Search

Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach suffers from undesirable byproducts and an inability to control product purity. Here we develop a system in human cells for programmable, DSB-free DNA integration using Type I CRISPR-associated transposons (CASTs). To adapt our previously described CAST systems, we optimized DNA targeting by the QCascade complex through a comprehensive assessment of protein design, and we developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase, TnsC, to genomic sites targeted by QCascade. After initial detection of plasmid-based transposition, we screened 15 homologous CAST systems from a wide range of bacterial hosts, identified a CAST homolog fromPseudoalteromonasthat exhibited improved activity, and increased integration efficiencies through parameter optimization. We further discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, and we propose that this critical accessory factor functions to drive active disassembly of the post-transposition CAST complex, akin to its demonstrated role in Mu transposition. Our work highlights the ability to functionally reconstitute complex, multi-component machineries in human cells, and establishes a strong foundation to realize the full potential of CRISPR-associated transposons for human genome engineering.

The role of the epicardial vasculature in supraventricular and ventricular arrhythmias was described in clinical studies as well as its treatment by intravascular point ablation or alcohol injection. We report on a case series of patients with different arrhythmias linked to an epicardial site of origin with evidence supporting transmural extensions that were targeted for ablation with successful outcomes.The records of patients who has catheter ablation for Supraventricular or Ventricular arrhythmias between 2015 and 2020 was searched for patients with (1) arrhythmias linked to the epicardial vasculature and (2) findings to support an endocardial connection to the epicardial vasculature by activation mapping, pace mapping, or differential pacing, and (3) were successfully ablated via an endocardial approach only.From the data searched, we identified five patients with the following arrhythmias left ventricular summit ectopy, peri-mitral atrial flutter, preexcitation with inducible atrioventricular reentry tachycardia (AVRT), and a concealed left side accessory pathway with inducible AVRT that were linked to the following vessels: Great Cardiac vein, persistent left superior vena cava, left coronary cusp, and left ventricular outflow tract. Endocardial connections were supported by a combination of electro anatomical activation mapping, pace-mapping, and differential pacing. Endocardial ablations performed in all patients were successful without complications.This report highlights a subset of patients with arrhythmias linked to the greater cardiac vascular system that can be safely and effectively ablated endocardially, given the limitations and possible complications of epicardial ablation within or in the proximity of the epicardial vasculature.

This paper examines the ethical behaviour of new lawyers from two contrasting points of view. First, we review the prevalence and type of ethical misconduct by lawyers in the Australian state of Vi...

Because of its complex root canal morphology, dens invaginatus (DI) is a developmental anomaly that represents an endodontic challenge to complete cleaning, shaping, and disinfecting the root canal system. The present case report describes the use of antimicrobial photodynamic therapy as an adjunctive treatment in the successful management of nonsurgical complex type II DI in a left maxillary lateral incisor with thin and fragile dentin walls associated with periradicular lesion.

Simultaneous multithreading (SMT) enables enhanced computing capacity by allowing multiple tasks to execute concurrently on the same computing core. Despite its benefits, its use has been largely eschewed in work on real-time systems due to concerns that tasks running on the same core may adversely interfere with each other. In this paper, the safety of using SMT in a mixed-criticality multicore context is considered in detail. To this end, a prior open-source framework called MC2 (mixedcriticality on multicore), which provides features for mitigating cache and memory interference, was re-implemented to support SMT on an SMT-capable multicore platform. The creation of this new, configurable $\mathrm{M}\mathrm{C}^{2}$ variant entailed producing the first operating-system implementations of several recently proposed real-time SMT schedulers and tying them together within a mixed-criticality context. These schedulers introduce new spatialisolation challenges, which required introducing isolation at both the L2 and L3 cache levels. The efficacy of the resulting $\mathrm{M}\mathrm{C}^{2}$ variant is demonstrated via three experimental efforts. The first involved obtaining execution data using a wide range of benchmark suites, including TACLeBench, DIS, SD-VBS, and synthetic microbenchmarks. The second involved conducting a large-scale overhead-aware schedulability study, parameterized by the collected benchmark data, to elucidate schedulability tradeoffs. The third involved experiments involving case-study task systems. In the schedulability study, the use of SMT proved capable of increasing platform capacity by an average factor of 1.32. In the case-study experiments, deadline misses of highly critical tasks were never observed.

Translocation renal cell carcinoma (tRCC) is an aggressive and poorly-characterized subtype of kidney cancer driven by MiT/TFE gene fusions. Here, we define the landmarks of tRCC through an integrative analysis of 152 tRCC patients identified across multiple genomic, clinical trial, and retrospective cohorts. Most tRCCs harbor few somatic alterations apart from MiT/TFE fusions and homozygous deletions at chromosome 9p21.3 (19.2% of cases). Transcriptionally, tRCCs display a heightened NRF2-driven antioxidant response that is associated with resistance to many targeted therapies. Consistently, we find that outcomes for tRCC patients treated with vascular endothelial growth factor receptor inhibitors (VEGFR-TKI) are worse than those treated with immune checkpoint inhibition (ICI). Multiparametric immunofluorescence confirmed the presence of CD8 + tumor-infiltrating T cells compatible with a clinical benefit from ICI and revealed an exhaustion immunophenotype distinct from clear cell RCC. Our findings comprehensively define the clinical and molecular features of tRCC and may inspire new therapeutic hypotheses.

SCHED_DEADLINE (DL for short) is an Earliest-Deadline-First (EDF) scheduler included in the Linux kernel. A question motivated by DL is how EDF should be implemented in the presence of CPU affinities to maintain optimal bounded tardiness guarantees. Recent works have shown that under arbitrary affinities, DL does not maintain such guarantees. Such works have also shown that repairing DL to maintain these guarantees would likely require an impractical overhaul of the existing code. In this work, we show that for the special case where affinities are semi-partitioned, DL can be modified to maintain tardiness guarantees with minor changes. We also draw attention to the fact that admission control is already broken in several respects in the existing DL implementation.

Sarcomatoid and rhabdoid (S/R) renal cell carcinoma (RCC) are highly aggressive tumors with limited molecular and clinical characterization. Emerging evidence suggests immune checkpoint inhibitors (ICI) are particularly effective for these tumors, although the biological basis for this property is largely unknown. Here, we evaluate multiple clinical trial and real-world cohorts of S/R RCC to characterize their molecular features, clinical outcomes, and immunologic characteristics. We find that S/R RCC tumors harbor distinctive molecular features that may account for their aggressive behavior, including BAP1 mutations, CDKN2A deletions, and increased expression of MYC transcriptional programs. We show that these tumors are highly responsive to ICI and that they exhibit an immune-inflamed phenotype characterized by immune activation, increased cytotoxic immune infiltration, upregulation of antigen presentation machinery genes, and PD-L1 expression. Our findings build on prior work and shed light on the molecular drivers of aggressivity and responsiveness to ICI of S/R RCC., Sarcomatoid and rhabdoid tumours are highly aggressive forms of renal cell carcinoma that are also responsive to immunotherapy. In this study, the authors perform a comprehensive molecular characterization of these tumours discovering an enrichment of specific alterations and an inflamed phenotype.