Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 28
Filtrar
1.
Hepatology ; 2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-38954820

RESUMEN

Alpha-1 antitrypsin deficiency (A1ATD) is a life-threatening condition caused by inheritance of the SERPINA1 'Z' genetic variant (PiZ) driving AAT protein misfolding in hepatocytes. There remain no approved medicines for this disease. Here, we report the results of a small molecule screen performed in patient derived iPSC-hepatocytes that identified Leucine-rich repeat kinase-2 (LRRK2) as a potentially new therapeutic target. Of the commercially available LRRK2 inhibitors tested, we identified CZC-25146, a candidate with favorable pharmacokinetic properties, as being capable of reducing polymer load, increasing normal AAT secretion, and reducing inflammatory cytokines in both cells and PiZ mice. Mechanistically, this effect was achieved through induction of autophagy. Our findings support the use of CZC-25146 and LRRK2 inhibitors in hepatic proteinopathy research and their further investigation as novel therapeutic candidates for A1ATD.

2.
iScience ; 26(2): 105928, 2023 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-36619367

RESUMEN

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

3.
STAR Protoc ; 3(2): 101355, 2022 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-35542177

RESUMEN

The colony formation assay is the gold-standard technique to assess cell viability after treatment with cytotoxic reagents, ionizing radiation, and cytotoxic combinatorial treatments. This protocol describes a high-throughput automated and high-content imaging approach to screen siRNA molecular libraries in HeLa cervical cancer cells in 96-well format. We detail reverse transfection of cells with siRNAs, followed by ionizing radiation, fixing, and staining of the plates for automated colony counting. This protocol can be used across a broad range of cell types. For complete details on the use and execution of this protocol, please refer to Tiwana et al. (2015).


Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Radiación Ionizante , Biblioteca de Genes , Ensayos Analíticos de Alto Rendimiento/métodos , ARN Interferente Pequeño/genética , Transfección
4.
Clin Infect Dis ; 74(7): 1208-1219, 2022 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-34216472

RESUMEN

BACKGROUND: Natural and vaccine-induced immunity will play a key role in controlling the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. SARS-CoV-2 variants have the potential to evade natural and vaccine-induced immunity. METHODS: In a longitudinal cohort study of healthcare workers (HCWs) in Oxfordshire, United Kingdom, we investigated the protection from symptomatic and asymptomatic polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infection conferred by vaccination (Pfizer-BioNTech BNT162b2, Oxford-AstraZeneca ChAdOx1 nCOV-19) and prior infection (determined using anti-spike antibody status), using Poisson regression adjusted for age, sex, temporal changes in incidence and role. We estimated protection conferred after 1 versus 2 vaccinations and from infections with the B.1.1.7 variant identified using whole genome sequencing. RESULTS: In total, 13 109 HCWs participated; 8285 received the Pfizer-BioNTech vaccine (1407 two doses), and 2738 the Oxford-AstraZeneca vaccine (49 two doses). Compared to unvaccinated seronegative HCWs, natural immunity and 2 vaccination doses provided similar protection against symptomatic infection: no HCW vaccinated twice had symptomatic infection, and incidence was 98% lower in seropositive HCWs (adjusted incidence rate ratio 0.02 [95% confidence interval {CI} < .01-.18]). Two vaccine doses or seropositivity reduced the incidence of any PCR-positive result with or without symptoms by 90% (0.10 [95% CI .02-.38]) and 85% (0.15 [95% CI .08-.26]), respectively. Single-dose vaccination reduced the incidence of symptomatic infection by 67% (0.33 [95% CI .21-.52]) and any PCR-positive result by 64% (0.36 [95% CI .26-.50]). There was no evidence of differences in immunity induced by natural infection and vaccination for infections with S-gene target failure and B.1.1.7. CONCLUSIONS: Natural infection resulting in detectable anti-spike antibodies and 2 vaccine doses both provide robust protection against SARS-CoV-2 infection, including against the B.1.1.7 variant.


Asunto(s)
COVID-19 , SARS-CoV-2 , Vacuna BNT162 , COVID-19/epidemiología , COVID-19/prevención & control , Vacunas contra la COVID-19 , ChAdOx1 nCoV-19 , Estudios de Cohortes , Personal de Salud , Humanos , Inmunoglobulinas , Incidencia , Estudios Longitudinales , Vacunación
5.
Oncogene ; 41(4): 476-488, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34773074

RESUMEN

We recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF-1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs.


Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Receptor IGF Tipo 1/metabolismo , Línea Celular Tumoral , Humanos , Transfección
6.
BMC Cancer ; 21(1): 896, 2021 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-34353313

RESUMEN

BACKGROUND: In clear cell renal cell carcinoma, 80% of cases have biallelic inactivation of the VHL gene, leading to constitutive activation of both HIF1α and HIF2α. As HIF2α is the driver of the disease promoting tumour growth and metastasis, drugs targeting HIF2α have been developed. However, resistance is common, therefore new therapies are needed. METHODS: We assessed the effect of the HIF2α antagonist PT2385 in several steps of tumour development and performed RNAseq to identify genes differentially expressed upon treatment. A drug screening was used to identify drugs with antiproliferative effects on VHL-mutated HIF2α-expressing cells and could increase effectiveness of PT2385. RESULTS: PT2385 did not reduce cell proliferation or clonogenicity but, in contrast to the genetic silencing of HIF2α, it reduced in vitro cell invasion. Many HIF-inducible genes were down-regulated upon PT2385 treatment, whereas some genes involved in cell migration or extracellular matrix were up-regulated. HIF2α was associated with resistance to statins, addition to PT2385 did not increase the sensitivity. CONCLUSIONS: this study shows key differences between inhibiting a target versus knockdown, which are potentially targetable.


Asunto(s)
Antineoplásicos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Silenciador del Gen , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Reposicionamiento de Medicamentos , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Perfilación de la Expresión Génica , Humanos , Indanos/farmacología , Indanos/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Sulfonas/farmacología , Sulfonas/uso terapéutico , Activación Transcripcional , Transcriptoma , Resultado del Tratamiento
7.
Clin Microbiol Infect ; 27(10): 1516.e7-1516.e14, 2021 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-34111577

RESUMEN

OBJECTIVES: We investigated determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike IgG responses in healthcare workers (HCWs) following one or two doses of Pfizer-BioNTech or Oxford-AstraZeneca vaccines. METHODS: HCWs participating in regular SARS-CoV-2 PCR and antibody testing were invited for serological testing prior to first and second vaccination, and 4 weeks post-vaccination if receiving a 12-week dosing interval. Quantitative post-vaccination anti-spike antibody responses were measured using the Abbott SARS-CoV-2 IgG II Quant assay (detection threshold: ≥50 AU/mL). We used multivariable logistic regression to identify predictors of seropositivity and generalized additive models to track antibody responses over time. RESULTS: 3570/3610 HCWs (98.9%) were seropositive >14 days post first vaccination and prior to second vaccination: 2706/2720 (99.5%) were seropositive after the Pfizer-BioNTech and 864/890 (97.1%) following the Oxford-AstraZeneca vaccines. Previously infected and younger HCWs were more likely to test seropositive post first vaccination, with no evidence of differences by sex or ethnicity. All 470 HCWs tested >14 days after the second vaccination were seropositive. Quantitative antibody responses were higher after previous infection: median (IQR) >21 days post first Pfizer-BioNTech 14 604 (7644-22 291) AU/mL versus 1028 (564-1985) AU/mL without prior infection (p < 0.001). Oxford-AstraZeneca vaccine recipients had lower readings post first dose than Pfizer-BioNTech recipients, with and without previous infection, 10 095 (5354-17 096) and 435 (203-962) AU/mL respectively (both p < 0.001 versus Pfizer-BioNTech). Antibody responses >21 days post second Pfizer vaccination in those not previously infected, 10 058 (6408-15 582) AU/mL, were similar to those after prior infection followed by one vaccine dose. CONCLUSIONS: SARS-CoV-2 vaccination leads to detectable anti-spike antibodies in nearly all adult HCWs. Whether differences in response impact vaccine efficacy needs further study.


