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Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.
Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9
Functionalizing self-assembled GaN quantum dot superlattices by Eu-implantation
Self-assembled GaN quantum dots (QDs) stacked in superlattices (SL) with AlN spacer layers were implanted with Europium ions to fluences of 1013, 1014, and 1015 cm−2. The damage level introduced in the QDs by the implantation stays well below that of thick GaN epilayers. For the lowest fluence, the structural properties remain unchanged after implantation and annealing while for higher fluences the implantation damage causes an expansion of the SL in the [0001] direction which increases with implantation fluence and is only partly reversed after thermal annealing at 1000 °C. Nevertheless, in all cases, the SL quality remains very good after implantation and annealing with Eu ions incorporated preferentially into near-substitutional cation sites. Eu3+ optical activation is achieved after annealing in all samples. In the sample implanted with the lowest fluence, the Eu3+ emission arises mainly from Eu incorporated inside the QDs while for the higher fluences only the emission from Eu inside the AlN-buffer, capping, and spacer layers is observed.
© 2010 American Institute of PhysicsFCT-PTDC/CTM/100756/2008program PESSOA EGIDE/GRICESFCT-SFRH/BD/45774/2008FCT-SFRH/BD/44635/200
Sudden cardiac death in the young (5-39 years) in the canton of Vaud, Switzerland.
Sudden cardiac death (SCD) among the young is a rare and devastating event, but its exact incidence in many countries remains unknown. An autopsy is recommended in every case because some of the cardiac pathologies may have a genetic origin, which can have an impact on the living family members. The aims of this retrospective study completed in the canton of Vaud, Switzerland were to determine both the incidence of SCD and the autopsy rate for individuals from 5 to 39 years of age.
The study was conducted from 2000 to 2007 on the basis of official statistics and analysis of the International Classification of Diseases codes for potential SCDs and other deaths that might have been due to cardiac disease.
During the 8 year study period there was an average of 292'546 persons aged 5-39 and there were a total of 1122 deaths, certified as potential SCDs in 3.6% of cases. The calculated incidence is 1.71/100'000 person-years (2.73 for men and 0.69 for women). If all possible cases of SCD (unexplained deaths, drowning, traffic accidents, etc.) are included, the incidence increases to 13.67/100'000 person-years. However, the quality of the officially available data was insufficient to provide an accurate incidence of SCD as well as autopsy rates. The presumed autopsy rate of sudden deaths classified as diseases of the circulatory system is 47.5%. For deaths of unknown cause (11.1% of the deaths), the autopsy was conducted in 13.7% of the cases according to codified data.
The incidence of presumed SCD in the canton of Vaud, Switzerland, is comparable to the data published in the literature for other geographic regions but may be underestimated as it does not take into account other potential SCDs, as unexplained deaths. Increasing the autopsy rate of SCD in the young, better management of information obtained from autopsies as well developing of structured registry could improve the reliability of the statistical data, optimize the diagnostic procedures, and the preventive measures for the family members
Sudden cardiac death among general population and sport related population in forensic experience.
PURPOSE: The goal of the study was to assess the causes and analyze the cases of sudden cardiac death (SCD) victims referred to the department of forensic medicine in Lausanne, with a particular focus on sports-related fatalities including also leisure sporting activities. To date, no such published assessment has been done nor for Switzerland nor for the central Europe.
METHODS: This is a retrospective study based on autopsy records of SCD victims, from 10 to 50 years of age, performed at the University Centre of Legal Medicine in Lausanne from 1995 to 2010. The study population was divided into two groups: sport-related (SR) and not sport-related (NSR) SCDs.
RESULTS: During the study period, 188 cases of SCD were recorded: 166 (88%) were NSR and 22 (12%) SR. The mean age of the 188 victims was 37.3 ± 10.1 years, with the majority of the cases being male (79%). A cause of death was established in 84%, and the pathology responsible for death varied according to the age of the victims. In the NSR group, the mean age was 38.2 ± 9.2 years and there was 82% of male. Coronary artery disease (CAD) was the main diagnosis in the victims aged 30-50 years. The majority of morphologically normal hearts were observed in the 15-29 year age range. There was no case in the 10-14 year age range. In the SR group, 91% of victims died during leisure sporting activities. In this group the mean age was 30.5 ± 13.5 years, with the majority being male (82%). The main cause of death was CAD, with 6 cases (27%) and a mean age of 40.8 ± 5.5 years. The youngest victim with CAD was 33 years old. A morphologically normal heart was observed in 5 cases (23%), with a mean age of 24.4 ± 14.9 years. The most frequently implicated sporting activities were hiking (26%) and swimming (17%).
CONCLUSION: In this study, CAD was the most common cause of death in both groups. Although this pathology most often affects adults over 35 years of age, there were also some victims under 35 years of age in both groups. SCDs during sport are mostly related to leisure sporting activities, for which preventive measures are not yet usually established. This study highlights also the need to inform both athletes and non athletes of the cardiovascular risks during sport activities and the role of a forensic autopsy and registries involving forensic pathologists for SR SCD
ACTN2 variant associated with a cardiac phenotype suggestive of left-dominant arrhythmogenic cardiomyopathy.
A novel LAMB2 gene mutation associated with a severe phenotype in a neonate with Pierson syndrome.
BACKGROUND: Pierson syndrome (PS) is a rare autosomal recessive disorder, caused by mutations in the laminin β2 (LAMB2) gene. It is characterized by congenital nephrotic syndrome, microcoria, and neurodevelopmental deficits. Several mutations with genotype-phenotype correlations have been reported, often with great clinical variability. We hereby report a novel homozygous nonsense mutation in the LAMB2 gene, associated with a severe phenotype presentation.
