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OBJECTIVE: Elevated levels of BAFF and APRIL are characteristic of patients with systemic lupus erythematosus (SLE). The reasons for enhanced cytokine production are not well understood. This study was undertaken to identify the cells responsible for the overproduction of these cytokines. METHODS: BAFF expression was analyzed on peripheral blood mononuclear cells by multiparameter flow cytometry and in tissue samples by immunofluorescence staining. The levels of BAFF and APRIL mRNA were quantified in sorted B cells. In vitro cultures were used to analyze whether B cell survival and differentiation was supported by autocrine BAFF and/or APRIL. RESULTS: Aberrant activation of B cells in patients with SLE was associated with a significant up-regulation of BAFF expression in naive, memory, and plasma cells. Furthermore, strong expression of BAFF and APRIL was found in plasma cells from the lymph node, bone marrow, and kidney. The levels of BAFF and APRIL mRNA in CD19+ B cells correlated both with the titer of anti-double stranded DNA antibodies and with the SLE Disease Activity Index. In vitro experiments demonstrated that B cells released functional BAFF/APRIL upon activation. CONCLUSION: Our data show that B cells contribute to the enhanced levels of circulating BAFF and APRIL. The aberrant up-regulation of these cytokines may initiate a vicious circle in which enhanced levels of BAFF and APRIL act in an autocrine manner to reinforce the systemic activation of the humoral immune system. [ABSTRACT FROM AUTHOR]

Doxycycline post-exposure prophylaxis (doxy-PEP) reduces bacterial sexually transmitted infections among men who have sex with men and transgender women. Although poised for widespread clinical implementation, the impact of doxy-PEP on antimicrobial resistance remains a primary concern as its effects on the gut microbiome and resistome, or the antimicrobial resistance genes (ARGs) present in the gut microbiome, are unknown. To investigate these effects, we studied participants from the DoxyPEP trial, a randomized clinical trial comparing doxy-PEP use, a one-time doxycycline 200-mg dose taken after condomless sex (DP arm, n = 100), to standard of care (SOC arm, n = 50) among men who have sex with men and transgender women. From self-collected rectal swabs at enrollment (day-0) and after 6 months (month-6), we performed metagenomic DNA sequencing (DNA-seq) or metatranscriptomic RNA sequencing (RNA-seq). DNA-seq data were analyzable from 127 samples derived from 89 participants, and RNA-seq data were analyzable from 86 samples derived from 70 participants. We compared the bacterial microbiome and resistome between the two study arms and over time. The median number of doxycycline doses taken since enrollment by participants with DNA-seq data was zero (interquartile range (IQR): 0-7 doses) for the SOC arm and 42 (IQR: 27-64 doses) for the DP arm. Tetracycline ARGs were detected in all day-0 DNA-seq samples and in 85% of day-0 RNA-seq samples. The proportional mass of tetracycline ARGs in the resistome increased between day-0 and month-6 in DP participants from 46% to 51% in the metagenome (P = 2.3 × 10 -2 ) and from 4% to 15% in the metatranscriptome (P = 4.5 × 10 -6 ), but no statistically significant increases in other ARG classes were observed. Exposure to a higher number of doxycycline doses correlated with proportional enrichment of tetracycline ARGs in the metagenome (Spearman's ρ = 0.23, P = 9.0 × 10 -3 ) and metatranscriptome (Spearman's ρ = 0.55, P = 3.7 × 10 -8 ). Bacterial microbiome alpha diversity, beta diversity and total bacterial mass did not differ between day-0 and month-6 samples from DP participants when assessed by either DNA-seq or RNA-seq. In an abundance-based correlation analysis, we observed an increase over time in the strength of the correlation between tetracycline ARGs and specific bacterial taxa, including some common human pathogens. In sum, doxy-PEP use over a 6-month period was associated with an increase in the proportion of tetracycline ARGs comprising the gut resistome and an increase in the expression of tetracycline ARGs. At 6 months of doxy-PEP use, no residual differences were observed in alpha and beta diversity or taxonomic composition of the gut microbiome. As doxy-PEP is implemented as a public health strategy, further studies and population-level surveillance of doxycycline-resistant pathogens are needed to understand the implications of these findings. ClinicalTrials.gov registration number: NCT03980223 ., (© 2024. The Author(s).)

Background: An escape room is a cooperative game that has been adapted into medical education and major academic conferences., Methods: We describe the design, development, and implementation of an educational ID-themed escape room activity entitled "Out-BREAK!" at an international conference, IDWeek 2023. An anonymous survey was conducted to collect demographic data, assess participant satisfaction with the escape room puzzles, and gauge participant interest in game-based learning., Results: Thirty escape room sessions were held over 3 days and included 201 participants. Escape room survey respondents (n = 132) were younger and more likely to be trainees compared with in-person IDWeek attendants. Among 131 responses, all respondents enjoyed the experience and would recommend the escape room activity to friends. Survey respondents enjoyed the puzzle solving (93%), medical content (92%), and team building (79%) components. Only 35% of the respondents had ever previously participated in game-based learning; 95% thought the escape room was a valuable teaching method. Among the 72 survey respondents involved in medical education, almost all (90%) said they were interested in incorporating escape rooms into medical education., Conclusions: The Out-BREAK! escape room at IDWeek 2023 was successfully implemented and well received. Despite only a third of participants having prior experience with game-based learning, almost all respondents perceived the escape room to be an effective teaching method., Competing Interests: Potential conflicts of interest. D.Z., S.D., V.T.C., N.G., K.L., J.B.S., E.H.R., A.B., J.B., and P.P. report no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Hematopoiesis is a continuous process of blood cell production driven by hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Proliferation and differentiation of HSPCs are regulated by complex transcriptional networks. In order to identify transcription factors with key roles in HSPC-mediated hematopoietic reconstitution, we developed an efficient and robust CRISPR/Cas9-based in vivo genetic screen. Using this experimental system, we identified the TFDP1 transcription factor to be essential for HSPC proliferation and post-transplant hematopoiesis. We further discovered that E2F4, an E2F transcription factor, serves as a binding partner of TFDP1 and is required for HSPC proliferation. Deletion of TFDP1 caused downregulation of genes associated with the cell cycle, with around 50% of these genes being identified as direct targets of TFDP1 and E2F4. Thus, our study expands the transcriptional network governing hematopoietic development through an in vivo CRISPR/Cas9-based genetic screen and identifies TFDP1/E2F4 as positive regulators of cell cycle genes in HSPCs., (© 2024. The Author(s).)

Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, often fatal immune deficiency characterized by severe systemic hyperinflammation. Although allogeneic bone marrow transplantation can be curative, more effective therapies are urgently needed. FHL is caused by inactivating mutations in proteins that regulate cellular immunity. Here, we used an adeno-associated virus-based CRISPR-Cas9 system with an inhibitor of nonhomologous end joining to repair such mutations in potentially long-lived T cells ex vivo. Repaired CD8 memory T cells efficiently cured lethal hyperinflammation in a mouse model of Epstein-Barr virus-triggered FHL2, a subtype caused by perforin-1 ( Prf1 ) deficiency. Furthermore, repair of PRF1 and Munc13-4 ( UNC13D )-whose deficiency causes the FHL subtype FHL3-in mutant memory T cells from two critically ill patients with FHL restored T cell cytotoxicity. These results provide a starting point for the treatment of genetic T cell immune dysregulation syndromes with repaired autologous T cells.

Antimicrobial resistant lower respiratory tract infections are an increasing public health threat and an important cause of global mortality. The lung microbiome can influence susceptibility of respiratory tract infections and represents an important reservoir for exchange of antimicrobial resistance genes. Studies of the gut microbiome have found an association between age and increasing antimicrobial resistance gene burden, however, corollary studies in the lung microbiome remain absent. We performed an observational study of children and adults with acute respiratory failure admitted to the intensive care unit. From tracheal aspirate RNA sequencing data, we evaluated age-related differences in detectable antimicrobial resistance gene expression in the lung microbiome. Using a multivariable logistic regression model, we find that detection of antimicrobial resistance gene expression was significantly higher in adults compared with children after adjusting for demographic and clinical characteristics. This association remained significant after additionally adjusting for lung bacterial microbiome characteristics, and when modeling age as a continuous variable. The proportion of adults expressing beta-lactam, aminoglycoside, and tetracycline antimicrobial resistance genes was higher compared to children. Together, these findings shape our understanding of the lung resistome in critically ill patients across the lifespan, which may have implications for clinical management and global public health., (© 2024. The Author(s).)

Two independent temporal-spatial clusters of hospital-onset Rhizopus infections were evaluated using whole-genome sequencing (WGS). Phylogenetic analysis confirmed that isolates within each cluster were unrelated despite epidemiological suspicion of outbreaks. The ITS1 region alone was insufficient for accurate analysis. WGS has utility for rapid rule-out of suspected nosocomial Rhizopus outbreaks.

An increasing number of cancer subtypes are treated with front-line immunotherapy. However, approaches to overcome primary and acquired resistance remain limited. Preclinical mouse models are often used to investigate resistance mechanisms, novel drug combinations, and delivery methods; yet most of these models lack the genetic diversity and mutational patterns observed in human tumors. Here we describe a series of 13 C57BL/6J melanoma cell lines to address this gap in the field. The Ohio State University-Moffitt Melanoma Exposed to Radiation (OSUMMER) cell lines are derived from mice expressing endogenous, melanocyte-specific, and clinically relevant Nras driver mutations (Q61R, Q61K, or Q61L). Exposure of these animals to a single, non-burning dose of ultraviolet B accelerates the onset of spontaneous melanomas with mutational patterns akin to human disease. Furthermore, in vivo irradiation selects against potent tumor antigens, which could prevent the outgrowth of syngeneic cell transfers. Each OSUMMER cell line possesses distinct in vitro growth properties, trametinib sensitivity, mutational signatures, and predicted antigenicity. Analysis of OSUMMER allografts shows a correlation between strong, predicted antigenicity and poor tumor outgrowth. These data suggest that the OSUMMER lines will be a valuable tool for modeling the heterogeneous responses of human melanomas to targeted and immune-based therapies., (© 2023 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

Prior research suggests that R&D intensive firms are especially vulnerable during crises due to their narrow specialization, high adjustment costs, increased distress risks, and higher sensitivity to financial distress. This paper exploits the difference in the research and development intensity as a quasi-natural experiment to examine the impact of the coronavirus pandemic on firm performance. Our study finds that the adverse consequences of COVID-19 on firms' profitability have been less pronounced for R&D intensive firms. R&D intensive firms are also able to record more positive changes in cash holdings as a response to the COVID-19 pandemic. As a result, R&D intensive firms are less likely to rely on external financing and record a lower level of increase in financing. Our results further highlight the potential function of R&D investment as a panacea for firm's growth during economic downturns., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

The CRISPR/Cas9 system is a powerful tool for gene repair that holds great potential for gene therapy to cure monogenic diseases. Despite intensive improvement, the safety of this system remains a major clinical concern. In contrast to Cas9 nuclease, Cas9 nickases with a pair of short-distance (38-68 bp) PAM-out single-guide RNAs (sgRNAs) preserve gene repair efficiency while strongly reducing off-target effects. However, this approach still leads to efficient unwanted on-target mutations that may cause tumorigenesis or abnormal hematopoiesis. We establish a precise and safe spacer-nick gene repair approach that combines Cas9 D10A nickase with a pair of PAM-out sgRNAs at a distance of 200-350 bp. In combination with adeno-associated virus (AAV) serotype 6 donor templates, this approach leads to efficient gene repair with minimal unintended on- and off-target mutations in human hematopoietic stem and progenitor cells (HSPCs). Here, we provide detailed protocols to use the spacer-nick approach for gene repair and to assess the safety of this system in human HSPCs. The spacer-nick approach enables efficient gene correction for repair of disease-causing mutations with increased safety and suitability for gene therapy. Graphical overview., Competing Interests: Competing interestsK.R., V.T.C., N.T.T. and R.K. are inventors on a patent application related to this protocol filed by the Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC) (European Patent Application Nr. 21192657.1-1111, filed 23 August 2021). All other authors declare no competing interests., (Copyright © 2023 The Authors.)

Introduction: The differentiation of B cells into antibody-secreting plasma cells depends on cell division-coupled, epigenetic and other cellular processes that are incompletely understood., Methods: We have developed a CRISPR/Cas9-based screen that models an early stage of T cell-dependent plasma cell differentiation and measures B cell survival or proliferation versus the formation of CD138+ plasmablasts. Here, we refined and extended this screen to more than 500 candidate genes that are highly expressed in plasma cells., Results: Among known genes whose deletion preferentially or mostly affected plasmablast formation were the transcription factors Prdm1 (BLIMP1), Irf4 and Pou2af1 (OBF-1), and the Ern1 gene encoding IRE1a, while deletion of XBP1, the transcriptional master regulator that specifies the expansion of the secretory program in plasma cells, had no effect. Defective plasmablast formation caused by Ern1 deletion could not be rescued by the active, spliced form of XBP1 whose processing is dependent on and downstream of IRE1a, suggesting that in early plasma cell differentiation IRE1a acts independently of XBP1. Moreover, we newly identified several genes involved in NF-kB signaling (Nfkbia), vesicle trafficking (Arf4, Preb) and epigenetic regulators that form part of the NuRD complex (Hdac1, Mta2, Mbd2) to be required for plasmablast formation. Deletion of ARF4, a small GTPase required for COPI vesicle formation, impaired plasmablast formation and blocked antibody secretion. After Hdac1 deletion plasmablast differentiation was consistently reduced by about 50%, while deletion of the closely related Hdac2 gene had no effect. Hdac1 knock-out led to strongly perturbed protein expression of antagonistic transcription factors that govern plasma cell versus B cell identity (by decreasing IRF4 and BLIMP1 and increasing BACH2 and PAX5)., Discussion: Taken together, our results highlight specific and non-redundant roles for Ern1, Arf4 and Hdac1 in the early steps of plasma cell differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xiong, Popp, Salomon, Mertins, Kocks, Rajewsky and Chu.)

