Citations

CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale . Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

Background Past research has shown how fires, heat and hot substances are important causes of health loss globally. Detailed estimates of the morbidity and mortality from these injuries could help drive preventative measures and improved access to care. Methods We used the Global Burden of Disease 2017 framework to produce three main results. First, we produced results on incidence, prevalence, years lived with disability, deaths, years of life lost and disability-adjusted life years from 1990 to 2017 for 195 countries and territories. Second, we analysed these results to measure mortality-to-incidence ratios by location. Third, we reported the measures above in terms of the cause of fire, heat and hot substances and the types of bodily injuries that result. Results Globally, there were 8 991 468 (7 481 218 to 10 740 897) new fire, heat and hot substance injuries in 2017 with 120 632 (101 630 to 129 383) deaths. At the global level, the age-standardised mortality caused by fire, heat and hot substances significantly declined from 1990 to 2017, but regionally there was variability in age-standardised incidence with some regions experiencing an increase (eg, Southern Latin America) and others experiencing a significant decrease (eg, High-income North America). Conclusions The incidence and mortality of injuries that result from fire, heat and hot substances affect every region of the world but are most concentrated in middle and lower income areas. More resources should be invested in measuring these injuries as well as in improving infrastructure, advancing safety measures and ensuring access to care.

Objectives: Immunotherapy (IO) has become standard of care (SOC) for many advanced malignancies, although identifying patients likely to benefit remains difficult. We sought to assess whether demographic factors are associated with response to IO, compared with SOC systemic therapy, using stratified meta-analysis. Methods: A systematic review of MEDLINE, PubMed, Embase, and Scopus from inception to October 2, 2018. Randomized controlled trials comparing IO to SOC in patients with advanced solid organ malignancies were included if results were stratified by age, performance status (PS), or race, assessing overall survival (OS). Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated for each group using random-effects models independently. Results: We identified 21 eligible randomized controlled trials, including 20 stratified by age, 17 by PS, and 4 by race. Patients with PS 0 (HR, 0.74; 95% CI, 0.63-0.86) and PS≥1 (HR, 0.75; 95% CI, 0.68-0.83) had similar OS benefits from IO compared with SOC (P=0.80). There was no difference on the basis of patient race (white vs. nonwhite) (P=0.46). IO demonstrated an OS benefit for younger (below 65 y: HR, 0.73; 95% CI, 0.65-0.82) and older (65 y and above: HR, 0.79; 95% CI, 0.71-0.88) patients with no difference between age groups (P=0.27). Among prespecified subgroup analyses, there was significant effect modification in 2 subgroups: younger patients in the first-line setting (P=0.03) and those receiving anti-CTLA-4 drugs (P=0.05). Conclusions: When examining OS using stratified meta-analysis, we did not demonstrate significant differences in IO efficacy according to patient age, PS or race, though data on race were sparse.

Objective IgG4‐related disease (IgG4‐ RD ) can cause fibroinflammatory lesions in nearly any organ. Correlation among clinical, serologic, radiologic, and pathologic data is required for diagnosis. This work was undertaken to develop and validate an international set of classification criteria for IgG4‐ RD . Methods An international multispecialty group of 86 physicians was assembled by the American College of Rheumatology ( ACR ) and the European League Against Rheumatism ( EULAR ). Investigators used consensus exercises, existing literature, derivation and validation cohorts of 1,879 subjects (1,086 cases, 793 mimickers), and multicriterion decision analysis to identify, weight, and test potential classification criteria. Two independent validation cohorts were included. Results A 3‐step classification process was developed. First, it must be demonstrated that a potential IgG4‐ RD case has involvement of at least 1 of 11 possible organs in a manner consistent with IgG4‐ RD . Second, exclusion criteria consisting of a total of 32 clinical, serologic, radiologic, and pathologic items must be applied; the presence of any of these criteria eliminates the patient from IgG4‐ RD classification. Third, 8 weighted inclusion criteria domains, addressing clinical findings, serologic results, radiology assessments, and pathology interpretations, are applied. In the first validation cohort, a threshold of 20 points had a specificity of 99.2% (95% confidence interval [95% CI ] 97.2–99.8%) and a sensitivity of 85.5% (95% CI 81.9–88.5%). In the second, the specificity was 97.8% (95% CI 93.7–99.2%) and the sensitivity was 82.0% (95% CI 77.0–86.1%). The criteria were shown to have robust test characteristics over a wide range of thresholds. Conclusion ACR / EULAR classification criteria for IgG4‐ RD have been developed and validated in a large cohort of patients. These criteria demonstrate excellent test performance and should contribute substantially to future clinical, epidemiologic, and basic science investigations.

