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Urinary tract infections (UTI) account for a substantial financial burden globally. Over 75% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which have demonstrated an extraordinarily rapid growth rate in vivo. This rapid growth rate appears paradoxical given that urine and the human urinary tract are relatively nutrient-restricted. Thus, we lack a fundamental understanding of how uropathogens propel growth in the host to fuel pathogenesis. Here, we used large in silico, in vivo, and in vitro screens to better understand the role of UPEC transport mechanisms and their contributions to uropathogenesis. In silico analysis of annotated transport systems indicated that the ATP-binding cassette (ABC) family of transporters was most conserved among uropathogenic bacterial species, suggesting their importance. Consistent with in silico predictions, we determined that the ABC family contributed significantly to fitness and virulence in the urinary tract: these were overrepresented as fitness factors in vivo (37.2%), liquid media (52.3%), and organ agar (66.2%). We characterized 12 transport systems that were most frequently defective in screening experiments by generating in-frame deletions. These mutant constructs were tested in urovirulence phenotypic assays and produced differences in motility and growth rate. However, deletion of multiple transport systems was required to achieve substantial fitness defects in the cochallenge murine model. This is likely due to genetic compensation among transport systems, highlighting the centrality of ABC transporters in these organisms. Therefore, these nutrient uptake systems play a concerted, critical role in pathogenesis and are broadly applicable candidate targets for therapeutic intervention.
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Transportadoras de Casetes de Unión a ATP , Escherichia coli Uropatógena , Humanos , Animales , Ratones , Transportadoras de Casetes de Unión a ATP/genética , Factores de Virulencia/genética , Escherichia coli Uropatógena/genética , Proteínas de Transporte de Membrana/genética , VirulenciaRESUMEN
INTRODUCTION: Fluorouracil (5FU) and capecitabine are metabolised by dihydropyrimidine dehydrogenase (DPD). Up to 9% of people have low levels of a working DPD enzyme and are at risk of severe toxicity from 5FU/capecitabine. In April 2020, the EMEA recommended patients undergo prospective screening for DPD deficiency before starting treatment, and this was introduced in our hospital. METHODS: We retrospectively reviewed records of all patients receiving 5FU/capecitabine in a tertiary Irish cancer centre from May 2020 to April 2021 (n = 197), and those starting first-line treatment in May 2019-April 2020 (n = 97). Our primary outcome was to estimate the prevalence of DPYD variant genes by prospective genotypic screening, with secondary outcomes including variant prevalence by prospective and reactive screening in patients receiving first-line treatment, and 5FU toxicity/tolerability in those with detected variants. RESULTS: In those treated 2020-2021, cancer subtypes included colorectal (n = 120, 61%), breast (n = 34, 17%), and biliary/pancreatic cancers (n = 21, 11%). Median patient age was 62 (range 25-86 years); 40% (n = 79) of patients were screened overall, with a prospective-screening deficiency prevalence of 6.8% (n = 3 of 44). Three patients had pathogenic DPYD-variants detected by prospective screening and tolerated treatment with 50% up-front dose reduction of 5FU, two had variants of uncertain significance detected by reactive screening. DISCUSSION: Other Irish studies estimated prevalence at 11-12%. As the number of variants detected was small, and screening rates were incomplete, our study may have underestimated prevalence. CONCLUSIONS: Approximately 6.8% of Irish patients may carry DPD deficiencies, prospective screening is essential to reduce the risk of life-threatening toxicity in these patients.
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AbstractThe sudden onset of severe traumatic brain injury (sTBI) is an event suffered by millions of individuals each year. Regardless of this frequency in occurrence, accurate prognostication remains difficult to achieve among physicians. There are many variables that affect this prognosis. Physicians are expected to assess the clinical indications of the brain injury while considering other factors such as patient quality of life, patient preferences, and environmental context. However, this lack of certainty in prognosis can ultimately affect treatment recommendations and prompt clinical ethical issues at the bedside, as it leaves room for physician bias and interpretation. In this article, we introduce data on neurosurgeon values that may shed light on the process physicians and patients involved in sTBI undergo. In doing so, we highlight the many nuances in decision-making for patients suffering from sTBI and discuss potential solutions to better patient-physician or surrogate-physician interactions.
