Directed evolution of hyperactive integrases for site specific insertion of transgenes.

The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites. By targeting att sites to the genome using technologies such as prime editing, integrases can target safe loci while avoiding double-strand breaks. We developed a method of phage-assisted continuous evolution we call IntePACE, that we used to rapidly perform hundreds of rounds of mutagenesis to systematically improve activity of PhiC31 and Bxb1 serine integrases. Novel hyperactive mutants were generated by combining synergistic mutations resulting in integration of a multi-gene cargo at rates as high as 80% of target chromosomes. Hyperactive integrases inserted a 15.7 kb therapeutic DNA cargo containing von Willebrand Factor. This technology could accelerate gene delivery therapeutics and our directed evolution strategy can easily be adapted to improve novel integrases from nature.

Lowering barriers to naloxone access through a student-led harm reduction program.

BACKGROUND: In recent years, the opioid crisis has had devastating effects on communities across the country. In the wake of 4 University of Southern California (USC) student overdose deaths in 2019, USC pharmacy students initiated a naloxone distribution program called NaloxoneSC. This program has distributed free naloxone kits, fentanyl test strips, and overdose prevention training to hundreds of students thus far. This report aims to provide a framework for the implementation of similar harm reduction programs at other universities. OBJECTIVES: NaloxoneSC partnered with USC Student Health to implement a naloxone distribution program that expands access to naloxone and increases opioid overdose education within the USC community. SETTING: USC undergraduate campus and Health Sciences Campus. METHODS: Students can follow the steps for obtaining a naloxone kit listed on the NaloxoneSC website. Watching a 10-minute training video or attending a naloxone training workshop completes the opioid overdose education. The kit is picked up by the student from 1 of 2 USC Student Health Center locations, Engemann or Eric Cohen. Each kit is packaged with Narcan nasal spray and fentanyl test strips. RESULTS: In 1 year, the program received at least 320 naloxone kit requests. Demand for naloxone kits increased over time, and roughly 4 times as many students requested kits from Engemann than from Eric Cohen. Approximately 600 students have received opioid overdose education through NaloxoneSC. CONCLUSIONS: In 1 year, more than 300 USC students voluntarily requested naloxone kits from NaloxoneSC. These findings suggest a need for increased access to this resource on college campuses. Peer-to-peer education and naloxone kit distribution are feasible and effective strategies that help address public health concerns and reduce harm among university students. NaloxoneSC can serve as an example for other college campuses or entities to newly implement or expand their own harm reduction measures.

Defluorination Capability of l-2-Haloacid Dehalogenases in the HAD-Like Hydrolase Superfamily Correlates with Active Site Compactness.

l-2-Haloacid dehalogenases, industrially and environmentally important enzymes that catalyse cleavage of the carbon-halogen bond in S-2-halocarboxylic acids, were known to hydrolyse chlorinated, brominated and iodinated substrates but no activity towards fluorinated compounds had been reported. A screen for novel dehalogenase activities revealed four l-2-haloacid dehalogenases capable of defluorination. We now report crystal structures for two of these enzymes, Bpro0530 and Rha0230, as well as for the related proteins PA0810 and RSc1362, which hydrolyse chloroacetate but not fluoroacetate, all at ∼2.2 Šresolution. Overall structure and active sites of these enzymes are highly similar. In molecular dynamics (MD) calculations, only the defluorinating enzymes sample more compact conformations, which in turn allow more effective interactions with the small fluorine atom. Structural constraints, based on X-ray structures and MD calculations, correctly predict the defluorination activity of the homologous enzyme ST2570.

Performance of different criteria for refractory myasthenia gravis.

BACKGROUND AND PURPOSE: Defining refractory myasthenia gravis is important, as this can drive clinical decision making, for example, by escalating treatments in refractory individuals. There are several definitions of refractory myasthenia, and their performances have not been compared. Having valid and reliable criteria can help select patients in whom more aggressive treatments may be needed. METHODS: We applied five different refractory myasthenia criteria (Drachman, Mantegazza, Suh, the International Consensus Guideline (ICG), and the randomised controlled trial of eculizumab in refractory, anti-acetylcholine receptor positive, generalised myasthenia gravis (REGAIN), to a cohort of 237 patients. We compared the proportion of refractory patients among different criteria and their scores on disease severity, fatigue, and quality-of-life (QoL) scales. We also assessed the agreement for each criterion between two trained assessors. RESULTS: The Drachman, Mantegazza, and Suh criteria resulted in high proportions of refractory individuals (40.1%, 39.2%, and 38.8%, respectively), compared with the ICG and REGAIN criteria (9.7% and 3.0%, respectively). Refractory patients by the ICG and REGAIN criteria had worse disease severity, QoL, and fatigue scores, compared with patients classified as refractory by other criteria. All criteria had high agreement between raters (between 70% and 100%). CONCLUSIONS: There is high variability in the proportion of refractory myasthenia gravis patients depending on the criteria used, with ICG and REGAIN criteria capturing patients with worse disease severity. This reflects conceptual differences as to what refractory means. Further multicenter studies are needed to determine appropriate criteria for refractory myasthenia gravis.

