The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption ...of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using “error-prone” DNA-repair machinery to generate precise edits.
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•DNA repair profiles of 223 sites in the human genome reveal nonrandom outcomes•The protospacer sequence, not genomic context, determines Cas9 DSB repair outcomes•Each DNA repair profile is composed of specific contributions from c-NHEJ and MMEJ•DNA repair profiling can be used to anticipate repair outcomes in primary cells
van Overbeek, Capurso et al. demonstrate that repair outcomes are nonrandom at S. pyogenes Cas9-mediated DSBs and are determined by the protospacer sequence rather than genomic context. DNA repair profiling reveals specific contributions of c-NHEJ and MMEJ at each site and an approach to generate precise edits without exogenous template.
3D printing has evolved quickly from rapid prototyping to true mass production. This is reflected in the use of the term additive manufacturing (AM) to emphasize the use of the technology in actual ...manufacturing of high‐value products and finished goods. Consequentially, the requirements on the materials and processes have become more stringent and challenging to satisfy. Furthermore, various manufacturing industries have their own unique sets of requirements, and these are difficult if not impossible to satisfy with neat polymers alone. Therefore, additives are playing an increasingly critical role for satisfying material requirements for AM applications. In this article, challenges are identified that will need to be addressed as AM matures toward manufacturing applications. These include speed of production, mechanical performance and durability of the products, their interaction with the environment, and readiness for manufacturing. The use of additives to overcome these challenges is discussed in terms of current trends and with the help of specific examples. These include, but are not limited to, reinforcements, thermal stabilizers, antioxidants, and flame retardants to improve performance and meet thermal, environmental, and flammability requirements in demanding applications like aerospace, defense, and automotive. Further, colors, pigments, and dyes are needed for aesthetics. Bioactive or bioinert materials are needed for specific medical applications, and electrically dissipative or thermally conductive materials are often needed for electronics applications. This article provides a critical look at the current state of AM and how the roles of additives in AM are expected to expand, including identification of future opportunities.
As 3D printing continues to be used in more and more challenging applications, the ability to meet requirements depends on the use of highly engineered materials incorporating additives, such as flow enhancers, fiber reinforcement, thermally or electrically functional materials, and antioxidants.
Biocatalysis has emerged as one of the most promising technologies to enable green synthesis of important chemicals, due to the ambient conditions generally applied for these reactions. Nonetheless, ...a general uptake of enzymatic transformations has been hindered by the perceived high cost of recombinant proteins. Recent interest in continuous flow from the synthetic chemistry community has now begun to spread to biotransformations, with protein immobilization playing a key part. As a consequence, continuous biotransformations using immobilized enzymes are becoming more accessible to nonexperts. This review will discuss several recent examples of continuous biotransformations that use immobilization, with a focus on examples involving fine chemical synthesis. It will also examine some of the issues that the community has as a whole, most importantly a lack of unified reporting tools to allow comparison and assessment of the different techniques.
The development of degradable polymers has commanded significant attention over the past half century. Approaches have predominantly relied on ring-opening polymerization of cyclic esters (e.g., ...lactones, lactides) and N-carboxyanhydrides, as well as radical ring-opening polymerizations of cyclic ketene acetals. In recent years, there has been a significant effort applied to expand the family of degradable polymers accessible via olefin metathesis polymerization. Given the excellent functional group tolerance of olefin metathesis polymerization reactions generally, a broad range of conceivable degradable moieties can be incorporated into appropriate monomers and thus into polymer backbones. This approach has proven particularly versatile in synthesizing a broad spectrum of degradable polymers including poly(ester), poly(amino acid), poly(acetal), poly(carbonate), poly(phosphoester), poly(phosphoramidate), poly(enol ether), poly(azobenzene), poly(disulfide), poly(sulfonate ester), poly(silyl ether), and poly(oxazinone) among others. In this review, we will highlight the main olefin metathesis polymerization strategies that have been used to access degradable polymers, including (i) acyclic diene metathesis polymerization, (ii) entropy-driven and (iii) enthalpy-driven ring-opening metathesis polymerization, as well as (iv) cascade enyne metathesis polymerization. In addition, the livingness or control of polymerization reactions via different strategies are highlighted and compared. Potential applications, challenges and future perspectives of this new library of degradable polyolefins are discussed. It is clear from recent and accelerating developments in this field that olefin metathesis polymerization represents a powerful synthetic tool towards degradable polymers with novel structures and properties inaccessible by other polymerization approaches.
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Diagnosis is one of the most important tasks performed by primary care physicians. The World Health Organization (WHO) recently prioritized patient safety areas in primary care, and included ...diagnostic errors as a high-priority problem. In addition, a recent report from the Institute of Medicine in the USA, ‘Improving Diagnosis in Health Care’, concluded that most people will likely experience a diagnostic error in their lifetime. In this narrative review, we discuss the global significance, burden and contributory factors related to diagnostic errors in primary care. We synthesize available literature to discuss the types of presenting symptoms and conditions most commonly affected. We then summarize interventions based on available data and suggest next steps to reduce the global burden of diagnostic errors. Research suggests that we are unlikely to find a ‘magic bullet’ and confirms the need for a multifaceted approach to understand and address the many systems and cognitive issues involved in diagnostic error. Because errors involve many common conditions and are prevalent across all countries, the WHO’s leadership at a global level will be instrumental to address the problem. Based on our review, we recommend that the WHO consider bringing together primary care leaders, practicing frontline clinicians, safety experts, policymakers, the health IT community, medical education and accreditation organizations, researchers from multiple disciplines, patient advocates, and funding bodies among others, to address the many common challenges and opportunities to reduce diagnostic error. This could lead to prioritization of practice changes needed to improve primary care as well as setting research priorities for intervention development to reduce diagnostic error.