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Lesiones Traumáticas del Encéfalo , Procedimientos Neuroquirúrgicos , Participación del Paciente , Médicos , Humanos , Lesiones Traumáticas del Encéfalo/cirugía , Relaciones Médico-Paciente , Pronóstico , Calidad de Vida , Incertidumbre , Toma de Decisiones Conjunta , Procedimientos Neuroquirúrgicos/efectos adversosRESUMEN
Elevated replication stress is evident at telomeres of about 10-15% of cancer cells, which maintain their telomeres via a homologous recombination (HR)-based mechanism, referred to as alternative lengthening of telomeres (ALT). How ALT cells resolve replication stress to support their growth remains incompletely characterized. Here, we report that CSB (also known as ERCC6) promotes recruitment of HR repair proteins (MRN, BRCA1, BLM and RPA32) and POLD3 to ALT telomeres, a process that requires the ATPase activity of CSB and is controlled by ATM- and CDK2-dependent phosphorylation. Loss of CSB stimulates telomeric recruitment of MUS81 and SLX4, components of the structure-specific MUS81-EME1-SLX1-SLX4 (MUS-SLX) endonuclease complex, suggesting that CSB restricts MUS-SLX-mediated processing of stalled forks at ALT telomeres. Loss of CSB coupled with depletion of SMARCAL1, a chromatin remodeler implicated in catalyzing regression of stalled forks, synergistically promotes not only telomeric recruitment of MUS81 but also the formation of fragile telomeres, the latter of which is reported to arise from fork stalling. These results altogether suggest that CSB-mediated HR repair and SMARCAL1-mediated fork regression cooperate to prevent stalled forks from being processed into fragile telomeres in ALT cells.
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Homeostasis del Telómero , Telómero , Reparación del ADN , Endonucleasas/metabolismo , Recombinación Homóloga , Telómero/genética , Telómero/metabolismo , Homeostasis del Telómero/genéticaRESUMEN
BACKGROUND: Palliative care has the potential to improve goal-concordant care in severe traumatic brain injury (sTBI). Our primary objective was to illuminate the demographic profiles of patients with sTBI who receive palliative care encounters (PCEs), with an emphasis on the role of race. Secondary objectives were to analyze PCE usage over time and compare health care resource utilization between patients with or without PCEs. METHODS: The National Inpatient Sample database was queried for patients age ≥ 18 who had a diagnosis of sTBI, defined by using International Classification of Diseases, 9th Revision codes. PCEs were defined by using International Classification of Diseases, 9th Revision code V66.7 and trended from 2001 to 2015. To assess factors associated with PCE in patients with sTBI, we performed unweighted generalized estimating equations regression. PCE association with decision making was modeled via its effect on rate of percutaneous endoscopic gastrostomy (PEG) tube placement. To quantify differences in PCE-related decisions by race, race was modeled as an effect modifier. RESULTS: From 2001 to 2015, the proportion of palliative care usage in patients with sTBI increased from 1.5 to 36.3%, with 41.6% White, 22.3% Black, and 25% Hispanic patients with sTBI having a palliative care consultation in 2015, respectively. From 2008 to 2015, we identified 17,673 sTBI admissions. White and affluent patients were more likely to have a PCE than Black, Hispanic, and low socioeconomic status patients. Across all races, patients receiving a PCE resulted in a lower rate of PEG tube placement; however, White patients exhibited a larger reduction of PEG tube placement than Black patients. Patients using palliative care had lower total hospital costs (median $16,368 vs. $26,442, respectively). CONCLUSIONS: Palliative care usage for sTBI has increased dramatically this century and it reduces resource utilization. This is true across races, however, its usage rate and associated effect on decision making are race-dependent, with White patients receiving more PCE and being more likely to decline the use of a PEG tube if they have had a PCE.