Direct Delivery of Antisense Oligonucleotides to the Middle and Inner Ear Improves Hearing and Balance in Usher Mice.

Usher syndrome is a syndromic form of hereditary hearing impairment that includes sensorineural hearing loss and delayed-onset retinitis pigmentosa (RP). Type 1 Usher syndrome (USH1) is characterized by congenital profound sensorineural hearing impairment and vestibular areflexia, with adolescent-onset RP. Systemic treatment with antisense oligonucleotides (ASOs) targeting the human USH1C c.216G>A splicing mutation in a knockin mouse model of USH1 restores hearing and balance. Herein, we explore the effect of delivering ASOs locally to the ear to treat hearing and vestibular dysfunction associated with Usher syndrome. Three localized delivery strategies were investigated in USH1C mice: inner ear injection, trans-tympanic membrane injection, and topical tympanic membrane application. We demonstrate, for the first time, that ASOs delivered directly to the ear correct Ush1c expression in inner ear tissue, improve cochlear hair cell transduction currents, restore vestibular afferent irregularity, spontaneous firing rate, and sensitivity to head rotation, and successfully recover hearing thresholds and balance behaviors in USH1C mice. We conclude that local delivery of ASOs to the middle and inner ear reach hair cells and can rescue both hearing and balance. These results also demonstrate the therapeutic potential of ASOs to treat hearing and balance deficits associated with Usher syndrome and other ear diseases.

Evaluating the impact of lymph node resampling on colorectal cancer nodal stage.

AIMS: Nodal staging in colorectal cancer (CRC) informs prognosis and guides adjuvant treatment decisions. A standard minimum of 12 lymph nodes is widely used, with additional sampling being performed as required. However, there are few data on how lymph node resampling in this context has an impact on nodal stage. The aims of this study were to evaluate the effectiveness of resampling in detecting metastases and tumour deposits, and the impact on stage. METHODS AND RESULTS: A retrospective cohort analysis was performed on CRC resections that underwent resampling because of an initial yield of <12 lymph nodes, from 2008 to 2018. Data relating to patient demographics, specimen, malignancy and prosection were collected. Slides were reviewed to quantify nodal metastases and tumour deposits before and after resampling. Among ≥pN1 cases, logistic regression analysis was performed to evaluate factors that predicted the finding of additional metastases and tumour deposits. The cohort comprised 395 cases: resampling identified nodal metastases and/or tumour deposits in 30 (7.6%) cases; nodal upstaging occurred in 20 (5.1%) cases; and eight (2.0%) cases changed from pN0 to ≥pN1. No factors predicted resampling of positive lymph nodes or tumour deposits, and pN upstaging occurred across a variety of cases. A subgroup analysis was performed to assess the impact of resampling on high-risk features in stage II cases (n = 117). There were 33 (8.5%) patients who no longer had any high-risk features after resampling. CONCLUSIONS: Lymph node resampling has an impact on nodal staging and possible treatment decisions in a considerable proportion of patients, and is recommended in all cases with <12 lymph nodes.

Kras mutant genetically engineered mouse models of human cancers are genomically heterogeneous.

KRAS mutant tumors are largely recalcitrant to targeted therapies. Genetically engineered mouse models (GEMMs) of Kras mutant cancer recapitulate critical aspects of this disease and are widely used for preclinical validation of targets and therapies. Through comprehensive profiling of exomes and matched transcriptomes of >200 KrasG12D-initiated GEMM tumors from one lung and two pancreatic cancer models, we discover that significant intratumoral and intertumoral genomic heterogeneity evolves during tumorigenesis. Known oncogenes and tumor suppressor genes, beyond those engineered, are mutated, amplified, and deleted. Unlike human tumors, the GEMM genomic landscapes are dominated by copy number alterations, while protein-altering mutations are rare. However, interspecies comparative analyses of the genomic landscapes demonstrate fidelity between genes altered in KRAS mutant human and murine tumors. Genes that are spontaneously altered during murine tumorigenesis are also among the most prevalent found in human indications. Using targeted therapies, we also demonstrate that this inherent tumor heterogeneity can be exploited preclinically to discover cancer-specific and genotype-specific therapeutic vulnerabilities. Focusing on Kras allelic imbalance, a feature shared by all three models, we discover that MAPK pathway inhibition impinges uniquely on this event, indicating distinct susceptibility and fitness advantage of Kras-mutant cells. These data reveal previously unknown genomic diversity among KrasG12D-initiated GEMM tumors, places them in context of human patients, and demonstrates how to exploit this inherent tumor heterogeneity to discover therapeutic vulnerabilities.