Human B lymphocytes are attractive targets for immunotherapies in autoantibody-mediated diseases. Gene editing technologies could provide a powerful tool to determine gene regulatory networks regulating B cell differentiation into plasma cells, and identify novel therapeutic targets for prevention and treatment of autoimmune disorders. Here, we describe a new approach that uses CRISPR-Cas9 technology to target genes in primary human B cells in vitro for identifying plasma cell regulators. We found that sgRNA and Cas9 components can be efficiently delivered into primary human B cells through RD114-pseudotyped retroviral vectors. Using this system, we achieved approximately 80% of gene knockout efficiency. We disrupted expression of a triad of transcription factors, IRF4, PRDM1, and XBP1, and showed that human B cell survival and plasma cell differentiation are severely impaired. Specifically, that IRF4, PRDM1, and XBP1 were expressed at different stages during plasma cell differentiation, IRF4 , PRDM1, and XBP1 -targeted B cells failed to progress to the pre-plasmablast, plasma cell state, and plasma cell survival, respectively. Our method opens a new avenue to study gene functions in primary human B cells and identify novel plasma cell regulators for therapeutic applications., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

While risk of fomite transmission of SARS-CoV-2 is considered low, there is limited environmental data within households. This January-April 2021 investigation describes frequency and types of surfaces positive for SARS-CoV-2 by real-time reverse transcription polymerase chain reaction (RT-PCR) among residences with ≥1 SARS-CoV-2 infection, and associations of household characteristics with surface RT-PCR and viable virus positivity. Of 1232 samples from 124 households, 27.8% (n = 342) were RT-PCR positive with nightstands (44.1%) and pillows (40.9%) most frequently positive. SARS-CoV-2 lineage, documented household transmission, greater number of infected persons, shorter interval between illness onset and sampling, total household symptoms, proportion of infected persons ≤12 years old, and persons exhibiting upper respiratory symptoms or diarrhea were associated with more positive surfaces. Viable virus was isolated from 0.2% (n = 3 samples from one household) of all samples. This investigation suggests that while SARS-CoV-2 on surfaces is common, fomite transmission risk in households is low., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Mitigation behaviors are key to preventing SARS-CoV-2 transmission. We identified the behaviors associated with secondary transmission from confirmed SARS-CoV-2 primary cases to household contacts and described the characteristics associated with reporting these behaviors., Methods: Households with confirmed SARS-CoV-2 infections were recruited in California and Colorado from January to April 2021. Self-reported behaviors and demographics were collected through interviews. We investigated behaviors associated with transmission and individual and household characteristics associated with behaviors using univariable and multivariable logistic regression with generalized estimating equations to account for household clustering., Results: Among household contacts of primary cases, 43.3% (133 of 307) became infected with SARS-CoV-2. When an adjusted analysis was conducted, household contacts who slept in the same bedroom with the primary case (AOR=2.19; 95% CI=1.25, 3.84) and ate food prepared by the primary case (AOR=1.98; 95% CI=1.02, 3.87) had increased odds of SARS-CoV-2 infection. Household contacts in homes ≤2,000 square feet had increased odds of sleeping in the same bedroom as the primary case compared with those in homes >2,000 square feet (AOR=3.97; 95% CI=1.73, 9.10). Parents, siblings, and other relationships (extended family, friends, or roommates) of the primary case had decreased odds of eating food prepared by the primary case compared with partners., Conclusions: Sleeping in the same bedroom as the primary case and eating food prepared by the primary case were associated with secondary transmission. Household dimension and relationship to the primary case were associated with these behaviors. Our findings encourage innovative means to promote adherence to mitigation measures that reduce household transmission., (© 2022 The Author(s).)

Background: In Spring 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.7 (Alpha) became the predominant variant in the United States. Research suggests that Alpha has increased transmissibility compared with non-Alpha lineages. We estimated household secondary infection risk (SIR), assessed characteristics associated with transmission, and compared symptoms of persons with Alpha and non-Alpha infections., Methods: We followed households with SARS-CoV-2 infection for 2 weeks in San Diego County and metropolitan Denver, January to April 2021. We collected epidemiologic information and biospecimens for serology, reverse transcription-polymerase chain reaction (RT-PCR), and whole-genome sequencing. We stratified SIR and symptoms by lineage and identified characteristics associated with transmission using generalized estimating equations., Results: We investigated 127 households with 322 household contacts; 72 households (56.7%) had member(s) with secondary infections. SIRs were not significantly higher for Alpha (61.0% [95% confidence interval, 52.4-69.0%]) than non-Alpha (55.6% [44.7-65.9%], P = .49). In households with Alpha, persons who identified as Asian or Hispanic/Latino had significantly higher SIRs than those who identified as White (P = .01 and .03, respectively). Close contact (eg, kissing, hugging) with primary cases was associated with increased transmission for all lineages. Persons with Alpha infection were more likely to report constitutional symptoms than persons with non-Alpha (86.9% vs 76.8%, P = .05)., Conclusions: Household SIRs were similar for Alpha and non-Alpha. Comparable SIRs may be due to saturation of transmission risk in households due to extensive close contact, or true lack of difference in transmission rates. Avoiding close contact within households may reduce SARS-CoV-2 transmission for all lineages among household members., (Published by Oxford University Press for the Infectious Diseases Society of America 2022.)

