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Excised signal circles (ESC) are circular DNA molecules generated during T‐ and B‐cell maturation. Previously considered biologically inert, recent work by Gao et al. now show that ESCs can replicate and accumulate in healthy lymphocytes. Moreover, the authors link higher levels of ESCs to an increased risk of relapse in B‐cell leukemia patients and propose that this phenomenon is due to the unique ability of ESCs to induce genome instability. Gao et al. report that circular DNA molecules created as by‐products of V(D)J recombination during lymphocyte maturation (ESCs) can replicate and be retained for much longer than previously thought in healthy cells. In BCP‐ALL cells, increased ESC abundance correlates with a greater chance of relapse likely mediated by their ability to induce genome instability.

The CRISPR- Cas9 system is a powerful tool to edit eukaryotic genomes that has recently been adapted for functional screens. Several of its applications—including the disruption of genes using Cas9 -nickase and the generation of large deletions—require co-expression of two distinct guide RNAs (gRNAs). However, the lack of experimental approaches to generate pools of paired gRNA vectors prevents these applications from being scalable. Here we report a simple, inexpensive, one-step method that allows for the rapid and efficient cloning of gRNA pairs into expression vectors. We show that this method can be used to generate pooled libraries and is therefore suitable for in vivo and in vitro functional screens. CRISPR-Cas9 is a powerful tool for genome editing; however, difficulties in generating pools of paired guide RNAs limit its applicability to large-scale screening experiments. Here the authors report a one-step method for rapid and efficient generation of pooled libraries of guide RNA pairs.

A tool for genome-wide design of CRISPR guide RNAs reduces off-target effects and facilitates targeting of the non-coding genome. We present GuideScan software for the design of CRISPR guide RNA libraries that can be used to edit coding and noncoding genomic regions. GuideScan produces high-density sets of guide RNAs (gRNAs) for single- and paired-gRNA genome-wide screens. We also show that the trie data structure of GuideScan enables the design of gRNAs that are more specific than those designed by existing tools.

In the last decades, several clinical scores have been developed and currently used to improve the diagnosis and risk management of patients with suspected acute appendicitis (AA). However, some of them exhibited different values of sensitivity and specificity. We conducted a systematic review and metanalysis of epidemiological studies, which compared RIPASA and Alvarado scores for the diagnosis of AA. This systematic review was conducted using PubMed and Web of Science databases. Selected studies had to compare RIPASA and Alvarado scores on patients with suspected AA and reported diagnostic parameters. Summary estimates of sensitivity and specificity were calculated by the Hierarchical Summary Receiver Operating Curve (HSROC) using STATA 17 (STATA Corp, College Station, TX) and MetaDiSc (version 1.4) software. We included a total of 33 articles, reporting data from 35 studies. For the Alvarado score, the Hierarchical Summary Receiver Operating Curve (HSROC) model produced a summary sensitivity of 0.72 (95%CI = 0.66-0.77), and a summary specificity of 0.77 (95%CI = 0.70-0.82). For the RIPASA score, the HSROC model produced a summary sensitivity of 0.95 (95%CI = 0.92-0.97), and a summary specificity of 0.71 (95%CI = 0.60-0.80). RIPASA score has higher sensitivity, but low specificity compared to Alvarado score. Since these scoring systems showed different sensitivity and specificity parameters, it is still necessary to develop novel scores for the risk assessment of patients with suspected AA.

In the last decades, several clinical scores have been developed and currently used to improve the diagnosis and risk management of patients with suspected acute appendicitis (AA). However, some of them exhibited different values of sensitivity and specificity. We conducted a systematic review and metanalysis of epidemiological studies, which compared RIPASA and Alvarado scores for the diagnosis of AA. This systematic review was conducted using PubMed and Web of Science databases. Selected studies had to compare RIPASA and Alvarado scores on patients with suspected AA and reported diagnostic parameters. Summary estimates of sensitivity and specificity were calculated by the Hierarchical Summary Receiver Operating Curve (HSROC) using STATA 17 (STATA Corp, College Station, TX) and MetaDiSc (version 1.4) software. We included a total of 33 articles, reporting data from 35 studies. For the Alvarado score, the Hierarchical Summary Receiver Operating Curve (HSROC) model produced a summary sensitivity of 0.72 (95%CI = 0.66-0.77), and a summary specificity of 0.77 (95%CI = 0.70-0.82). For the RIPASA score, the HSROC model produced a summary sensitivity of 0.95 (95%CI = 0.92-0.97), and a summary specificity of 0.71 (95%CI = 0.60-0.80). RIPASA score has higher sensitivity, but low specificity compared to Alvarado score. Since these scoring systems showed different sensitivity and specificity parameters, it is still necessary to develop novel scores for the risk assessment of patients with suspected AA.

