Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea
. Target interference relies on a multi-subunit, RNA-guided complex called Cascade
, which recruits a ...trans-acting helicase-nuclease, Cas3, for target degradation
. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain
and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.
The loading of oligomeric helicases onto replication origins marks an essential step in replisome assembly. In cells, dedicated AAA+ ATPases regulate loading, however, the mechanism by which these ...factors recruit and deposit helicases has remained unclear. To better understand this process, we determined the structure of the ATPase region of the bacterial helicase loader DnaC from
Aquifex aeolicus to 2.7 Å resolution. The structure shows that DnaC is a close paralog of the bacterial replication initiator, DnaA, and unexpectedly shares an ability to form a helical assembly similar to that of ATP-bound DnaA. Complementation and ssDNA-binding assays validate the importance of homomeric DnaC interactions, while pull-down experiments show that the DnaC and DnaA AAA+ domains interact in a nucleotide-dependent manner. These findings implicate DnaC as a molecular adaptor that uses ATP-activated DnaA as a docking site for regulating the recruitment and correct spatial deposition of the DnaB helicase onto origins.
The loading of oligomeric helicases onto replication origins marks an essential step in replisome assembly. In cells, dedicated AAA+ ATPases regulate loading, however, the mechanism by which these ...factors help recruit and deposit helicases has remained unclear. To better understand this process, we determined the structure of the ATPase region of the bacterial helicase loader DnaC from
Aquifex aeolicus
to 2.7 Å resolution. The structure shows that DnaC is a close paralog of the bacterial replication initiator, DnaA, that unexpectedly forms a right-handed helical assembly similar to the quaternary state adopted by ATP-bound DnaA. Complementation and ssDNA-binding assays validate the importance of homomeric DnaC interactions, while pull-down experiments show that the AAA+ domains of DnaC and DnaA interact in a nucleotide-dependent manner. These findings implicate DnaC as a molecular adaptor that uses ATP-activated DnaA as a docking site for regulating the recruitment and correct spatial deposition of the DnaB helicase onto origins.
Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective ...inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Targeting the human Nav1.7 channel involved in pain perception, we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4), where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. Residues from the S2 and S3 helices are key determinants of isoform selectivity, and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists.
Multiple lines of evidence indicate that mitochondrial dysfunction is central to Parkinson's disease. Here we investigate the mechanism by which parkin, an E3 ubiquitin ligase, and USP30, a ...mitochondrion-localized deubiquitylase, regulate mitophagy. We find that mitochondrial damage stimulates parkin to assemble Lys 6, Lys 11 and Lys 63 chains on mitochondria, and that USP30 is a ubiquitin-specific deubiquitylase with a strong preference for cleaving Lys 6- and Lys 11-linked multimers. Using mass spectrometry, we show that recombinant USP30 preferentially removes these linkage types from intact ubiquitylated mitochondria and counteracts parkin-mediated ubiquitin chain formation in cells. These results, combined with a series of chimaera and localization studies, afford insights into the mechanism by which a balance of ubiquitylation and deubiquitylation regulates mitochondrial homeostasis, and suggest a general mechanism for organelle autophagy.
Patients receiving complementary and alternative medicine (CAM) therapies often report shifts in well-being that go beyond resolution of the original presenting symptoms. We undertook a research ...program to develop and evaluate a patient-centered outcome measure to assess the multidimensional impacts of CAM therapies, utilizing a novel mixed methods approach that relied upon techniques from the fields of anthropology and psychometrics. This tool would have broad applicability, both for CAM practitioners to measure shifts in patients' states following treatments, and conventional clinical trial researchers needing validated outcome measures. The US Food and Drug Administration has highlighted the importance of valid and reliable measurement of patient-reported outcomes in the evaluation of conventional medical products. Here we describe Phase I of our research program, the iterative process of content identification, item development and refinement, and response format selection. Cognitive interviews and psychometric evaluation are reported separately.
From a database of patient interviews (n = 177) from six diverse CAM studies, 150 interviews were identified for secondary analysis in which individuals spontaneously discussed unexpected changes associated with CAM. Using ATLAS.ti, we identified common themes and language to inform questionnaire item content and wording. Respondents' language was often richly textured, but item development required a stripping down of language to extract essential meaning and minimize potential comprehension barriers across populations. Through an evocative card sort interview process, we identified those items most widely applicable and covering standard psychometric domains. We developed, pilot-tested, and refined the format, yielding a questionnaire for cognitive interviews and psychometric evaluation.
