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1.
Cell ; 160(6): 1072-86, 2015 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-25768904

RESUMEN

The mechanisms by which transcription factor haploinsufficiency alters the epigenetic and transcriptional landscape in human cells to cause disease are unknown. Here, we utilized human induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) to show that heterozygous nonsense mutations in NOTCH1 that cause aortic valve calcification disrupt the epigenetic architecture, resulting in derepression of latent pro-osteogenic and -inflammatory gene networks. Hemodynamic shear stress, which protects valves from calcification in vivo, activated anti-osteogenic and anti-inflammatory networks in NOTCH1(+/+), but not NOTCH1(+/-), iPSC-derived ECs. NOTCH1 haploinsufficiency altered H3K27ac at NOTCH1-bound enhancers, dysregulating downstream transcription of more than 1,000 genes involved in osteogenesis, inflammation, and oxidative stress. Computational predictions of the disrupted NOTCH1-dependent gene network revealed regulatory nodes that, when modulated, restored the network toward the NOTCH1(+/+) state. Our results highlight how alterations in transcription factor dosage affect gene networks leading to human disease and reveal nodes for potential therapeutic intervention.


Asunto(s)
Epigénesis Genética , Redes Reguladoras de Genes , Receptor Notch1/genética , Células Endoteliales/metabolismo , Femenino , Haploinsuficiencia , Código de Histonas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/metabolismo , Masculino , Osteogénesis , Linaje , Receptor Notch1/metabolismo , Estrés Mecánico , Transcripción Genética
2.
Nucleic Acids Res ; 52(2): 831-843, 2024 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-38084901

RESUMEN

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.


Asunto(s)
Empaquetamiento del Genoma Viral , Ensamble de Virus , Ensamble de Virus/genética , Bacteriófago lambda/genética , Endodesoxirribonucleasas/metabolismo , ADN , ADN Viral/metabolismo , Empaquetamiento del ADN
3.
Cell Mol Life Sci ; 81(1): 283, 2024 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-38963422

RESUMEN

Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.


Asunto(s)
AMP Cíclico , Factores de Intercambio de Guanina Nucleótido , Sumoilación , Enzimas Ubiquitina-Conjugadoras , Proteínas de Unión al GTP rap1 , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/química , Humanos , AMP Cíclico/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Proteínas de Unión al GTP rap1/metabolismo , Proteínas de Unión al GTP rap1/genética , Células HEK293 , Simulación de Dinámica Molecular , Complejo Shelterina/metabolismo , Transducción de Señal , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap/genética , Respuesta al Choque Térmico , Secuencia de Aminoácidos , Unión Proteica
4.
Nucleic Acids Res ; 51(16): 8850-8863, 2023 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-37486760

RESUMEN

The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the 5' end of the genome functions as a switch to transition from viral translation to replication by interacting with host poly(C)-binding protein 2 (PCBP2) and the viral 3CDpro protein. We determined the structures of cloverleaf RNA from coxsackievirus and poliovirus. Cloverleaf RNA folds into an H-type four-way junction and is stabilized by a unique adenosine-cytidine-uridine (A•C-U) base triple involving the conserved pyrimidine mismatch region. The two PCBP2 binding sites are spatially proximal and are located on the opposite end from the 3CDpro binding site on cloverleaf. We determined that the A•C-U base triple restricts the flexibility of the cloverleaf stem-loops resulting in partial occlusion of the PCBP2 binding site, and elimination of the A•C-U base triple increases the binding affinity of PCBP2 to the cloverleaf RNA. Based on the cloverleaf structures and biophysical assays, we propose a new mechanistic model by which enteroviruses use the cloverleaf structure as a molecular switch to transition from viral protein translation to genome replication.


