RESUMEN
At least two global "Snowball Earth" glaciations occurred during the Neoproterozoic Era (1000-538.8 million years ago). Post-glacial surface environments during this time are recorded in cap carbonates: layers of limestone or dolostone that directly overlie glacial deposits. Postulated environmental conditions that created the cap carbonates lack consensus largely because single hypotheses fail to explain the cap carbonates' global mass, depositional timescales, and geochemistry of parent waters. Here, we present a global geologic carbon cycle model before, during, and after the second glaciation (i.e. the Marinoan) that explains cap carbonate characteristics. We find a three-stage process for cap carbonate formation: (1) low-temperature seafloor weathering during glaciation generates deep-sea alkalinity; (2) vigorous post-glacial continental weathering supplies alkalinity to a carbonate-saturated freshwater layer, rapidly precipitating cap carbonates; (3) mixing of post-glacial meltwater with deep-sea alkalinity prolongs cap carbonate deposition. We suggest how future geochemical data and modeling refinements could further assess our hypothesis.
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Recent advances in native mass spectrometry (MS) and denatured intact protein MS have made these techniques essential for biotherapeutic characterization. As MS analysis has increased in throughput and scale, new data analysis workflows are needed to provide rapid quantitation from large datasets. Here, we describe the UniDec processing pipeline (UPP) for the analysis of batched biotherapeutic intact MS data. UPP is built into the UniDec software package, which provides fast processing, deconvolution, and peak detection. The user and programming interfaces for UPP read a spreadsheet that contains the data file names, deconvolution parameters, and quantitation settings. After iterating through the spreadsheet and analyzing each file, it returns a spreadsheet of results and HTML reports. We demonstrate the use of UPP to measure the correct pairing percentage on a set of bispecific antibody data and to measure drug-to-antibody ratios from antibody-drug conjugates. Moreover, because the software is free and open-source, users can easily build on this platform to create customized workflows and calculations. Thus, UPP provides a flexible workflow that can be deployed in diverse settings and for a wide range of biotherapeutic applications.
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Análisis de Datos , Programas Informáticos , Espectrometría de Masas/métodos , Flujo de TrabajoRESUMEN
KRAS, which is mutated in â¼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulating MAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit.
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Carcinogénesis/metabolismo , Dimerización , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Fosforilación/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas ras/genéticaRESUMEN
The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.
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Anticuerpos/química , Péptidos/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas Ubiquitinadas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Células Cultivadas , Cristalografía por Rayos X/métodos , Humanos , Espectrometría de Masas/métodos , Unión Proteica , Proteómica/métodos , Conejos , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/inmunología , UbiquitinaciónRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Neddylation is the post-translational protein modification most closely related to ubiquitination. Whereas the ubiquitin-like protein NEDD8 is well studied for its role in activating cullin-RING E3 ubiquitin ligases, little is known about other substrates. We developed serial NEDD8-ubiquitin substrate profiling (sNUSP), a method that employs NEDD8 R74K knock-in HEK293 cells, allowing discrimination of endogenous NEDD8- and ubiquitin-modification sites by MS after Lys-C digestion and K-εGG-peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1, implying that many non-cullin proteins are neddylated. Among the candidates, we characterized lysine 112 of the actin regulator cofilin as a novel neddylation event. Global inhibition of neddylation in developing neurons leads to cytoskeletal defects, altered actin dynamics and neurite growth impairments, whereas site-specific neddylation of cofilin at K112 regulates neurite outgrowth, suggesting that cofilin neddylation contributes to the regulation of neuronal actin organization.
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Actinas/metabolismo , Cofilina 1/metabolismo , Proteína NEDD8/metabolismo , Neuronas/metabolismo , Animales , Línea Celular , Células Cultivadas , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Proteína NEDD8/genética , Neuronas/citología , Mutación Puntual , Ratas , Ratas Sprague-Dawley , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: Myxoid liposarcoma typically presents as a deep-seated mass in the lower extremity of adults. Presentation as a primary subcutaneous tumor is rare. Here we discuss clinicopathologic characteristics of three such cases and their differential diagnosis to alert dermatopathologists to this unusual clinical presentation of a potentially aggressive entity. METHODS: Cases of myxoid liposarcoma were retrieved from archives and consultation files. Inclusion required location above the subcutaneous fascia with no evidence of a metastatic origin. Clinicopathologic features were retrospectively reviewed. Fluorescence in situ hybridization for DDIT3 (CHOP) gene rearrangement was performed on all cases. RESULTS: The tumors affected young adults (two males and one female, mean 36 years, range 32-40 years). No prior history of myxoid liposarcoma or deep soft tissue mass was identified. The tumors occurred in the foot, thigh and hand. All demonstrated multilobular architecture with abundant myxoid stroma, prominent branching capillary vascular network and lipoblastic differentiation. No dermal involvement was seen. Round cell features were identified in one case and represented <5% of the tumor. All patients remain disease-free following local excision only at 6, 8 and 13 months. CONCLUSIONS: Myxoid liposarcoma can rarely present as a primary subcutaneous mass and should be considered in the differential diagnosis of cutaneous myxoid tumors in adults.
