RESUMEN
The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors.
Asunto(s)
Proteínas de Unión al ADN , Recombinasas , Humanos , ADN/genética , Reparación del ADN , Replicación del ADN , ADN Cruciforme , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Recombinasas/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismoRESUMEN
Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.
Asunto(s)
Agammaglobulinemia/inmunología , Linfocitos B/inmunología , Proteínas de Transporte de Catión/inmunología , Zinc/inmunología , Agammaglobulinemia/genética , Agammaglobulinemia/metabolismo , Animales , Linfocitos B/metabolismo , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Preescolar , Citosol/inmunología , Citosol/metabolismo , Modelos Animales de Enfermedad , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Lactante , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Linaje , Zinc/metabolismoRESUMEN
Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.
Asunto(s)
Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/metabolismo , Nucleosomas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , ADN Helicasas/genética , Replicación del ADN/efectos de los fármacos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de Unión al ADN/genética , Recombinación Homóloga/efectos de los fármacos , Ratones , Ratones Noqueados , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Nucleosomas/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
BACKGROUND: The efficacy and safety of risankizumab as compared with ustekinumab in patients with Crohn's disease are unknown. METHODS: In this phase 3b, multicenter, open-label, randomized, controlled trial with blinded assessment of end points, patients with moderate-to-severe Crohn's disease who had had an inadequate response to anti-tumor necrosis factor (TNF) therapy or unacceptable side effects with such therapy were randomly assigned to receive risankizumab or ustekinumab at standard doses for 48 weeks. The two primary end points, which were tested sequentially, were clinical remission at week 24 (defined as a Crohn's Disease Activity Index score of <150 [range, 0 to 600, with higher scores indicating more severe disease activity]), which was analyzed in the first 50% of patients to complete the week 24 visit, with a noninferiority margin of 10 percentage points; and endoscopic remission at week 48 (defined as a score of ≤4, a decrease of ≥2 points from baseline, and no subscore >1 in any individual variable on the Simple Endoscopic Score for Crohn's Disease [range, 0 to 56, with higher scores indicating more severe disease]), which was analyzed for superiority in 100% of the patients. Safety was assessed in all patients who received at least one dose of risankizumab or ustekinumab. RESULTS: In the full intention-to-treat population for the efficacy analysis, 230 of 255 patients (90.2%) who received risankizumab and 193 of 265 patients (72.8%) who received ustekinumab completed all the assigned treatments. Both primary end points were met; risankizumab was noninferior to ustekinumab with respect to clinical remission at week 24 (58.6% vs. 39.5%; adjusted difference, 18.4 percentage points; 95% confidence interval [CI], 6.6 to 30.3) and superior to ustekinumab with respect to endoscopic remission at week 48 (31.8% vs. 16.2%; adjusted difference, 15.6 percentage points; 95% CI, 8.4 to 22.9; P<0.001). The incidence of adverse events appeared to be similar in the two groups. CONCLUSIONS: In this head-to-head clinical trial of risankizumab and ustekinumab involving patients with moderate-to-severe Crohn's disease who had had unacceptable side effects with anti-TNF therapy or an inadequate response to such therapy, risankizumab was noninferior to ustekinumab with respect to clinical remission at week 24 and superior with respect to endoscopic remission at week 48. (Funded by AbbVie; ClinicalTrials.gov number, NCT04524611.).
