Quantification of CRISPR-Cas9 diffusion dynamics in Escherichia coli

What do adaptive immunity, genetic engineering and antimicrobials have in common? CRISPR-Cas9, the popular enzymatic complex that produces DNA double-strand breaks when associated with a guide-RNA. Hundreds of labs routinely use this system to edit genomic DNA; however, some of the mechanisms by which it interacts with nucleic acid remain unclear. In my lab, we developed an expertise in the study of DNA recombination, DNA repair and DNA interactions in Escherichia coli. We use single-molecule fluorescent microscopy to collect images in real time, in vivo. During my PhD, I harnessed this expertise to follow the behaviour of the Cas9 protein under different conditions: various expression levels; various gRNAs; and various genomic targets. By observing the diffusion dynamics of the protein, I was able to quantify how different DNA interactions were impacting the motion of the protein in the cytoplasm and inferred that actual ON-target interactions were very rare throughout the lifetime of the protein. In contrast, the protein was mainly involved in non-specific OFF-target DNA interactions, in search of its actual target. Additionally, my results reveal the presence of a large fraction of non-specific interactions, hitherto not reported in the literature, owing to their absence of DNA modification. In total, this work offers a collection of highly quantitative measurements on the behaviour of a protein whose activity is central to many biologists, while shedding a new light on the importance of Cas9 searching and targeting mechanisms. Finally, it opens a discussion on the role of DNA recognition in the context of gene editing and antimicrobial resistance

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Etude de la formation de la barriere de Schotiky Au-InSe par photoemission angulaire

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Behavioral Own-Body-Transformations in Children and Adolescents With Typical Development, Autism Spectrum Disorder, and Developmental Coordination Disorder

International audienc

Behavioral Own-Body-Transformations in Children and Adolescents With Typical Development, Autism Spectrum Disorder, and Developmental Coordination Disorder

Background: In motor imitation, taking a partner's perspective often involves a mental body transformation from an embodied, ego-centered viewpoint to a disembodied, hetero-centered viewpoint. Impairments of both own-body-transformation (OBT) and abnormalities in visual-spatial processing have been reported in patients with neurodevelopmental disorders including autism spectrum disorder (ASD). In the context of a visual-motor interactive task, studying OBT impairments while disentangling the contribution of visual-spatial impairments associated with motor coordination problems has not been investigated.Methods: 85 children and adolescents (39 controls with typical development, TD; 29 patients with ASD; 17 patients with developmental coordination disorder, DCD), aged 6–19 years, participated in a behavioral paradigm in which participants interacted with a virtual tightrope walker (TW) standing and moving with him. The protocol enables to distinguish ego-centered and hetero-centered perspectives.Results: We show that (1) OBT was possible but difficult for children with neurodevelopmental disorders, as well as for TD children, when the task required the participant to perform a mental rotation in order to adopt a hetero-centered perspective. (2) Using multivariate models, hetero-centered perspective score was significantly associated with age, TW orientation, latency, and diagnosis. ASD and TD groups' performances were close and significantly correlated with age. However, it was not the case for DCD, since this group was specifically handicapped by visual-spatial impairments. (3) ASD and DCD did not perform similarly: motor performance as shown by movement amplitude was better in DCD than ASD. ASD motor response was more ambiguous and hardly readable.Conclusion: Changing perspective in a spatial environment is possible for patients with ASD although delayed compared with TD children. In patients with DCD, their visual-spatial impairments negatively modulated their performances in the experiment

Semi-automatic behavioral change-point detection: a case study analyzing children interactions with a social agent

The study of human behaviors in cognitive sciences provides clues to understand and describe people’s personal and interpersonal functioning. In particular, the temporal analysis of behavioral dynamics can be a powerful tool to reveal events, correlations and causalities but also to discover abnormal behaviors. However, the annotation of these dynamics can be expensive in terms of temporal and human resources. To tackle this challenge, this paper proposes a methodology to semi-automatically annotate behavioral data. Behavioral dynamics can be expressed as sequences of simple dynamical processes: transitions between such processes are generally known as change-points. This paper describes the necessary steps to detect and classify change-points in behavioral data by using a dataset collected in a real use-case scenario. This dataset includes motor observations from children with typical development and with neuro-developmental disorders. Abnormal movements which are present in such disorders are useful to validate the system in conditions that are challenging even for experienced annotators. Results show that the system: can be effective in the semi-automated annotation task; can be efficient in presence of abnormal behaviors; may achieve good performance when trained with limited manually annotated data

Semi-automatic behavioral change-point detection: a case study analyzing children interactions with a social agent

International audienc

Acute Kidney Injury Predicts Major Adverse Outcomes in Diabetes: Synergic Impact With Low Glomerular Filtration Rate and Albuminuria

International audienc