Background: COVID-19 vaccination reduces SARS-CoV-2 infection and transmission. However, evidence is emerging on the degree of protection across variants and in high-transmission settings. To better understand the protection afforded by vaccination specifically in a high-transmission setting, we examined household transmission of SARS-CoV-2 during a period of high community incidence with predominant SARS-CoV-2 B.1.1.7 (Alpha) variant, among vaccinated and unvaccinated contacts., Methods: We conducted a household transmission investigation in San Diego County, California, and Denver, Colorado, during January-April 2021. Households were enrolled if they had at least one person with documented SARS-CoV-2 infection. We collected nasopharyngeal swabs, blood, demographic information, and vaccination history from all consenting household members. We compared infection risks (IRs), RT-PCR cycle threshold values, SARS-CoV-2 culture results, and antibody statuses among vaccinated and unvaccinated household contacts., Results: We enrolled 493 individuals from 138 households. The SARS-CoV-2 variant was identified from 121/138 households (88%). The most common variants were Alpha (75/121, 62%) and Epsilon (19/121, 16%). There were no households with discordant lineages among household members. One fully vaccinated secondary case was symptomatic (13%); the other 5 were asymptomatic (87%). Among unvaccinated secondary cases, 105/108 (97%) were symptomatic. Among 127 households with a single primary case, the IR for household contacts was 45% (146/322; 95% Confidence Interval [CI] 40-51%). The observed IR was higher in unvaccinated (130/257, 49%, 95% CI 45-57%) than fully vaccinated contacts (6/26, 23%, 95% CI 11-42%). A lower proportion of households with a fully vaccinated primary case had secondary cases (1/5, 20%) than households with an unvaccinated primary case (66/108, 62%)., Conclusions: Although SARS-CoV-2 infections in vaccinated household contacts were reported in this high transmission setting, full vaccination protected against SARS-CoV-2 infection. These findings further support the protective effect of COVID-19 vaccination and highlight the need for ongoing vaccination among eligible persons., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Objective: To assess the household secondary infection risk (SIR) of B.1.1.7 (Alpha) and non-Alpha lineages of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among children., Study Design: During January to April 2021, we prospectively followed households with a SARS-CoV-2 infection. We collected questionnaires, serial nasopharyngeal swabs for reverse transcription polymerase chain reaction testing and whole genome sequencing, and serial blood samples for serology testing. We calculated SIRs by primary case age (pediatric vs adult), household contact age, and viral lineage. We evaluated risk factors associated with transmission and described symptom profiles among children., Results: Among 36 households with pediatric primary cases, 21 (58%) had secondary infections. Among 91 households with adult primary cases, 51 (56%) had secondary infections. SIRs among pediatric and adult primary cases were 45% and 54%, respectively (OR, 0.79; 95% CI, 0.41-1.54). SIRs among pediatric primary cases with Alpha and non-Alpha lineage were 55% and 46%, respectively (OR, 1.52; 95% CI, 0.51-4.53). SIRs among pediatric and adult household contacts were 55% and 49%, respectively (OR, 1.01; 95% CI, 0.68-1.50). Among pediatric contacts, no significant differences in the odds of acquiring infection by demographic or household characteristics were observed., Conclusions: Household transmission of SARS-CoV-2 from children and adult primary cases to household members was frequent. The risk of secondary infection was similar among child and adult household contacts. Among children, household transmission of SARS-CoV-2 and the risk of secondary infection was not influenced by lineage. Continued mitigation strategies (eg, masking, physical distancing, vaccination) are needed to protect at-risk groups regardless of virus lineage circulating in communities., (Published by Elsevier Inc.)

Background and Objectives: There have been numerous reports of neurologic manifestations identified in hospitalized patients infected with SARS-CoV-2, the virus that causes COVID-19. Here, we identify the spectrum of associated neurologic symptoms and diagnoses, define the time course of their development, and examine readmission rates and mortality risk posthospitalization in a multiethnic urban cohort., Methods: We identify the occurrence of new neurologic diagnoses among patients with laboratory-confirmed SARS-CoV-2 infection in New York City. A retrospective cohort study was performed on 532 cases (hospitalized patients with new neurologic diagnoses within 6 weeks of positive SARS-CoV-2 laboratory results between March 1, 2020, and August 31, 2020). We compare demographic and clinical features of the 532 cases with 532 controls (hospitalized COVID-19 patients without neurologic diagnoses) in a case-control study with one-to-one matching and examine hospital-related data and outcomes of death and readmission up to 6 months after acute hospitalization in a secondary case-only analysis., Results: Among the 532 cases, the most common new neurologic diagnoses included encephalopathy (478, 89.8%), stroke (66, 12.4%), and seizures (38, 7.1%). In the case-control study, cases were more likely than controls to be male (58.6% vs 52.8%, p = 0.05), had baseline neurologic comorbidities (36.3% vs 13.0%, p < 0.0001), and were to be treated in an intensive care unit (62.0% vs 9.6%, p < 0.0001). Of the 394 (74.1%) cases who survived acute hospitalization, more than half (220 of 394, 55.8%) were readmitted within 6 months, with a mortality rate of 23.2% during readmission., Discussion: Hospitalized patients with SARS-CoV-2 and new neurologic diagnoses have significant morbidity and mortality postdischarge. Further research is needed to define the effect of neurologic diagnoses during acute hospitalization on longitudinal post-COVID-19-related symptoms including neurocognitive impairment., (© 2022 American Academy of Neurology.)

We compared paired serum specimens from household contacts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases with detectable SARS-CoV-2 seroconversion with contacts who remained seronegative. No protection from SARS-CoV-2 infection was associated with human coronavirus antibodies; however, an increase in common betacoronavirus antibodies was associated with seroconversion to SARS-CoV-2 in mild to moderately ill cases., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2022.)

Importance: As self-collected home antigen tests become widely available, a better understanding of their performance during the course of SARS-CoV-2 infection is needed., Objective: To evaluate the diagnostic performance of home antigen tests compared with reverse transcription-polymerase chain reaction (RT-PCR) and viral culture by days from illness onset, as well as user acceptability., Design, Setting, and Participants: This prospective cohort study was conducted from January to May 2021 in San Diego County, California, and metropolitan Denver, Colorado. The convenience sample included adults and children with RT-PCR-confirmed infection who used self-collected home antigen tests for 15 days and underwent at least 1 nasopharyngeal swab for RT-PCR, viral culture, and sequencing., Exposures: SARS-CoV-2 infection., Main Outcomes and Measures: The primary outcome was the daily sensitivity of home antigen tests to detect RT-PCR-confirmed cases. Secondary outcomes included the daily percentage of antigen test, RT-PCR, and viral culture results that were positive, and antigen test sensitivity compared with same-day RT-PCR and cultures. Antigen test use errors and acceptability were assessed for a subset of participants., Results: This study enrolled 225 persons with RT-PCR-confirmed infection (median [range] age, 29 [1-83] years; 117 female participants [52%]; 10 [4%] Asian, 6 [3%] Black or African American, 50 [22%] Hispanic or Latino, 3 [1%] Native Hawaiian or Other Pacific Islander, 145 [64%] White, and 11 [5%] multiracial individuals) who completed 3044 antigen tests and 642 nasopharyngeal swabs. Antigen test sensitivity was 50% (95% CI, 45%-55%) during the infectious period, 64% (95% CI, 56%-70%) compared with same-day RT-PCR, and 84% (95% CI, 75%-90%) compared with same-day cultures. Antigen test sensitivity peaked 4 days after illness onset at 77% (95% CI, 69%-83%). Antigen test sensitivity improved with a second antigen test 1 to 2 days later, particularly early in the infection. Six days after illness onset, antigen test result positivity was 61% (95% CI, 53%-68%). Almost all (216 [96%]) surveyed individuals reported that they would be more likely to get tested for SARS-CoV-2 infection if home antigen tests were available over the counter., Conclusions and Relevance: The results of this cohort study of home antigen tests suggest that sensitivity for SARS-CoV-2 was moderate compared with RT-PCR and high compared with viral culture. The results also suggest that symptomatic individuals with an initial negative home antigen test result for SARS-CoV-2 infection should test again 1 to 2 days later because test sensitivity peaked several days after illness onset and improved with repeated testing.