Asunto(s)
Anticuerpos Monoclonales , Enfermedad de Crohn , Ustekinumab , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/efectos adversos , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/tratamiento farmacológico , Análisis de Intención de Tratar , Inducción de Remisión , Índice de Severidad de la Enfermedad , Ustekinumab/uso terapéutico , Ustekinumab/efectos adversos , Endoscopía GastrointestinalRESUMEN
Protein ubiquitination at sites of DNA double-strand breaks (DSBs) by RNF168 recruits BRCA1 and 53BP11,2, which are mediators of the homologous recombination and non-homologous end joining DSB repair pathways, respectively3. Non-homologous end joining relies on 53BP1 binding directly to ubiquitinated lysine 15 on H2A-type histones (H2AK15ub)4,5 (which is an RNF168-dependent modification6), but how RNF168 promotes BRCA1 recruitment and function remains unclear. Here we identify a tandem BRCT-domain-associated ubiquitin-dependent recruitment motif (BUDR) in BRCA1-associated RING domain protein 1 (BARD1) (the obligate partner protein of BRCA1) that, by engaging H2AK15ub, recruits BRCA1 to DSBs. Disruption of the BUDR of BARD1 compromises homologous recombination and renders cells hypersensitive to PARP inhibition and cisplatin. We further show that BARD1 binds nucleosomes through multivalent interactions: coordinated binding of H2AK15ub and unmethylated H4 lysine 20 by its adjacent BUDR and ankyrin repeat domains, respectively, provides high-affinity recognition of DNA lesions in replicated chromatin and promotes the homologous recombination activities of the BRCA1-BARD1 complex. Finally, our genetic epistasis experiments confirm that the need for BARD1 chromatin-binding activities can be entirely relieved upon deletion of RNF168 or 53BP1. Thus, our results demonstrate that by sensing DNA-damage-dependent and post-replication histone post-translation modification states, BRCA1-BARD1 complexes coordinate the antagonization of the 53BP1 pathway with promotion of homologous recombination, establishing a simple paradigm for the governance of the choice of DSB repair pathway.
Asunto(s)
Recombinación Homóloga , Lisina/química , Lisina/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Adulto , Secuencias de Aminoácidos , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Cromatina/metabolismo , Cisplatino/farmacología , Roturas del ADN de Doble Cadena , Daño del ADN/efectos de los fármacos , Femenino , Células HCT116 , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Masculino , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Dominios Proteicos , Reparación del ADN por Recombinación , Proteínas Supresoras de Tumor/química , Proteína 1 de Unión al Supresor Tumoral P53/deficiencia , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/deficienciaRESUMEN
Genomic instability is a hallmark of cancer, and has a central role in the initiation and development of breast cancer1,2. The success of poly-ADP ribose polymerase inhibitors in the treatment of breast cancers that are deficient in homologous recombination exemplifies the utility of synthetically lethal genetic interactions in the treatment of breast cancers that are driven by genomic instability3. Given that defects in homologous recombination are present in only a subset of breast cancers, there is a need to identify additional driver mechanisms for genomic instability and targeted strategies to exploit these defects in the treatment of cancer. Here we show that centrosome depletion induces synthetic lethality in cancer cells that contain the 17q23 amplicon, a recurrent copy number aberration that defines about 9% of all primary breast cancer tumours and is associated with high levels of genomic instability4-6. Specifically, inhibition of polo-like kinase 4 (PLK4) using small molecules leads to centrosome depletion, which triggers mitotic catastrophe in cells that exhibit amplicon-directed overexpression of TRIM37. To explain this effect, we identify TRIM37 as a negative regulator of centrosomal pericentriolar material. In 17q23-amplified cells that lack centrosomes, increased levels of TRIM37 block the formation of foci that comprise pericentriolar material-these foci are structures with a microtubule-nucleating capacity that are required for successful cell division in the absence of centrosomes. Finally, we find that the overexpression of TRIM37 causes genomic instability by delaying centrosome maturation and separation at mitotic entry, and thereby increases the frequency of mitotic errors. Collectively, these findings highlight TRIM37-dependent genomic instability as a putative driver event in 17q23-amplified breast cancer and provide a rationale for the use of centrosome-targeting therapeutic agents in treating these cancers.
