RESUMO
Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC, including cohesins, FACT, STAGA, and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B cell state and highlights Burkitt latency reversal therapeutic targets.
Assuntos
Linfoma de Burkitt/patologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ativação Viral , Latência Viral , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B/virologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/virologia , Proliferação de Células , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Feminino , Regulação Viral da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma, the leading childhood cancer in sub-Saharan Africa. Burkitt cells retain aspects of germinal center B-cell physiology with MYC-driven B-cell hyperproliferation; however, little is presently known about their iron metabolism. CRISPR/Cas9 analysis highlighted the little-studied ferrireductase CYB561A3 as critical for Burkitt proliferation but not for that of the closely related EBV-transformed lymphoblastoid cells or nearly all other Cancer Dependency Map cell lines. Burkitt CYB561A3 knockout induced profound iron starvation, despite ferritinophagy ad plasma membrane transferrin upregulation. Elevated concentrations of ascorbic acid, a key CYB561 family electron donor, or the labile iron source ferrous citrate rescued Burkitt CYB561A3 deficiency. CYB561A3 knockout caused catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. Conversely, lymphoblastoid B cells with the transforming EBV latency III program were instead dependent on the STEAP3 ferrireductase. These results highlight CYB561A3 as an attractive therapeutic Burkitt lymphoma target.
Assuntos
Linfoma de Burkitt/patologia , Citocromos b/genética , Regulação Neoplásica da Expressão Gênica , Lisossomos/patologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfoma de Burkitt/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Infecções por Vírus Epstein-Barr/complicações , FMN Redutase/genética , Células HEK293 , Herpesvirus Humano 4/isolamento & purificação , Humanos , Lisossomos/genética , Mitocôndrias/genética , Mitocôndrias/patologiaRESUMO
Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with multiple human malignancies. EBV drives B-cell proliferation, which contributes to the pathogenesis of multiple lymphomas. Yet, knowledge of how EBV subverts host biosynthetic pathways to transform resting lymphocytes into activated lymphoblasts remains incomplete. Using a temporal proteomic dataset of EBV primary human B-cell infection, we identified that cholesterol and fatty acid biosynthetic pathways were amongst the most highly EBV induced. Epstein-Barr nuclear antigen 2 (EBNA2), sterol response element binding protein (SREBP) and MYC each had important roles in cholesterol and fatty acid pathway induction. Unexpectedly, HMG-CoA reductase inhibitor chemical epistasis experiments revealed that mevalonate pathway production of geranylgeranyl pyrophosphate (GGPP), rather than cholesterol, was necessary for EBV-driven B-cell outgrowth, perhaps because EBV upregulated the low-density lipoprotein receptor in newly infected cells for cholesterol uptake. Chemical and CRISPR genetic analyses highlighted downstream GGPP roles in EBV-infected cell small G protein Rab activation. Rab13 was highly EBV-induced in an EBNA3-dependent manner and served as a chaperone critical for latent membrane protein (LMP) 1 and 2A trafficking and target gene activation in newly infected and in lymphoblastoid B-cells. Collectively, these studies identify highlight multiple potential therapeutic targets for prevention of EBV-transformed B-cell growth and survival.