Recent fire seasons in the western United States are some of the most damaging and costly on record. Wildfires in the wildland-urban interface on the Colorado Front Range, resulting in thousands of ...homes burned and civilian fatalities, although devastating, are not without historical reference. These fires are consistent with the characteristics of large, damaging, interface fires that threaten communities across much of the western United States. Wildfires are inevitable, but the destruction of homes, ecosystems, and lives is not. We propose the principles of risk analysis to provide land management agencies, first responders, and affected communities who face the inevitability of wildfires the ability to reduce the potential for loss. Overcoming perceptions of wildland-urban interface fire disasters as a wildfire control problem rather than a home ignition problem, determined by home ignition conditions, will reduce home loss.
RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using ...Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
Summary Background The outcome of patients with ruptured abdominal aortic aneurysm (rAAA) varies by country. Study of practice differences might allow the formulation of pathways to improve care. ...Methods We compared data from the Hospital Episode Statistics for England and the Nationwide Inpatient Sample for the USA for patients admitted to hospital with rAAA from 2005 to 2010. Primary outcomes were in-hospital mortality, mortality after intervention, and decision to follow non-corrective treatment. In-hospital mortality and the rate of non-corrective treatment were analysed by binary logistic regression for each health-care system, after adjustment for age, sex, year, and Charlson comorbidity index. Findings The study included 11 799 patients with rAAA in England and 23 838 patients with rAAA in the USA. In-hospital mortality was lower in the USA than in England (53·05% 95% CI 51·26–54·85 vs 65·90%; p<0·0001). Intervention (open or endovascular repair) was offered to a greater proportion of cases in the USA than in England (19 174 80·43% vs 6897 58·45%; p<0·0001) and endovascular repair was more common in the USA than in England (4003 20·88% vs 589 8·54%; p<0·0001). Postintervention mortality was similar in both countries (41·77% for England and 41·65% for USA). These observations persisted in age-matched and sex-matched comparisons. In both countries, reduced mortality was associated with increased use of endovascular repair, increased hospital caseload (volume) for rAAA, high hospital bed capacity, hospitals with teaching status, and admission on a weekday. Interpretation In-hospital survival from rAAA, intervention rates, and uptake of endovascular repair are lower in England than in the USA. In England and the USA, the lowest mortality for rAAA was seen in teaching hospitals with larger bed capacities and doing a greater proportion of cases with endovascular repair. These common factors suggest strategies for improving outcomes for patients with rAAA. Funding None.
Objective Stent graft coverage of the left subclavian artery (LSA) may be required to achieve an adequate landing zone in up to 40% of descending thoracic aneurysms (DTAs). The Valiant Mona LSA ...Thoracic Stent Graft System (Medtronic, Santa Rosa, Calif) consists of a main stent graft and a branch stent graft designed to maintain LSA patency while diverting circulation through the encroaching aneurysm. Participating in the United States Food and Drug Administration's new Innovation Pathway, this first-in-human, early feasibility study evaluates early clinical experience of the Valiant Mona LSA Stent Graft System in patients with DTAs where the proximal landing zone necessitates LSA coverage. Methods This premarket, nonrandomized, single-arm prospective study recruited nine patients (age 72.9 ± 7.6 years). Primary end points were aneurysm-related mortality, stroke, paraplegia, left arm/hand ischemia, and treatment success. Neurologic events were assessed by imaging and by independent neurologists. Inclusion criteria required patients with a DTA or penetrating aortic ulcer to have a distance of ≥10 mm between the left common carotid artery and the LSA. Mean aneurysm diameter was 53.7 ± 10.0 mm. The indication for repair was for saccular DTA in five patients or fusiform DTA in four. Seven patients were at American Society of Anesthesiologists Physical Status Classification III/IV. Results All nine patients received one main stent graft (diameter 28 to 42 mm) and one branch stent graft (diameter 10 to 14 mm). To extend device coverage distally, a commercial Valiant Captivia device was implanted in seven patients. No uncorrected endoleaks were observed at the end of the implant procedure. Four endoleaks developed before discharge in four of eight evaluable patients. Two were identified as type II and two were of undetermined type. No endoleak resulted in a secondary intervention. Technical success, defined by the clinical investigational plan, was achieved in all nine patients intraoperatively, and treatment success was achieved in all eight evaluable patients at 1 month. There were no major, disabling strokes. Four minor nondisabling strokes were reported in three patients ≤30 days (days 1, 1, 5, and 24). To date, there have been no reports of death, left arm ischemia, paraplegia, rupture, conversion to open surgery, or secondary endovascular procedure. No patients required surgical LSA revascularization. Conclusions The Valiant Mona LSA system demonstrates technical and treatment success, relative safety, and early durability in its initial use in DTA patients. Midterm results are expected as patients are monitored at regular intervals to 5 years.
New municipalism is a nascent global social movement aiming to democratically transform the local state and economy – but what, precisely, is so new about it? I situate new municipalism in its ...geographical, political-economic and historical contexts, by comparison with earlier waves of municipal socialism and international municipalism, arguing that it re-politicises traditions of transnationalism, based not on post-political policy mobilities but on urban solidarities in contesting neoliberal austerity urbanism and platform capitalism. This article identifies three new municipalisms – platform, autonomist, managed – whose characteristics, contradictions, interconnections and potentials are explored in terms of state-space restructuring, urban-capitalist crisis and cycles of contention.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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