Objective. Osteochondral allograft (OCA) transplantation has demonstrated good long-term outcomes in treatment of cartilage defects. Viability, a key factor in clinical success, decreases with peri-implantation storage at 4°C during pathogen testing, matching logistics, and transportation. Modern, physiologic storage conditions may improve viability and enhance outcomes. Design. Osteochondral specimens from total knee arthroplasty patients (6 males, 5 females, age 56.4 ± 2.2 years) were stored in media and incubated at normoxia (21% O 2 ) at 22°C or 37°C, and hypoxia (2% O 2 ) at 37°C. Histology, live-dead staining, and quantitative polymerase chain reaction (qPCR) was performed 24 hours after harvest and following 7 days of incubation. Tissue architecture, cell viability, and gene expression were analyzed. Results. No significant viability or gene expression deterioration of cartilage was observed 1-week postincubation at 37°C, with or without hypoxia. Baseline viable cell density (VCD) was 94.0% ± 2.7% at day 1. At day 7, VCD was 95.1% (37°C) with normoxic storage and 92.2% (37°C) with hypoxic storage ( P ≥ 0.27). Day 7 VCD (22°C) incubation was significantly lower than both the baseline and 37°C storage values (65.6%; P < 0.01). COL1A1, COL1A2, and ACAN qPCR expression was unchanged from baseline ( P < 0.05) for all storage conditions at day 7, while CD163 expression, indicative of inflammatory macrophages and monocytes, was significantly lower in the 37°C groups ( P < 0.01). Conclusion. Physiologic storage at 37°C demonstrates improved chondrocyte viability and metabolism, and maintained collagen expression compared with storage at 22°C. These novel findings guide development of a method to optimize short-term fresh OCA storage, which may lead to improved clinical results.

Background: Calcaneus fractures have serious potential complications. Tongue type calcaneus fractures, in particular, are at risk for secondary soft tissue breakdown due in part to the tension placed on the soft tissue by the forces of the Achilles tendon on the superior fragment of the posterior calcaneal tuberosity. The current literature is limited regarding outcomes in pure tongue type calcaneus fractures. The purpose of this study is to report injury details, treatment, and complications in this group of fractures. Methods: The study is a retrospective study of patients between 2007 and 2015 with tongue type calcaneus fractures. Demographic data and comorbidities were collected for each patient. Clinical data including details of the injury, time to surgery, length of hospital stay, wound complications, additional surgical procedures, and follow-up data were evaluated. Results: There were 25 patients with 26 fractures included in this study, including 7 open fractures. Wound complications occurred in 7 (27%) patients, and fixation failure occurred in 4 (21%) patients. Patients with open fractures reached full healing in less time (2.8±0.4 mo) compared with patients with closed fractures (4±1.3 mo). The difference in time to full healing was not statistically significant (P>0.05) when comparing smokers and nonsmokers. Conclusions: The literature is limited regarding outcomes of pure tongue type calcaneus fractures. We found these fractures had similar or even higher rates of wound complications compared with other studies investigating intra-articular calcaneus fractures. This is one of the first studies to report on the complication rates associated specifically with these fractures. Level of Evidence: Level IV.

AbstractIntroductionOur research aim was to develop a novel clinimetric scale sensitive enough to detect disease progression in primary lateral sclerosis (PLS).MethodsA prototype of the PLS Functional Rating Scale (PLSFRS) was generated. Seventy‐seven participants with PLS were enrolled and evaluated at 21 sites that comprised the PLSFRS study group. Participants were assessed using the PLSFRS, Neuro‐Quality of Life (QoL), Schwab‐England Activities of Daily Living (ADL), and the Clinical Global Impression of Change scales. Participants completed telephone assessments at 12, 24, and 48 weeks after enrollment.ResultsThe PLSFRS demonstrated internal consistency as well as intrarater, interrater, telephone test‐retest reliability, and construct validity. Significant changes in disease progression were detected at 6 and 12 months; changes measured by the PLSFRS vs the ALSFRS‐R were significantly higher.DiscussionThe PLSFRS is a valid tool to assess the natural history of PLS in a shorter study period.

Introduction: Medical schools have an enduring need to provide ongoing faculty development and to foster educational alliances between teachers and learners, so that feedback provided to learners is both frequent and of high quality. We hypothesized that medical students trained as academic detailers with a mission to increase the emphasis on feedback could serve in this role during our clerkship, while still being evaluated as students in our clerkship rotation. Methods: The family medicine clerkship at Mayo Clinic School of Medicine launched a revised curriculum in 2016 in which students were taught how they might build an educational alliance with preceptors, were taught characteristics of high-quality feedback, and practiced requesting more useful feedback when initial quality was poor. After utilizing a clerkship-specific curriculum with small group sessions on receiving feedback, and training students and preceptors on the SNAPPs model, students were then directed to request feedback from their preceptors and model successful feedback conversations for preceptors. The study evaluated the medical students’ summative evaluations to compare the rate from the preintervention year (2015-2016) and the intervention year (2016-2017) at which preceptors added comments on students’ use of feedback. Results: Preceptors’ written comments about students seeking feedback increased at about a four-fold rate (74.4% vs 18.8%, P&lt;.001) after implementing a suite of changes to our clerkship curriculum. Conclusions: Using medical students to change preceptor behaviors was an important part of our suite of interventions. This intervention directed preceptor attention toward our instructional goal of increased medical student feedback.

Replication of negative-strand RNA viruses relies on two components: a helical ribonucleocapsid and an RNA-dependent RNA polymerase composed of a catalytic subunit, the L protein, and a cofactor, the P protein. We show that the measles virus (MeV) C protein is an additional component of the replication complex. We provide evidence that the C protein is recruited to the ribonucleocapsid by the P protein and map the interacting segments of both C and P proteins. We conclude that the primary function of MeV C is to improve polymerase processivity and accuracy, rather than uniquely to antagonize the type I interferon response. Since most viruses of the Paramyxoviridae family express C proteins, their primary function may be conserved.