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Pie/patología , Mano/patología , Liposarcoma Mixoide/patología , Muslo/patología , Adulto , Diagnóstico Diferencial , Femenino , Humanos , Liposarcoma Mixoide/diagnóstico , Liposarcoma Mixoide/genética , Liposarcoma Mixoide/cirugía , Masculino , Estudios Retrospectivos , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismoRESUMEN
Primary hyperparathyroidism is surgically correctable and frequently presents with mild hypercalcemia. The symptoms of hyperparathyroidism are nonspecific often leading to a delay in diagnosis until patients present with an acute condition. Literature suggests that up to 20 per cent of patients presenting to the emergency department (ED) found to have hypercalcemia are ultimately diagnosed with hyperparathyroidism. We performed a retrospective review from 2012 to 2013 of patients with hypercalcemia in our ED and analyzed their characteristics. One hundred sixty-eight patients were identified with hypercalcemia. Patient medical history, chief complaint, review of symptoms, discharge disposition, and primary care physician (PCP) status were evaluated. Eighty-four per cent were classified as mild (10.8 to 11.9 mg/dL), 11 per cent as moderate (12 to 14 mg/dL), and five per cent as severe (greater than 14 mg/dL). A definitive diagnosis of hyperparathyroidism was identified in 3.5 per cent (six of 168). Documentation of hypercalcemia as a diagnosis was present in all patients in the severe and 78 per cent in the moderate categories. However, only 21 per cent of patients with mild hypercalcemia had documentation addressing this diagnosis. Of concern, 24 per cent (41 of 168) of patients were identified with mild hypercalcemia and discharged from the ED with no definitive plan based on lack of a PCP. Additionally, 81 per cent of these patients had symptoms referable to hypercalcemia. Mild hypercalcemia found during ED workup rarely requires immediate medical treatment. However, a significant number of those patients will have hyperparathyroidism amendable to surgical correction. Therefore, an appropriate mechanism for outpatient hypercalcemia workup should be integrated into the patient's ED discharge plan.
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Servicio de Urgencia en Hospital , Hipercalcemia/diagnóstico , Hiperparatiroidismo Primario/diagnóstico , Femenino , Humanos , Hipercalcemia/epidemiología , Hiperparatiroidismo Primario/epidemiología , Masculino , Persona de Mediana Edad , Prevalencia , Estudios Retrospectivos , Tennessee/epidemiologíaRESUMEN
We report a case of a primary cutaneous epithelioid rhabdomyosarcoma that presented as a single raised pink-purple lesion (3.3 × 2.2 cm) on the left base of neck in a 75-year-old man. Histopathologic examination revealed an exophytic, nodular tumor within the dermis and superficial subcutis with overlying ulceration. The tumor exhibited sheet-like growth, infiltration of adjacent structures, and was composed of uniform epithelioid cells with abundant eosinophilic cytoplasm and eccentrically placed vesicular nuclei with irregular nuclear contours and prominent central nucleoli. Numerous mitotic figures were present [28/10 high power fields (HPF)] but only mild cytologic pleomorphism was identified. By immunohistochemistry, tumor cells were diffusely and strongly positive for desmin and MYOD1. Focal positive staining for myogenin and cytokeratin CK903 was identified. Stains for Melan-A, S-100, SOX10, p63 and CK5/6 were negative. These histopathologic and immunophenotypic features support a diagnosis of epithelioid rhabdomyosarcoma. No evidence of a deep soft tissue primary lesion was identified. In summary, epithelioid rhabdomyosarcoma can present as a primary cutaneous lesion and dermatopathologists should be aware of this entity.
