGPNP is freely available at http://forensicdna.kr/cpgpnp/.
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Islas de CpG/genética , Metilación de ADN , Cartilla de ADN , Programas Informáticos , Algoritmos , Genética Forense , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
This paper describes the development of a novel paper-based capillary electrophoresis (pCE) microdevice using mineral paper, which is durable, oil and tear resistant, and waterproof. The pCE device is inexpensive (~$1.6 per device for materials), simple to fabricate, lightweight, and disposable, so it is more adequate for point-of-care (POC) pathogen diagnostics than a conventional CE device made of glass, quartz, silicon or polymer. In addition, the entire fabrication process can be completed within 1h without using expensive clean room facilities and cumbersome photolithography procedures. A simple cross-designed pCE device was patterned on the mineral paper by using a plotter, and assembled with an OHP film via a double-sided adhesive film. After filling the microchannel with polyacrylamide gel, the injection, backbiasing, and separation steps were sequentially operated to differentiate single-stranded DNA (ssDNA) with 4 bp resolution in a 2.9cm-long CE separation channel. Furthermore, we successfully demonstrated the identification of the PCR amplicons of two target genes of Escherichia coli O157:H7 (rrsH gene, 121 bp) and Staphylococcus aureus (glnA gene, 225 bp). For accurate assignment of the peaks in the electropherogram, two bracket ladders (80 bp for the shortest and 326 bp for the longest) were employed, so the two amplicons of the pathogens were precisely identified on a pCE chip within 3min using the relative migration time ratio without effect of the CE environments. Thus, we believe that the pCE microdevice could be very useful for the separation of nucleic acids, amino acids, and ions as an analytical tool for use in the medical applications in the resource-limited environments as well as fundamental research fields.
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Electroforesis Capilar/instrumentación , Escherichia coli O157/aislamiento & purificación , Papel , Sistemas de Atención de Punto , Staphylococcus aureus/aislamiento & purificación , Técnicas Biosensibles/instrumentación , ADN Bacteriano/análisis , ADN Bacteriano/genética , Diseño de Equipo , Infecciones por Escherichia coli/microbiología , Escherichia coli O157/genética , Humanos , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genéticaRESUMEN
We have developed an integrated direct loop-mediated isothermal amplification (Direct LAMP) microdevice incorporated with an immunochromatographic strip (ICS) to identify bacteria contaminated in real samples. The Direct LAMP is a novel isothermal DNA amplification technique which does not require thermal cycling steps as well as any sample preparation steps such as cell lysis and DNA extraction for amplifying specific target genes. In addition, the resultant amplicons were colorimetrically detected on the ICS, thereby enabling the entire genetic analysis process to be simplified. The two functional units (Direct LAMP and ICS) were integrated on a single device without use of the tedious and complicated microvalve and tubing systems. The utilization of a slidable plate allows us to manipulate the fluidic control in the microchannels manually and the sequential operation of the Direct LAMP and ICS detection could be performed by switching the slidable plate to each functional unit. Thus, the combination of the direct isothermal amplification without any sample preparation and thermal cycling steps, the ICS based amplicon detection by naked eyes, and the slidable plate to eliminate the microvalves in the integrated microdevice would be an ideal platform for point-of-care DNA diaganotics. On the integrated Direct LAMP-ICS microdevice, we could analyze Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) contaminated in human whole blood or milk at a single-cell level within 1h.