While CRISPR-Cas9 is key for the development of gene therapy, its potential off-target mutations are still a major concern. Here, we establish a "spacer-nick" gene correction approach that combines the Cas9 D10A nickase with a pair of PAM-out sgRNAs at a distance of 200 to 350 bp. In combination with adeno-associated virus (AAV) serotype 6 template delivery, our approach led to efficient HDR in human hematopoietic stem and progenitor cells (HSPCs including long-term HSCs) and T cells, with minimal NHEJ-mediated on-target mutations. Using spacer-nick, we developed an approach to repair disease-causing mutations occurring in the HBB , ELANE , IL7R , and PRF1 genes. We achieved gene correction efficiencies of 20 to 50% with minimal NHEJ-mediated on-target mutations. On the basis of in-depth off-target assessment, frequent unintended genetic alterations induced by classical CRISPR-Cas9 were significantly reduced or absent in the HSPCs treated with spacer-nick. Thus, the spacer-nick gene correction approach provides improved safety and suitability for gene therapy.

Background: Although a human adenovirus (HAdV) vaccine is available for military use, officers-in-training are not routinely vaccinated. We describe an HAdV-associated respiratory outbreak among unvaccinated cadets at the US Coast Guard Academy and its impact on cadet training., Methods: We defined a case as a cadet with new onset cough or sore throat during August 1-October 4, 2019. We reviewed medical records and distributed a questionnaire to identify cases and to estimate impact on cadet training. We performed real-time polymerase chain reaction testing on patient and environmental samples and whole genome sequencing on a subset of positive patient samples., Results: Among the 1072 cadets, 378 (35%) cases were identified by medical records (n = 230) or additionally by the questionnaire (n = 148). Of the 230 cases identified from medical records, 138 (60%) were male and 226 (98%) had no underlying conditions. From questionnaire responses, 113 of 228 (50%) cases reported duty restrictions. Of cases with respiratory specimens, 36 of 50 (72%) were HAdV positive; all 14 sequenced specimens were HAdV-4a1. Sixteen (89%) of 18 environmental specimens from the cadet dormitory were HAdV-positive., Conclusions: The HAdV-4-associated outbreak infected a substantial number of cadets and significantly impacted cadet training. Routine vaccination could prevent HAdV respiratory outbreaks in this population., (Published by Oxford University Press for the Infectious Diseases Society of America 2021.)

Background: The evidence base for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is nascent. We sought to characterize SARS-CoV-2 transmission within US households and estimate the household secondary infection rate (SIR) to inform strategies to reduce transmission., Methods: We recruited patients with laboratory-confirmed SARS-CoV-2 infection and their household contacts in Utah and Wisconsin during 22 March 2020-25 April 2020. We interviewed patients and all household contacts to obtain demographics and medical histories. At the initial household visit, 14 days later, and when a household contact became newly symptomatic, we collected respiratory swabs from patients and household contacts for testing by SARS-CoV-2 real-time reverse-transcription polymerase chain reaction (rRT-PCR) and sera for SARS-CoV-2 antibodies testing by enzyme-linked immunosorbent assay (ELISA). We estimated SIR and odds ratios (ORs) to assess risk factors for secondary infection, defined by a positive rRT-PCR or ELISA test., Results: Thirty-two (55%) of 58 households secondary infection among household contacts. The SIR was 29% (n = 55/188; 95% confidence interval [CI], 23%-36%) overall, 42% among children (aged <18 years) of the COVID-19 patient and 33% among spouses/partners. Household contacts to COVID-19 patients with immunocompromised conditions and household contacts who themselves had diabetes mellitus had increased odds of infection with ORs 15.9 (95% CI, 2.4-106.9) and 7.1 (95% CI: 1.2-42.5), respectively., Conclusions: We found substantial evidence of secondary infections among household contacts. People with COVID-19, particularly those with immunocompromising conditions or those with household contacts with diabetes, should take care to promptly self-isolate to prevent household transmission., (Published by Oxford University Press for the Infectious Diseases Society of America 2020.)

Background: Optimized symptom-based COVID-19 case definitions that guide public health surveillance and individual patient management in the community may assist pandemic control., Methods: We assessed diagnostic performance of existing cases definitions (e.g. influenza-like illness, COVID-like illness) using symptoms reported from 185 household contacts to a PCR-confirmed case of COVID-19 in Wisconsin and Utah, United States. We stratified analyses between adults and children. We also constructed novel case definitions for comparison., Results: Existing COVID-19 case definitions generally showed high sensitivity (86-96%) but low positive predictive value (PPV) (36-49%; F-1 score 52-63) in this community cohort. Top performing novel symptom combinations included taste or smell dysfunction and improved the balance of sensitivity and PPV (F-1 score 78-80). Performance indicators were generally lower for children (< 18 years of age)., Conclusions: Existing COVID-19 case definitions appropriately screened in household contacts with COVID-19. Novel symptom combinations incorporating taste or smell dysfunction as a primary component improved accuracy. Case definitions tailored for children versus adults should be further explored., (© 2021. The Author(s).)