Assuntos
Linfócitos B/virologia , Ácidos Graxos/biossíntese , Herpesvirus Humano 4/patogenicidade , Ácido Mevalônico/metabolismo , Alquil e Aril Transferases/metabolismo , Linfócitos B/patologia , Proliferação de Células , Sobrevivência Celular , Colesterol/biossíntese , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Redes e Vias Metabólicas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Proteínas Virais/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
INTRODUCTION & OBJECTIVE: Children with cognitive delay often experience challenges with obtaining hearing thresholds through behavioral audiometry (BA). This necessitates sedated Auditory Brainstem Response (sABR) testing. This study aimed to evaluate diagnostic and hearing patterns in children with Down Syndrome (DS), Autism Spectrum Disorder (ASD), Global Developmental delay (GDD), and Cerebral Palsy (CP) who were unable to complete reliable BA testing due to severe cognitive delay. METHODS: Retrospective chart review on a cohort of children aged 0.5-18 years with a diagnosis of DS, ASD, GDD, or CP who underwent sABR due to unsuccessful BA testing. This was performed at a tertiary care institution from 2014 to 2019. Testing patterns and audiometric data were collected. RESULTS: Across 15 DS, 39 ASD, 10 GDD, and 11 CP patients, the average time from first nondiagnostic BA to sABR ranged from 8.6 months (in GDD) to 21.8 months (in DS). The average number of BAs performed before sABR ranged from 1.6 (in ASD and GDD) to 2.7 (in DS). Hearing loss (HL) was diagnosed in 10%, 13%, 36% and 46% of patients with GDD, ASD, CP and DS respectively. Up to 75% of the HL was sensorineural (in CP patients). CONCLUSION: In children with significant cognitive delays, a high incidence of HL (especially SNHL) was identified, therefore high suspicion for HL should be held in these patients. Multiple unsuccessful BAs contribute to prolonged time to diagnosis and treatment, thus prompt sABR should be performed in patients whose severe cognitive delay inhibits reliable testing with BA.
Assuntos
Audiometria/métodos , Diagnóstico Tardio , Deficiências do Desenvolvimento , Perda Auditiva Neurossensorial/diagnóstico , Adolescente , Transtorno do Espectro Autista/complicações , Paralisia Cerebral , Criança , Pré-Escolar , Diagnóstico Tardio/prevenção & controle , Deficiências do Desenvolvimento/complicações , Síndrome de Down/complicações , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Perda Auditiva Neurossensorial/complicações , Perda Auditiva Neurossensorial/epidemiologia , Humanos , Incidência , Lactente , Masculino , Estudos RetrospectivosRESUMO
Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.IMPORTANCE Epstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to â¼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and â¼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Virais/metabolismo , Linfócitos B/virologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Células HEK293 , Herpesvirus Humano 4/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genética , Proteínas Virais/genéticaAssuntos
Leucemia Linfocítica Crônica de Células B/genética , Subunidade p50 de NF-kappa B/fisiologia , Senilidade Prematura/genética , Animais , Cruzamentos Genéticos , Modelos Animais de Doenças , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Contagem de Leucócitos , Camundongos , Camundongos Knockout , Subunidade p50 de NF-kappa B/deficiência , Subunidade p50 de NF-kappa B/genética , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Tamanho do Órgão , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Baço/patologia , Carga TumoralRESUMO
BACKGROUND: We tested whether proton pump inhibitors (PPIs) are associated with enteric infections among those with inflammatory bowel disease (IBD), after adequately accounting for baseline differences between PPI users and nonusers. METHODS: This was a self-controlled case series, with each patient serving as their own control. Ambulatory patients with IBD were included if they were tested for enteric infection by multiplex polymerase chain reaction testing panel (GIPCR) and/or Clostridoides difficile toxin PCR from 2015 to 2019 and received PPIs for some but not all of this period. Rates of enteric infections were compared between the PPI-exposed period vs pre- and post-PPI periods identical in duration to the exposed period. Conditional Poisson regression was used to adjust for time-varying factors. RESULTS: Two hundred twenty-one IBD patients were included (49% ulcerative colitis, 46% Crohn's disease, and 5% indeterminate colitis). The median PPI duration was 7 months (interquartile range 4 to 11 months). A total of 25 (11%) patients had a positive GIPCR or C. difficile test in the PPI period, 9 (4%) in the pre-PPI period, and 8 (4%) in the post-PPI period. Observed incidence rates for enteric infections were 2.5, 7.4, and 2.2 per 100 person years for the pre-PPI, PPI, and post-PPI periods, respectively (adjusted incidence rate ratios, 2.8; 95% confidence interval [CI] 1.3-6.0) for PPI vs pre-PPI and 2.9 (95% CI, 1.3-6.4) for PPI vs post-PPI). The adjusted absolute excess risk associated with PPIs was 4.9 infections per 100 person years. CONCLUSIONS: Proton pump inhibitors were associated with a 3-fold increased risk for enteric infection among those with IBD but had a modest absolute risk.