Key Components of Traditional Consultation Letters and Their Relevance to Electronic Consultation Replies: A Systematic Review.

Background: Effective communication between primary care providers (PCPs) and specialists plays a key role in providing high-quality patient care. A high-quality referral process should involve referral letters containing all information that is necessary to support shared care between primary and specialty care. Introduction: There is no consensus on the optimal components of specialist-to-PCP communication after a face-to-face patient encounter or in the context of the emerging field of electronic consultations (eConsult). In this study, we aimed at synthesizing the evidence on key components of a traditional consultation letter and at determining whether they can be applied to eConsult replies. Methods: We conducted a systematic review by using a narrative synthesis approach. We searched Pubmed and Embase from inception to January/March 2016 (English). Included studies focused on features of specialists' responses to PCPs. We extracted components of a consultation letter that were identified to be of importance to PCPs and attempted to relate their applicability to eConsult replies. Results: The search revealed 744 potentially relevant citations, of which 65 were deemed eligible for full-text review. Forty-one papers were excluded on full-text review, resulting in 24 studies included in the final synthesis. Important components of consultation letters that were applicable to eConsults included: answering a direct question, providing a diagnosis, providing treatment options, providing education around the case, providing a prognosis, and arranging follow-up, clarity, and organization. Key differences between traditional and eConsult replies included the history and physical investigations, impression, plan, and rationale for plan/education. Conclusion: When seeking to improve the quality of specialist reply letters in both traditional and eConsult replies, one should consider differences in how information is collected and accessed, the role of each provider, and factors that impact specialist-to-PCP communication.

Tools for Enhancement and Quality Improvement of Peer Assessment and Clinical Care in Endocrinology and Metabolism.

Members of the College of Physicians and Surgeons of Ontario Endocrinology and Metabolism Peer Review Network have been involved in a quality improvement project to help standardize the peer assessment of physicians practicing in endocrinology and metabolism. This has included developing state-of-the-art summaries of common endocrine problems by Canadian experts in endocrinology and metabolism. These tools have been developed in response to the educational needs, as identified by peer reviewers, of practicing endocrinologists in Ontario. These pedagogical tools aim not only to standardize the documentation of the clinical performance of endocrinologists but also to make the process more transparent and to improve the quality of patient care in Ontario. This article summarizes the project and also provides the tools developed for the endocrinology and metabolism section of the College of Physicians and Surgeons of Ontario.

Fatigue is a relevant outcome in patients with myasthenia gravis.

INTRODUCTION: Patients with myasthenia gravis often experience fatigue, but its effect on quality of life (QoL) is underestimated, and fatigue is rarely measured in clinical trials. METHODS: Two hundred fifty-seven myasthenic patients completed the Neuro-QoL-Fatigue and measures of disease severity and QoL. We studied the relationship between fatigue and clinical and demographic variables. Finally, we studied the responsiveness of the Neuro-QoL-Fatigue in 95 patients receiving treatments for myasthenia and estimated the minimal important difference (MID). RESULTS: Fatigue correlated with greater disease severity (r = 0.52-0.69, P < 0.0001) and worse QoL (r = 0.65-0.75, P < 0.0001). Patients in remission, with minimal manifestations, and pure ocular symptoms reported minimal fatigue. Regression modeling showed that, in addition to its relationship with disease severity, fatigue was worse in females, patients with generalized disease, and those with anxiety/depression. Fatigue improved after immunomodulation (P < 0.0001), and the MID was 5.3 points. DISCUSSION: Fatigue in myasthenia correlates with disease severity, affects QoL, and can improve after treatment. Muscle Nerve 58: 197-203, 2018.

Predictors and clinical outcomes of inpatient versus ambulatory management after an emergency department visit for atrial fibrillation: A population-based study.