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Lesiones Traumáticas del Encéfalo , Cuidados Paliativos , Lesiones Traumáticas del Encéfalo/terapia , Hispánicos o Latinos , Humanos , Derivación y Consulta , Estudios RetrospectivosRESUMEN
The clonal proliferation of antigen-specific T cells during an immune response critically depends on the differential response to growth factors, such as IL-2. While activated T cells proliferate robustly in response to IL-2 stimulation, naïve (quiescent) T cells are able to ignore the potent effects of growth factors because they possess chromatin that is tightly condensed such that transcription factors, such as STAT5, cannot access DNA. Activation via the T cell receptor (TCR) induces a rapid decondensation of chromatin, permitting STAT5-DNA engagement and ultimately promoting proliferation of only antigen-specific T cells. Previous work demonstrated that the mobilization of intracellular calcium following TCR stimulation is a key event in the decondensation of chromatin. Here we examine PKC-dependent signaling mechanisms to determine their role in activation-induced chromatin decondensation and the subsequent acquisition of competence to respond to IL-2 stimulation. We found that a calcium-dependent PKC contributes to activation-induced chromatin decondensation and that the p38 MAPK and NFκB pathways downstream of PKC each contribute to regulating the proper decondensation of chromatin. Importantly, we found that p44/42 MAPK activity is required for peripheral T cells to gain competence to properly respond to IL-2 stimulation. Our findings shed light on the mechanisms that control the clonal proliferation of antigen-specific peripheral T cells during an immune response.
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Ensamble y Desensamble de Cromatina/fisiología , Interleucina-2/inmunología , Activación de Linfocitos/inmunología , Proteína Quinasa C/metabolismo , Linfocitos T/inmunología , Animales , Proliferación Celular/fisiología , Células Cultivadas , Cromatina/metabolismo , Selección Clonal Mediada por Antígenos/inmunología , ADN/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/inmunología , Factores de Transcripción/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.
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Inhibidores Enzimáticos/farmacología , Epigénesis Genética/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Línea Celular Tumoral , Cristalografía por Rayos X , Reparación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/química , Compuestos Heterocíclicos de 4 o más Anillos/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación/efectos de los fármacos , Modelos Moleculares , Estructura MolecularRESUMEN
TRF1, a duplex telomeric DNA-binding protein, plays an important role in telomere metabolism. We have previously reported that a fraction of endogenous TRF1 can stably exist free of telomere chromatin when it is phosphorylated at T371 by Cdk1; however, the role of this telomere-free (pT371)TRF1 has yet to be fully characterized. Here we show that phosphorylated (pT371)TRF1 is recruited to sites of DNA damage, forming damage-induced foci in response to ionizing radiation (IR), etoposide and camptothecin. We find that IR-induced (pT371)TRF1 foci formation is dependent on the ATM- and Mre11/Rad50/Nbs1-mediated DNA damage response. While loss of functional BRCA1 impairs the formation of IR-induced (pT371)TRF1 foci, depletion of either 53BP1 or Rif1 stimulates IR-induced (pT371)TRF1 foci formation. In addition, we show that TRF1 depletion or the lack of its phosphorylation at T371 impairs DNA end resection and repair of nontelomeric DNA double-strand breaks by homologous recombination. The lack of TRF1 phosphorylation at T371 also hampers the activation of the G2/M checkpoint and sensitizes cells to PARP inhibition, IR and camptothecin. Collectively, these results reveal a novel but important function of phosphorylated (pT371)TRF1 in facilitating DNA double-strand break repair and the maintenance of genome integrity.
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Roturas del ADN de Doble Cadena , Reparación del ADN por Recombinación , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/fisiología , Línea Celular , Puntos de Control de la Fase G2 del Ciclo Celular , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Fosforilación , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
The majority of Cockayne syndrome (CS) patients carry a mutation in Cockayne Syndrome group B (CSB), a large nuclear protein implicated in DNA repair, transcription and chromatin remodeling. However, whether CSB may play a role in telomere metabolism has not yet been characterized. Here, we report that CSB physically interacts with TRF2, a duplex telomeric DNA binding protein essential for telomere protection. We find that CSB localizes at a small subset of human telomeres and that it is required for preventing the formation of telomere dysfunction-induced foci (TIF) in CS cells. We find that CS cells or CSB knockdown cells accumulate telomere doublets, the suppression of which requires CSB. We find that overexpression of CSB in CS cells promotes telomerase-dependent telomere lengthening, a phenotype that is associated with a decrease in the amount of telomere-bound TRF1, a negative mediator of telomere length maintenance. Furthermore, we show that CS cells or CSB knockdown cells exhibit misregulation of TERRA, a large non-coding telomere repeat-containing RNA important for telomere maintenance. Taken together, these results suggest that CSB is required for maintaining the homeostatic level of TERRA, telomere length and integrity. These results further imply that CS patients carrying CSB mutations may be defective in telomere maintenance.
