Outcome prediction of cardiac arrest with automatically computed gray-white matter ratio on computed tomography images.

BACKGROUND: This study aimed to develop an automated method to measure the gray-white matter ratio (GWR) from brain computed tomography (CT) scans of patients with out-of-hospital cardiac arrest (OHCA) and assess its significance in predicting early-stage neurological outcomes. METHODS: Patients with OHCA who underwent brain CT imaging within 12 h of return of spontaneous circulation were enrolled in this retrospective study. The primary outcome endpoint measure was a favorable neurological outcome, defined as cerebral performance category 1 or 2 at hospital discharge. We proposed an automated method comprising image registration, K-means segmentation, segmentation refinement, and GWR calculation to measure the GWR for each CT scan. The K-means segmentation and segmentation refinement was employed to refine the segmentations within regions of interest (ROIs), consequently enhancing GWR calculation accuracy through more precise segmentations. RESULTS: Overall, 443 patients were divided into derivation N=265, 60% and validation N=178, 40% sets, based on age and sex. The ROI Hounsfield unit values derived from the automated method showed a strong correlation with those obtained from the manual method. Regarding outcome prediction, the automated method significantly outperformed the manual method in GWR calculation (AUC 0.79 vs. 0.70) across the entire dataset. The automated method also demonstrated superior performance across sensitivity, specificity, and positive and negative predictive values using the cutoff value determined from the derivation set. Moreover, GWR was an independent predictor of outcomes in logistic regression analysis. Incorporating the GWR with other clinical and resuscitation variables significantly enhanced the performance of prediction models compared to those without the GWR. CONCLUSIONS: Automated measurement of the GWR from non-contrast brain CT images offers valuable insights for predicting neurological outcomes during the early post-cardiac arrest period.

The Influencing Factors of Implementation in Emergency Medical Service Systems - A Scoping Review.

OBJECTIVES: Emergency medical services (EMS) provide health care in situations with limited time and resources. Challenges arise when introducing novel medications, treatments, or technologies or modifying existing practices in these settings. Effective implementation strategies are pivotal for their success. This study aims to identify and categorize potential facilitators and barriers in the implementation of prehospital EMS through a review of relevant research articles. METHODS: We searched PubMed and EMbase to identify studies published before December 2023, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for our search strategy and scoping review. We included original articles written in English that report on the factors that influence the implementation in prehospital settings. We extracted and categorized the factors into different themes. RESULTS: Out of the 371 retrieved papers, we selected 19 (5%) for inclusion in this review. We extracted 46 influencing factors from the selected articles and categorized them into ten themes: (1) Outer system, (2) Inner system, (3) Practitioner characteristics, (4) Resources, (5) Communication and collaboration, (6) Patient factors, (7) Intervention characteristics, (8) De-implementation of prior practices, (9) Logistical issues, and (10) Quality improvement. CONCLUSIONS: This study examined the literature on EMS implementation factors and proposed a 10-theme EMS model framework. Key factors include training/education, equipment/tools, communication with hospitals, and practitioners' attitudes.

Clinical Validation of Explainable Deep Learning Model for Predicting the Mortality of In-Hospital Cardiac Arrest Using Diagnosis Codes of Electronic Health Records.

Background: Using deep learning for disease outcome prediction is an approach that has made large advances in recent years. Notwithstanding its excellent performance, clinicians are also interested in learning how input affects prediction. Clinical validation of explainable deep learning models is also as yet unexplored. This study aims to evaluate the performance of Deep SHapley Additive exPlanations (D-SHAP) model in accurately identifying the diagnosis code associated with the highest mortality risk. Methods: Incidences of at least one in-hospital cardiac arrest (IHCA) for 168,693 patients as well as 1,569,478 clinical records were extracted from Taiwan's National Health Insurance Research Database. We propose a D-SHAP model to provide insights into deep learning model predictions. We trained a deep learning model to predict the 30-day mortality likelihoods of IHCA patients and used D-SHAP to see how the diagnosis codes affected the model's predictions. Physicians were asked to annotate a cardiac arrest dataset and provide expert opinions, which we used to validate our proposed method. A 1-to-4-point annotation of each record (current decision) along with four previous records (historical decision) was used to validate the current and historical D-SHAP values. Results: A subset consisting of 402 patients with at least one cardiac arrest record was randomly selected from the IHCA cohort. The median age was 72 years, with mean and standard deviation of 69 ± 17 years. Results indicated that D-SHAP can identify the cause of mortality based on the diagnosis codes. The top five most important diagnosis codes, namely respiratory failure, sepsis, pneumonia, shock, and acute kidney injury were consistent with the physician's opinion. Some diagnoses, such as urinary tract infection, showed a discrepancy between D-SHAP and clinical judgment due to the lower frequency of the disease and its occurrence in combination with other comorbidities. Conclusions: The D-SHAP framework was found to be an effective tool to explain deep neural networks and identify most of the important diagnoses for predicting patients' 30-day mortality. However, physicians should always carefully consider the structure of the original database and underlying pathophysiology.