Asunto(s)
Enterovirus , Genoma Viral , Poliovirus , ARN Viral , Humanos , Enterovirus/genética , Enterovirus/fisiología , Células HeLa , Conformación de Ácido Nucleico , Poliovirus/genética , Poliovirus/fisiología , Biosíntesis de Proteínas , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/genética
5.
J Biol Chem ; 298(11): 102536, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36174675

RESUMEN

The cellular response to hypoxia is regulated through enzymatic oxygen sensors, including the prolyl hydroxylases, which control degradation of the well-known hypoxia inducible factors (HIFs). Other enzymatic oxygen sensors have been recently identified, including members of the KDM histone demethylase family. Little is known about how different oxygen-sensing pathways interact and if this varies depending on the form of hypoxia, such as chronic or intermittent. In this study, we investigated how two proposed cellular oxygen-sensing systems, HIF-1 and KDM4A, KDM4B, and KDM4C, respond in cells exposed to rapid forms of intermittent hypoxia (minutes) and compared to chronic hypoxia (hours). We found that intermittent hypoxia increases HIF-1α protein through a pathway distinct from chronic hypoxia, involving the KDM4A, KDM4B, and KDM4C histone lysine demethylases. Intermittent hypoxia increases the quantity and activity of KDM4A, KDM4B, and KDM4C, resulting in a decrease in histone 3 lysine 9 (H3K9) trimethylation near the HIF1A locus. We demonstrate that this contrasts with chronic hypoxia, which decreases KDM4A, KDM4B, and KDM4C activity, leading to hypertrimethylation of H3K9 globally and at the HIF1A locus. Altogether, we found that demethylation of histones bound to the HIF1A gene in intermittent hypoxia increases HIF1A mRNA expression, which has the downstream effect of increasing overall HIF-1 activity and expression of HIF target genes. This study highlights how multiple oxygen-sensing pathways can interact to regulate and fine tune the cellular hypoxic response depending on the period and length of hypoxia.


Asunto(s)
Histonas , Subunidad alfa del Factor 1 Inducible por Hipoxia , Procesamiento Proteico-Postraduccional , Humanos , Desmetilación , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Oxígeno/metabolismo
6.
Nucleic Acids Res ; 49(11): 6474-6488, 2021 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-34050764

RESUMEN

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases.


Asunto(s)
Adenosina Trifosfatasas/química , Empaquetamiento del Genoma Viral , Proteínas Virales/química , Adenosina/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Arginina/química , Fagos de Bacillus/enzimología , Dominio Catalítico , Cristalografía por Rayos X , Simulación de Dinámica Molecular , Fosfatos/química , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Proteínas Virales/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(37): 23140-23147, 2020 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-32868422

RESUMEN

In higher plants, molecular responses to exogenous hypoxia are driven by group VII ethylene response factors (ERF-VIIs). These transcriptional regulators accumulate in the nucleus under hypoxia to activate anaerobic genes but are destabilized in normoxic conditions through the action of oxygen-sensing plant cysteine oxidases (PCOs). The PCOs catalyze the reaction of oxygen with the conserved N-terminal cysteine of ERF-VIIs to form cysteine sulfinic acid, triggering degradation via the Cys/Arg branch of the N-degron pathway. The PCOs are therefore a vital component of the plant oxygen signaling system, connecting environmental stimulus with cellular and physiological response. Rational manipulation of PCO activity could regulate ERF-VII levels and improve flood tolerance, but requires detailed structural information. We report crystal structures of the constitutively expressed PCO4 and PCO5 from Arabidopsis thaliana to 1.24 and 1.91 Å resolution, respectively. The structures reveal that the PCOs comprise a cupin-like scaffold, which supports a central metal cofactor coordinated by three histidines. While this overall structure is consistent with other thiol dioxygenases, closer inspection of the active site indicates that other catalytic features are not conserved, suggesting that the PCOs may use divergent mechanisms to oxidize their substrates. Conservative substitution of two active site residues had dramatic effects on PCO4 function both in vitro and in vivo, through yeast and plant complementation assays. Collectively, our data identify key structural elements that are required for PCO activity and provide a platform for engineering crops with improved hypoxia tolerance.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Oxígeno/metabolismo , Cisteína-Dioxigenasa/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Oxidación-Reducción , Transducción de Señal/fisiología , Factores de Transcripción
8.
Proc Natl Acad Sci U S A ; 117(30): 17992-18001, 2020 07 28.
Artículo en Inglés | MEDLINE | ID: mdl-32669438