The resulting questionnaire contained 18 items, in visual analog scale format, in which each line was anchored by the positive and negative extremes relevant to the experiential domain. Because of frequent informant allusions to response set shifts from before to after CAM therapies, we chose a retrospective pretest format. Items cover physical, emotional, cognitive, social, spiritual, and whole person domains.
This paper reports the success of a novel approach to the development of outcome instruments, in which items are extracted from patients' words instead of being distilled from pre-existing theory. The resulting instrument, focused on measuring shifts in patients' perceptions of health and well-being along pre-specified axes, is undergoing continued testing, and is available for use by cooperating investigators.
Mortality in pulmonary arterial hypertension (PAH) remains high and referral to palliative or supportive care (P/SC) specialist services is recommended when appropriate. However, access to P/SC is ...frequently a challenge for patients with a noncancer diagnosis and few patients living with PAH report P/SC involvement in their care. A modified Delphi process of three questionnaires completed by a multidisciplinary panel (N = 15) was used to develop expert consensus statements regarding the use of P/SC to support patients with PAH. Panelists rated their agreement with each statement on a Likert scale. There was a strong consensus that patients should be referred to P/SC when disease symptoms become unmanageable or for end‐of‐life care. Services that achieved consensus were pain management techniques, end‐of‐life care, and psychosocial recommendations. Palliative or supportive care should be discussed with patients, preferably in‐person, when disease symptoms become unmanageable, when starting treatment, when treatment‐related adverse events occur or become refractory to initial intervention. Care partners and patient support groups were considered important in improving a patient's overall health outcomes, treatment adherence, and perception of care. Most patients with PAH experience cognitive and/or psychosocial changes and those who receive psychosocial management have better persistence and/or compliance with their treatment. These consensus statements provide guidance to healthcare providers on the “who and when” of referral to palliative care services, as well as the importance of focusing on the psychosocial aspects of patient care and quality of life.
Neuromuscular diseases (NMD) account for a significant proportion of infant and childhood mortality and devastating chronic disease. Determining the specific diagnosis of NMD is challenging due to ...thousands of unique or rare genetic variants that result in overlapping phenotypes. We present four unique childhood myopathy cases characterized by relatively mild muscle weakness, slowly progressing course, mildly elevated creatine phosphokinase (CPK), and contractures. We also present two additional cases characterized by severe prenatal/neonatal myopathy. Prior extensive genetic testing and histology of these cases did not reveal the genetic etiology of disease. Here, we applied whole exome sequencing (WES) and bioinformatics to identify likely causal pathogenic variants in each pedigree. In two cases, we identified novel pathogenic variants in COL6A3. In a third case, we identified novel likely pathogenic variants in COL6A6 and COL6A3. We identified a novel splice variant in EMD in a fourth case. Finally, we classify two cases as calcium channelopathies with identification of novel pathogenic variants in RYR1 and CACNA1S. These are the first cases of myopathies reported to be caused by variants in COL6A6 and CACNA1S. Our results demonstrate the utility and genetic diagnostic value of WES in the broad class of NMD phenotypes.
In this article, we describe six diagnostic odyssey cases of myopathy and the novel genetic variants that we identified as the probable cause of neuromuscular disease. In particular we have identified variants in CACNA1S as the cause of a new, severe form of central core disease. In addition, we identify novel pathogenic variants in COL6A3 (images shown above), COL6A6, EMD, and RYR1.
This article reports the use of the continuous quality improvement (CQI) process to improve patient outcomes. The FADE method (focus, analyze, develop, and execute) was used to focus on vascular ...access planning, analyze data concerning intravenous (i.v.) therapy, develop a vascular access planning algorithm, and execute implementation of the algorithm. An evaluation study revealed that patients whose vascular access planning was consistent with the algorithm reported fewer i.v.s, less difficulty starting i.v.s, and less stress; waited significantly less time until central venous line (CVL) placement (for those who received CVLs); and had significantly shorter lengths of stay.
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