We tested whether proton pump inhibitors (PPIs) are associated with enteric infections among those with inflammatory bowel disease (IBD) by using a case-controlled series method, which allows for controlling of residual confounding. We studied ambulatory IBD patients who were tested for enteric infection from 2015 to 2019 and received PPIs for some of this period. Rates of enteric infections were compared between the PPI exposed period vs pre- and post-PPI periods identical in duration to the exposed period. We found that PPIs were associated with a 3-fold increased risk for enteric infection among those with IBD but had a modest absolute risk.
Assuntos
Clostridioides difficile , Doença de Crohn , Doenças Inflamatórias Intestinais , Humanos , Inibidores da Bomba de Prótons/efeitos adversos , Fatores de Risco , Doenças Inflamatórias Intestinais/complicações , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/induzido quimicamente , Doença de Crohn/complicações , Doença de Crohn/tratamento farmacológico , Doença de Crohn/induzido quimicamenteRESUMO
We present an updated version of the Predicting Protein-Protein Interactions (PrePPI) webserver which predicts PPIs on a proteome-wide scale. PrePPI combines structural and non-structural evidence within a Bayesian framework to compute a likelihood ratio (LR) for essentially every possible pair of proteins in a proteome; the current database is for the human interactome. The structural modeling (SM) component is derived from template-based modeling and its application on a proteome-wide scale is enabled by a unique scoring function used to evaluate a putative complex. The updated version of PrePPI leverages AlphaFold structures that are parsed into individual domains. As has been demonstrated in earlier applications, PrePPI performs extremely well as measured by receiver operating characteristic curves derived from testing on E. coli and human protein-protein interaction (PPI) databases. A PrePPI database of â¼1.3 million human PPIs can be queried with a webserver application that comprises multiple functionalities for examining query proteins, template complexes, 3D models for predicted complexes, and related features (https://honiglab.c2b2.columbia.edu/PrePPI). PrePPI is a state-of-the-art resource that offers an unprecedented structure-informed view of the human interactome.
Assuntos
Bases de Dados de Proteínas , Mapeamento de Interação de Proteínas , Proteoma , Humanos , Teorema de Bayes , Escherichia coli/metabolismo , Proteoma/metabolismoRESUMO
We present an updated version of the Predicting Protein-Protein Interactions (PrePPI) webserver which predicts PPIs on a proteome-wide scale. PrePPI combines structural and non-structural clues within a Bayesian framework to compute a likelihood ratio (LR) for essentially every possible pair of proteins in a proteome; the current database is for the human interactome. The structural modeling (SM) clue is derived from templatebased modeling and its application on a proteome-wide scale is enabled by a unique scoring function used to evaluate a putative complex. The updated version of PrePPI leverages AlphaFold structures that are parsed into individual domains. As has been demonstrated in earlier applications, PrePPI performs extremely well as measured by receiver operating characteristic curves derived from testing on E. coli and human protein-protein interaction (PPI) databases. A PrePPI database of ~1.3 million human PPIs can be queried with a webserver application that comprises multiple functionalities for examining query proteins, template complexes, 3D models for predicted complexes, and related features ( https://honiglab.c2b2.columbia.edu/PrePPI ). PrePPI is a state-of- the-art resource that offers an unprecedented structure-informed view of the human interactome.
