Recently, the Cas9-CRISPR system has proven to be a useful tool for genome editing in eukaryotes, which repair the double stranded breaks made by Cas9 with non-homologous end joining or homologous recombination. Escherichia coli lacks non-homologous end joining and has a very low homologous recombination rate, effectively rendering targeted Cas9 activity lethal. We have developed a heat curable, serializable, plasmid based system for selectionless Cas9 editing in arbitrary E. coli strains that uses synthetic CRISPRs for targeting and -red to effect repairs of double stranded breaks. We have demonstrated insertions, substitutions, and multi-target deletions with our system, which we have tested in several strains.

Introduction

Advance care planning (ACP) is a process that allows patients to identify their goals for medical care. Traditionally, ACP has focused on completing advance directives; however, we have expanded the ACP paradigm to also prepare patients to communicate their wishes and make informed decisions. To this end, we created an ACP website called PREPARE (http://www.prepareforyourcare.org) to prepare diverse English-speaking and Spanish-speaking older adults for medical decision-making. Here, we describe the study protocol for a randomised controlled efficacy trial of PREPARE in a safety-net setting. The goal is to determine the efficacy of PREPARE to engage diverse English-speaking and Spanish-speaking older adults in a full spectrum of ACP behaviours.

Methods and analysis

We include English-speaking and Spanish-speaking adults from an urban public hospital who are ≥55 years old, have ≥2 chronic medical conditions and have seen a primary care physician ≥2 times in the last year. Participants are randomised to the PREPARE intervention (review PREPARE and an easy-to-read advance directive) or the control arm (only the easy-to-read advance directive). The primary outcome is documentation of an advance directive and/or ACP discussion. Secondary outcomes include ACP behaviour change processes measured with validated surveys (eg, self-efficacy, readiness) and a broad range of ACP actions (eg, choosing a surrogate, identifying goals for care, discussing ACP with clinicians and/or surrogates). Using blinded outcome ascertainment, outcomes will be measured at 1 week and at 3, 6 and 12 months, and compared between study arms using mixed-effects logistic regression and mixed-effects linear, Poisson or negative binomial regression.

Ethics and dissemination

This study has been approved by the appropriate Institutional Review Boards and is guided by input from patient and clinical advisory boards and a data safety monitoring board. The results of this study will be disseminated to academic and community stakeholders.

Trial registration numbers

NCT01990235; NCT02072941; Pre-results.

Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. Meeting these challenges will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression-repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repress transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Using a bioinformatics approach, we identified 54 orthologous systems from various bacteria, and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.

Introduction

A common view is that head tremor (HT) in cervical dystonia (CD) decreases when the head assumes an unopposed dystonic posture and increases when the head is held at midline. However, this has not been examined with objective measures in a large, multicenter cohort.

Methods

For 80 participants with CD and HT, we analyzed videos from examination segments in which participants were instructed to 1) let their head drift to its most comfortable position (null point) and then 2) hold their head straight at midline. We used our previously developed Computational Motor Objective Rater (CMOR) to quantify changes in severity, amplitude, and frequency between the two postures.

Results

Although up to 9% of participants had exacerbated HT in midline, across the whole cohort, paired t-tests reveal no significant changes in overall severity (t = -0.23, p = 0.81), amplitude (t = -0.80, p = 0.43), and frequency (t = 1.48, p = 0.14) between the two postures.

Conclusions

When instructed to first let their head drift to its null point and then to hold their head straight at midline, most patient's changes in HT were below the thresholds one would expect from the sensitivity of clinical rating scales. Counter to common clinical impression, CMOR objectively showed that HT does not consistently increase at midline posture in comparison to the null posture.

To compare the inter-rater reliability (IRR) of five clinical rating scales for video-based assessment of hemifacial spasm (HFS) motor severity. We evaluated the video recordings of 45 HFS participants recruited through the Dystonia Coalition. In Round 1, six clinicians with expertise in HFS assessed the participants motor severity with five scales used to measure motor severity of HFS: the Jankovic rating scale (JRS), Hemifacial Spasm Grading Scale (HSGS), Samsung Medical Center (SMC) grading system for severity of HFS spasms (Lees scale), clinical grading of spasm intensity (Chens scale), and a modified version of the Abnormal Involuntary Movement Scale (Tuncs scale). In Round 2, clinicians rated the same cohort with simplified scale wording after consensus training. For each round, we evaluated the IRR using the intraclass correlation coefficient [ICC (2,1) single-rater, absolute-agreement, 2-way random model]. The scales exhibited IRR that ranged from poor to moderate; the mean ICCs were 0.41, 0.43, 0.47, 0.43, and 0.65 for the JRS, HSGS, Lees, Chens, and Tuncs scales, respectively, for Round 1. In Round 2, the corresponding IRRs increased to 0.63, 0.60, 0.59, 0.53, and 0.71. In both rounds, Tuncs scale exhibited the highest IRR. For clinical assessments of HFS motor severity based on video observations, we recommend using Tuncs scale because of its comparative reliability and because clinicians interpret the scale easily without modifications or the need for consensus training.