BACKGROUND: There is substantial variation in the management of atrial fibrillation (AF) in the emergency department (ED), particularly whether these patients are admitted to hospital. We sought to identify factors that predict admission and to examine the relationship between AF admission and outcomes. METHODS: We performed a retrospective cohort analysis of patients ≥20 years of age who had an index ED visit with a primary diagnosis of AF from between April 1, 2005, and March 31, 2010, in Ontario, Canada. We excluded patients who died during the index ED visit or hospitalization. A hierarchical logistic regression model was used to determine predictors of hospital admission during the index ED visit. A propensity-matched analysis was used to test for associations between hospital admission and 1-year outcomes. RESULTS: The cohort consisted of 33,699 patients, of whom 16,270 (48.3%) were admitted to hospital. Substantial variation was seen across the 154 hospitals, with admission rates ranging from 3.0% to 91.0%. Admitted patients had higher rates of comorbidities compared to discharged patients. Mortality rates at 1 year were significantly higher in matched admitted versus discharged patients (hazard ratio 1.45, 95% CI 1.33-1.57, P < .001), as were all-cause hospitalizations (hazard ratio 1.18, 95% CI 1.13-1.22, P < .001). CONCLUSIONS: Wide practice variation was observed between hospitals in terms of the proportion of patients admitted. Our data suggest that selected patients when discharged have similar or improved outcomes compared to those who are initially admitted. Future research is needed to better standardize admission/discharge decisions for AF patients in the ED.

eCONSULTS TO ENDOCRINOLOGISTS IMPROVE ACCESS AND CHANGE PRIMARY CARE PROVIDER BEHAVIOR.

OBJECTIVE: To describe the impact of an eConsult service on access to endocrinologists along with its influence on changing primary care provider (PCP) course of action and referral behaviors. METHODS: Established in 2011, the Champlain BASE (Building Access to Specialist Care via eConsult) service allows PCPs to access specialist care in lieu of traditional face-to-face referrals. We conducted a cross-sectional study of eConsult cases submitted to endocrinologists by PCPs between April 15, 2011 and January 31, 2015. Usage data and PCP responses to a mandatory closeout survey were analyzed to determine eConsult response times, PCP practice behavior, referral outcomes, and provider satisfaction. Each eConsult was coded according to clinical topic and question type based on established taxonomies. RESULTS: A total of 180 PCPs submitted 464 eConsults to endocrinology during the study period. Specialist median response time was 7 hours, with 90% of responses occurring within 3 days. PCPs received a new or additional course of action in 62% of submitted cases. An unnecessary face-to-face referral was avoided in 44% of all eConsults and in 67% of cases where the PCP initially contemplated requesting a referral. Over 95% of cases were rated at least 4 out of 5 in value for PCPs and their patients. CONCLUSION: The use of eConsult improves access to endocrinologists by providing timely, highly rated practice-changing clinical advice while reducing the need for patients to attend face-to-face office visits. ABBREVIATIONS: BASE = Building Access to Specialist Advice through eConsult PCP = primary care physician UCSF = University of California San Francisco.

Oncogenic RAS pathway activation promotes resistance to anti-VEGF therapy through G-CSF-induced neutrophil recruitment.

Granulocyte-colony stimulating factor (G-CSF) promotes mobilization of CD11b(+)Gr1(+) myeloid cells and has been implicated in resistance to anti-VEGF therapy in mouse models. High G-CSF production has been associated with a poor prognosis in cancer patients. Here we show that activation of the RAS/MEK/ERK pathway regulates G-CSF expression through the Ets transcription factor. Several growth factors induced G-CSF expression by a MEK-dependent mechanism. Inhibition of G-CSF release with a MEK inhibitor markedly reduced G-CSF production in vitro and synergized with anti-VEGF antibodies to reduce CD11b(+)Ly6G(+) neutrophil mobilization and tumor growth and led to increased survival in animal models of cancer, including a genetically engineered mouse model of pancreatic adenocarcinoma. Analysis of biopsies from pancreatic cancer patients revealed increased phospho-MEK, G-CSF, and Ets expression and enhanced neutrophil recruitment compared with normal pancreata. These results provide insights into G-CSF regulation and on the mechanism of action of MEK inhibitors and point to unique anticancer strategies.

Thyroid-stimulating hormone acutely increases monocyte gene expression in vivo.