RESUMEN

Dengue virus (DENV) was designated as a top 10 public health threat by the World Health Organization in 2019. No clinically approved anti-DENV drug is currently available. Here we report the high-resolution cocrystal structure (1.5 Å) of the DENV-2 capsid protein in complex with an inhibitor that potently suppresses DENV-2 but not other DENV serotypes. The inhibitor induces a "kissing" interaction between two capsid dimers. The inhibitor-bound capsid tetramers are assembled inside virions, resulting in defective uncoating of nucleocapsid when infecting new cells. Resistant DENV-2 emerges through one mutation that abolishes hydrogen bonds in the capsid structure, leading to a loss of compound binding. Structure-based analysis has defined the amino acids responsible for the inhibitor's inefficacy against other DENV serotypes. The results have uncovered an antiviral mechanism through inhibitor-induced tetramerization of the viral capsid and provided essential structural and functional knowledge for rational design of panserotype DENV capsid inhibitors.


Asunto(s)
Antivirales/química , Proteínas de la Cápside/química , Virus del Dengue , Modelos Moleculares , Conformación Proteica , Secuencia de Aminoácidos , Antivirales/farmacología , Sitios de Unión , Proteínas de la Cápside/genética , Virus del Dengue/efectos de los fármacos , Mutación , Nucleocápside/química , Nucleocápside/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad
9.
Death Stud ; 47(5): 618-623, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-35939644

RESUMEN

Cannabis use has been indicated as a risk factor for suicide in veterans. This study of Gulf War veterans tested the relationship between self-report past year cannabis use and (a) past year suicidal ideation and (b) risk for suicidal behavior. Data were from a national sample (N = 1126) of Gulf War veterans. Logistic regression models indicated cannabis use was associated with past year suicidal ideation and elevated risk for suicidal behavior, independent of key covariates. In corroboration with research on other military populations, this study indicates a potentially concerning association between cannabis use and suicide risk in Gulf War veterans.


Asunto(s)
Cannabis , Trastornos por Estrés Postraumático , Suicidio , Veteranos , Humanos , Cannabis/efectos adversos , Guerra del Golfo , Ideación Suicida , Factores de Riesgo
10.
Gut ; 71(12): 2502-2517, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-35477539

RESUMEN

OBJECTIVE: Stroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy. DESIGN: To address the lack of efficacious therapeutic options for patients with stroma-rich CC, we stratified our human tumour cohorts according to stromal content, enabling identification of the biology underpinning relapse and potential therapeutic vulnerabilities specifically within stroma-rich tumours that could be exploited clinically. Following human tumour-based discovery and independent clinical validation, we use a series of in vitro and stroma-rich in vivo models to test and validate the therapeutic potential of elevating the biology associated with reduced relapse in human tumours. RESULTS: By performing our analyses specifically within the stroma-rich/high-fibroblast (HiFi) subtype of CC, we identify and validate the clinical value of a HiFi-specific prognostic signature (HPS), which stratifies tumours based on STAT1-related signalling (High-HPS v Low-HPS=HR 0.093, CI 0.019 to 0.466). Using in silico, in vitro and in vivo models, we demonstrate that the HPS is associated with antigen processing and presentation within discrete immune lineages in stroma-rich CC, downstream of double-stranded RNA and viral response signalling. Treatment with the TLR3 agonist poly(I:C) elevated the HPS signalling and antigen processing phenotype across in vitro and in vivo models. In an in vivo model of stroma-rich CC, poly(I:C) treatment significantly increased systemic cytotoxic T cell activity (p<0.05) and reduced liver metastases (p<0.0002). CONCLUSION: This study reveals new biological insight that offers a novel therapeutic option to reduce relapse rates in patients with the worst prognosis CC.