Objective

Deviated head posture is a defining characteristic of cervical dystonia (CD). Head posture severity is typically quantified with clinical rating scales such as the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS). Because clinical rating scales are inherently subjective, they are susceptible to variability that reduces their sensitivity as outcome measures. The variability could be circumvented with methods to measure CD head posture objectively. However, previously used objective methods require specialized equipment and have been limited to studies with a small number of cases. The objective of this study was to evaluate a novel software system-the Computational Motor Objective Rater (CMOR)-to quantify multi-axis directionality and severity of head posture in CD using only conventional video camera recordings.

Methods

CMOR is based on computer vision and machine learning technology that captures 3D head angle from video. We used CMOR to quantify the axial patterns and severity of predominant head posture in a retrospective, cross-sectional study of 185 patients with isolated CD recruited from 10 sites in the Dystonia Coalition.

Results

The predominant head posture involved more than one axis in 80.5% of patients and all three axes in 44.4%. CMOR's metrics for head posture severity correlated with severity ratings from movement disorders neurologists using both the TWSTRS-2 and an adapted version of the Global Dystonia Rating Scale (rho = 0.59-0.68, all p <0.001).

Conclusions

CMOR's convergent validity with clinical rating scales and reliance upon only conventional video recordings supports its future potential for large scale multisite clinical trials.

Objective

Dystonia is a complex movement disorder. Research progress has been difficult, particularly in developing widely effective therapies. This is a review of the current state of knowledge, research gaps, and proposed research priorities.

Methods

The NIH convened leaders in the field for a 2-day workshop. The participants addressed the natural history of the disease, the underlying etiology, the pathophysiology, relevant research technologies, research resources, and therapeutic approaches and attempted to prioritize dystonia research recommendations.

Results

The heterogeneity of dystonia poses challenges to research and therapy development. Much can be learned from specific genetic subtypes, and the disorder can be conceptualized along clinical, etiology, and pathophysiology axes. Advances in research technology and pooled resources can accelerate progress. Although etiologically based therapies would be optimal, a focus on circuit abnormalities can provide a convergent common target for symptomatic therapies across dystonia subtypes. The discussions have been integrated into a comprehensive review of all aspects of dystonia.

Conclusion

Overall research priorities include the generation and integration of high-quality phenotypic and genotypic data, reproducing key features in cellular and animal models, both of basic cellular mechanisms and phenotypes, leveraging new research technologies, and targeting circuit-level dysfunction with therapeutic interventions. Collaboration is necessary both for collection of large data sets and integration of different research methods.

Background

Head tremor (HT) is a common feature of cervical dystonia (CD), usually quantified by subjective observation. Technological developments offer alternatives for measuring HT severity that are objective and amenable to automation.

Objectives

Our objectives were to develop CMOR (Computational Motor Objective Rater; a computer vision-based software system) to quantify oscillatory and directional aspects of HT from video recordings during a clinical examination and to test its convergent validity with clinical rating scales.

Methods

For 93 participants with isolated CD and HT enrolled by the Dystonia Coalition, we analyzed video recordings from an examination segment in which participants were instructed to let their head drift to its most comfortable dystonic position. We evaluated peak power, frequency, and directional dominance, and used Spearman's correlation to measure the agreement between CMOR and clinical ratings.

Results

Power averaged 0.90 (SD 1.80) deg²/Hz, and peak frequency 1.95 (SD 0.94) Hz. The dominant HT axis was pitch (antero/retrocollis) for 50%, roll (laterocollis) for 6%, and yaw (torticollis) for 44% of participants. One-sided t-tests showed substantial contributions from the secondary (t = 18.17, p < 0.0001) and tertiary (t = 12.89, p < 0.0001) HT axes. CMOR's HT severity measure positively correlated with the HT item on the Toronto Western Spasmodic Torticollis Rating Scale-2 (Spearman's rho = 0.54, p < 0.001).

Conclusions

We demonstrate a new objective method to measure HT severity that requires only conventional video recordings, quantifies the complexities of HT in CD, and exhibits convergent validity with clinical severity ratings.

Background

Clinical characteristics of isolated idiopathic cervical dystonia such as onset site and spread to and from additional body regions have been addressed in single-site studies with limited data and incomplete or variable dissociation of focal and segmental subtypes. The objectives of this study were to characterize the clinical characteristics and demographics of isolated idiopathic cervical dystonia in the largest standardized multicenter cohort.

Methods

The Dystonia Coalition, through a consortium of 37 recruiting sites in North America, Europe, and Australia, recruited 1477 participants with focal (60.7%) or segmental (39.3%) cervical dystonia on examination. Clinical and demographic characteristics were evaluated in terms of the body region of dystonia onset and spread.

Results

Site of dystonia onset was: (1) focal neck only (78.5%), (2) focal onset elsewhere with later segmental spread to neck (13.3%), and (3) segmental onset with initial neck involvement (8.2%). Frequency of spread from focal cervical to segmental dystonia (22.8%) was consistent with prior reports, but frequency of segmental onset with initial neck involvement was substantially higher than the 3% previously reported. Cervical dystonia with focal neck onset, more than other subtypes, was associated with spread and tremor of any type. Sensory tricks were less frequent in cervical dystonia with segmental components, and segmental cervical onset occurred at an older age.

Conclusions

Subgroups had modest but significant differences in the clinical characteristics that may represent different clinical entities or pathophysiologic subtypes. These findings are critical for design and implementation of studies to describe, treat, or modify disease progression in idiopathic isolated cervical dystonia. © 2016 International Parkinson and Movement Disorder Society.