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Síndrome de Cockayne/genética , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Homeostasis del Telómero , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Línea Celular , ADN Helicasas/análisis , ADN Helicasas/genética , Enzimas Reparadoras del ADN/análisis , Enzimas Reparadoras del ADN/genética , Humanos , Mutación , Proteínas de Unión a Poli-ADP-Ribosa , ARN Largo no Codificante/metabolismo , Telomerasa/metabolismo , Telómero/químicaRESUMEN
BACKGROUND AND OBJECTIVES: Racial and socioeconomic disparities in spine surgery for degenerative lumbar spondylolisthesis persist in the United States, potentially contributing to unequal health-related quality of life (HRQoL) outcomes. This is important as lumbar spondylolisthesis is one of the most common causes of surgical low back pain, and low back pain is the largest disabler of individuals worldwide. Our objective was to assess the relationship between race, socioeconomic factors, treatment utilization, and outcomes in patients with lumbar spondylolisthesis. METHODS: This cohort study analyzed prospectively collected data from 9941 patients diagnosed with lumbar spondylolisthesis between 2015 and 2020 at 5 academic hospitals. Exposures were race, socioeconomic status, health coverage, and HRQoL measures. Main outcomes and measures included treatment utilization rates between racial groups and the association between race and treatment outcomes using logistic regression, adjusting for patient characteristics, socioeconomic status, health coverage, and HRQoL measures. RESULTS: Of the 9941 patients included (mean [SD] age, 67.37 [12.40] years; 63% female; 1101 [11.1%] Black, Indigenous, and People of Color [BIPOC]), BIPOC patients were significantly less likely to use surgery than White patients (odds ratio [OR] = 0.68; 95% CI, 0.62-0.75). Furthermore, BIPOC race was associated with significantly lower odds of reaching the minimum clinically important difference for physical function (OR = 0.74; 95% CI, 0.60; 0.91) and pain interference (OR = 0.77; 95% CI, 0.62-0.97). Medicaid beneficiaries were significantly less likely (OR = 0.65; 95% CI, 0.46-0.92) to reach a clinically important improvement in HRQoL when accounting for race. CONCLUSION: This study found that BIPOC patients were less likely to use spine surgery for degenerative lumbar spondylolisthesis despite reporting higher pain interference, suggesting an association between race and surgical utilization. These disparities may contribute to unequal HRQoL outcomes for patients with lumbar spondylolisthesis and warrant further investigation to address and reduce treatment disparities.
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Disparidades en Atención de Salud , Vértebras Lumbares , Calidad de Vida , Espondilolistesis , Humanos , Espondilolistesis/cirugía , Espondilolistesis/etnología , Masculino , Femenino , Anciano , Persona de Mediana Edad , Disparidades en Atención de Salud/estadística & datos numéricos , Disparidades en Atención de Salud/etnología , Vértebras Lumbares/cirugía , Estudios de Cohortes , Estados Unidos , Etnicidad/estadística & datos numéricos , Resultado del Tratamiento , Dolor de la Región Lumbar/cirugía , Dolor de la Región Lumbar/etnología , Estudios Prospectivos , Factores SocioeconómicosRESUMEN
Healthcare disparities and racism are finally being addressed in medical education. Medical schools are working to implement anti-racist programming; however, this can have a negative impact on the mental health of doctors and medical students of color. Continually hearing about people of one's racial group suffering from inequity can have a negative personal and performance impact. Specifically, providers of color want to be a part of the anti-racist movement, but it takes a toll on their mental and emotional wellbeing in the process. Through the support of their respective institutions, it is important for these providers to prioritize self-care such as engaging in physical activity, coaching and therapy. By caring for themselves in and outside of the work environment, providers of color will be better able to fight for anti-racism in healthcare. In this essay, we discuss the importance of health disparity education, challenges in implementing successful healthcare disparity curricula, and possible solutions to support providers and trainees of color.