Asunto(s)
Neoplasias de la Mama/genética , Centrosoma/metabolismo , Centrosoma/patología , Cromosomas Humanos Par 17/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Centrosoma/efectos de los fármacos , Femenino , Fase G2 , Inestabilidad Genómica , Humanos , Mitosis/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Antibiotics are compounds that have a particular mode of action upon the microorganism they are targeting. However, discovering and developing new antibiotics is a challenging and timely process. Antibiotic development process can take up to 10-15 years and over $1billion to develop a single new therapeutic product. Rapid screening tools to understand the mode of action of the new antimicrobial agent are considered one of the main bottle necks in the antimicrobial agent development process. Classical approaches require multifarious microbiological methods and they do not capture important biochemical and organism therapeutic-interaction mechanisms. This work aims to provide a rapid antibiotic-antimicrobial biochemical diagnostic tool to reduce the timeframes of therapeutic development, while also generating new biochemical insight into an antimicrobial-therapeutic screening assay in a complex matrix. The work evaluates the effect of antimicrobial action through "traditional" microbiological analysis techniques with a high-throughput rapid analysis method using UV-VIS spectroscopy and chemometrics. Bacteriostatic activity from tetracycline and bactericidal activity from amoxicillin were evaluated on a system using non-resistant Escherichia coli O157:H7 by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and UV-VIS spectroscopy (high-throughput analysis). The data were analysed using principal component analysis (PCA) and support vector machine (SVM) classification. The rapid diagnostic technique could easily identify differences between bacteriostatic and bactericidal mechanisms and was considerably quicker than the "traditional" methods tested.
Asunto(s)
Antiinfecciosos , Escherichia coli O157 , Inteligencia Artificial , Antibacterianos/farmacología , Antiinfecciosos/farmacología , Análisis Espectral , Aprendizaje Automático , Pruebas de Sensibilidad MicrobianaRESUMEN
The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Reparación del ADN , Proteínas Nucleares/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fosforilación , Estructura Terciaria de Proteína , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Alineación de SecuenciaRESUMEN
53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination1,2, and reduced fidelity of long-range V(D)J recombination3. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres4, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis5-7. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive8, unlike 53BP1-deficient cells9,10, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin-a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)-as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.
Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN/química , ADN/metabolismo , Proteínas Mad2/metabolismo , Complejos Multiproteicos/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Roturas del ADN de Doble Cadena , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Cambio de Clase de Inmunoglobulina/genética , Proteínas Mad2/deficiencia , Proteínas Mad2/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Complejos Multiproteicos/química , Mutación , Proteína 1 de Unión al Supresor Tumoral P53/deficiencia , Recombinación V(D)J/genéticaRESUMEN
The tumor suppressor protein 53BP1, a pivotal regulator of DNA double-strand break (DSB) repair, was first identified as a p53-interacting protein over two decades ago. However, its direct contributions to p53-dependent cellular activities remain undefined. Here, we reveal that 53BP1 stimulates genome-wide p53-dependent gene transactivation and repression events in response to ionizing radiation (IR) and synthetic p53 activation. 53BP1-dependent p53 modulation requires both auto-oligomerization and tandem-BRCT domain-mediated bivalent interactions with p53 and the ubiquitin-specific protease USP28. Loss of these activities results in inefficient p53-dependent cell-cycle checkpoint and exit responses. Furthermore, we demonstrate 53BP1-USP28 cooperation to be essential for normal p53-promoter element interactions and gene transactivation-associated events, yet dispensable for 53BP1-dependent DSB repair regulation. Collectively, our data provide a mechanistic explanation for 53BP1-p53 cooperation in controlling anti-tumorigenic cell-fate decisions and reveal these activities to be distinct and separable from 53BP1's regulation of DNA double-strand break repair pathway choice.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Proteína p53 Supresora de Tumor/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Ubiquitina Tiolesterasa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Proteína 9 Asociada a CRISPR , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endonucleasas/genética , Endonucleasas/metabolismo , Rayos gamma , Edición Génica , Regulación de la Expresión Génica , Humanos , Células MCF-7 , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/química , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/metabolismoRESUMEN