Introduction The ACCM/PALS guidelines address early correction of paediatric septic shock using conventional measures. In the evolution of these recommendations, indirect measures of the balance between systemic oxygen delivery and demands using central venous or superior vena cava oxygen saturation (ScvO 2  ≥ 70%) in a goal-directed approach have been added. However, while these additional goal-directed endpoints are based on evidence-based adult studies, the extrapolation to the paediatric patient remains unvalidated. Objective The purpose of this study was to compare treatment according to ACCM/PALS guidelines, performed with and without ScvO 2 goal-directed therapy, on the morbidity and mortality rate of children with severe sepsis and septic shock. Design, participants and interventions Children and adolescents with severe sepsis or fluid-refractory septic shock were randomly assigned to ACCM/PALS with or without ScvO 2 goal-directed resuscitation. Measurements Twenty-eight-day mortality was the primary endpoint. Results Of the 102 enrolled patients, 51 received ACCM/PALS with ScvO 2 goal-directed therapy and 51 received ACCM/PALS without ScvO 2 goal-directed therapy. ScvO 2 goal-directed therapy resulted in less mortality (28-day mortality 11.8% vs. 39.2%, p  = 0.002), and fewer new organ dysfunctions ( p  = 0.03). ScvO 2 goal-directed therapy resulted in more crystalloid (28 (20–40) vs. 5 (0–20) ml/kg, p  < 0.0001), blood transfusion (45.1% vs. 15.7%, p  = 0.002) and inotropic (29.4% vs. 7.8%, p  = 0.01) support in the first 6 h. Conclusions This study supports the current ACCM/PALS guidelines. Goal-directed therapy using the endpoint of a ScvO 2  ≥ 70% has a significant and additive impact on the outcome of children and adolescents with septic shock.

Rare diseases are characterized by a wide diversity of signs and symptoms and vary not only from disease to disease but also from person to person, and living with a disease leads patients to peculiar experiences, without limits of time and space, as they extend to various environments and relationships of their lives. Therefore, the objective of this study is the theoretical interaction between value co-creation (VC) and the stakeholder theory (ST) with the shared decision-making (SDM) health care theory, to enable the analysis of the relationships between patients and their stakeholders in the co-creation of value for decision-making focused on the patient’s quality of life. It is configured as a multi-paradigmatic proposal by enabling the analysis of multiple perspectives of different stakeholders in health care. Thus, co-created decision-making (CDM) emerges with emphasis on interactivity of the relationships. As previous studies have already highlighted the importance of holistic care, seeing the patient as a whole and not just the body, studies with CDM will be beneficial for analyses that go beyond the clinical office and doctor–patient relationships, extending to all environments and interactions that add value to the patient’s treatment. It was concluded that the essence of this new theory proposed here is neither in patient-centered care nor in patient self-care, but in co-created relationships with and between stakeholders, including non-health care environments that are important to the patient, such as relationships with friends, family, other patients with the same disease, social media, public policies, and the practice of pleasurable activities.

We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly design and analysis of individual gRNAs and gRNA libraries for targeting coding and non-coding regions in custom genomes. GuideScan2 analysis identifies widespread confounding effects of low-specificity gRNAs in published CRISPR screens and enables construction of a gRNA library that reduces off-target effects in a gene essentiality screen. GuideScan2 also enables the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome. GuideScan2 will facilitate CRISPR experiments across a wide range of applications.

MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34-deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation in vitro, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34-deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc-initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.

The widespread application of high-throughput sequencing methods is resulting in the identification of a rapidly growing number of novel gene fusions caused by tumour-specific chromosomal rearrangements, whose oncogenic potential remains unknown. Here we describe a strategy that builds upon recent advances in genome editing and combines ex vivo and in vivo chromosomal engineering to rapidly and effectively interrogate the oncogenic potential of genomic rearrangements identified in human brain cancers. We show that one such rearrangement, an microdeletion resulting in a fusion between Brevican (BCAN) and Neurotrophic Receptor Tyrosine Kinase 1 (NTRK1), is a potent oncogenic driver of high-grade gliomas and confers sensitivity to the experimental TRK inhibitor entrectinib. This work demonstrates that BCAN-NTRK1 is a bona fide human glioma driver and describes a general strategy to define the oncogenic potential of novel glioma-associated genomic rearrangements and to generate accurate preclinical models of this lethal human cancer. The use of mouse models has been an invaluable resource in cancer research but their generation is lengthy and costly. Here the authors describe an approach to generate engineered mouse models carrying specific gene fusions and, as a proof of principle, show that Bcan-Ntrk1 fusion leads to glioblastomas.