OBJECTIVES: Thyroid-stimulating hormone (TSH) acts in an extra-thyroidal fashion and induces a pro-inflammatory, pro-coagulant state. Blood monocytes can be activated by vascular stress, but it is not known if this occurs upon TSH administration. Our aim was to determine if recombinant human (rh) TSH, administered acutely to patients being screened for thyroid cancer recurrence, alters blood monocyte gene expression. DESIGN AND SETTING: Patients (14 women, 1 man) had a mean (±SD) age of 48±10 years, a body mass index of 26±6 kg/m2, a history of total thyroidectomy and radioablation for thyroid cancer, and were on L-thyroxine therapy at a university teaching hospital. They received 2 intramuscular doses of rhTSH (0.9 mg), administered on days 1 and 2. Blood samples were obtained at baseline on day1, and on days 3 and 5. RESULTS: Monocyte MCP-1 mRNA (mean±SE) increased significantly by 1.7±0.3 fold on day 5 following rhTSH stimulation (p=0.03, n=15). IL-1ß and CD36 mRNA expression also increased on day 5 (1.9±0.4 fold, p=0.07, n=14) and 2.5±0.4 fold, p=0.1, n=10), respectively, although did not quite reach statistical significance. Significant correlations were detected between the BMI of patients and their TSH-stimulated monocyte mRNA responses at day 5 for CD11a, (r=0.66, n=14, p=0.01); CD14 (r=0.638, n=13, p=0.019), and CD16, r=0.84, n=13, p=0.0003). CONCLUSION: TSH administration increases pro-atherogenic monocyte gene expression.

Impact of Question Content on e-Consultation Outcomes.

BACKGROUND: By facilitating direct communication of primary care providers (PCPs) with specialists for advice, electronic consult (e-consult) services can reduce the need for patients to wait for and travel to face-to-face consultations with specialists. An association between avoiding face-to-face referrals using an e-consult service and specific content within each e-consult has not been rigorously explored. MATERIALS AND METHODS: Cases submitted to the Champlain Building Access to Specialists through eConsultation service between April 2011 to May 2013 were evaluated. Factors analyzed include question type (e.g., diagnosis or management), formulation (if interventions or outcomes were specified), and the addressed specialty. An avoided referral was present if the PCP indicated so in a mandatory close-out survey. A discrepancy was present if the PCP made a referral when the specialist did not indicate one was necessary, or if the PCP did not request a referral despite the specialist recommending one. RESULTS: There were 426 (40%) avoided referrals among 1,055 cases analyzed. Questions associated with the highest avoided referral rates included ones pertaining to diagnosis (44%), nonspecific requests for direction (44%), questions without specified interventions or outcomes (47%), and dermatology cases (49.5%). Specialists agreed on the need for a referral in 82% of cases, with most discrepancies due to the PCP making a referral without the specialist recommending one. CONCLUSIONS: Referral outcomes are associated with the type of question being asked, the formulation of each question, and the specialty being addressed. Discrepancies among PCPs and specialists regarding which patients require face-to-face referrals may help identify knowledge gaps and guide professional development.

Designing a touchless physical examination for a virtual Objective Structured Clinical Examination.

Purpose: Given the COVID-19 pandemic, many Objective Structured Clinical Examinations (OSCEs) have been adapted to virtual formats without addressing whether physical examination maneuvers can or should be assessed virtually. In response, we developed a novel touchless physical examination station for a virtual OSCE and gathered validity evidence for its use. Methods: We used a touchless physical examination OSCE station pilot-tested in a virtual OSCE in which Internal Medicine residents had to verbalize their approach to the physical examination, interpret images and videos of findings provided upon request, and make a diagnosis. We explored differences in performance by training year using ANOVA. In addition, we analyzed data using elements of Bloom's taxonomy of learning, i.e. knowledge, understanding, and synthesis. Results: Sixty-seven residents (PGY1-3) participated in the OSCE. Scores on the pilot station were significantly different between training levels (F=3.936, p = 0.024, ηp2 = 0.11). The pilot station-total correlation (STC) was r = 0.558, and the item-station correlations ranged from r = 0.115-0.571, with the most discriminating items being those that assessed application of knowledge (interpretation and synthesis) rather than recall. Conclusion: This touchless physical examination station was feasible, had acceptable psychometric characteristics, and discriminated between residents at different levels of training.