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Actin assemblies are important in motile cells such as leukocytes, which form dynamic plasma membrane extensions or podia. L-plastin (LCP1) is a leukocyte-specific calcium-dependent actin-bundling protein that, in mammals, is known to affect immune cell migration. Previously, we generated CRISPR/Cas9 engineered zebrafish lacking L-plastin (lcp1-/-) and reported that they had reduced survival to adulthood, suggesting that lack of this actin-bundler might negatively affect the immune system. To test this hypothesis, we examined the distribution and migration of neutrophils and macrophages in the transparent tail of early zebrafish larvae using cell-specific markers and an established wound-migration assay. Knockout larvae were similar to their heterozygous siblings in having equal body sizes and comparable numbers of neutrophils in caudal hematopoietic tissue at 2 days postfertilization, indicating no gross defect in neutrophil production or developmental migration. When stimulated by a tail wound, all genotypes of neutrophils were equally migratory in a two-hour window. However, for macrophages we observed both migration defects and morphological differences. L-plastin knockout macrophages (lcp1 -/-) still homed to wounds but were slower, less directional and had a star-like morphology with many leading and trailing projections. In contrast, heterozygous macrophages lcp1 (+/-) were faster, more directional, and had a streamlined, slug-like morphology. Overall, these findings show that in larval zebrafish L-plastin knockout primarily affects the macrophage response with possible consequences for organismal immunity. Consistent with our observations, we propose a model in which cytoplasmic L-plastin negatively regulates macrophage integrin adhesion by holding these transmembrane heterodimers in a "clasped," inactive form and is a necessary part of establishing macrophage polarity during chemokine-induced motility.
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Actinas , Pez Cebra , Animales , Calcio , Movimiento Celular/genética , Quimiocinas , Integrinas , Leucocitos , Mamíferos , Glicoproteínas de Membrana , Proteínas de Microfilamentos , Pez Cebra/genéticaRESUMEN
Using electronic nicotine delivery systems (ENDS) has become a stepping stone for smokers in their cessation of tobacco use. Students within Doctor of Pharmacy and other health care programs have expressed varying responses as to how likely they are to recommend ENDS based on their knowledge of these devices. Because the amount of education on these products provided by PharmD programs varies, one study shows student pharmacists were less likely to recommend the use of ENDS to current cigarette smokers. This commentary suggests why some student pharmacists support such recommendations and therefore require more adequate ENDS education to better equip themselves for future patient education counseling sessions.
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Educación en Farmacia , Sistemas Electrónicos de Liberación de Nicotina , Cese del Hábito de Fumar , Curriculum , Atención a la Salud , HumanosRESUMEN
Objective. To implement an advanced elective compounding course where pharmacy students conduct investigations to improve compounding-related issues that were subsequently evaluated in a required compounding course. Methods. The elective compounding course required students to engage in self-directed learning, critical thinking, creation and evaluation of laboratory data, and self- and group reflection. Students researched and developed "solutions" to compounded preparation problems, and their solutions were tested in the next iteration of a required compounding course. For example, students in the elective course identified sources of potency variability in a ketoprofen Pluronic organogel (PLO) emulsion preparation. The students identified six variables and executed an investigative action plan. They considered all data collected and proposed a method to reduce potency variation. The recommended solution was implemented in the next offering of a required compounding course and the potency variability results were compared to the previous required course's results. Results. The mean ketoprofen PLO emulsion potency achieved in the required course prior to implementing the elective course recommendation was 129% (SD 21%), n=158. After the recommended change from elective course was implemented, the mean potency was 118% (SD 21%), n=131. Conclusion. The teaching methods and activities conducted in the elective course provided students with a deeper level of learning and understanding of compounding science, while providing practical experience in scientific research methodology. The course also provided a cyclic quality improvement feedback mechanism for the required course.