Asunto(s)
Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Vacuna BNT162 , COVID-19/sangre , ChAdOx1 nCoV-19 , Femenino , Personal de Salud , Humanos , Inmunogenicidad Vacunal , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Vacunación
8.
BMC Infect Dis ; 21(1): 187, 2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33602152

RESUMEN

BACKGROUND: Thresholds for SARS-CoV-2 antibody assays have typically been determined using samples from symptomatic, often hospitalised, patients. In this setting the sensitivity and specificity of the best performing assays can both exceed 98%. However, antibody assay performance following mild infection is less clear. METHODS: We assessed quantitative IgG responses in a cohort of healthcare workers in Oxford, UK, with a high pre-test probability of Covid-19, in particular the 991/11,475(8.6%) who reported loss of smell/taste. We use anosmia/ageusia and other risk factors as probes for Covid-19 infection potentially undiagnosed by immunoassays by investigating their relationship with antibody readings either side of assay thresholds. RESULTS: The proportion of healthcare workers reporting anosmia/ageusia increased at antibody readings below diagnostic thresholds using an in-house ELISA (n = 9324) and the Abbott Architect chemiluminescent microparticle immunoassay (CMIA; n = 11,324): 426/906 (47%) reported anosmia/ageusia with a positive ELISA, 59/449 (13.1%) with high-negative and 326/7969 (4.1%) with low-negative readings. Similarly, by CMIA, 518/1093 (47.4%) with a positive result reported anosmia/ageusia, 106/686 (15.5%) with a high-negative and 358/9563 (3.7%) with a low-negative result. Adjusting for the proportion of staff reporting anosmia/ageusia suggests the sensitivity of both assays in mild infection is lower than previously reported: Oxford ELISA 89.8% (95%CI 86.6-92.8%) and Abbott CMIA 79.3% (75.9-82.7%). CONCLUSION: Following mild SARS-CoV-2 infection 10-30% of individuals may have negative immunoassay results. While lowered diagnostic thresholds may result in unacceptable specificity, our findings have implications for epidemiological analyses and result interpretation in individuals with a high pre-test probability. Samples from mild PCR-confirmed infections should be included in SARS-CoV-2 immunoassay evaluations.


Asunto(s)
Anticuerpos Antivirales/análisis , Prueba Serológica para COVID-19/normas , COVID-19/diagnóstico , Inmunoglobulina G/análisis , Adulto , Ageusia/virología , Anosmia/virología , Infecciones Asintomáticas , Ensayo de Inmunoadsorción Enzimática/normas , Femenino , Personal de Salud , Humanos , Inmunoensayo/normas , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Enfermedades no Diagnosticadas , Reino Unido
9.
Clin Infect Dis ; 73(3): e699-e709, 2021 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-33400782

RESUMEN

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) antibody measurements can be used to estimate the proportion of a population exposed or infected and may be informative about the risk of future infection. Previous estimates of the duration of antibody responses vary. METHODS: We present 6 months of data from a longitudinal seroprevalence study of 3276 UK healthcare workers (HCWs). Serial measurements of SARS-CoV-2 anti-nucleocapsid and anti-spike IgG were obtained. Interval censored survival analysis was used to investigate the duration of detectable responses. Additionally, Bayesian mixed linear models were used to investigate anti-nucleocapsid waning. RESULTS: Anti-spike IgG levels remained stably detected after a positive result, for example, in 94% (95% credibility interval [CrI] 91-96%) of HCWs at 180 days. Anti-nucleocapsid IgG levels rose to a peak at 24 (95% CrI 19-31) days post first polymerase chain reaction (PCR)-positive test, before beginning to fall. Considering 452 anti-nucleocapsid seropositive HCWs over a median of 121 days from their maximum positive IgG titer, the mean estimated antibody half-life was 85 (95% CrI 81-90) days. Higher maximum observed anti-nucleocapsid titers were associated with longer estimated antibody half-lives. Increasing age, Asian ethnicity, and prior self-reported symptoms were independently associated with higher maximum anti-nucleocapsid levels and increasing age and a positive PCR test undertaken for symptoms with longer anti-nucleocapsid half-lives. CONCLUSIONS: SARS-CoV-2 anti-nucleocapsid antibodies wane within months and fall faster in younger adults and those without symptoms. However, anti-spike IgG remains stably detected. Ongoing longitudinal studies are required to track the long-term duration of antibody levels and their association with immunity to SARS-CoV-2 reinfection.


Asunto(s)
COVID-19 , SARS-CoV-2 , Adulto , Anticuerpos Antivirales , Formación de Anticuerpos , Teorema de Bayes , Personal de Salud , Humanos , Inmunoglobulina G , Estudios Seroepidemiológicos
10.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-33429995

RESUMEN

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.