RESUMO
We describe the Predicting Protein-Compound Interactions (PrePCI) database which comprises over 5 billion predicted interactions between 6.8 million chemical compounds and 19,797 human proteins. PrePCI relies on a proteome-wide database of structural models based on both traditional modeling techniques and the AlphaFold Protein Structure Database. Sequence- and structural similarity-based metrics are established between template proteins, T, in the Protein Data Bank that bind compounds, C, and query proteins in the model database, Q. When the metrics exceed threshold values, it is assumed that C also binds to Q with a likelihood ratio (LR) derived from machine learning. If the relationship is based on structural similarity, the LR is based on a scoring function that measures the extent to which C is compatible with the binding site of Q as described in the LT-scanner algorithm. For every predicted complex derived in this way, chemical similarity based on the Tanimoto coefficient identifies other small molecules that may bind to Q. An overall LR for the binding of C to Q is obtained from Naive Bayesian statistics. The PrePCI database can be queried by entering a UniProt ID or gene name for a protein to obtain a list of compounds predicted to bind to it along with associated LRs. Alternatively, entering an identifier for the compound outputs a list of proteins it is predicted to bind. Specific applications of the database to lead discovery, elucidation of drug mechanism of action, and biological function annotation are described.
Assuntos
Bases de Dados de Compostos Químicos , Proteínas , Humanos , Teorema de Bayes , Proteínas/química , Algoritmos , Bases de Dados de ProteínasRESUMO
OBJECTIVES: To evaluate clinical characteristics and outcomes of patients diagnosed with anterior (ASB) or lateral skull base (LSB) spontaneous cerebrospinal fluid (sCSF) leak. METHODS: Single center retrospective review of patients diagnosed with sCSF leaks of ASB or LSB between 1/1/2009 and 11/1/2019 (n = 69). Body mass index (BMI), gender, age at diagnosis, origin of CSF leak (ASB vs LSB), surgical approach, lumbar drain use, recurrence, pre-operative diagnosis of diabetes mellitus (DM), and obstructive sleep apnea (OSA) were collected. RESULTS: 69 patients included in this study met criteria for sCSF leak without a traumatic or iatrogenic cause (Female: 51 (74%); average BMI: 37.0 ± 7.9). Forty-eight (70.0%) presented with sCSF leaks of the lateral skull base. All ASB leaks were treated with an endoscopic transnasal approach. Eleven (22.9%) LSB leak patients were treated using transmastoid approaches and 35 (72.9%) patients with a middle cranial fossa approach. Eleven patients (15.9%) reported sCSF leak recurrence. Two patients (9.5%) with anterior skull base and 9 patients (18.8%) with lateral skull base leaks had recurrence. LSB sCSF leaks had a relative risk of 2.192 of recurrence compared to ASB leaks (95% CI: 0.431-11.157, P = .483). A 5.017 times increased risk (95% CI: 1.285-19.583, P = .020) was reported for patients with OSA, while the risks for DM and BMI were 1.351 (95% CI: 0.67-9.105, P = .177) and 1.026 (95% CI: 0.963-1.094, P = .426) respectively. Patients with sCSF leak recurrence had significantly lower lumbar drain use (33.3%) than those without recurrence (72.7%) (P = .049). CONCLUSION: Spontaneous CSF leak recurrence is complex and multifactorial, and while patients with both DM and OSA had the higher risk of recurrence, OSA is likely an independent clinical risk factor for sCSF leak recurrence in this patient population.