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Curriculum/normas , Composición de Medicamentos/normas , Educación en Farmacia/métodos , Mejoramiento de la Calidad/normas , Evaluación Educacional/normas , Retroalimentación , Humanos , Laboratorios/normas , Aprendizaje , Estudiantes de FarmaciaRESUMEN
XPF-ERCC1, a structure-specific endonuclease, is involved in nucleotide excision repair, crosslink repair and homologous recombination. XPF-ERCC1 is also found to interact with TRF2, a duplex telomeric DNA binding protein. We have previously shown that XPF-ERCC1 is required for TRF2-promoted telomere shortening. However, whether XPF-ERCC1 by itself has a role in telomere length maintenance has not been determined. Here we report that overexpression of XPF induces telomere shortening in XPF-proficient cells whereas XPF complementation suppresses telomere lengthening in XPF-deficient cells. These results suggest that XPF-ERCC1 can function as a negative mediator of telomere length maintenance. In addition, we find that introduction of wild type XPF into XPF-deficient cells leads to over 40% reduction in TRF2 association with telomeric DNA, indicating that XPF-ERCC1 negatively regulates TRF2 binding to telomeric DNA. Furthermore, we show that XPF carrying mutations in the conserved nuclease domain fails to control TRF2 association with telomeric DNA but it is competent for modulating telomere length maintenance. These results imply that XPF-ERCC1 controls TRF2 and telomere length maintenance through two distinctive mechanisms, with the former requiring its nuclease activity. Our results further imply that TRF2 association with telomeres may be deregulated in cells derived from XPF patients.
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Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Regulación de la Expresión Génica , Recombinación Genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Alelos , Animales , Senescencia Celular , Inmunoprecipitación de Cromatina , Humanos , Inmunoprecipitación , Ratones , Modelos Biológicos , Telómero/ultraestructura , beta-Galactosidasa/metabolismoRESUMEN
Regulation of the cytoskeleton is essential for cell migration in health and disease. Lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) is a hematopoietic-specific actin-bundling protein that is highly conserved in zebrafish, mice and humans. In addition, L-plastin expression is documented as both a genetic marker and a cellular mechanism contributing to the invasiveness of tumors and transformed cell lines. Despite L-plastin's role in both immunity and cancer, in zebrafish there are no direct studies of its function, and no mutant, knockout or reporter lines available. Using CRISPR-Cas9 genome editing, we generated null alleles of zebrafish lcp1 and examined the phenotypes of these fish throughout the life cycle. Our editing strategy used gRNA to target the second exon of lcp1, producing F0 mosaic fish that were outcrossed to wild types to confirm germline transmission. F1 heterozygotes were then sequenced to identify three unique null alleles, here called 'Charlie', 'Foxtrot' and 'Lima'. In silico, each allele truncates the endogenous protein to less than 5% normal size and removes both essential actin-binding domains (ABD1 and ABD2). Although none of the null lines express detectable LCP1 protein, homozygous mutant zebrafish (-/-) can develop and reproduce normally, a finding consistent with that of the L-plastin null mouse (LPL -/-). However, such mice do have a profound immune defect when challenged by lung bacteria. Interestingly, we observed reduced long-term survival of zebrafish lcp1 -/- homozygotes (~30% below the expected numbers) in all three of our knockout lines, with greatest mortality corresponding to the period (4-6 weeks post-fertilization) when the innate immune system is functional, but the adaptive immune system is not yet mature. This suggests that null zebrafish may have reduced capacity to combat opportunistic infections, which are more easily transmissible in the aquatic environment. Overall, our novel mutant lines establish a sound genetic model and an enhanced platform for further studies of L-plastin gene function in hematopoiesis and cancer.
