CRISPR-induced double-strand breaks trigger recombination between homologous chromosome arms

CRISPR–Cas9–based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms—often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention

Sleep Disruption and Daytime Sleepiness Correlating with Disease Severity and Insulin Resistance in Non-Alcoholic Fatty Liver Disease: A Comparison with Healthy Controls

BACKGROUND & AIMS: Sleep disturbance is associated with the development of obesity, diabetes and hepatic steatosis in murine models. Hepatic triglyceride accumulation oscillates in a circadian rhythm regulated by clock genes, light-dark cycle and feeding time in mice. The role of the sleep-wake cycle in the pathogenesis of human non-alcoholic fatty liver disease (NAFLD) is indeterminate. We sought to detail sleep characteristics, daytime sleepiness and meal times in relation to disease severity in patients with NAFLD. METHODS: Basic Sleep duration and latency, daytime sleepiness (Epworth sleepiness scale), Pittsburgh sleep quality index, positive and negative affect scale, Munich Chronotype Questionnaire and an eating habit questionnaire were assessed in 46 patients with biopsy-proven NAFLD and 22 healthy controls, and correlated with biochemical and histological parameters. RESULTS: In NAFLD compared to healthy controls, time to fall asleep was vastly prolonged (26.9 vs. 9.8 min., p = 0.0176) and sleep duration was shortened (6.3 vs. 7.2 hours, p = 0.0149). Sleep quality was poor (Pittsburgh sleep quality index 8.2 vs. 4.7, p = 0.0074) and correlated with changes in affect. Meal frequency was shifted towards night-times (p = 0.001). In NAFLD but not controls, daytime sleepiness significantly correlated with liver enzymes (ALAT [r = 0.44, p = 0.0029], ASAT [r = 0.46, p = 0.0017]) and insulin resistance (HOMA-IR [r = 0.5, p = 0.0009]) independent of cirrhosis. In patients with fibrosis, daytime sleepiness correlated with the degree of fibrosis (r = 0.364, p = 0.019). CONCLUSIONS: In NAFLD sleep duration was shortened, sleep onset was delayed and sleep quality poor. Food-intake was shifted towards the night. Daytime sleepiness was positively linked to biochemical and histologic surrogates of disease severity. The data may indicate a role for sleep-wake cycle regulation and timing of food-intake in the pathogenesis of human NAFLD as suggested from murine models

Two-hour algorithm for triage toward rule-out and rule-in of acute myocardial infarction using high-sensitivity cardiac troponin T

BACKGROUND: The early triage of patients toward ruleout and rule-in of acute myocardial infarction (AMI) is challenging. Therefore, we aimed to develop a 2-h algorithm that uses high-sensitivity cardiac troponin I (hs-cTnI). METHODS: We prospectively enrolled 1435 (derivation cohort) and 1194 (external validation cohort) patients presenting with suspected AMI to the emergency department. The final diagnosis was adjudicated by 2 independent cardiologists. hs-cTnI was measured at presentation and after 2 h in a blinded fashion. We derived and validated a diagnostic algorithm incorporating hscTnI values at presentation and absolute changes within the first 2 h. RESULTS: AMI was the final diagnosis in 17% of patients in the derivation and 13% in the validation cohort. The 2-h algorithm developed in the derivation cohort classified 56% of patients as rule-out, 17% as rule-in, and 27% as observation. Resulting diagnostic sensitivity and negative predictive value (NPV) were 99.2% and 99.8% for rule-out; specificity and positive predictive value (PPV) were 95.2% and 75.8% for rule-in. Applying the 2-h algorithm in the external validation cohort, 60% of patients were classified as rule-out, 13% as rule-in, and 27% as observation. Diagnostic sensitivity and NPV were 98.7% and 99.7% for rule-out; specificity and PPV were 97.4% and 82.2% for rule-in. Thirty-day survival was 100% for rule-out patients in both cohorts. CONCLUSIONS: A simple algorithm incorporating hscTnI baseline values and absolute 2-h changes allowed a triage toward safe rule-out or accurate rule-in of AMI in the majority of patients

Assessing Spinal Cerebrospinal Fluid Leaks in Spontaneous Intracranial Hypotension With a Scoring System Based on Brain Magnetic Resonance Imaging Findings.

Importance Various signs may be observed on brain magnetic resonance imaging (MRI) in patients with spontaneous intracranial hypotension (SIH). However, the lack of a classification system integrating these findings limits decision making in clinical practice. Objective To develop a probability score based on the most relevant brain MRI findings to assess the likelihood of an underlying spinal cerebrospinal fluid (CSF) leak in patients with SIH. Design, Setting, and Participants This case-control study in consecutive patients investigated for SIH was conducted at a single hospital department from February 2013 to October 2017. Patients with missing brain MRI data were excluded. Three blinded readers retrospectively reviewed the brain MRI scans of patients with SIH and a spinal CSF leak, patients with orthostatic headache without a CSF leak, and healthy control participants, evaluating 9 quantitative and 7 qualitative signs. A predictive diagnostic score based on multivariable backward logistic regression analysis was then derived. Its performance was validated internally in a prospective cohort of patients who had clinical suspicion for SIH. Main Outcomes and Measures Likelihood of a spinal CSF leak based on the proposed diagnostic score. Results A total of 152 participants (101 female [66.4%]; mean [SD] age, 46.1 [14.3] years) were studied. These included 56 with SIH and a spinal CSF leak, 16 with orthostatic headache without a CSF leak, 60 control participants, and 20 patients in the validation cohort. Six imaging findings were included in the final scoring system. Three were weighted as major (2 points each): pachymeningeal enhancement, engorgement of venous sinus, and effacement of the suprasellar cistern of 4.0 mm or less. Three were considered minor (1 point each): subdural fluid collection, effacement of the prepontine cistern of 5.0 mm or less, and mamillopontine distance of 6.5 mm or less. Patients were classified into groups at low, intermediate, or high probability of having a spinal CSF leak, with total scores of 2 points or fewer, 3 to 4 points, and 5 points or more, respectively, on a scale of 9 points. The discriminatory ability of the proposed score could be demonstrated in the validation cohort. Conclusions and Relevance This 3-tier predictive scoring system is based on the 6 most relevant brain MRI findings and allows assessment of the likelihood (low, intermediate, or high) of a positive spinal imaging result in patients with SIH. It may be useful in identifying patients with SIH who are leak positive and in whom further invasive myelographic examinations are warranted before considering targeted therapy

Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

A new study reveals a functional rule for N-terminal acetylation in higher eukaryotes called the (X)PX rule and describes a generic method that prevents this modification to allow the study of N-terminal acetylation in any given protein

The impact of regional origin on the incidence of gestational diabetes mellitus in a multiethnic European cohort

IntroductionWomen with migration background present specific challenges related to risk stratification and care of gestational diabetes mellitus (GDM). Therefore, this study aims to investigate the role of ethnic origin on the risk of developing GDM in a multiethnic European cohort.MethodsPregnant women were included at a median gestational age of 12.9 weeks and assigned to the geographical regions of origin: Caucasian Europe (n = 731), Middle East and North Africa countries (MENA, n = 195), Asia (n = 127) and Sub-Saharan Africa (SSA, n = 48). At the time of recruitment maternal characteristics, glucometabolic parameters and dietary habits were assessed. An oral glucose tolerance test was performed in mid-gestation for GDM diagnosis.ResultsMothers with Caucasian ancestry were older and had higher blood pressure and an adverse lipoprotein profile as compared to non-Caucasian mothers, whereas non-Caucasian women (especially those from MENA countries) had a higher BMI and were more insulin resistant. Moreover, we found distinct dietary habits. Non-Caucasian mothers, especially those from MENA and Asian countries, had increased incidence of GDM as compared to the Caucasian population (OR 1.87, 95%CI 1.40 to 2.52, p < 0.001). Early gestational fasting glucose and insulin sensitivity were consistent risk factors across different ethnic populations, however, pregestational BMI was of particular importance in Asian mothers.DiscussionPrevalence of GDM was higher among women from MENA and Asian countries, who already showed adverse glucometabolic profiles at early gestation. Fasting glucose and early gestational insulin resistance (as well as higher BMI in women from Asia) were identified as important risk factors in Caucasian and non-Caucasian patients

A conserved regulatory program drives emergence of the lateral plate mesoderm

Cardiovascular cell lineages emerge with kidney, smooth muscle, and limb skeleton progenitors from the lateral plate mesoderm (LPM). How the LPM emerges during development and how it has evolved to form key lineages of the vertebrate body plan remain unknown. Here, we captured LPM formation by transgenic in toto imaging and lineage tracing using the first pan-LPM enhancer element from the zebrafish gene draculin (drl). drl LPM enhancer-based reporters are specifically active in LPM-corresponding territories of several chordate species, uncovering a universal LPM-specific gene program. Distinct from other mesoderm, we identified EomesA, FoxH1, and MixL1 with BMP/Nodal-controlled Smad activity as minimally required factors to drive drl-marked LPM formation. Altogether, our work provides a developmental and mechanistic framework for LPM emergence and the in vitro differentiation of cardiovascular cell types. Our findings suggest that the LPM may represent an ancient cell fate domain that predates ancestral vertebrates

Risk stratification in patients with acute chest pain using three high-sensitivity cardiac troponin assays

Aims Several high-sensitivity cardiac troponin (hs-cTn) assays have recently been developed. It is unknown which hs-cTn provides the most accurate prognostic information and to what extent early changes in hs-cTn predict mortality. Methods and results In a prospective, international multicentre study, cTn was simultaneously measured with three novel [high-sensitivity cardiac Troponin T (hs-cTnT), Roche Diagnostics; hs-cTnI, Beckman-Coulter; hs-cTnI, Siemens] and a conventional assay (cTnT, Roche Diagnostics) in a blinded fashion in 1117 unselected patients with acute chest pain. Patients were followed up 2 years regarding mortality. Eighty-two (7.3%) patients died during the follow-up. The 2-year prognostic accuracy of hs-cTn was most accurate for hs-cTnT [area under the receivers operating characteristic curve (AUC) 0.78 (95% CI: 0.73-0.83) and outperformed both hs-cTnI (Beckman-Coulter, 0.71 (95% CI: 0.65-0.77; P = 0.001 for comparison), hs-cTnI (Siemens) 0.70 (95% CI: 0.64-0.76; P < 0.001 for comparison)] and cTnT 0.67 (95% CI: 0.61-0.74; P < 0.001 for comparison). Absolute changes of hs-cTnT were more accurate than relative changes in predicting mortality, but inferior to presentation values of hs-cTnT. Combining changes of hs-cTnT within the first 6 h with their presentation values did not further improve prognostic accuracy. Similar results were obtained for both hs-cTnI assays regarding the incremental value of changes. Hs-cTn concentrations remained predictors of death in clinically challenging subgroups such as patients with pre-existing coronary artery disease, impaired renal function, and patients older than 75 years. Conclusion High-sensitivity cardiac Troponin T is more accurate than hs-cTnI in the prediction of long-term mortality. Changes of hs-cTn do not seem to further improve risk stratification beyond initial presentation value

Conserved enhancers control notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development