Assuntos
Base do Crânio , Apneia Obstrutiva do Sono , Humanos , Feminino , Base do Crânio/cirurgia , Vazamento de Líquido Cefalorraquidiano/epidemiologia , Vazamento de Líquido Cefalorraquidiano/etiologia , Vazamento de Líquido Cefalorraquidiano/cirurgia , Fossa Craniana Média , Estudos Retrospectivos , Apneia Obstrutiva do Sono/diagnóstico , Apneia Obstrutiva do Sono/epidemiologia , Apneia Obstrutiva do Sono/cirurgia , Resultado do TratamentoRESUMO
Epstein-Barr virus (EBV) infects 95% of adults worldwide and causes infectious mononucleosis. EBV is associated with endemic Burkitt lymphoma, Hodgkin lymphoma, posttransplant lymphomas, nasopharyngeal and gastric carcinomas. In these cancers and in most infected B-cells, EBV maintains a state of latency, where nearly 80 lytic cycle antigens are epigenetically suppressed. To gain insights into host epigenetic factors necessary for EBV latency, we recently performed a human genome-wide CRISPR screen that identified the chromatin assembly factor CAF1 as a putative Burkitt latency maintenance factor. CAF1 loads histones H3 and H4 onto newly synthesized host DNA, though its roles in EBV genome chromatin assembly are uncharacterized. Here, we found that CAF1 depletion triggered lytic reactivation and virion secretion from Burkitt cells, despite also strongly inducing interferon-stimulated genes. CAF1 perturbation diminished occupancy of histones 3.1 and 3.3 and of repressive histone 3 lysine 9 and 27 trimethyl (H3K9me3 and H3K27me3) marks at multiple viral genome lytic cycle regulatory elements. Suggestive of an early role in establishment of latency, EBV strongly upregulated CAF1 expression in newly infected primary human B-cells prior to the first mitosis, and histone 3.1 and 3.3 were loaded on the EBV genome by this time point. Knockout of CAF1 subunit CHAF1B impaired establishment of latency in newly EBV-infected Burkitt cells. A nonredundant latency maintenance role was also identified for the DNA synthesis-independent histone 3.3 loader histone regulatory homologue A (HIRA). Since EBV latency also requires histone chaperones alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler (ATRX) and death domain-associated protein (DAXX), EBV coopts multiple host histone pathways to maintain latency, and these are potential targets for lytic induction therapeutic approaches.IMPORTANCE Epstein-Barr virus (EBV) was discovered as the first human tumor virus in endemic Burkitt lymphoma, the most common childhood cancer in sub-Saharan Africa. In Burkitt lymphoma and in 200,000 EBV-associated cancers per year, epigenetic mechanisms maintain viral latency, during which lytic cycle factors are silenced. This property complicated EBV's discovery and facilitates tumor immunoevasion. DNA methylation and chromatin-based mechanisms contribute to lytic gene silencing. Here, we identified histone chaperones CAF1 and HIRA, which have key roles in host DNA replication-dependent and replication-independent pathways, respectively, as important for EBV latency. EBV strongly upregulates CAF1 in newly infected B-cells, where viral genomes acquire histone 3.1 and 3.3 variants prior to the first mitosis. Since histone chaperones ATRX and DAXX also function in maintenance of EBV latency, our results suggest that EBV coopts multiple histone pathways to reprogram viral genomes and highlight targets for lytic induction therapeutic strategies.
Assuntos
Linfócitos B/virologia , Proteínas de Ciclo Celular/genética , Fator 1 de Modelagem da Cromatina/genética , Epigênese Genética , Herpesvirus Humano 4/fisiologia , Chaperonas de Histonas/genética , Fatores de Transcrição/genética , Ativação Viral/genética , Linhagem Celular Tumoral , Cromatina/genética , Infecções por Vírus Epstein-Barr , Regulação Viral da Expressão Gênica , Inativação Gênica , Interações entre Hospedeiro e Microrganismos/genética , HumanosRESUMO
During the coronavirus 2019 pandemic, there has been a surge in production of remote learning materials for continued otolaryngology resident education. Medical students traditionally rely on elective and away subinternship experiences for exposure to the specialty. Delays and cancellation of clinical rotations have forced medical students to pursue opportunities outside of the traditional learning paradigm. In this commentary, we discuss the multi-institutional development of a robust syllabus for medical students using a multimodal collection of resources. Medical students collaborated with faculty and residents from 2 major academic centers to identify essential otolaryngology topics. High-quality, publicly available, and open-access content from multiple sources were incorporated into a curriculum that appeals to a variety of learners. Multimodal remote education strategies can be used as a foundation for further innovation aimed at developing tomorrow's otolaryngologists.