CASE DIAGNOSIS: We describe a term male infant born from consanguineous parents. The mother previously lost three children in the neonatal period, secondary to undefined renal disease, had two spontaneous abortions, and gave birth to one healthy daughter. The index case presented at birth with bilateral microcoria, severe hypotonia, respiratory distress, and congenital nephrotic syndrome associated with anuria and severe renal failure requiring peritoneal dialysis. The patients' clinical follow-up was unfavorable, and the newborn died at 7 days of life, after withdrawal of life support. Genetic analysis revealed a homozygous nonsense mutation at position c.2890C>T causing a premature stop codon (p.R964*) in LAMB2 gene.
CONCLUSION: We here describe a novel nonsense homozygous mutation in LAMB2 gene causing a severe neonatal presentation of Pierson syndrome. This new mutation expands the genotype-phenotype spectrum of this rare disease and confirms that truncating mutations might be associated with severe clinical features
Process Oriented Collaboration in Grid-Environments: A Case Study in the Construction Industry
This paper addresses the process-oriented collaboration based on a grid-based platform for the support of virtual organizations (VO), illustrated on the example of the construction industry. Distributed, organizational and IT-structures of teams involved in vintage complex projects cannot be managed with conventional methods in an appropriate manner. Both using a grid platform and grid-based services, in conjunction with semantic methods for consistency saving and goal-oriented process management can increase the efficiency of collaboration processes in large-scale projects. A hybrid grid- and web service-based architecture for the next generation of VO service and a gateway solution was developed integrating the process-oriented perspective and prototypically implemented. The problem, as well as the solution on the basis of the hybrid system architecture combing the benefits of the cutting-edge technologies, the methodical concept for modeling VO processes and their automated execution on a grid platform are discussed in detail
Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy
shRNAs can trigger effective silencing of gene expression in mammalian cells, thereby providing powerful tools for genetic studies, as well as potential therapeutic strategies. Specific shRNAs can interfere with the replication of pathogenic viruses and are currently being tested as antiviral therapies in clinical trials. However, this effort is hindered by our inability to systematically and accurately identify potent shRNAs for viral genomes. Here we apply a recently developed highly parallel sensor assay to identify potent shRNAs for HIV, hepatitis C virus (HCV), and influenza. We observe known and previously unknown sequence features that dictate shRNAs efficiency. Validation using HIV and HCV cell culture models demonstrates very high potency of the top-scoring shRNAs. Comparing our data with the secondary structure of HIV shows that shRNA efficacy is strongly affected by the secondary structure at the target RNA site. Artificially introducing secondary structure to the target site markedly reduces shRNA silencing. In addition, we observe that HCV has distinct sequence features that bias HCV-targeting shRNAs toward lower efficacy. Our results facilitate further development of shRNA based antiviral therapies and improve our understanding and ability to predict efficient shRNAs
Efficacy and safety of abrocitinib in patients with moderate‐to‐severe atopic dermatitis and comorbid allergies
Background: Abrocitinib efficacy by comorbidity status in patients with moderate‐to‐severe atopic dermatitis (AD) has not been previously assessed. This post hoc analysis evaluated the efficacy and safety of abrocitinib in patients with AD and allergic comorbidities.
Methods: Data were pooled from patients who received abrocitinib 200 mg, 100 mg, or placebo in phase 2b (NCT02780167) and phase 3 (NCT03349060, NCT03575871) monotherapy trials. Patients with and without allergic comorbidities (allergic asthma, rhinitis, conjunctivitis, or food allergy) were evaluated for Investigator's Global Assessment (IGA) response (clear [0] or almost clear [1]), ≥75% improvement in the Eczema Area and Severity Index (EASI‐75), ≥4‐point improvement in Peak Pruritus Numerical Rating Scale (PP‐NRS4), and Dermatology Life Quality Index (DLQI) response (<2 with baseline score ≥2). Other outcomes were Patient‐Oriented Eczema Measure (POEM), SCORing Atopic Dermatitis (SCORAD), Pruritus and Symptoms Assessment for Atopic Dermatitis (PSAAD), and treatment‐emergent adverse events (TEAEs).
Results: Of 942 patients, 498 (53%) reported at least one allergic comorbidity (asthma only, 33%; conjunctivitis only or rhinitis only or both, 17%; food allergies only, 15%; >1 allergic comorbidity, 34%). Regardless of comorbidity status, from Week 2 to Week 12, higher percentages of patients treated with either abrocitinib dose achieved IGA 0/1, EASI‐75, PP‐NRS4, or DLQI 0/1 versus placebo‐treated patients. Changes from baseline in POEM, SCORAD, and PSAAD were greater with abrocitinib than with placebo in patients with and without allergic comorbidities. Most TEAEs were mild or moderate.
Conclusions: Efficacy and safety data support abrocitinib use to manage AD in patients with or without allergic comorbidities
Monoclonal antibody-conjugated dendritic nanostructures for siRNA delivery
Small interfering RNA (siRNA) is a promising tool for gene therapy-based disease treatments. However, delivery of siRNA to the target cells requires a specific and reliable carrier system. Herein we describe a targeted carrier system that can deliver siRNA to cancer cells overexpressing the human epidermal growth factor 2 (HER2) receptor. Trastuzumab-conjugated poly(amido)amine dendrimers can be synthesized using the protocols described here
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