Asunto(s)
Biomarcadores de Tumor , Neoplasias del Colon , Humanos , Biomarcadores de Tumor/genética , Células del Estroma/patología , Recurrencia Local de Neoplasia/prevención & control , Recurrencia Local de Neoplasia/patología , Neoplasias del Colon/patología , Pronóstico
11.
J Hum Evol ; 165: 103153, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35299090

RESUMEN

Studies of flake tools in the British Lower Paleolithic are rare owing to lower quantities of flake tools than handaxes and the perception that flake tool technology became more important in the succeeding Middle Paleolithic. In Britain, and Europe more broadly, MIS 9 (328-301 ka) has been characterized as a period of technological transition owing to the presence of early prepared core technology and the status of the period as the final interglacial prior to the onset of the Middle Paleolithic. It has been argued that the period demonstrates an increase in both the numbers and importance of flake tools, possibly showing emerging Middle Paleolithic behaviors. This study presents the results of a technological examination of flake tools in Britain during MIS 9, focusing on 25 sites, including 15 assemblages previously recorded as having higher quantities of flake tools. We use these assemblages to assess whether the flake tools of MIS 9 represent a transition toward the technology of the Middle Paleolithic. We consider factors including collection history, site formation, function, reduction, and cultural groups. We argue that in Britain the archaeological record of MIS 9 does not show an increase in the use of flake tools and demonstrates more continuity than change in relation to earlier periods of the Lower Paleolithic. There is a technological background of ad hoc retouch of simple flake tools with occasional evidence of more invasively retouched scrapers. Furthermore, aside from the introduction of Levallois technology, flake tools change little in the Early Middle Paleolithic. These results are contextualized within the broader evidence from Europe and comparisons to the longer sequences at key sites. We conclude that the major changes in technology began between MIS 13 and MIS 11 and these merely became cemented during MIS 9 and the following Middle Paleolithic.


Asunto(s)
Arqueología , Hominidae , Animales , Europa (Continente) , Tecnología , Reino Unido
12.
J Nurs Scholarsh ; 54(6): 750-761, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35568964

RESUMEN

PURPOSE: In Ireland, there is a regulatory focus on restraint minimisation in elderly residential care facilities. Consistent with this focus, this study aimed to explore and identify the relationship between nurses' knowledge levels, attitudes and intentions regarding physical restraint use in two large Irish elderly residential care facilities. DESIGN: A correlational and cross-sectional survey design was used to collect data on variables including nurses' education levels, years of experience and intentions toward restraint utilization. METHODS: Data was obtained from a sample of 83 nurses in early 2020 via an anonymous, adapted survey measuring knowledge, attitude and intentions. RESULTS: Results showed high knowledge levels, negative attitudes toward restraint implementation, and moderate mean intention scores. A significant positive relationship existed between knowledge and attitudes, with both variables negatively predicting intentions regarding restraint. Education was significant in predicting knowledge and attitudes; however, years of experience reported no such findings. CONCLUSION: Knowledge and attitudes negatively predict nurses' intentions toward restraint, with attitude being the stronger predictor of intentions. Falls risk caused the greatest variation in intention scores. CLINICAL RELEVANCE: This research offered a seminal study providing insight into the use of restraints in an Irish context with findings that are in line with international research. It highlights the importance of knowledge and attitudes along with education with understanding intentions to use restraints. Furthermore this research demonstrates a useful instrument in the assessment of nurses' knowledge, attitudes, and intentions in Irish elderly residential care facilities, which can possibly be used in other settings.


Asunto(s)
Enfermeras y Enfermeros , Restricción Física , Humanos , Anciano , Intención , Conocimientos, Actitudes y Práctica en Salud , Actitud del Personal de Salud , Estudios Transversales , Competencia Clínica , Encuestas y Cuestionarios
13.
Environ Monit Assess ; 194(8): 578, 2022 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-35819550