Epstein-Barr virus subverts mevalonate and fatty acid pathways to promote infected B-cell proliferation and survival.

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.

Epstein-Barr Virus LMP1-Mediated Oncogenicity.

Epstein-Barr virus latent membrane protein 1 (LMP1) is expressed in multiple human malignancies, including nasopharyngeal carcinoma and Hodgkin and immunosuppression-associated lymphomas. LMP1 mimics CD40 signaling to activate multiple growth and survival pathways, in particular, NF-κB. LMP1 has critical roles in Epstein-Barr virus (EBV)-driven B-cell transformation, and its expression causes fatal lymphoproliferative disease in immunosuppressed mice. Here, we review recent developments in studies of LMP1 signaling, LMP1-induced host dependency factors, mouse models of LMP1 lymphomagenesis, and anti-LMP1 immunotherapy approaches.

Identification of Slug and SOX7 as transcriptional repressors binding to the hepatitis B virus core promoter.

BACKGROUND & AIMS: The Hepatitis B Virus (HBV) may gain entry into non-liver cells but does not actively replicate in them. We investigated the possibility that these cells possess mechanisms that block HBV core promoter (HBVCP) transcription, specifically absent in liver cells, which together with other liver-specific mechanisms, such as sodium-taurocholate cotransporting polypeptide-mediated entry, enable liver cells to effectively produce HBV. METHODS: Liver and non-liver cell lines were screened for their capacity to activate the HBVCP and synthesize pre-genomic RNA (pgRNA). Transcription regulators differentially expressed between cells with active or inactive HBVCP were determined by human transcriptome array. Slug (SNAI2) and SRY-related HMG box 7 (SOX7) transcriptional repressors were identified and shown to bind specifically to the HBVCP by electrophoretic mobility shift assay. The resultant inhibitory effect on HBVCP transcription was validated using luciferase reporter and assays for pgRNA, HBcAg and cccDNA accumulation in cells with HBV replicon and HBV infection models. To further confirm their specific activity, short peptide mimetics generated from Slug zinc-finger domains and SOX7 HMG-box were generated. RESULTS: The HBVCP was found to be active in liver and selected non-liver cells. These cells have low/negligible expression of Slug and SOX7, which inhibit HBVCP transcription specifically by binding at the pgRNA initiator site and competitively displacing hepatocyte nuclear factor 4α, respectively. Overexpression of Slug and/or SOX7 specifically reduced HBVCP transcription, significantly diminishing pgRNA synthesis, HBcAg and cccDNA accumulation in HBV-infected primary human hepatocytes. Similar results were obtained with Slug and SOX7 stapled peptides individually, which were even more potent in combination. CONCLUSIONS: Slug and SOX7 are transcriptional repressors that bind specifically to the HBVCP. Their absence or weak expression in liver cells contribute to the favorable host environment for the active and efficient production of HBV. LAY SUMMARY: Hepatitis B virus (HBV) replication occurs efficiently in human liver because of the specificity of viral uptake receptors and presence of numerous liver-enriched transcription activators. Herein, we show that the specific lack of transcriptional inhibitory mechanisms in liver cells also contribute to effective HBV production. HBV replication is kept low in non-liver cells as transcriptional repressors Slug and SRY-related HMG box 7 (SOX7) actively bind to the transcriptional initiator and displace transcription activators, respectively, within the HBV core promoter.