Assuntos
Betacoronavirus , Infecções por Coronavirus/epidemiologia , Currículo , Educação de Graduação em Medicina/métodos , Internato e Residência/métodos , Otolaringologia/educação , Pneumonia Viral/epidemiologia , COVID-19 , Humanos , Pandemias , SARS-CoV-2 , Inquéritos e QuestionáriosRESUMO
CD40 has major roles in B cell development, activation, and germinal center responses. CD40 hypoactivity causes immunodeficiency whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B cell autonomous CD40 regulators, we use CRISPR/Cas9 genome-scale screens in Daudi B cells stimulated by multimeric CD40 ligand. These highlight known CD40 pathway components and reveal multiple additional mechanisms regulating CD40. The nuclear ubiquitin ligase FBXO11 supports CD40 expression by targeting repressors CTBP1 and BCL6. FBXO11 knockout decreases primary B cell CD40 abundance and impairs class-switch recombination, suggesting that frequent lymphoma monoallelic FBXO11 mutations may balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controls exon splicing critical for CD40 activity, while the N6-adenosine methyltransferase WTAP negatively regulates CD40 mRNA abundance. At the protein level, ESCRT negatively regulates activated CD40 levels while the negative feedback phosphatase DUSP10 limits downstream MAPK responses. These results serve as a resource for future studies and highlight potential therapeutic targets.
Assuntos
Linfócitos B/metabolismo , Antígenos CD40/biossíntese , Sistemas CRISPR-Cas , Sistema de Sinalização das MAP Quinases , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Linfócitos B/citologia , Antígenos CD40/genética , Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Humanos , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismoRESUMO
Epstein-Barr virus (EBV) causes Burkitt, Hodgkin, and post-transplant B cell lymphomas. How EBV remodels metabolic pathways to support rapid B cell outgrowth remains largely unknown. To gain insights, primary human B cells were profiled by tandem-mass-tag-based proteomics at rest and at nine time points after infection; >8,000 host and 29 viral proteins were quantified, revealing mitochondrial remodeling and induction of one-carbon (1C) metabolism. EBV-encoded EBNA2 and its target MYC were required for upregulation of the central mitochondrial 1C enzyme MTHFD2, which played key roles in EBV-driven B cell growth and survival. MTHFD2 was critical for maintaining elevated NADPH levels in infected cells, and oxidation of mitochondrial NADPH diminished B cell proliferation. Tracing studies underscored contributions of 1C to nucleotide synthesis, NADPH production, and redox defense. EBV upregulated import and synthesis of serine to augment 1C flux. Our results highlight EBV-induced 1C as a potential therapeutic target and provide a new paradigm for viral onco-metabolism.
Assuntos
Aminoidrolases/metabolismo , Linfócitos B/metabolismo , Linfócitos B/virologia , Transformação Celular Viral , Infecções por Vírus Epstein-Barr/metabolismo , Ácido Fólico/metabolismo , Herpesvirus Humano 4/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Enzimas Multifuncionais/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Feminino , Glicólise , Células HEK293 , Humanos , Ativação Linfocitária , Mitocôndrias/metabolismo , NADP/biossíntese , Oxirredução , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Serina/biossínteseRESUMO
PURPOSE OF REVIEW: Surgical mission trips in head and neck surgery are common. There are an increasing number of surgical groups performing complex reconstructions in low and middle-income countries (LMIC). Consideration of reconstructive options that are location and patient specific are critical for optimum patient care and local physician education. RECENT FINDINGS: The pectoralis muscle regional flap is a versatile and reliable option and has been shown to reconstruct defects in nearly every head and neck subsite. Additional regional flap options described are the supraclavicular island flap and submental island flap. There are 15 published case series describing experience with performing microvascular reconstructions in LMIC. The average success rate was 87.1%. Both loupe and microscope magnification are used. SUMMARY: Complex reconstructions are successfully being performed in LMIC. Although microvascular reconstruction is being utilized by some groups, local and patient-specific factors should be carefully considered, as many regional and local flap options available provide the same reconstruction benefit while minimizing operating room time, resources, and postoperative care needs.