RESUMEN

For pesticide registrations in the USA under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), as implemented by the United States Environmental Protection Agency, drinking water risk assessments for groundwater sources are based on standard scenario modeling concentration estimates. The conceptual model for the drinking water protection goals is defined in terms of (1) a rural well in or near a relatively high pesticide use area, a shallow well (4-10 m); (2) long-term, single-station weather data; (3) soils characterized as highly leachable; (4) upper-end or surrogate, worst-case environmental fate parameters; and (5) maximum, annual use rates repeated every year. To date, monitoring data have not been quantitatively incorporated into FIFRA drinking water risk assessment; even though considerable, US national-scale temporal and spatial data for some chemistries exists. Investigations into drinking water monitoring data development have historically focused on single-source efforts that may not represent wide geographies and/or time periods, whereas Safe Drinking Water Act groundwater monitoring data are focused on a community-level scale rather than an individual, shallow, rural well. In the current case study, US national-scale, rural well data for the herbicide atrazine was collected, quality controlled, and combined into a single database from mixed sources (termed the atrazine rural well database) to (1) characterize differences between exposure estimates from standard EPA modeling approaches for specific characterization, (2) evaluate monitoring data toward direct use in US drinking water risk assessments to compliment or supersede standard modeling approaches to define risk, and (3) evaluate monitoring trends a function of time relative to label changes implemented as part of the registration review process. Of the 75,665 drinking water samples collected from groundwater, atrazine was only detected in 3185, a 4% detection rate.


Asunto(s)
Atrazina , Agua Potable , Agua Subterránea , Plaguicidas , Atrazina/análisis , Monitoreo del Ambiente , Plaguicidas/análisis , Estados Unidos
14.
Biochemistry ; 60(40): 2987-3006, 2021 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-34605636

RESUMEN

During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Receptores de AMP Cíclico/metabolismo , Algoritmos , Regulación Alostérica , Sitios de Unión , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli/química , Concentración de Iones de Hidrógeno , Modelos Químicos , Unión Proteica , Conformación Proteica , Dominios Proteicos , Receptores de AMP Cíclico/química , Termodinámica
15.
Cancer Immunol Immunother ; 70(12): 3525-3540, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33903974

RESUMEN

Immune checkpoint inhibitors (ICIs) have emerged as promising therapies for the treatment of cancer. However, existing ICIs, namely PD-(L)1 and CTLA-4 inhibitors, generate durable responses only in a subset of patients. TIGIT is a co-inhibitory receptor and member of the DNAM-1 family of immune modulating proteins. We evaluated the prevalence of TIGIT and its cognate ligand, PVR (CD155), in human cancers by assessing their expression in a large set of solid tumors. TIGIT is expressed on CD4+ and CD8+ TILs and is upregulated in tumors compared to normal tissues. PVR is expressed on tumor cells and tumor-associated macrophages from multiple solid tumors. We explored the therapeutic potential of targeting TIGIT by generating COM902, a fully human anti-TIGIT hinge-stabilized IgG4 monoclonal antibody that binds specifically to human, cynomolgus monkey, and mouse TIGIT, and disrupts the binding of TIGIT with PVR. COM902, either alone or in combination with a PVRIG (COM701) or PD-1 inhibitor, enhances antigen-specific human T cell responses in-vitro. In-vivo, a mouse chimeric version of COM902 in combination with an anti-PVRIG or anti-PD-L1 antibody inhibited tumor growth and increased survival in two syngeneic mouse tumor models. In summary, COM902 enhances anti-tumor immune responses and is a promising candidate for the treatment of advanced malignancies.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígeno B7-H1/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Receptores de Superficie Celular/inmunología , Receptores Inmunológicos/inmunología , Transducción de Señal/inmunología , Animales , Línea Celular Tumoral , Proliferación Celular/fisiología , Femenino , Humanos , Inmunoglobulina G/inmunología , Inmunoterapia/métodos , Células Jurkat , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C
16.
Eur Radiol ; 31(7): 5312-5323, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-33452627