Approximately 67% of U.S. households have pets. Limited data are available on SARS-CoV-2 in pets. We assessed SARS-CoV-2 infection in pets during a COVID-19 household transmission investigation. Pets from households with ≥1 person with laboratory-confirmed COVID-19 were eligible for inclusion from April-May 2020. We enrolled 37 dogs and 19 cats from 34 households. All oropharyngeal, nasal, and rectal swabs tested negative by rRT-PCR; one dog's fur swabs (2%) tested positive by rRT-PCR at the first sampling. Among 47 pets with serological results, eight (17%) pets (four dogs, four cats) from 6/30 (20%) households had detectable SARS-CoV-2 neutralizing antibodies. In households with a seropositive pet, the proportion of people with laboratory-confirmed COVID-19 was greater (median 79%; range: 40-100%) compared to households with no seropositive pet (median 37%; range: 13-100%) ( p = 0.01). Thirty-three pets with serologic results had frequent daily contact (≥1 h) with the index patient before the person's COVID-19 diagnosis. Of these 33 pets, 14 (42%) had decreased contact with the index patient after diagnosis and none were seropositive; of the 19 (58%) pets with continued contact, four (21%) were seropositive. Seropositive pets likely acquired infection after contact with people with COVID-19. People with COVID-19 should restrict contact with pets and other animals.

Serologic assays developed for SARS-CoV-2 detect different antibody subtypes and are based on different target antigens. Comparison of the performance of a SARS-CoV-2 Spike-Protein ELISA and the nucleocapsid-based Abbott ArchitectTM SARS-CoV-2 IgG assay indicated that the assays had high concordance, with rare paired discordant tests results., Competing Interests: The authors have declared that no competing interests exist.

During December 3, 2020-January 31, 2021, CDC, in collaboration with the University of Utah Health and Economic Recovery Outreach Project,* Utah Department of Health (UDOH), Salt Lake County Health Department, and one Salt Lake county school district, offered free, in-school, real-time reverse transcription-polymerase chain reaction (RT-PCR) saliva testing as part of a transmission investigation of SARS-CoV-2, the virus that causes COVID-19, in elementary school settings. School contacts † of persons with laboratory-confirmed SARS-CoV-2 infection, including close contacts, were eligible to participate (1). Investigators approached parents or guardians of student contacts by telephone, and during January, using school phone lines to offer in-school specimen collection; the testing procedures were explained in the preferred language of the parent or guardian. Consent for participants was obtained via an electronic form sent by e-mail. Analyses examined participation (i.e., completing in-school specimen collection for SARS-CoV-2 testing) in relation to factors § that were programmatically important or could influence likelihood of SARS-CoV-2 testing, including race, ethnicity, and SARS-CoV-2 incidence in the community (2). Crude prevalence ratios (PRs) were calculated using univariate log-binomial regression. ¶ This activity was reviewed by CDC and was conducted consistent with federal law and CDC policy.*., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Mary Hill, Ilene Risk, and Nathan LaCross report grant support from the Federal Government Coronavirus Aid, Relief, and Economic Security Act during the course of the study. No other potential conflicts of interest were disclosed.

Objectives: In late June 2020, a large outbreak of coronavirus disease 2019 (COVID-19) occurred at a sleep-away youth camp in Georgia, affecting primarily persons ≤21 years. We conducted a retrospective cohort study among campers and staff (attendees) to determine the extent of the outbreak and assess factors contributing to transmission., Methods: Attendees were interviewed to ascertain demographic characteristics, known exposures to COVID-19 and community exposures, and mitigation measures before, during, and after attending camp. COVID-19 case status was determined for all camp attendees on the basis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test results and reported symptoms. We calculated attack rates and instantaneous reproduction numbers and sequenced SARS-CoV-2 viral genomes from the outbreak., Results: Among 627 attendees, the median age was 15 years (interquartile range: 12-16 years); 56% (351 of 627) of attendees were female. The attack rate was 56% (351 of 627) among all attendees. On the basis of date of illness onset or first positive test result on a specimen collected, 12 case patients were infected before arriving at camp and 339 case patients were camp associated. Among 288 case patients with available symptom information, 45 (16%) were asymptomatic. Despite cohorting, 50% of attendees reported direct contact with people outside their cabin cohort. On the first day of camp session, the instantaneous reproduction number was 10. Viral genomic diversity was low., Conclusions: Few introductions of SARS-CoV-2 into a youth congregate setting resulted in a large outbreak. Testing strategies should be combined with prearrival quarantine, routine symptom monitoring with appropriate isolation and quarantine, cohorting, social distancing, mask wearing, and enhanced disinfection and hand hygiene. Promotion of mitigation measures among younger populations is needed., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2021 by the American Academy of Pediatrics.)

School closures affected more than 55 million students across the United States when implemented as a strategy to prevent the transmission of SARS-CoV-2, the virus that causes COVID-19 (1). Reopening schools requires balancing the risks for SARS-CoV-2 infection to students and staff members against the benefits of in-person learning (2). During December 3, 2020-January 31, 2021, CDC investigated SARS-CoV-2 transmission in 20 elementary schools (kindergarten through grade 6) that had reopened in Salt Lake County, Utah. The 7-day cumulative number of new COVID-19 cases in Salt Lake County during this time ranged from 290 to 670 cases per 100,000 persons. † Susceptible § school contacts ¶ (students and staff members exposed to SARS-CoV-2 in school) of 51 index patients** (40 students and 11 staff members) were offered SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) testing. Among 1,041 susceptible school contacts, 735 (70.6%) were tested, and five of 12 cases identified were classified as school-associated; the secondary attack rate among tested susceptible school contacts was 0.7%. Mask use among students was high (86%), and the median distance between students' seats in classrooms was 3 ft. Despite high community incidence and an inability to maintain ≥6 ft of distance between students at all times, SARS-CoV-2 transmission was low in these elementary schools. The results from this investigation add to the increasing evidence that in-person learning can be achieved with minimal SARS-CoV-2 transmission risk when multiple measures to prevent transmission are implemented (3,4)., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Ilene Risk and Mary Hill report grant funding from the Federal Government Coronavirus Aid, Relief, and Economic Security (CARES) Act, during the conduct of the study. No other potential conflicts of interest were disclosed.