Application of strong transverse magneto-optical Kerr effect on high sensitive surface plasmon grating sensors.

A high sensitive sensor is demonstrated by exploiting strong transverse magneto-optical Kerr effect on a ferromagnetic surface plasmon grating. The surface plasmon grating, made of a hybridized Au/Fe/Au layer, exhibits a very dispersive Kerr parameter variation near the surface plasmon polariton (SPP) wavelength via coherent scattering of the SPP on the grating structure. Interrogating this Kerr parameter can be utilized for detecting chemical or biological objects in a fluid medium. The experiment results show the minimal detectable mass concentration of sodium chloride in a saline solution is 4.27 × 10(-3) %, corresponding to a refractive index change of 7.60 × 10(-6) RIU. For an avidin-biotin interaction experiment, the sensitivity of avidin detection in PBS solution is 1.97 nM, which is limited by the index fluctuation of flowing media during measurement.

Bevacizumab is associated with a higher gastrointestinal/genitourinary fistula or perforation risk in cervical cancer patients undergoing pelvic radiotherapy.

BACKGROUND: Bevacizumab serves as an effective treatment in cervical cancer patients with metastatic, recurrent, or advanced disease. However, gastrointestinal (GI)/genitourinary (GU) toxicities have been observed after bevacizumab treatment. Radiotherapy (RT) is the mainstay of treatment of cervical cancer. OBJECTIVES: To investigate the risk of GI/GU toxicities with bevacizumab plus RT compared with RT alone in cervical cancer patients. SEARCH STRATEGY: In this meta-analysis, PubMed, Embase, Web of Science, and Cochrane databases were searched from inception to September 25, 2022. SELECTION CRITERIA: Cohort studies evaluating the association between bevacizumab and GI/GU fistula or perforation in irradiated metastatic, recurrent, or advanced cervical cancer patients. DATA COLLECTION AND ANALYSIS: Results are expressed as odds ratios (OR) with 95% confidence intervals (CI). The inconsistency test (I2) was used to assess heterogeneity. Egger's regression test with a two-tailed P value was used to evaluate publication bias. MAIN RESULTS: Four cohort studies met the inclusion criteria with a total of 597 women included. There was a significant association between GI fistula/perforation and GU fistula/perforation in irradiated cervical cancer patients receiving bevacizumab (OR 4.03 [95% CI: 1.76-9.20] and OR 4.71 [95% CI: 1.51-14.70], respectively). CONCLUSIONS: The bevacizumab-containing regimen was associated with an increased risk of GI or GU toxicities in cervical cancer individuals undergoing pelvic RT. These results suggest the bevacizumab-associated benefits and risk should be better weighted to reach an optimal treatment strategy. Further investigation on optimal dosage and timing of bevacizumab and RT is vital to minimize the adverse events and maximize the benefits.

Counterproductive effects of anti-CD38 and checkpoint inhibitor for the treatment of NK/T cell lymphoma.

Introduction: Natural killer/T cell lymphoma (NKTL) is an aggressive malignancy associated with poor prognosis. This is largely due to limited treatment options, especially for relapsed patients. Immunotherapies like immune checkpoint inhibitors (ICI) and anti-CD38 therapies have shown promising but variable clinical efficacies. Combining these therapies has been suggested to enhance efficacy. Methods: We conducted a case study on a relapsed NKTL patient treated sequentially with anti-CD38 followed by ICI (anti-PD1) using cytometry analyses. Results and Discussion: Our analysis showed an expected depletion of peripheral CD38+ B cells following anti-CD38 treatment. Further analysis indicated that circulating anti-CD38 retained their function for up to 13 weeks post-administration. Anti-PD1 treatment triggered re-activation and upregulation of CD38 on the T cells. Consequently, these anti-PD1-activated T cells were depleted by residual circulating anti-CD38, rendering the ICI treatment ineffective. Finally, a meta-analysis confirmed this counterproductive effect, showing a reduced efficacy in patients undergoing combination therapy. In conclusion, our findings demonstrate that sequential anti-CD38 followed by anti-PD1 therapy leads to a counterproductive outcome in NKTL patients. This suggests that the treatment sequence is antithetic and warrants re-evaluation for optimizing cancer immunotherapy strategies.