RESUMEN

OBJECTIVES: We examined whether providing a quantitative report (QReport) of regional brain volumes improves radiologists' accuracy and confidence in detecting volume loss, and in differentiating Alzheimer's disease (AD) and frontotemporal dementia (FTD), compared with visual assessment alone. METHODS: Our forced-choice multi-rater clinical accuracy study used MRI from 16 AD patients, 14 FTD patients, and 15 healthy controls; age range 52-81. Our QReport was presented to raters with regional grey matter volumes plotted as percentiles against data from a normative population (n = 461). Nine raters with varying radiological experience (3 each: consultants, registrars, 'non-clinical image analysts') assessed each case twice (with and without the QReport). Raters were blinded to clinical and demographic information; they classified scans as 'normal' or 'abnormal' and if 'abnormal' as 'AD' or 'FTD'. RESULTS: The QReport improved sensitivity for detecting volume loss and AD across all raters combined (p = 0.015* and p = 0.002*, respectively). Only the consultant group's accuracy increased significantly when using the QReport (p = 0.02*). Overall, raters' agreement (Cohen's κ) with the 'gold standard' was not significantly affected by the QReport; only the consultant group improved significantly (κs 0.41➔0.55, p = 0.04*). Cronbach's alpha for interrater agreement improved from 0.886 to 0.925, corresponding to an improvement from 'good' to 'excellent'. CONCLUSION: Our QReport referencing single-subject results to normative data alongside visual assessment improved sensitivity, accuracy, and interrater agreement for detecting volume loss. The QReport was most effective in the consultants, suggesting that experience is needed to fully benefit from the additional information provided by quantitative analyses. KEY POINTS: • The use of quantitative report alongside routine visual MRI assessment improves sensitivity and accuracy for detecting volume loss and AD vs visual assessment alone. • Consultant neuroradiologists' assessment accuracy and agreement (kappa scores) significantly improved with the use of quantitative atrophy reports. • First multi-rater radiological clinical evaluation of visual quantitative MRI atrophy report for use as a diagnostic aid in dementia.


Asunto(s)
Enfermedad de Alzheimer , Demencia Frontotemporal , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/diagnóstico por imagen , Atrofia , Demencia Frontotemporal/diagnóstico por imagen , Sustancia Gris , Humanos , Imagen por Resonancia Magnética , Persona de Mediana Edad
17.
Eur Radiol ; 31(1): 34-44, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32749588

RESUMEN

OBJECTIVES: Hippocampal sclerosis (HS) is a common cause of temporal lobe epilepsy. Neuroradiological practice relies on visual assessment, but quantification of HS imaging biomarkers-hippocampal volume loss and T2 elevation-could improve detection. We tested whether quantitative measures, contextualised with normative data, improve rater accuracy and confidence. METHODS: Quantitative reports (QReports) were generated for 43 individuals with epilepsy (mean age ± SD 40.0 ± 14.8 years, 22 men; 15 histologically unilateral HS; 5 bilateral; 23 MR-negative). Normative data was generated from 111 healthy individuals (age 40.0 ± 12.8 years, 52 men). Nine raters with different experience (neuroradiologists, trainees, and image analysts) assessed subjects' imaging with and without QReports. Raters assigned imaging normal, right, left, or bilateral HS. Confidence was rated on a 5-point scale. RESULTS: Correct designation (normal/abnormal) was high and showed further trend-level improvement with QReports, from 87.5 to 92.5% (p = 0.07, effect size d = 0.69). Largest magnitude improvement (84.5 to 93.8%) was for image analysts (d = 0.87). For bilateral HS, QReports significantly improved overall accuracy, from 74.4 to 91.1% (p = 0.042, d = 0.7). Agreement with the correct diagnosis (kappa) tended to increase from 0.74 ('fair') to 0.86 ('excellent') with the report (p = 0.06, d = 0.81). Confidence increased when correctly assessing scans with the QReport (p < 0.001, η2p = 0.945). CONCLUSIONS: QReports of HS imaging biomarkers can improve rater accuracy and confidence, particularly in challenging bilateral cases. Improvements were seen across all raters, with large effect sizes, greatest for image analysts. These findings may have positive implications for clinical radiology services and justify further validation in larger groups. KEY POINTS: • Quantification of imaging biomarkers for hippocampal sclerosis-volume loss and raised T2 signal-could improve clinical radiological detection in challenging cases. • Quantitative reports for individual patients, contextualised with normative reference data, improved diagnostic accuracy and confidence in a group of nine raters, in particular for bilateral HS cases. • We present a pre-use clinical validation of an automated imaging assessment tool to assist clinical radiology reporting of hippocampal sclerosis, which improves detection accuracy.