Background and Objectives: Limited data exist on severe acute respiratory syndrome coronavirus 2 in children. We described infection rates and symptom profiles among pediatric household contacts of individuals with coronavirus disease 2019., Methods: We enrolled individuals with coronavirus disease 2019 and their household contacts, assessed daily symptoms prospectively for 14 days, and obtained specimens for severe acute respiratory syndrome coronavirus 2 real-time reverse transcription polymerase chain reaction and serology testing. Among pediatric contacts (<18 years), we described transmission, assessed the risk factors for infection, and calculated symptom positive and negative predictive values. We compared secondary infection rates and symptoms between pediatric and adult contacts using generalized estimating equations., Results: Among 58 households, 188 contacts were enrolled (120 adults; 68 children). Secondary infection rates for adults (30%) and children (28%) were similar. Among households with potential for transmission from children, child-to-adult transmission may have occurred in 2 of 10 (20%), and child-to-child transmission may have occurred in 1 of 6 (17%). Pediatric case patients most commonly reported headache (79%), sore throat (68%), and rhinorrhea (68%); symptoms had low positive predictive values, except measured fever (100%; 95% confidence interval [CI]: 44% to 100%). Compared with symptomatic adults, children were less likely to report cough (odds ratio [OR]: 0.15; 95% CI: 0.04 to 0.57), loss of taste (OR: 0.21; 95% CI: 0.06 to 0.74), and loss of smell (OR: 0.29; 95% CI: 0.09 to 0.96) and more likely to report sore throat (OR: 3.4; 95% CI: 1.04 to 11.18)., Conclusions: Children and adults had similar secondary infection rates, but children generally had less frequent and severe symptoms. In two states early in the pandemic, we observed possible transmission from children in approximately one-fifth of households with potential to observe such transmission patterns., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2021 by the American Academy of Pediatrics.)

Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. In this study, we use CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)6 as a donor template delivery system to repair the ELANE L172P mutation in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs). We used a single guide RNA (sgRNA) specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, ∼34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide a proof of principle for using CRISPR-Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

There is increasing evidence that children and adolescents can efficiently transmit SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19) (1-3). During July-August 2020, four state health departments and CDC investigated a COVID-19 outbreak that occurred during a 3-week family gathering of five households in which an adolescent aged 13 years was the index and suspected primary patient; 11 subsequent cases occurred., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Coronavirus disease 2019 (COVID-19), the respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, China and has since become pandemic. In response to the first cases identified in the United States, close contacts of confirmed COVID-19 cases were investigated to enable early identification and isolation of additional cases and to learn more about risk factors for transmission. Close contacts of nine early travel-related cases in the United States were identified and monitored daily for development of symptoms (active monitoring). Selected close contacts (including those with exposures categorized as higher risk) were targeted for collection of additional exposure information and respiratory samples. Respiratory samples were tested for SARS-CoV-2 by real-time reverse transcription polymerase chain reaction at the Centers for Disease Control and Prevention. Four hundred four close contacts were actively monitored in the jurisdictions that managed the travel-related cases. Three hundred thirty-eight of the 404 close contacts provided at least basic exposure information, of whom 159 close contacts had ≥1 set of respiratory samples collected and tested. Across all actively monitored close contacts, two additional symptomatic COVID-19 cases (i.e., secondary cases) were identified; both secondary cases were in spouses of travel-associated case patients. When considering only household members, all of whom had ≥1 respiratory sample tested for SARS-CoV-2, the secondary attack rate (i.e., the number of secondary cases as a proportion of total close contacts) was 13% (95% CI: 4-38%). The results from these contact tracing investigations suggest that household members, especially significant others, of COVID-19 cases are at highest risk of becoming infected. The importance of personal protective equipment for healthcare workers is also underlined. Isolation of persons with COVID-19, in combination with quarantine of exposed close contacts and practice of everyday preventive behaviors, is important to mitigate spread of COVID-19., Competing Interests: The authors have declared that no competing interests exist.

Limited data are available about transmission of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), among youths. During June 17-20, an overnight camp in Georgia (camp A) held orientation for 138 trainees and 120 staff members; staff members remained for the first camp session, scheduled during June 21-27, and were joined by 363 campers and three senior staff members on June 21. Camp A adhered to the measures in Georgia's Executive Order* that allowed overnight camps to operate beginning on May 31, including requiring all trainees, staff members, and campers to provide documentation of a negative viral SARS-CoV-2 test ≤12 days before arriving. Camp A adopted most † components of CDC's Suggestions for Youth and Summer Camps § to minimize the risk for SARS-CoV-2 introduction and transmission. Measures not implemented were cloth masks for campers and opening windows and doors for increased ventilation in buildings. Cloth masks were required for staff members. Camp attendees were cohorted by cabin and engaged in a variety of indoor and outdoor activities, including daily vigorous singing and cheering. On June 23, a teenage staff member left camp A after developing chills the previous evening. The staff member was tested and reported a positive test result for SARS-CoV-2 the following day (June 24). Camp A officials began sending campers home on June 24 and closed the camp on June 27. On June 25, the Georgia Department of Public Health (DPH) was notified and initiated an investigation. DPH recommended that all attendees be tested and self-quarantine, and isolate if they had a positive test result., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

We describe the contact investigation for an early confirmed case of coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in the United States. Contacts of the case-patient were identified, actively monitored for symptoms, interviewed for a detailed exposure history, and tested for SARS-CoV-2 infection by real-time reverse transcription PCR (rRT-PCR) and ELISA. Fifty contacts were identified and 38 (76%) were interviewed, of whom 11 (29%) reported unprotected face-to-face interaction with the case-patient. Thirty-seven (74%) had respiratory specimens tested by rRT-PCR, and all tested negative. Twenty-three (46%) had ELISA performed on serum samples collected ≈6 weeks after exposure, and none had detectable antibodies to SARS-CoV-2. Among contacts who were tested, no secondary transmission was identified in this investigation, despite unprotected close interactions with the infectious case-patient.

Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis., Competing Interests: The authors declare no competing interest.

Mutations that accumulate in self-renewing hematopoietic stem and progenitor cells (HSPCs) can cause severe blood disorders. To model such disorders in mice, we developed a CRISPR/Cas9/adeno-associated virus (AAV)-based system to knock in and repair genes by homologous recombination in mouse HSPCs. Here, we provide a step-by-step protocol to achieve high efficiency of gene knockin in mouse HSPCs, while maintaining engraftment capacity. This approach enables the functional study of hematopoietic disease mutations in vivo , without requiring germline mutagenesis. For complete details on the use and execution of this protocol, please refer to Tran et al. (2019)., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