Assuntos
Países em Desenvolvimento , Procedimentos de Cirurgia Plástica/economia , Retalhos Cirúrgicos/irrigação sanguínea , Ferimentos e Lesões/cirurgia , Países em Desenvolvimento/economia , Cabeça/cirurgia , Humanos , Pescoço/cirurgia , Pobreza/economia , Procedimentos de Cirurgia Plástica/métodos , Classe Social , Ferimentos e Lesões/etiologiaRESUMO
Epstein-Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease-deficient Cas9 (dCas9)-VP64 (Cas9 associated with CRISPRa) or dCas9-KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus. The ribonucleoprotein complex formed by the dCas9 molecule and its cognate sgRNA enables sequence-specific binding at a promoter or enhancer of interest to affect the expression of genes regulated by the targeted promoter or enhancer. © 2018 by John Wiley & Sons, Inc.
Assuntos
Linfócitos B/metabolismo , Linfócitos B/virologia , Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Herpesvirus Humano 4/genética , RNA Guia de Cinetoplastídeos/genética , Linhagem Celular Tumoral , Clonagem Molecular/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Lentivirus/genética , Regiões Promotoras Genéticas , Ribonucleoproteínas/genética , Transcrição Gênica , Transdução Genética/métodosRESUMO
Epstein-Barr virus (EBV) efficiently transforms primary human B cells into immortalized lymphoblastoid cell lines (LCLs), which are extensively used in human genetic, immunological and virological studies. LCLs provide unlimited sources of DNA for genetic investigation, but can be difficult to manipulate, for instance because low retroviral or lentiviral transduction frequencies hinder experiments that require co-expression of multiple components. This unit details Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 engineering for robust LCL genome editing. We describe the generation and delivery of single-guide RNAs (sgRNAs), or dual-targeting sgRNAs, via lentiviral transduction of LCLs that stably express Cas9 protein. CRISPR/Cas9 editing allows LCL loss-of-function studies, including knock-out of protein-coding genes or deletion of DNA regulatory elements, and can be adapted for large-scale screening approaches. Low transfection efficiencies are a second barrier to performing CRISPR editing in LCLs, which are not typically lipid-transfectable. To circumvent this barrier, we provide an optimized protocol for LCL nucleofection of Cas9/sgRNA ribonucleoprotein complexes (RNPs) as an alternative route to achieve genome editing in LCLs. These editing approaches can also be employed in other B-cell lines, including Burkitt lymphoma and diffuse large B-cell lymphoma cells, and are highly reproducible. © 2018 by John Wiley & Sons, Inc.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Herpesvirus Humano 4/genética , RNA Guia de Cinetoplastídeos/genética , Transformação Genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/virologia , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , HumanosRESUMO
Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma (BL) and immunosuppression-related lymphomas. These B cell malignancies arise by distinct transformation pathways and have divergent viral and host expression programs. To identify host dependency factors resulting from these EBV+, B cell-transformed cell states, we performed parallel genome-wide CRISPR/Cas9 loss-of-function screens in BL and lymphoblastoid cell lines (LCLs). These highlighted 57 BL and 87 LCL genes uniquely important for their growth and survival. LCL hits were enriched for EBV-induced genes, including viral super-enhancer targets. Our systematic approach uncovered key mechanisms by which EBV oncoproteins activate the PI3K/AKT pathway and evade tumor suppressor responses. LMP1-induced cFLIP was found to be critical for LCL defense against TNFα-mediated programmed cell death, whereas EBV-induced BATF/IRF4 were critical for BIM suppression and MYC induction in LCLs. Finally, EBV super-enhancer-targeted IRF2 protected LCLs against Blimp1-mediated tumor suppression. Our results identify viral transformation-driven synthetic lethal targets for therapeutic intervention.