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Técnicas Biosensibles , Cromatografía de Afinidad/métodos , Escherichia coli O157/aislamiento & purificación , Staphylococcus aureus/aislamiento & purificación , Animales , Sangre/microbiología , Escherichia coli O157/patogenicidad , Humanos , Límite de Detección , Leche/microbiología , Staphylococcus aureus/patogenicidadRESUMEN
Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase is an enzyme that converts GL-7-ACA to 7-aminocephalosporanic acid, a starting material for semisynthetic cephalosporin antibiotics. In this study, optimal conditions for the immobilization of GL-7-ACA acylase were determined by experimental observations and statistical methods. The optimal conditions were as follows: 1.1 M phosphate buffer (pH 8.3) as buffer solution, immobilization temperature of 20 degrees C, and immobilization time of 120 min. Unreacted aldehyde groups were quenched by reaction with a low-molecular-weight material such as L-lysine, glycine, and ethanolamine after immobilization in order to enhance the activity of immobilized GL-7-ACA acylase. The activities of immobilized GL-7-ACA acylase obtained by using the low-molecular-weight materials were higher than those obtained by immobilized GL-7-ACA acylase not treated with low-molecular-weight materials. In particular, the highest activity of immobilized GL-7-ACA acylase was obtained using 0.4% (v/v) ethanolamine. We also investigated the effect of sodium cyanoborohydride in order to increase the stability of the linkage between the enzyme and the support. The effect on operational stability was obvious: the activity of immobilized GL-7-ACA acylase treated with 4% (w/w) sodium cyanoborohydride remained almost 100% after 20 times of reuse.
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Enzimas Inmovilizadas/química , Modelos Químicos , Penicilina Amidasa/química , Dióxido de Silicio/química , Borohidruros/química , Cefalosporinas/síntesis química , Cefalosporinas/química , Activación Enzimática , Estabilidad de Enzimas , Etanolamina/química , Concentración de Iones de Hidrógeno , Control de Calidad , Gel de Sílice , TemperaturaRESUMEN
Regulator of calcineurin 1 (RCAN1; also referred as DSCR1 or MCIP1) is located in close proximity to a Down syndrome critical region of human chromosome 21. Although RCAN1 is an endogenous inhibitor of calcineurin signaling that controls lymphocyte activation, apoptosis, heart development, skeletal muscle differentiation, and cardiac function, it is not yet clear whether RCAN1 might be involved in other cellular activities. In this study, we explored the extra-functional roles of RCAN1 by searching for novel RCAN1-binding partners. Using a yeast two-hybrid assay, we found that RCAN1 (RCAN1-1S) interacts with histone deacetylase 3 (HDAC3) in mammalian cells. We also demonstrate that HDAC3 deacetylates RCAN1. In addition, HDAC3 increases RCAN1 protein stability by inhibiting its poly-ubiquitination. Furthermore, HDAC3 promotes RCAN1 nuclear translocation. These data suggest that HDAC3, a new binding regulator of RCAN1, affects the protein stability and intracellular localization of RCAN1.
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Transporte Activo de Núcleo Celular , Histona Desacetilasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/metabolismo , Acetilación , Línea Celular , Proteínas de Unión al ADN , Expresión Génica , Histona Desacetilasas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Musculares/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Especificidad por Sustrato , UbiquitinaciónRESUMEN
Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) catalyzes the hydrolysis of glycerophospholipids at the sn-2 position to liberate fatty acids. Although cPLA(2)alpha has been implicated in various cellular processes, the detailed mechanism of its expression remains to be elucidated. Here we report that phorbol 12-myristate 13-acetate (PMA) up-regulates cPLA(2)alpha in A549 airway epithelium cells, and that this effect is sensitive to rottlerin, a potent inhibitor of protein kinase Cdelta (PKCdelta). Consistent with this observation, a dominant negative mutant of PKCdelta reduced cPLA(2)alpha induction in response to PMA. Up-regulation of cPLA(2)alpha by PMA was also inhibited by PDTC, an inhibitor of nuclear factor-kappa B (NF-kappaB), and degradation of IkappaB and subsequent activation of NF-kappaB occurred in response to PMA treatment. These findings indicate that PMA induces expression of cPLA(2)alpha at the transcriptional level via an NF-kappaB-dependent mechanism. In addition, activation of the NF-kappaB promoter by PMA was diminished by pretreatment with DPI, a flavoenzyme inhibitor as well as by rottlerin, suggesting a role for reactive oxygen species (ROS) as well as PKCdelta. Consistent with this, PMA stimulated the production of ROS and this was blocked by inhibiting PKCdelta. Our results suggest that PKCdelta and ROS lie upstream of NF-kappaB, and we conclude that a PKCdelta-ROS-NF-kappaB cascade plays a pivotal role in cPLA(2)alpha induction by PMA.