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Biomarcadores de Tumor/biosíntesis , Proteínas de Neoplasias/biosíntesis , Rabdomiosarcoma , Neoplasias Cutáneas , Anciano , Humanos , Masculino , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
BACKGROUND: Rhabdomyosarcoma is a malignant mesenchymal tumor with skeletal muscle differentiation. Primary cutaneous rhabdomyosarcoma is rare. We report a series of 11 cases of primary cutaneous rhabdomyosarcoma. METHODS: Cases diagnosed as rhabdomyosarcoma arising in the dermis/subcutis with no identified primary tumor elsewhere were retrospectively reviewed. Follow-up was obtained. RESULTS: The tumors occurred in five children and six adults. The adult subset consisted of pleomorphic, epithelioid and not otherwise specified (NOS) subtypes while the pediatric subset showed alveolar and embryonal subtypes. All cases showed immunohistochemical staining consistent with the diagnosis of rhabdomyosarcoma. Three adult cases showed immunoreactivity for cytokeratins (one pleomorphic, one epithelioid and one NOS. CONCLUSIONS: Primary cutaneous rhabdomyosarcoma shows a bimodal age distribution and male predominance, correlating with rhabdomyosarcoma in deep soft tissue. Follow-up, available on all patients, showed aggressive behavior in both children and adults. Primary cutaneous rhabdomyosarcoma should be considered in the differential diagnosis of tumors with abundant eosinophilic cytoplasm and those with "small round blue cell" morphology. Desmin, myogenin and MYOD1 are a trio of markers with high sensitivity and specificity for primary cutaneous rhabdomyosarcoma. Cytokeratin immunoreactivity in primary cutaneous rhabdomyosarcoma represents a potential diagnostic pitfall in the differential diagnosis with sarcomatoid carcinoma.
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Biomarcadores de Tumor/metabolismo , Citoplasma , Dermis , Queratinas/metabolismo , Proteína MioD/metabolismo , Proteínas de Neoplasias/metabolismo , Rabdomiosarcoma , Neoplasias Cutáneas , Adolescente , Factores de Edad , Anciano , Anciano de 80 o más Años , Preescolar , Citoplasma/metabolismo , Citoplasma/patología , Dermis/metabolismo , Dermis/patología , Diagnóstico Diferencial , Femenino , Estudios de Seguimiento , Humanos , Lactante , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/patología , Factores Sexuales , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
The CBL ubiquitin ligase targets a variety of activated tyrosine kinases (TKs) for degradation. Many TKs are mutationally or autocrine activated and/or often overexpressed at the mRNA and protein levels in acute leukemias. We hypothesized that CBL is mutated in patients with acute myeloid leukemia (AML). Four of 12 patients and the MOLM-13 cell line harbored c-CBL mutations, either RNA splicing mutations, missense mutations, or a nucleotide insertion. Additionally, 1 of the 12 patients harbored a missense mutation in the related CBL-b gene. Each c-CBL mutation involves the structurally important alpha-helix within the linker region, while the mutation in CBL-b was located in the Ub-E2 protein-binding RING finger. Short-interfering RNA knockdown of mutant c-CBL present in MOLM-13 cells was growth inhibitory. In summary, novel mutations in c-CBL and CBL-b have been identified in human AML and may represent potential targets for novel therapeutics.
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Proteínas Adaptadoras Transductoras de Señales/genética , Leucemia Mieloide Aguda/genética , Mutación Missense , Proteínas Proto-Oncogénicas c-cbl/genética , Empalme del ARN/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular , Línea Celular Tumoral , Femenino , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Masculino , Estructura Secundaria de Proteína/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
In humans, T cells differentiate in thymus and B cells develop in bone marrow (BM), but the natural killer (NK) precursor cell(s) and site(s) of NK development are unclear. The CD56bright NK subset predominates in lymph nodes (LN) and produces abundant cytokines compared to the cytolytic CD56dim NK cell that predominates in blood. Here, we identify a novel CD34dimCD45RA(+) hematopoietic precursor cell (HPC) that is integrin alpha4beta7bright. CD34dimCD45RA(+)beta7bright HPCs constitute <1% of BM CD34(+) HPCs and approximately 6% of blood CD34(+) HPCs, but >95% of LN CD34(+) HPCs. They reside in the parafollicular T cell regions of LN with CD56bright NK cells, and when stimulated by IL-15, IL-2, or activated LN T cells, they become CD56bright NK cells. The data identify a new NK precursor and support a model of human NK development in which BM-derived CD34dimCD45RA(+)beta7bright HPCs reside in LN where endogenous cytokines drive their differentiation to CD56bright NK cells in vivo.