Multi-omic Analysis of Human B-cell Activation Reveals a Key Lysosomal BCAT1 Role in mTOR Hyperactivation by B-cell receptor and TLR9.

B-lymphocytes play major adaptive immune roles, producing antibody and driving T-cell responses. However, how immunometabolism networks support B-cell activation and differentiation in response to distinct receptor stimuli remains incompletely understood. To gain insights, we systematically investigated acute primary human B-cell transcriptional, translational and metabolomic responses to B-cell receptor (BCR), Toll-like receptor 9 (TLR9), CD40-ligand (CD40L), interleukin-4 (IL4) or combinations thereof. T-independent BCR/TLR9 co-stimulation, which drives malignant and autoimmune B-cell states, jointly induced PD-L1 plasma membrane expression, supported by NAD metabolism and oxidative phosphorylation. BCR/TLR9 also highly induced the transaminase BCAT1, which localized to lysosomal membranes to support branched chain amino acid synthesis and mTORC1 hyperactivation. BCAT1 inhibition blunted BCR/TLR9, but not CD40L/IL4-triggered B-cell proliferation, IL10 expression and BCR/TLR pathway-driven lymphoma xenograft outgrowth. These results provide a valuable resource, reveal receptor-mediated immunometabolism remodeling to support key B-cell phenotypes including PD-L1 checkpoint signaling, and identify BCAT1 as a novel B-cell therapeutic target.

Targeting Metabolic Vulnerabilities in Epstein-Barr Virus-Driven Proliferative Diseases.

The metabolism of cancer cells and Epstein-Barr virus (EBV) infected cells have remarkable similarities. Cancer cells frequently reprogram metabolic pathways to augment their ability to support abnormal rates of proliferation and promote intra-organismal spread through metastatic invasion. On the other hand, EBV is also capable of manipulating host cell metabolism to enable sustained growth and division during latency as well as intra- and inter-individual transmission during lytic replication. It comes as no surprise that EBV, the first oncogenic virus to be described in humans, is a key driver for a significant fraction of human malignancies in the world (~1% of all cancers), both in terms of new diagnoses and attributable deaths each year. Understanding the contributions of metabolic pathways that underpin transformation and virus replication will be important for delineating new therapeutic targets and designing nutritional interventions to reduce disease burden. In this review, we summarise research hitherto conducted on the means and impact of various metabolic changes induced by EBV and discuss existing and potential treatment options targeting metabolic vulnerabilities in EBV-associated diseases.

Human Milk Antibodies after BNT162b2 Vaccination Exhibit Reduced Binding against SARS-CoV-2 Variants of Concern.

SARS-CoV-2-specific antibody responses are engendered in human milk after BNT162b2 vaccination. However, the emergence of variants of concern (VOCs) raises concerns about the specificity of and potential cross-protection mediated by milk antibody responses, which are crucial for passive immunity transferred from breastfeeding mothers to their infants. In this study, we collected milk samples at three different time points pre- and post-vaccination, and measured milk IgA antibody binding to the receptor binding domain (RBD) of the original Wuhan-Hu-1 strain, and the four VOCs, namely Alpha, Beta, Gamma and Delta. We report a significant level of anti-RBD IgA in milk collected at 4-6 weeks after the second dose of vaccination compared to pre-vaccination. We observed around a 30% reduction in binding to most VOCs, including the major circulating Delta variant, compared to the original Wuhan-Hu-1 strain. As COVID-19 vaccines may take some time to be approved for infants, these individuals remain at risk for severe disease and rely mainly on transferred passive immunity. Our findings support the current recommendations for vaccinating lactating women with the aim of transferring mucosal immunity to breastfeeding infants.

Immune and pathophysiologic profiling of antenatal coronavirus disease 2019 in the GIFT cohort: A Singaporean case-control study.