The use of the immunomodulator OmniGen- AF® (OMN) feed supplement, where the response in milk yield, health, and reproduction is uncertain, allows for an application of type I (use the product when it is not profitable) and type II (do not use the product when it is profitable) analysis to aid decision making regarding its profitability. This study applies a type I and II error analysis to quantify the economic risk of investing in OMN feed supplementation using data from a controlled study and a field data set collected on dairy farms. Four prediction models were applied considering the expected value and standard deviation of the response in milk yield; milk and health; milk and reproduction; milk, health, and reproduction. Uncertainty in the milk response was modeled following a normal distribution with mean 0.15 kg/cow/d and standard deviation 0.81 kg/cow/d. This distribution was left-truncated at 0 kg, which resulted in a mean (expected) response of 0.7 kg/cow/d. Based on the field data set, the reduction in health costs and reproduction were estimated at $0.086 and $0.33/cow/day. All 3 responses were assumed to be present or not in the first 90 DIM. The increase in milk yields necessary to pay for the OMN supplementation considering a response in milk yield only, milk and health, milk and reproduction, and milk, health, and reproduction were 0.45, 0.19, -0.56, and -0.82 kg/cow/d respectively. The break-even OMN cost per cow per day when considering the increases in milk yield, milk and health, and milk and reproduction, milk, health, and reproduction were $0.23, $0.31, $0.56, and $0.65 respectively. Production responses resulted in costs exceeding revenue with a type I error cost of $2.62 for milk and $0.47 for milk and health response for the broke even. There was no type I error cost associated when considering the responses in milk and reproduction, and the combination of milk, health and reproduction which implies that there is no risk of using OMN when it is not profitable ( = the product is profitable for certain). Type II costs were associated with returns that are unrealized because of failing to use OMN when it is profitable with an opportunity cost of $9.92, $15.53, $37.23, and $45.08 per cow per day for the responses in milk yield; milk and health; milk and reproduction; and milk, health, and reproduction respectively. We concluded that a type I and II error analysis applied to the use of the OMN feed additive provided additional insights beyond a break-even analysis to support economic decision-making when the response on dairy farms is uncertain. The type I and II error analysis can aid in decision-making process to evaluate the adoption of a new technology.
RESUMEN
Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas Mad2/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Reparación del ADN por Recombinación , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/genética , Histonas/metabolismo , Humanos , Cambio de Clase de Inmunoglobulina/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mad2/deficiencia , Proteínas Mad2/genética , Ratones , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The appropriate execution of DNA double-strand break (DSB) repair is critical for genome stability and tumor avoidance. 53BP1 and BRCA1 directly influence DSB repair pathway choice by regulating 5' end resection, but how this is achieved remains uncertain. Here we report that Rif1(-/-) mice are severely compromised for 53BP1-dependent class switch recombination (CSR) and fusion of dysfunctional telomeres. The inappropriate accumulation of RIF1 at DSBs in S phase is antagonized by BRCA1, and deletion of Rif1 suppresses toxic nonhomologous end joining (NHEJ) induced by PARP inhibition in Brca1-deficient cells. Mechanistically, RIF1 is recruited to DSBs via the N-terminal phospho-SQ/TQ domain of 53BP1, and DSBs generated by ionizing radiation or during CSR are hyperresected in the absence of RIF1. Thus, RIF1 and 53BP1 cooperate to block DSB resection to promote NHEJ in G1, which is antagonized by BRCA1 in S phase to ensure a switch of DSB repair mode to homologous recombination.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/genética , ADN/metabolismo , Proteínas de Unión a Telómeros/genética , Animales , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Ratones , Recombinación Genética , Telómero/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Transfección , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
BACKGROUND: Intimate partner violence (IPV) is a major public health problem with harmful consequences. In Australia, there is no national standard screening tool and screening practice is variable across states. The objectives of this study were to assess in the antenatal healthcare setting: i) the validity of a new IPV brief screening tool and ii) women's preference for screening response format, screening frequency and comfort level. METHODS: One thousand sixty-seven antenatal patients in a major metropolitan Victorian hospital in Australia completed a paper-based, self-administered survey. The survey included four screening items about whether they were Afraid/Controlled/Threatened/Slapped or physically hurt (ACTS) by a partner or ex-partner in the last 12 months; and the Composite Abuse Scale (reference standard). The ACTS screen was presented firstly with a binary yes/no response format and then with a five-point ordinal frequency format from 'never' (0) to 'very frequently' (4). The main outcome measures were test statistics of the four-item ACTS screening tool (sensitivity, specificity, predictive values, and area under the curve) against the reference standard and women's screening preferences. RESULTS: Twelve-month IPV prevalence varied depending on the ACTS response format with 8% (83) positive on ACTS yes/no format, 12.8% (133) positive on ACTS ordinal frequency format and 10.5% (108) on the reference Composite Abuse Scale. Overall, the ACTS screening tool demonstrated clinical utility for the ordinal frequency format (AUC, 0.80; 95% CI = 0.76 to 0.85) and the binary yes/no format (AUC, 0.74, 95% CI = 0.69 to 0.79). The frequency scale (66%) had greater sensitivity than the yes/no scale (51%). The positive and negative predictive values were 56 and 96% for the frequency scale and 68 and 95% for the yes/no scale. Specificity was high regardless of screening question response options. Half (53%) of the women categorised as abused preferred the yes/no scale. Around half of the women (48%, 472) thought health care providers should ask pregnant women about IPV at every visit. CONCLUSIONS: The four-item ACTS tool (using the frequency scale and a cut-off of one on any item) is recommended for written self-administered screening of women to identify those experiencing IPV to enable first-line response and follow-up.