Asunto(s)
Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Bibliotecas de Moléculas Pequeñas/química , Humanos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-Actividad
11.
Cancer Res ; 81(8): 2128-2141, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33509941

RESUMEN

Inhibition of IGF receptor (IGF1R) delays repair of radiation-induced DNA double-strand breaks (DSB), prompting us to investigate whether IGF1R influences endogenous DNA damage. Here we demonstrate that IGF1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. In cancer cells, inhibition or depletion of IGF1R delayed replication fork progression accompanied by activation of ATR-CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflected unanticipated regulation of global replication by IGF1 mediated via AKT, MEK/ERK, and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. Consequently, inhibition or depletion of IGF1R downregulated RRM2, compromising RNR function and perturbing dNTP supply. The resulting delay in fork progression and hallmarks of replication stress were rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF1 regulates replication. Suspecting existence of a backup pathway protecting from toxic sequelae of replication stress, targeted compound screens in breast cancer cells identified synergy between IGF inhibition and ATM loss. Reciprocal screens of ATM-proficient/deficient fibroblasts identified an IGF1R inhibitor as the top hit. IGF inhibition selectively compromised growth of ATM-null cells and spheroids and caused regression of ATM-null xenografts. This synthetic-lethal effect reflected conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. Overall, these data implicate IGF1R in alleviating replication stress, and the reciprocal IGF:ATM codependence we identify provides an approach to exploit this effect in ATM-deficient cancers. SIGNIFICANCE: This study identifies regulation of ribonucleotide reductase function and dNTP supply by IGFs and demonstrates that IGF axis blockade induces replication stress and reciprocal codependence on ATM. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2128/F1.large.jpg.


Asunto(s)
Roturas del ADN de Doble Cadena , Daño del ADN , Replicación del ADN , Receptor IGF Tipo 1/antagonistas & inhibidores , Ribonucleósido Difosfato Reductasa/metabolismo , Ribonucleótido Reductasas/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Reparación del ADN , Desoxirribonucleósidos/metabolismo , Regulación hacia Abajo , Fibroblastos , Xenoinjertos , Histonas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Células MCF-7 , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Receptores Nucleares Huérfanos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptor IGF Tipo 1/metabolismo , Puntos de Control de la Fase S del Ciclo Celular , Esferoides Celulares
12.
N Engl J Med ; 384(6): 533-540, 2021 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-33369366

RESUMEN

BACKGROUND: The relationship between the presence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the risk of subsequent reinfection remains unclear. METHODS: We investigated the incidence of SARS-CoV-2 infection confirmed by polymerase chain reaction (PCR) in seropositive and seronegative health care workers attending testing of asymptomatic and symptomatic staff at Oxford University Hospitals in the United Kingdom. Baseline antibody status was determined by anti-spike (primary analysis) and anti-nucleocapsid IgG assays, and staff members were followed for up to 31 weeks. We estimated the relative incidence of PCR-positive test results and new symptomatic infection according to antibody status, adjusting for age, participant-reported gender, and changes in incidence over time. RESULTS: A total of 12,541 health care workers participated and had anti-spike IgG measured; 11,364 were followed up after negative antibody results and 1265 after positive results, including 88 in whom seroconversion occurred during follow-up. A total of 223 anti-spike-seronegative health care workers had a positive PCR test (1.09 per 10,000 days at risk), 100 during screening while they were asymptomatic and 123 while symptomatic, whereas 2 anti-spike-seropositive health care workers had a positive PCR test (0.13 per 10,000 days at risk), and both workers were asymptomatic when tested (adjusted incidence rate ratio, 0.11; 95% confidence interval, 0.03 to 0.44; P = 0.002). There were no symptomatic infections in workers with anti-spike antibodies. Rate ratios were similar when the anti-nucleocapsid IgG assay was used alone or in combination with the anti-spike IgG assay to determine baseline status. CONCLUSIONS: The presence of anti-spike or anti-nucleocapsid IgG antibodies was associated with a substantially reduced risk of SARS-CoV-2 reinfection in the ensuing 6 months. (Funded by the U.K. Government Department of Health and Social Care and others.).