Asunto(s)
Epilepsia del Lóbulo Temporal , Epilepsia , Adulto , Epilepsia/patología , Epilepsia del Lóbulo Temporal/diagnóstico por imagen , Epilepsia del Lóbulo Temporal/patología , Hipocampo/diagnóstico por imagen , Hipocampo/patología , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Esclerosis/diagnóstico por imagen , Esclerosis/patología
18.
Nature ; 522(7557): 502-6, 2015 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-26083743

RESUMEN

Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases. The active site environment is dominated by π systems, which assist phosphate-C1' bond breakage following FMN reduction, leading to formation of the N5-C1' bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3'-C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires.


Asunto(s)
Biocatálisis , Carboxiliasas/metabolismo , Dimetilaliltranstransferasa/metabolismo , Flavinas/metabolismo , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/metabolismo , Ubiquinona/biosíntesis , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Aspergillus niger/enzimología , Aspergillus niger/genética , Carboxiliasas/química , Carboxiliasas/genética , Dominio Catalítico , Cristalografía por Rayos X , Reacción de Cicloadición , Descarboxilación , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/genética , Transporte de Electrón , Mononucleótido de Flavina/metabolismo , Flavinas/biosíntesis , Flavinas/química , Modelos Moleculares , Pseudomonas aeruginosa/genética
19.
Nature ; 522(7557): 497-501, 2015 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-26083754

RESUMEN

The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis or microbial biodegradation of aromatic compounds, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad1. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.


Asunto(s)
Biocatálisis , Carboxiliasas/metabolismo , Reacción de Cicloadición , Alquenos/química , Alquenos/metabolismo , Aspergillus niger/enzimología , Aspergillus niger/genética , Carboxiliasas/química , Carboxiliasas/genética , Cristalografía por Rayos X , Descarboxilación , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flavinas/biosíntesis , Flavinas/química , Flavinas/metabolismo , Isomerismo , Ligandos , Modelos Moleculares , Ubiquinona/biosíntesis
20.
Environ Monit Assess ; 193(12): 827, 2021 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-34796399

RESUMEN

Inclusion of pesticide monitoring data in pesticide risk assessment is important yet challenging for several reasons, including infrequent or irregular data collection, disparate sources procedures and associated monitoring periods, and interpretation of the data itself in a policy context. These challenges alone, left unaddressed, will likely introduce unintentional and unforeseen risk assessment conclusions. While individual water quality monitoring programs report standard operating procedures and quality control practices for their own data, cross-checking data for duplicated data from one database to another does not routinely occur. Consequently, we developed a novel quality control and assurance methodology to identify errors and duplicated records toward creating an aggregated, single pesticide database toward use in ecological risk assessment. This methodology includes (1) standardization and reformatting practices, (2) data error and duplicate record identification protocols, (3) missing or inconsistent limit of detection and quantification reporting, and (4) site metadata scoring and ranking procedures to flag likely duplicate records. We applied this methodology to develop an aggregated (multiple-source), national-scale database for atrazine from a diverse set of surface water monitoring programs. The resultant database resolved and/or removed approximately 31% of the total ~ 385,000 records that were due to duplicated records. Identification of sample replicates was also developed. While the quality control and assurances methodologies developed in this work were applied to atrazine, they generally demonstrate how a properly constructed and aggregated single pesticide database would benefit from the methods described herein before use in subsequent statistical and data analysis or risk assessment.


Asunto(s)
Atrazina , Plaguicidas , Atrazina/análisis , Monitoreo del Ambiente , Plaguicidas/análisis , Control de Calidad , Estándares de Referencia
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