Asunto(s)
Violencia de Pareja , Maltrato Conyugal , Estudios Transversales , Atención a la Salud , Femenino , Humanos , Embarazo , Atención PrenatalRESUMEN
Feeding yeast culture fermentation products has been associated with improved feed intake and milk yield in transition dairy cows. These improvements in performance have been further described in terms of rumen characteristics, metabolic profile, and immune response. The objective of this study was to evaluate the effects of a commercial yeast culture product (YC; Culture Classic HD, Phibro Animal Health) on performance, blood biomarkers, rumen fermentation, and rumen bacterial population in dairy cows from -30 to 50 d in milk (DIM). Forty Holstein dairy cows were enrolled in a randomized complete block design from -30 to 50 DIM and blocked according to expected calving day, parity, previous milk yield, and genetic merit. At -30 DIM, cows were assigned to either a basal diet plus 114 g/d of ground corn (control; n = 20) or a basal diet plus 100 g/d of ground corn and 14 g/d of YC (n = 20), fed as a top-dress. Cows received the same close-up diet from 30 d prepartum until calving [1.39 Mcal/kg of dry matter (DM) and 12.3% crude protein (CP)] and lactation diet from calving to 50 DIM (1.60 Mcal/kg of DM and 15.6% CP). Blood samples and rumen fluid were collected at various time points from -30 to 50 d relative to calving. Cows fed YC compared with control showed a trend for increased energy-corrected milk (+3.2 kg/d). Lower somatic cell counts were observed in YC cows than in control. We detected a treatment × time interaction in nonesterified fatty acids (NEFA) that could be attributed to a trend for greater NEFA in YC cows than control at 7 DIM, followed by lower NEFA in YC cows than control at 14 and 30 DIM. In the rumen, YC contributed to mild changes in rumen fermentation, mainly increasing postpartal valerate while decreasing prepartal isovalerate. This was accompanied by alterations in rumen microbiota, including a greater abundance of cellulolytic (Fibrobacter succinogenes) and lactate-utilizing bacteria (Megasphaera elsdenii). These results describe the potential benefits of supplementing yeast culture during the late pregnancy through early lactation, at least in terms of rumen environment and performance.
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Rumen , Saccharomyces cerevisiae , Animales , Biomarcadores/metabolismo , Bovinos , Dieta/veterinaria , Suplementos Dietéticos , Femenino , Fermentación , Fibrobacter , Lactancia , Leche , Embarazo , Rumen/metabolismoRESUMEN
The continuous trend for a narrowing margin between feed cost and milk prices across dairy farms in the United States highlights the need to improve and maintain feed efficiency. Yeast culture products are alternative supplements that have been evaluated in terms of milk performance and feed efficiency; however, less is known about their potential effects on altering rumen microbial populations and consequently rumen fermentation. Therefore, the objective of this study was to evaluate the effect of yeast culture supplementation on lactation performance, rumen fermentation profile, and abundance of major species of ruminal bacteria in lactating dairy cows. Forty mid-lactation Holstein dairy cows (121 ± 43 days in milk; mean ± standard deviation; 32 multiparous and 8 primiparous) were used in a randomized complete block design with a 7-d adaptation period followed by a 60-d treatment period. Cows were blocked by parity, days in milk, and previous lactation milk yield and assigned to a basal total mixed ration (TMR; 1.6 Mcal/kg of dry matter, 14.6% crude protein, 21.5% starch, and 38.4% neutral detergent fiber) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.). Treatments were top-dressed over the TMR once a day. Cows were individually fed 1 × /d throughout the trial. Blood and rumen fluid samples were collected in a subset of cows (n = 10/treatment) at 0, 30, and 60 d of the treatment period. Rumen fluid sampled via esophageal tubing was analyzed for ammonia-N, volatile fatty acids (VFA), and ruminal bacteria populations via quantitative PCR amplification of 16S ribosomal DNA genes. Milk yield was not affected by treatment effects. Energy balance was lower in YC