Asunto(s)
Anticuerpos Antivirales/sangre , COVID-19/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Personal de Salud , Inmunoglobulina G/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/diagnóstico , COVID-19/epidemiología , Prueba de Ácido Nucleico para COVID-19 , Prueba Serológica para COVID-19 , Femenino , Humanos , Inmunoglobulina G/sangre , Incidencia , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Recurrencia , SARS-CoV-2/aislamiento & purificación , Seroconversión , Reino Unido , Adulto Joven
13.
Euro Surveill ; 25(42)2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33094717

RESUMEN

SARS-CoV-2 IgG screening of 1,000 antenatal serum samples in the Oxford area, United Kingdom, between 14 April and 15 June 2020, yielded a 5.3% seroprevalence, mirroring contemporaneous regional data. Among the 53 positive samples, 39 showed in vitro neutralisation activity, correlating with IgG titre (Pearson's correlation p<0.0001). While SARS-CoV-2 seroprevalence in pregnancy cohorts could potentially inform population surveillance, clinical correlates of infection and immunity in pregnancy, and antenatal epidemiology evolution over time need further study.


Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Betacoronavirus/inmunología , Infecciones por Coronavirus/epidemiología , Inmunoglobulina G/sangre , Pandemias , Neumonía Viral/epidemiología , Vigilancia de la Población , Complicaciones Infecciosas del Embarazo/sangre , Primer Trimestre del Embarazo/sangre , Adolescente , Adulto , COVID-19 , Estudios de Cohortes , Infecciones por Coronavirus/sangre , Inglaterra/epidemiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Persona de Mediana Edad , Neumonía Viral/sangre , Embarazo , Diagnóstico Prenatal , Prevalencia , SARS-CoV-2 , Estudios Seroepidemiológicos , Método Simple Ciego , Adulto Joven
14.
Thorax ; 75(11): 1004-1008, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32943495

RESUMEN

Malignant pleural mesothelioma (MPM) is an aggressive cancer, associated with poor prognosis. We assessed the feasibility of patient-derived cell cultures to serve as an ex vivo model of MPM. Patient-derived MPM cell cultures (n=16) exhibited stemness features and reflected intratumour and interpatient heterogeneity. A subset of the cells were subjected to high-throughput drug screening and coculture assays with cancer-specific cytotoxic T cells and showed diverse responses. Some of the biphasic MPM cells were capable of processing and presenting the neoantigen SSX-2 endogenously. In conclusion, patient-derived MPM cell cultures are a promising and faithful ex vivo model of MPM.


Asunto(s)
Biomarcadores de Tumor/análisis , Mesotelioma Maligno/patología , Neoplasias Pleurales/patología , Células Tumorales Cultivadas/citología , Técnicas de Cultivo de Célula , Genes Supresores de Tumor , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunoterapia , Mesotelioma Maligno/terapia , Mutación , Neoplasias Pleurales/terapia , Secuenciación Completa del Genoma
15.
Elife ; 92020 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-32820721

RESUMEN

We conducted voluntary Covid-19 testing programmes for symptomatic and asymptomatic staff at a UK teaching hospital using naso-/oro-pharyngeal PCR testing and immunoassays for IgG antibodies. 1128/10,034 (11.2%) staff had evidence of Covid-19 at some time. Using questionnaire data provided on potential risk-factors, staff with a confirmed household contact were at greatest risk (adjusted odds ratio [aOR] 4.82 [95%CI 3.45-6.72]). Higher rates of Covid-19 were seen in staff working in Covid-19-facing areas (22.6% vs. 8.6% elsewhere) (aOR 2.47 [1.99-3.08]). Controlling for Covid-19-facing status, risks were heterogenous across the hospital, with higher rates in acute medicine (1.52 [1.07-2.16]) and sporadic outbreaks in areas with few or no Covid-19 patients. Covid-19 intensive care unit staff were relatively protected (0.44 [0.28-0.69]), likely by a bundle of PPE-related measures. Positive results were more likely in Black (1.66 [1.25-2.21]) and Asian (1.51 [1.28-1.77]) staff, independent of role or working location, and in porters and cleaners (2.06 [1.34-3.15]).