COVID-19 can be severe in pregnant women, and have adverse consequences for the subsequent infant. We profiled the post-infectious immune responses in maternal and child blood as well as breast milk in terms of antibody and cytokine expression and performed histopathological studies on placentae obtained from mothers convalescent from antenatal COVID-19. Seventeen mother-child dyads (8 cases of antenatal COVID-19 and 9 healthy unrelated controls; 34 individuals in total) were recruited to the Gestational Immunity For Transfer (GIFT) study. Maternal and infant blood, and breast milk samples were collected over the first year of life. All samples were analyzed for IgG and IgA against whole SARS-CoV-2 spike protein, the spike receptor-binding domain (RBD), and previously reported immunodominant epitopes, as well as cytokine levels. The placentae were examined microscopically. The study is registered at clinicaltrials.gov under the identifier NCT04802278. We found high levels of virus-specific IgG in convalescent mothers and similarly elevated titers in newborn children. Thus, antenatal SARS-CoV-2 infection led to high plasma titers of virus-specific antibodies in infants postnatally. However, this waned within 3-6 months of life. Virus neutralization by plasma was not uniformly achieved, and the presence of antibodies targeting known immunodominant epitopes did not assure neutralization. Virus-specific IgA levels were variable among convalescent individuals' sera and breast milk. Antibody transfer ratios and the decay of transplacentally transferred virus-specific antibodies in neonatal circulation resembled that for other pathogens. Convalescent mothers showed signs of chronic inflammation marked by persistently elevated IL17RA levels in their blood. Four placentae presented signs of acute inflammation, particularly in the subchorionic region, marked by neutrophil infiltration even though > 50 days had elapsed between virus clearance and delivery. Administration of a single dose of BNT162b2 mRNA vaccine to mothers convalescent from antenatal COVID-19 increased virus-specific IgG and IgA titers in breast milk, highlighting the importance of receiving the vaccine even after natural infection with the added benefit of enhanced passive immunity.

Codominant IgG and IgA expression with minimal vaccine mRNA in milk of BNT162b2 vaccinees.

Lactating women can produce protective antibodies in their milk after vaccination, which has informed antenatal vaccination programs for diseases such as influenza and pertussis. However, whether SARS-CoV-2-specific antibodies are produced in human milk as a result of COVID-19 vaccination is still unclear. In this study, we show that lactating mothers who received the BNT162b2 vaccine secreted SARS-CoV-2-specific IgA and IgG antibodies into milk, with the most significant increase at 3-7 days post-dose 2. Virus-specific IgG titers were stable out to 4-6 weeks after dose 2. In contrast, SARS-CoV-2-specific IgA levels showed substantial decay. Vaccine mRNA was detected in few milk samples (maximum of 2 ng/ml), indicative of minimal transfer. Additionally, infants who consumed post-vaccination human milk had no reported adverse effects up to 28 days post-ingestion. Our results define the safety and efficacy profiles of the vaccine in this demographic and provide initial evidence for protective immunity conferred by milk-borne SARS-CoV-2-specific antibodies. Taken together, our study supports recommendations for uninterrupted breastfeeding subsequent to mRNA vaccination against COVID-19.

Data-Driven Analysis of COVID-19 Reveals Persistent Immune Abnormalities in Convalescent Severe Individuals.

Severe SARS-CoV-2 infection can trigger uncontrolled innate and adaptive immune responses, which are commonly associated with lymphopenia and increased neutrophil counts. However, whether the immune abnormalities observed in mild to severely infected patients persist into convalescence remains unclear. Herein, comparisons were drawn between the immune responses of COVID-19 infected and convalescent adults. Strikingly, survivors of severe COVID-19 had decreased proportions of NKT and Vδ2 T cells, and increased proportions of low-density neutrophils, IgA+/CD86+/CD123+ non-classical monocytes and hyperactivated HLADR+CD38+ CD8+ T cells, and elevated levels of pro-inflammatory cytokines such as hepatocyte growth factor and vascular endothelial growth factor A, long after virus clearance. Our study suggests potential immune correlates of "long COVID-19", and defines key cells and cytokines that delineate true and quasi-convalescent states.

CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation.

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.

Epstein-Barr-Virus-Induced One-Carbon Metabolism Drives B Cell Transformation.

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.

Modulating Gene Expression in Epstein-Barr Virus (EBV)-Positive B Cell Lines with CRISPRa and CRISPRi.

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.

CRISPR/Cas9-Mediated Genome Editing in Epstein-Barr Virus-Transformed Lymphoblastoid B-Cell Lines.

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.

A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells.

Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.