cows than CON, which was partially explain by the trend for lower dry matter intake as % body weight in YC cows than CON. Cows fed YC had greater overall ruminal pH and greater total VFA (mM) at 60 d of treatment period. There was a contrasting greater molar proportion of isovalerate and lower acetate proportion in YC-fed cows compared with CON cows. Although the ruminal abundance of specific fiber-digesting bacteria, including Eubacterium ruminantium and Ruminococcus flavefaciens, was increased in YC cows, others such as Fibrobacter succinogenes were decreased. The abundance of amylolytic bacteria such as Ruminobacter amylophilus and Succinimonas amylolytica were decreased in YC cows than CON. Our results indicate that the yeast culture supplementation seems to promote some specific fiber-digesting bacteria while decreasing amylolytic bacteria, which might have partially promoted more neutral rumen pH, greater total VFA, and isovalerate.
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Lactancia , Rumen , Alimentación Animal/análisis , Animales , Bovinos , Dieta/veterinaria , Suplementos Dietéticos , Digestión , Eubacterium , Femenino , Fermentación , Fibrobacter , Leche , Embarazo , Rumen/metabolismo , Ruminococcus , Saccharomyces cerevisiae , SuccinivibrionaceaeRESUMEN
The creation of pain as the fifth vital sign in 2001 led to an unforeseen and dramatic increase in postoperative narcotic use. It became clear that chronic opioid use was associated with overdoses and deaths, and state medical licensing boards began to require completion of narcotic Continuing Medical Education courses to maintain licensure. Despite the overwhelming evidence of adverse effects of narcotic usage in both the pre- and postoperative periods, this continues to be a persistent problem in all areas of orthopaedic surgery. The magnitude of the problem is significant and now opioid-specific training is a mandated component of the American Board of Orthopaedic Surgery Maintenance of Certification for their Web-based Longitudinal Assessment of continuing medical education. Large database studies are helpful in identifying trends and factors that influence outcomes, potentially cut cost of care, and hopefully help us find a way out of this ongoing dilemma. This dilemma has taken a long time to create and will require a concerted disciplined effort to eliminate.
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Trastornos Relacionados con Opioides , Lesiones del Manguito de los Rotadores , Analgésicos Opioides/uso terapéutico , Artroscopía , Humanos , Manguito de los Rotadores , Lesiones del Manguito de los Rotadores/tratamiento farmacológicoRESUMEN
BACKGROUND AND PURPOSE: Previous studies have reported conflicting results regarding possible anticipation in familial E200K Creutzfeldt-Jakob disease (fCJD). Our objective was to use a large database to assess the age of disease onset (AODO) in CJD. METHODS: The study population included 477 CJD patients [266 with fCJD, 145 with sporadic CJD (sCJD) and 66 patients of Libyan origin but negative family history] from the Israeli registry of CJD conducted since 1954. In all patients, AODO in relatives and family trees was documented. Comparison of AODO was done using a paired t test and regression using Pearson correlation for birth and year of onset. RESULTS: The initial analysis in 52/73 families in which more than one generation was affected revealed an AODO of 63.30 ± 9.44 in the first generation compared to 56.96 ± 8.99 in the second generation (P < 0.001). However, inspection of individual AODO values plotted by year of birth showed a clear rhomboid methodological artifact generated by missing data of many young onset CJD patients who died before the database began to function in 1954 and of many late onset CJD patients missing at the present time since they will only develop the disease in the future. The 'generation' effect completely disappears if analysis is performed by year of disease onset or for the periods in which complete data are available. CONCLUSIONS: In this very large dataset, true anticipation in fCJD patients was not detected. It is plausible that previous reports supporting the presence of anticipation are biased by a rhomboid-shaped data availability artifact.