Asunto(s)
Infecciones por Coronavirus/epidemiología , Personal de Salud/estadística & datos numéricos , Neumonía Viral/epidemiología , Adolescente , Adulto , Factores de Edad , Anciano , Infecciones Asintomáticas/epidemiología , Betacoronavirus/aislamiento & purificación , COVID-19 , Infecciones por Coronavirus/transmisión , Infecciones por Coronavirus/virología , Femenino , Hospitales de Enseñanza/estadística & datos numéricos , Humanos , Incidencia , Transmisión de Enfermedad Infecciosa de Paciente a Profesional/estadística & datos numéricos , Unidades de Cuidados Intensivos/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/transmisión , Neumonía Viral/virología , Riesgo , SARS-CoV-2 , Encuestas y Cuestionarios , Reino Unido/epidemiología , Adulto Joven
16.
EMBO J ; 38(20): e101443, 2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31424118

RESUMEN

Cyclins are central engines of cell cycle progression in conjunction with cyclin-dependent kinases (CDKs). Among the different cyclins controlling cell cycle progression, cyclin F does not partner with a CDK, but instead forms via its F-box domain an SCF (Skp1-Cul1-F-box)-type E3 ubiquitin ligase module. Although various substrates of cyclin F have been identified, the vulnerabilities of cells lacking cyclin F are not known. Thus, we assessed viability of cells lacking cyclin F upon challenging them with more than 180 different kinase inhibitors. The screen revealed a striking synthetic lethality between Chk1 inhibition and cyclin F loss. Chk1 inhibition in cells lacking cyclin F leads to DNA replication catastrophe. Replication catastrophe depends on accumulation of the transcription factor E2F1 in cyclin F-depleted cells. We find that SCF-cyclin F controls E2F1 ubiquitylation and degradation during the G2/M phase of the cell cycle and upon challenging cells with Chk1 inhibitors. Thus, Cyclin F restricts E2F1 activity during the cell cycle and upon checkpoint inhibition to prevent DNA replication stress. Our findings pave the way for patient selection in the clinical use of checkpoint inhibitors.


Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Ciclinas/metabolismo , Factor de Transcripción E2F1/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis , Proteínas Ligasas SKP Cullina F-box/metabolismo , Mutaciones Letales Sintéticas , Ciclo Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Ciclinas/genética , Replicación del ADN , Factor de Transcripción E2F1/genética , Células HeLa , Humanos , Fosforilación , Unión Proteica , Proteínas Ligasas SKP Cullina F-box/genética , Ubiquitinación
17.
Stem Cell Reports ; 10(3): 693-702, 2018 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-29478892

RESUMEN

Use of hepatocytes derived from induced pluripotent stem cells (i-Heps) is limited by their functional differences in comparison with primary cells. Extracellular niche factors likely play a critical role in bridging this gap. Using image-based characterization (high content analysis; HCA) of freshly isolated hepatocytes from 17 human donors, we devised and validated an algorithm (Hepatocyte Likeness Index; HLI) for comparing the hepatic properties of cells against a physiological gold standard. The HLI was then applied in a targeted screen of extracellular niche factors to identify substrates driving i-Heps closer to the standard. Laminin 411, the top hit, was validated in two additional induced pluripotent stem cell (iPSC) lines, primary tissue, and an in vitro model of α1-antitrypsin deficiency. Cumulatively, these data provide a reference method to control and screen for i-Hep differentiation, identify Laminin 411 as a key niche protein, and underscore the importance of combining substrates, soluble factors, and HCA when developing iPSC applications.


Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Laminina/metabolismo , Adolescente , Adulto , Diferenciación Celular/fisiología , Femenino , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Masculino , alfa 1-Antitripsina/metabolismo
18.
Angew Chem Int Ed Engl ; 56(49): 15555-15559, 2017 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-28976073

RESUMEN

Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell-permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure-activity relationships, leading to the development of a small molecule with around 75-fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.


Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Proteínas F-Box/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Proteínas F-Box/metabolismo , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Estructura Molecular , Relación Estructura-Actividad
19.
Nat Commun ; 8: 14773, 2017 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-28382930

RESUMEN

The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Histona Demetilasas con Dominio de Jumonji/metabolismo , Espectrometría de Masas , Proteolisis , Relación Estructura-Actividad , Especificidad por Sustrato
20.
Artículo en Inglés | MEDLINE | ID: mdl-28265301

RESUMEN

BACKGROUND: Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active 'probe' molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells. RESULTS: A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition. CONCLUSIONS: High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors.


Asunto(s)
Inhibidores Enzimáticos/metabolismo , Histona Demetilasas/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Biocatálisis , Dominio Catalítico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Células HeLa , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histonas/metabolismo , Humanos , Concentración 50 Inhibidora , Metilación/efectos de los fármacos , Microscopía Fluorescente , Mutagénesis , Paclitaxel/toxicidad , Filogenia , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad Proteica/efectos de los fármacos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...