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IMPORTANCE: Shigella species cause bacillary dysentery, the second leading cause of diarrheal deaths worldwide. There is a pressing need to identify novel molecular drug targets. Shigella virulence phenotypes are controlled by the transcriptional regulator, VirB. We show that VirB belongs to a fast-evolving, plasmid-borne clade of the ParB superfamily, which has diverged from versions with a distinct cellular role-DNA partitioning. We report that, like classic members of the ParB family, VirB binds a highly unusual ligand, CTP. Mutants predicted to be defective in CTP binding are compromised in a variety of virulence attributes controlled by VirB, likely because these mutants cannot engage DNA. This study (i) reveals that VirB binds CTP, (ii) provides a link between VirB-CTP interactions and Shigella virulence phenotypes, (iii) provides new insight into VirB-CTP-DNA interactions, and (iv) broadens our understanding of the ParB superfamily, a group of bacterial proteins that play critical roles in many bacteria.
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Proteínas de Unión al ADN , Shigella , Virulencia/genética , Proteínas de Unión al ADN/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Ligandos , Shigella flexneri , Shigella/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
Complex DNA double-strand break (DSB), which is defined as a DSB coupled with additional strand breaks within 10 bp in this study, induced after ionizing radiation or X-rays, is recognized as fatal damage which can induce cell death with a certain probability. In general, a DSB site inside the nucleus of live cells can be experimentally detected using the γ-H2AX focus formation assay. DSB complexity is believed to be detected by analyzing the focus size using such an assay. However, the relationship between focus size and DSB complexity remains uncertain. In this study, using Monte Carlo (MC) track-structure simulation codes, i.e., an in-house WLTrack code and a Particle and Heavy Ion Transport code System (PHITS), we developed an analytical method for qualifying the DSB complexity induced by photon irradiation from the microscopic image of γ-H2AX foci. First, assuming that events (i.e., ionization and excitation) potentially induce DNA strand breaks, we scored the number of events in a water cube (5.03 × 5.03 × 5.03 nm3) along electron tracks. Second, we obtained the relationship between the number of events and the foci size experimentally measured by the γ-H2AX focus formation assay. Third, using this relationship, we evaluated the degree of DSB complexity induced after photon irradiation for various X-ray spectra using the foci size, and the experimental DSB complexity was compared to the results estimated by the well-verified DNA damage estimation model in the PHITS code. The number of events in a water cube was found to be proportional to foci size, suggesting that the number of events intrinsically related to DSB complexity at the DNA scale. The developed method was applicable to focus data measured for various X-ray spectral situations (i.e., diagnostic kV X-rays and therapeutic MV X-rays). This method would contribute to a precise understanding of the early biological impacts of photon irradiation by means of the γ-H2AX focus formation assay.
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Núcleo Celular , Daño del ADN , Relación Dosis-Respuesta en la Radiación , Núcleo Celular/metabolismo , Rayos X , ADN/metabolismoRESUMEN
Mycoplasma contamination of cell culture represents a serious problem in research and decontamination from cell-propagated obligate intracellular bacteria has proven challenging. Here, we presented an optimized protocol to remove Mycoplasma from contaminated Chlamydia trachomatis culture. A stepwise procedure of Mycoplasma removal entails (i) incubation in nonionic detergent-containing solution and (ii) separation of viable chlamydial organisms by fluorescence-activated cell sorting (FACS), followed by subcloning using a focus-forming assay. We also adapted a polymerase chain reaction (PCR) assay using paired universal and Mycoplasma-specific primers, which are distinguishable from the C. trachomatis counterparts, in combination with Sanger sequencing to determine the presence of mycoplasmas' 16S rRNA genes. These integrated approaches allow for full removal of Mycoplasma, as verified by the improved PCR assay, without compromising the capacity of viable C. trachomatis to adapt to new infection in epithelial cells. Some pitfalls during the Mycoplasma decontamination process are discussed.