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Eliminación de Gen , Glicoproteínas de Membrana/genética , Proteínas de Microfilamentos/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Alelos , Secuencia de Aminoácidos , Animales , Clonación Molecular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Ratones , Homología de Secuencia de AminoácidoRESUMEN
TRF1, a component of the shelterin complex, plays a key role in both telomerase-dependent telomere maintenance and alternative lengthening of telomeres, the latter also known as ALT. Characteristics of ALT cells include C-circles and ALT-associated PML bodies, referred to as APBs. The function of TRF1 is tightly regulated by post-translational modification including phosphorylation, however TRF1 phosphorylation sites have yet to be fully characterized. Here we report a novel TRF1 phosphorylation site threonine 271. We show that a nonphosphorylatable mutation of T271A impairs TRF1 binding to telomeric DNA in vivo and renders TRF1 defective in inhibiting telomerase-dependent telomere elongation. On the other hand, TRF1 carrying a phosphomimic mutation of T271D is competent in not only binding to telomeric DNA but also inhibiting telomerase-mediated telomere lengthening. These results suggest that TRF1 phosphorylation on T271 negatively regulates telomerase-mediated telomere maintenance. We find that in telomerase-negative ALT cells, TRF1 carrying either a T271A or T271D mutation is able to promote C-circle production but fails to support APB formation. These results suggest that TRF1 phosphorylation on T271 is necessary for APB formation but dispensable for C-circle production. These results further imply that APB formation can be mechanistically separated from C-circle production.
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Cuerpos de Inclusión Intranucleares/metabolismo , Proteína de la Leucemia Promielocítica/metabolismo , Telomerasa/metabolismo , Telómero/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Tirosina/química , Línea Celular Tumoral , Células HeLa , Homeostasis , Humanos , Mutación , Fosforilación , Unión Proteica , Homeostasis del Telómero , Proteína 1 de Unión a Repeticiones Teloméricas/química , Proteína 1 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
Antigen engagement of the T-cell receptor (TCR) induces a rapid and dramatic decondensation of chromatin that is necessary for T-cell activation. This decondensation makes T-cells competent to respond to interleukin-2 providing a mechanism to ensure clonotypic proliferation during an immune response. Using murine T-cells, we investigated the mechanism by which TCR signaling can initiate chromatin decondensation, focusing on the role of calcium mobilization. During T-cell activation, calcium is first released from intracellular stores, followed by influx of extracellular calcium via store operated calcium entry. We show that mobilization of intracellular calcium is required for TCR-induced chromatin decondensation. However, the decondensation is not dependent on the activity of the downstream transcription factor NFAT. Furthermore, we show that the influx of extracellular calcium is dispensable for initiating chromatin decondensation. Finally, we show that mobilization of calcium from intracellular stores is sufficient to induce decondensation, independent of TCR engagement. Collectively, our data suggest that chromatin decondensation in peripheral T-cells is controlled by modulating intracellular calcium levels.
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Señalización del Calcio , Cromatina/metabolismo , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Animales , Calcio/metabolismo , Diglicéridos/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Espacio Intracelular/metabolismo , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/metabolismoRESUMEN
Methylation of N-terminal arginines of the shelterin component TRF2 is important for cellular proliferation. While TRF2 is found at telomeres, where it plays an essential role in maintaining telomere integrity, little is known about the cellular localization of methylated TRF2. Here we report that the majority of methylated TRF2 is resistant to extraction by high salt buffer and DNase I treatment, indicating that methylated TRF2 is tightly associated with the nuclear matrix. We show that methylated TRF2 drastically alters its nuclear staining as normal human primary fibroblast cells approach and enter replicative senescence. This altered nuclear staining, which is found to be overwhelmingly associated with misshapen nuclei and abnormal nuclear matrix folds, can be suppressed by hTERT and it is barely detectable in transformed and cancer cell lines. We find that dysfunctional telomeres and DNA damage, both of which are potent inducers of cellular senescence, promote the altered nuclear staining of methylated TRF2, which is dependent upon the ATM-mediated DNA damage response. Collectively, these results suggest that the altered nuclear staining of methylated TRF2 may represent ATM-mediated nuclear structural alteration associated with cellular senescence. Our data further imply that methylated TRF2 can serve as a potential biomarker for cellular senescence.