Objet: Compte tenu de la pandémie de COVID-19, de nombreux examens cliniques objectifs structurés (ECOS) ont été adaptés vers un format virtuel sans que l'on se questionne à savoir si les manœuvres d'examen physique peuvent ou doivent être évaluées virtuellement. Conséquemment, nous avons développé une nouvelle station d'examen physique sans contact pour un ECOS virtuel et recueilli des preuves de validité concernant son utilisation. Méthodes: Nous avons utilisé une station d'examen physique sans contact testée dans le cadre d'un ECOS virtuel pendant lequel les résidents en médecine interne devaient verbaliser leur approche concernant l'examen physique, interpréter des images et des vidéos d'examens fournis sur demande, et poser un diagnostic. Nous avons étudié les différences de rendement en fonction de l'année de formation à l'aide de l'ANOVA. En outre, nous avons analysé les données en utilisant les éléments de la taxonomie de l'apprentissage de Bloom, c'est-à-dire la connaissance, la compréhension et la synthèse. Résultats: Soixante-sept résidents (PGY1-3) ont participé à l'ECOS. Les scores de la station pilote étaient significativement différents entre les niveaux de formation (F=3.936, p=0.024, ηp2=0.11). La corrélation totale de la station pilote (STC) était de r=0,558, et les corrélations question-station variaient de r=0,115-0,571, les questions les plus discriminantes étant celles qui évaluaient l'application (interprétation et synthèse) plutôt que le rappel de connaissances. Conclusion: Cette station d'examen physique sans contact était réalisable, a présenté des caractéristiques psychométriques acceptables et a permis d'établir une discrimination entre les résidents de différents niveaux de formation.

The Ansa Hypoglossi: Quantifying Axonal Density of a Donor Nerve for Facial Reinnervation.

Background: There are a number of nerve grafting options for facial reanimation and the ansa hypoglossi (AH) may be considered in select situations. Objective: To compare axonal density, area, and diameter of AH with other nerves more usually used for facial reanimation. Methods: AH specimens from patients undergoing neck dissections were submitted in formalin. Proximal to distal cross sections, nerve diameters, and the number of axons per nerve, proximally and distally, were measured and counted. Results: Eighteen nerve specimens were analyzed. The average manual axon count for the distal and proximal nerve sections was 1378 ± 333 and 1506 ± 306, respectively. The average QuPath counts for the proximal and distal nerve sections were 1381 ± 325 and 1470 ± 334, respectively. The mean nerve area of the proximal and distal nerve sections was 0.206 ± 0.01 and 0.22 ± 0.064 mm2, respectively. The mean nerve diameter for the proximal and distal nerve sections were 0.498 ± 0.121 and 0.526 ± 0.75 mm, respectively. Conclusion: The histological characteristics of the AH support clinical examination of outcomes as a promising option in facial reanimation.

The use of virtual nominal groups in healthcare research: An extended scoping review.

INTRODUCTION: The Nominal Group Technique (NGT) is a consensus group method used to synthesize expert opinions. Given the global shift to virtual meetings, the extent to which researchers leveraged virtual platforms is unclear. This scoping review explores the use of the vNGT in healthcare research during the COVID-19 pandemic. METHODS: Following the Arksey and O'Malley's framework, eight cross-disciplinary databases were searched (January 2020-July 2022). Research articles that reported all four vNGT stages (idea generation, round robin sharing, clarification, voting) were included. Media Synchronicity Theory informed analysis. Corresponding authors were surveyed for additional information. RESULTS: Of 2,589 citations, 32 references were included. Articles covered healthcare (27/32) and healthcare education (4/32). Platforms used most were Zoom, MS Teams and GoTo but was not reported in 44% of studies. Only 22% commented on the benefits/challenges of moving the NGT virtually. Among authors who responded to our survey (16/32), 80% felt that the vNGT was comparable or superior. CONCLUSIONS: The vNGT provides several advantages such as the inclusion of geographically dispersed participants, scheduling flexibility and cost savings. It is a promising alternative to the traditional in-person meeting, but researchers should carefully describe modifications, potential limitations, and impact on results.

Directed evolution of hyperactive integrases for site specific insertion of transgenes.

The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites. By targeting att sites to the genome using technologies such as prime editing, integrases can target safe loci while avoiding double-strand breaks. We developed a method of phage-assisted continuous evolution we call IntePACE, that we used to rapidly perform hundreds of rounds of mutagenesis to systematically improve activity of PhiC31 and Bxb1 serine integrases. Novel hyperactive mutants were generated by combining synergistic mutations resulting in integration of a multi-gene cargo at rates as high as 80% of target chromosomes. Hyperactive integrases inserted a 15.7 kb therapeutic DNA cargo containing Von Willebrand Factor. This technology could accelerate gene delivery therapeutics and our directed evolution strategy can easily be adapted to improve novel integrases from nature.