In December 2019, a cluster of cases of pneumonia emerged in Wuhan City in central China's Hubei Province. Genetic sequencing of isolates obtained from patients with pneumonia identified a novel coronavirus (2019-nCoV) as the etiology (1). As of February 4, 2020, approximately 20,000 confirmed cases had been identified in China and an additional 159 confirmed cases in 23 other countries, including 11 in the United States (2,3). On January 17, CDC and the U.S. Department of Homeland Security's Customs and Border Protection began health screenings at U.S. airports to identify ill travelers returning from Wuhan City (4). CDC activated its Emergency Operations Center on January 21 and formalized a process for inquiries regarding persons suspected of having 2019-nCoV infection (2). As of January 31, 2020, CDC had responded to clinical inquiries from public health officials and health care providers to assist in evaluating approximately 650 persons thought to be at risk for 2019-nCoV infection. Guided by CDC criteria for the evaluation of persons under investigation (PUIs) (5), 210 symptomatic persons were tested for 2019-nCoV; among these persons, 148 (70%) had travel-related risk only, 42 (20%) had close contact with an ill laboratory-confirmed 2019-nCoV patient or PUI, and 18 (9%) had both travel- and contact-related risks. Eleven of these persons had laboratory-confirmed 2019-nCoV infection. Recognizing persons at risk for 2019-nCoV is critical to identifying cases and preventing further transmission. Health care providers should remain vigilant and adhere to recommended infection prevention and control practices when evaluating patients for possible 2019-nCoV infection (6). Providers should consult with their local and state health departments when assessing not only ill travelers from 2019-nCoV-affected countries but also ill persons who have been in close contact with patients with laboratory-confirmed 2019-nCoV infection in the United States., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Mutations accumulating in hematopoietic stem and progenitor cells (HSPCs) during development can cause severe hematological disorders. Modeling these mutations in mice is essential for understanding their functional consequences. Here, we describe an efficient CRISPR/Cas9-based system to knock in and repair genes in mouse HSPCs. CRISPR/Cas9 ribonucleoproteins, in combination with recombinant adeno-associated virus (rAAV)-DJ donor templates, led to gene knockin efficiencies of up to 30% in the Lmnb1 and Actb loci of mouse HSPCs in vitro. The targeted HSPCs engraft and reconstitute all immune cell lineages in the recipient mice. Using this approach, we corrected a neomycin-disrupted Rag2 gene. The Rag2-corrected HSPCs restore B and T cell development in vivo, confirming the functionality of the approach. Our method provides an efficient strategy to study gene function in the hematopoietic system and model hematological disorders in vivo, without the need for germline mutagenesis., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Patients with Rett syndrome (RTT) have severe mental and physical disabilities. The majority of RTT patients carry a heterozygous mutation in methyl-CpG binding protein 2 (MECP2), an X-linked gene encoding an epigenetic factor crucial for normal nerve cell function. No curative therapy for RTT syndrome exists, and cellular mechanisms are incompletely understood. Here, we developed a CRISPR/Cas9-mediated system that targets and corrects the disease relevant regions of the MECP2 exon 4 coding sequence. We achieved homologous recombination (HR) efficiencies of 20% to 30% in human cell lines and iPSCs. Furthermore, we successfully introduced a MECP2 R270X mutation into the MECP2 gene in human induced pluripotent stem cells (iPSCs). Consequently, using CRISPR/Cas9, we were able to repair such mutations with high efficiency in human mutant iPSCs. In summary, we provide a new strategy for MECP2 gene targeting that can be potentially translated into gene therapy or for iPSCs-based disease modeling of RTT syndrome.

The CRISPR-Cas9 system is used for genome editing in mammalian cells by introducing double-strand breaks (DSBs) which are predominantly repaired via non-homologous end joining (NHEJ) or to lesser extent by homology-directed repair (HDR). To enhance HDR for improving the introduction of precise genetic modifications, we tested fusion proteins of Cas9 nuclease with HDR effectors to enforce their localization at DSBs. Using a traffic-light DSB repair reporter (TLR) system for the quantitative detection of HDR and NHEJ events in human HEK cells we found that Cas9 fusions with CtIP, Rad52, and Mre11, but not Rad51C promote HDR up to twofold in human cells and significantly reduce NHEJ events. We further compared, as an alternative to the direct fusion with Cas9, two components configurations that associate CtIP fusion proteins with a Cas9-SunTag fusion or with guide RNA that includes MS2 binding loops. We found that the Cas9-CtIP fusion and the MS2-CtIP system, but not the SunTag approach increase the ratio of HDR/NHEJ 4.5-6-fold. Optimal results are obtained by the combined use of Cas9-CtIP and MS2-CtIP, shifting the HDR/NHEJ ratio by a factor of 14.9. Thus, our findings provide a simple and effective tool to promote precise gene modifications in mammalian cells.

B2 cells engage in classical antibody responses, whereas B1 cells are considered carriers of innate immunity, biased toward recognizing epitopes present on the surfaces of common pathogens and self antigens. To explore the role of B cell antigen receptor (BCR) specificity in driving B1 cell differentiation, we developed a transgenic system allowing us to change BCR specificity in B cells in an inducible and programmed manner. Mature B2 cells differentiated into bona fide B1 cells upon acquisition of a B1 cell-typical self-reactive BCR through a phase of proliferative expansion. Thus, B2 cells have B1 cell differentiation potential in addition to their classical capacity to differentiate into memory and plasma cells, and B1 differentiation can be instructed by BCR-mediated self-reactivity, in the absence of B1-lineage precommitment., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence. Here, we discovered that two short sequence motifs at the 3' end of the targeting sequence are almost exclusively present in inefficient sgRNAs of published sgRNA-activity datasets. By specific knock-in of sgRNA target sequences with or without these motifs and quantitative measurement of knockout efficiency, we show that the presence of these motifs in sgRNAs per se results in a 10-fold reduction of gene knockout frequencies. Mechanistically, the cause of the low efficiency differs between the two motifs. These sequence motifs are relevant for future sgRNA design approaches and studies of Cas9-DNA interactions., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The germinal center reaction is essential for efficient humoral immunity, but it can also give rise to B cell lymphomas. Cre/loxP-mediated conditional gene knock-out or knock-in can be used for the genetic manipulation of germinal center B cells in vivo. Here we present a novel allele, Cγ1-CreERT2, that allows for timed activation of Cre recombinase in a small fraction of germinal center B cells. This allele will be useful to study normal and malignant germinal center B cell development in vivo., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Forkhead box class O1 (FOXO1) acts as a tumor suppressor in solid tumors. The oncogenic phosphoinositide-3-kinase (PI3K) pathway suppresses FOXO1 transcriptional activity by enforcing its nuclear exclusion upon AKT-mediated phosphorylation. We show here abundant nuclear expression of FOXO1 in Burkitt lymphoma (BL), a germinal center (GC) B-cell-derived lymphoma whose pathogenesis is linked to PI3K activation. Recurrent FOXO1 mutations, which prevent AKT targeting and lock the transcription factor in the nucleus, are used by BL to circumvent mutual exclusivity between PI3K and FOXO1 activation. Using genome editing in human and mouse lymphomas in which MYC and PI3K cooperate synergistically in tumor development, we demonstrate proproliferative and antiapoptotic activity of FOXO1 in BL and identify its nuclear localization as an oncogenic event in GC B-cell-derived lymphomagenesis., (© 2018 by The American Society of Hematology.)