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Técnicas de Cultivo de Célula , Chlamydia trachomatis/crecimiento & desarrollo , Descontaminación/métodos , Mycoplasma/crecimiento & desarrollo , Células Cultivadas , Chlamydia trachomatis/genética , HumanosRESUMEN
Kinases play important roles in diverse cellular processes, including signaling, differentiation, proliferation, and metabolism. They are frequently mutated in cancer and are the targets of a large number of specific inhibitors. Surveys of cancer genome atlases reveal that kinase domains, which consist of 300 amino acids, can harbor numerous (150 to 200) single-point mutations across different patients in the same disease. This preponderance of mutations-some activating, some silent-in a known target protein make clinical decisions for enrolling patients in drug trials challenging since the relevance of the target and its drug sensitivity often depend on the mutational status in a given patient. We show through computational studies using molecular dynamics (MD) as well as enhanced sampling simulations that the experimentally determined activation status of a mutated kinase can be predicted effectively by identifying a hydrogen bonding fingerprint in the activation loop and the αC-helix regions, despite the fact that mutations in cancer patients occur throughout the kinase domain. In our study, we find that the predictive power of MD is superior to a purely data-driven machine learning model involving biochemical features that we implemented, even though MD utilized far fewer features (in fact, just one) in an unsupervised setting. Moreover, the MD results provide key insights into convergent mechanisms of activation, primarily involving differential stabilization of a hydrogen bond network that engages residues of the activation loop and αC-helix in the active-like conformation (in >70% of the mutations studied, regardless of the location of the mutation).
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Quinasa de Linfoma Anaplásico/química , Aprendizaje Automático , Simulación de Dinámica Molecular , Mutación , Quinasa de Linfoma Anaplásico/deficiencia , Activación Enzimática/genética , Humanos , Conformación Proteica en Hélice alfaRESUMEN
Advanced techniques allow investigating cellular DNA damage measurements. Ionizing radiation produces multiple DNA damages. Among them, DNA double strand breaks are most toxic to cells. DSBs can form mutations, chromosome aberrations, and cell killing. Although DSBs in cells can be detected directly by neutral elution, pulse field gel electrophoresis, and premature chromosome condensation, recent technologies like cellular immunocytochemistry-based fluorescence detection allow us to visualize the DSBs in cells. Here, we describe gamma-H2AX and Rad51 focus formation assay, which play an important role in DNA damage responses.
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Bioensayo/métodos , Daño del ADN , Histonas/metabolismo , Humanos , Rayos XRESUMEN
DNA double-strand breaks (DSBs) are deleterious DNA lesions that must be properly repaired to maintain genome stability. Agents, generated both exogenously (environmental radiation, dental X-rays, etc.) and endogenously (reactive oxygen species, DNA replication, V(D)J recombination, etc.), induce numerous DSBs every day. To counter these DSBs, there are two major repair pathways in mammalian cells, nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ directly mediates the religation of the broken DNA molecule and is active in all phases of the cell cycle. HR directs repair via the use of a homologous DNA sequence as a template and is primarily active in only S/G2 phases owing to the availability of a DNA template via a sister chromatid. As NHEJ and HR are active in multiple cell cycle phases, there is significant interest in how a cell chooses between the two DSB repair pathways. Therefore, it is essential to utilize assays to study DSB repair that can distinguish between the two DSB repair pathways and the different phases of the cell cycle. In this chapter, we describe methods to measure the contribution of DNA repair pathways in different phases of the cell cycle. These methods are simple, can be applied to most mammalian cell lines, and can be used as a broad utility to monitor cell cycle-dependent DSB repair.