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Antígenos CD34/inmunología , Diferenciación Celular/inmunología , Células Asesinas Naturales/citología , Ganglios Linfáticos/citología , Subgrupos Linfocitarios/citología , Antígenos CD34/metabolismo , Antígeno CD56 , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Inmunohistoquímica , Células Asesinas Naturales/inmunología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunologíaRESUMEN
OBJECTIVE: A telepathology connection between Richmond VAMC and Beckley VAMC using dynamic robotic telepathology to provide pathology services remotely was established. MATERIALS AND METHODS: This study reports a 14-month experience using telepathology to diagnose surgical specimens obtained from patients at the Beckley VA Medical Center and viewed in Richmond 250 miles away. Over 14 months, 2325 slides representing 1000 cases were viewed. RESULTS: Discrepancies were observed in 20 of 2325 slides, or 0.86% of the total. None of the patients, where a discrepancy was found, were adversely affected by the preliminary report given. CONCLUSIONS: This study demonstrates that telepathology is a reliable and cost-effective alternative to on-site pathology services and reviews advantages and disadvantages of the system.
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Hospitales de Veteranos , Servicio de Patología en Hospital , Consulta Remota , Robótica , Telepatología , Costos de Hospital , Hospitales de Veteranos/economía , Humanos , Servicio de Patología en Hospital/economía , Enfermedades de la Piel/patología , Telepatología/economía , Virginia , West VirginiaRESUMEN
BACKGROUND/PURPOSE: This is a retrospective review of the pediatric all-terrain vehicle trauma victims who presented to the five major trauma centers serving the state of West Virginia during the 5-year period from January 1991 to December 1995. The purpose of this research is to characterize the nature of the injuries and the individuals injured to better appreciate the magnitude of the problem of ATV-related injuries in the pediatric population. METHODS: This study is a retrospective review of these 218 consecutive pediatric patients from trauma registry data and their medical records. RESULTS: Two hundred eighteen patients between the ages of 2 years and 16 years presented during the study period. Boys outnumbered girls three to one. The average Injury Severity Score (ISS) was 8.76, the average Glasgow Coma Score (GCS) was 14.4, and the average Trauma Score (TS) was 15.2. The most common injuries were orthopedic followed by head and facial injuries. The majority of the children did not wear helmets, and their injuries resulted in an average hospital length of stay of 4.3 days. Thirty-eight percent of the children required surgery. There were a total of four deaths for a mortality rate of 1.8%. The estimated total hospitalization cost for the 218 patients was $1,918,400.00. CONCLUSIONS: All-terrain vehicle-related trauma remains an ongoing safety concern facing society today. Every physician who cares for children should address this important issue when talking to children and parents about safety issues and injury prevention.
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Vehículos a Motor Todoterreno/estadística & datos numéricos , Heridas y Lesiones/epidemiología , Accidentes/mortalidad , Accidentes/estadística & datos numéricos , Adolescente , Niño , Preescolar , Traumatismos Craneocerebrales/epidemiología , Femenino , Escala de Coma de Glasgow , Dispositivos de Protección de la Cabeza/estadística & datos numéricos , Costos de Hospital , Humanos , Puntaje de Gravedad del Traumatismo , Masculino , Estudios Retrospectivos , West Virginia/epidemiologíaRESUMEN
BACKGROUND: This study evaluated academic advisement from the perspectives of RN-to-BSN students in relation to the advisement elements of function, style, and outcome. METHOD: Using multistage sampling, data were collected from 323 students enrolled in either a beginning, mid-way, or final course in the RN-to-BSN curriculum. RESULTS: All aspects of advisor style were perceived to have been employed and important. None of the outcomes were perceived to have been achieved nor important. There was a significant difference (p < 0.05) between students from ADN and Diploma educational backgrounds in relation to their perceptions of the importance of advisement functions and outcomes. Students rated their perceptions in accordance with andragogical learning principles that stressed the importance of individuality. The researcher developed Academic Advisement Questionnaires have content validity and reliability for internal consistency at 0.94 by Cronbach's coefficient alpha. CONCLUSION: Advisement in RN-to-BSN educational programs can positively influence student retention rates by providing advisement that is conducive to the needs and values of adult learner students.