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Ciclo Celular , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas/metabolismoRESUMEN
Malignant pleural mesothelioma (MPM) is characterized by dissemination and aggressive growth in the thoracic cavity. Podoplanin (PDPN) is an established diagnostic marker for MPM, but the function of PDPN in MPM is not fully understood. The purpose of this study was to determine the pathogenetic function of PDPN in MPM. Forty-seven of 52 tumors (90%) from Japanese patients with MPM and 3/6 (50%) MPM cell lines tested positive for PDPN. Knocking down PDPN in PDPN-high expressing MPM cells resulted in decreased cell motility. In contrast, overexpression of PDPN in PDPN-low expressing MPM cells enhanced cell motility. PDPN stimulated motility was mediated by activation of the RhoA/ROCK pathway. Moreover, knocking down PDPN with short hairpin (sh) RNA in PDPN-high expressing MPM cells resulted in decreased development of a thoracic tumor in mice with severe combined immune deficiency (SCID). In sharp contrast, transfection of PDPN in PDPN-low expressing MPM cells resulted in an increase in the number of Ki-67-positive proliferating tumor cells and it promoted progression of a thoracic tumor in SCID mice. Interestingly, PDPN promoted focus formation in vitro, and a low level of E-cadherin expression and YAP1 activation was observed in PDPN-high MPM tumors. These findings indicate that PDPN is a diagnostic marker as well as a pathogenetic regulator that promotes MPM progression by increasing cell motility and inducing focus formation. Therefore, PDPN might be a pathogenetic determinant of MPM dissemination and aggressive growth and may thus be an ideal therapeutic target.
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Movimiento Celular/genética , Glicoproteínas de Membrana/genética , Mesotelioma/genética , Neoplasias Pleurales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Western Blotting , Cadherinas/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Glicoproteínas de Membrana/metabolismo , Mesotelioma/metabolismo , Mesotelioma/patología , Ratones SCID , Fosfoproteínas/metabolismo , Neoplasias Pleurales/metabolismo , Neoplasias Pleurales/patología , Interferencia de ARN , Transducción de Señal , Factores de Transcripción , Trasplante Heterólogo , Proteínas Señalizadoras YAP , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
BACKGROUND: The ability to image DNA repair in cancer cells after irradiation, as well as its inhibition by potential therapeutic agents, is important for the further development of effective cancer therapy. 53BP1 is a DNA repair protein that is overexpressed and forms foci when double-stranded DNA breaks occur in DNA. MATERIALS AND METHODS: The re-localization of green fluorescent protein (GFP) fused to the chromatin-binding domain of 53BP1 to form foci was imaged after UVC irradiation of breast and pancreatic cancer cells expressing 53BP1-GFP using confocal microscopy. RESULTS: During live-cell imaging, 53BP1-GFP focus formation was observed within 10 minutes after UVC irradiation. Most 53BP1 foci resolved by 100 minutes. To block UVC-induced double-strand break repair in cancer cells, poly(ADP-ribose) polymerase (PARP) was targeted with ABT-888 (veliparib). PARP inhibition markedly enhanced UVC-irradiation-induced persistence of 53BP1-foci, even beyond 100 minutes after UVC irradiation, and reduced proliferation of breast and pancreatic cancer cells. CONCLUSION: Confocal microscopy of 53BP1-GFP is a powerful method for imaging UVC-induced DNA damage and repair, as well as inhibition of repair.
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Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/terapia , Poli(ADP-Ribosa) Polimerasa-1/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Bencimidazoles/administración & dosificación , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Proteínas Fluorescentes Verdes/química , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Fotoquimioterapia , Poli(ADP-Ribosa) Polimerasa-1/biosíntesis , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Proteína 1 de Unión al Supresor Tumoral P53/biosíntesis , Rayos UltravioletaRESUMEN
The response to DNA damage during mitosis was visualized using real-time fluorescence imaging of focus formation by the DNA-damage repair (DDR) response protein 53BP1 linked to green fluorescent protein (GFP) (53BP1-GFP) in the MiaPaCa-2(Tet-On) pancreatic cancer cell line. To observe 53BP1-GFP foci during mitosis, MiaPaCa-2(Tet-On) 53BP1-GFP cells were imaged every 30 min by confocal microscopy. Time-lapse imaging demonstrated that 11.4 ± 2.1% of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells had increased focus formation over time. Non-mitotic cells did not have an increase in 53BP1-GFP focus formation over time. Some of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells with focus formation became apoptotic. The results of the present report suggest that DNA strand breaks occur during mitosis and undergo repair, which may cause some of the mitotic cells to enter apoptosis in a phenomenon possibly related to mitotic catastrophe.