Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites

Interaction with smart assistants using alternative and augmentative communication

Smart assistants utilize speech recognition, sensing, artificial intelligence, and networking technologies to enable improved human-machine interaction. However, there remain use cases where smart assistants are not easily usable by humans. For example, voice-activated assistants are not accessible to those that are hearing or speech impaired. Touchscreen based assistants are not usable by those who lack fine motor skills and/or reading ability. This disclosure adds to the modalities by which humans can control and communicate with smart assistants by enabling use of physical objects, facial expressions, gross motor skills, body movements, etc. to provide commands. Collectively, these techniques of control and communication are referred to as alternative and augmentative communication (AAC)

The relative contribution of target-site mutations in complex acaricide resistant phenotypes as assessed by marker assisted backcrossing in Tetranychus urticae

The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo

C-reactive protein testing to guide antibiotic prescribing for COPD exacerbations

BACKGROUND: Point-of-care testing of C-reactive protein (CRP) may be a way to reduce unnecessary use of antibiotics without harming patients who have acute exacerbations of chronic obstructive pulmonary disease (COPD). METHODS: We performed a multicenter, open-label, randomized, controlled trial involving patients with a diagnosis of COPD in their primary care clinical record who consulted a clinician at 1 of 86 general medical practices in England and Wales for an acute exacerbation of COPD. The patients were assigned to receive usual care guided by CRP point-of-care testing (CRP-guided group) or usual care alone (usual-care group). The primary outcomes were patient-reported use of antibiotics for acute exacerbations of COPD within 4 weeks after randomization (to show superiority) and COPD-related health status at 2 weeks after randomization, as measured by the Clinical COPD Questionnaire, a 10-item scale with scores ranging from 0 (very good COPD health status) to 6 (extremely poor COPD health status) (to show noninferiority). RESULTS: A total of 653 patients underwent randomization. Fewer patients in the CRP-guided group reported antibiotic use than in the usual-care group (57.0% vs. 77.4%; adjusted odds ratio, 0.31; 95% confidence interval [CI], 0.20 to 0.47). The adjusted mean difference in the total score on the Clinical COPD Questionnaire at 2 weeks was −0.19 points (two-sided 90% CI, −0.33 to −0.05) in favor of the CRP-guided group. The antibiotic prescribing decisions made by clinicians at the initial consultation were ascertained for all but 1 patient, and antibiotic prescriptions issued over the first 4 weeks of follow-up were ascertained for 96.9% of the patients. A lower percentage of patients in the CRP-guided group than in the usual-care group received an antibiotic prescription at the initial consultation (47.7% vs. 69.7%, for a difference of 22.0 percentage points; adjusted odds ratio, 0.31; 95% CI, 0.21 to 0.45) and during the first 4 weeks of follow-up (59.1% vs. 79.7%, for a difference of 20.6 percentage points; adjusted odds ratio, 0.30; 95% CI, 0.20 to 0.46). Two patients in the usual-care group died within 4 weeks after randomization from causes considered by the investigators to be unrelated to trial participation. CONCLUSIONS: CRP-guided prescribing of antibiotics for exacerbations of COPD in primary care clinics resulted in a lower percentage of patients who reported antibiotic use and who received antibiotic prescriptions from clinicians, with no evidence of harm

General practitioner use of a C-reactive protein point-of-care test to help target antibiotic prescribing in patients with acute exacerbations of chronic obstructive pulmonary disease (the PACE study) : study protocol for a randomised controlled trial

BACKGROUND: Most patients presenting with acute exacerbations of chronic obstructive pulmonary disease (AECOPD) in primary care are prescribed an antibiotic, which may not always be appropriate and may cause harm. C-reactive protein (CRP) is an acute-phase biomarker that can be rapidly measured at the point of care and may predict benefit from antibiotic treatment in AECOPD. It is not clear whether the addition of a CRP point-of-care test (POCT) to clinical assessment leads to a reduction in antibiotic consumption without having a negative impact on COPD health status. METHODS/DESIGN: This is a multicentre, individually randomised controlled trial (RCT) aiming to include 650 participants with a diagnosis of AECOPD in primary care. Participants will be randomised to be managed according to usual care (control) or with the addition of a CRP POCT to guide antibiotic prescribing. Antibiotic consumption for AECOPD within 4 weeks post randomisation and COPD health status (total score) measured by the Clinical COPD Questionnaire (CCQ) at 2 weeks post randomisation will be co-primary outcomes. Primary analysis (by intention-to-treat) will determine differences in antibiotic consumption for superiority and COPD health status for non-inferiority. Secondary outcomes include: COPD health status, CCQ domain scores, use of other COPD treatments (weeks 1, 2 and 4), EQ-5D utility scores (weeks 1, 2 and 4 and month 6), disease-specific, health-related quality of life (HRQoL) at 6 months, all-cause antibiotic consumption (antibiotic use for any condition) during first 4 weeks post randomisation, total antibiotic consumption (number of days during first 4 weeks of antibiotic consumed for AECOPD/any reason), antibiotic prescribing at the index consultation and during following 4 weeks, adverse effects over the first 4 weeks, incidence of pneumonia (weeks 4 and 6 months), health care resource use and cost comparison over the 6 months following randomisation. Prevalence and resistance profiles of bacteria will be assessed using throat and sputum samples collected at baseline and 4-week follow-up. A health economic evaluation and qualitative process evaluation will be carried out. DISCUSSION: If shown to be effective (i.e. leads to a reduction in antibiotic use with no worse COPD health status), the use of the CRP POCT could lead to better outcomes for patients with AECOPD and help reduce selective pressures driving the development of antimicrobial resistance. PACE will be one of the first studies to evaluate the cost-effectiveness of a POCT biomarker to guide clinical decision-making in primary care on patient-reported outcomes, antibiotic prescribing and antibiotic resistance for AECOPD. TRIAL REGISTRATION: ISRCTN registry, ID: ISRCTN24346473 . Registered on 20 August 2014

C-reactive protein point-of-care testing for safely reducing antibiotics for acute exacerbations of chronic obstructive pulmonary disease: the PACE RCT

Most patients presenting with acute exacerbations of chronic obstructive pulmonary disease (AECOPD) in primary care are prescribed antibiotics, but these may not be beneficial, and they can cause side effects and increase the risk of subsequent resistant infections. Point-of-care tests (POCTs) could safely reduce inappropriate antibiotic prescribing and antimicrobial resistance. To determine whether or not the use of a C-reactive protein (CRP) POCT to guide prescribing decisions for AECOPD reduces antibiotic consumption without having a negative impact on chronic obstructive pulmonary disease (COPD) health status and is cost-effective. A multicentre, parallel-arm, randomised controlled open trial with an embedded process, and a health economic evaluation. General practices in Wales and England. A UK NHS perspective was used for the economic analysis. Adults (aged ≥ 40 years) with a primary care diagnosis of COPD, presenting with an AECOPD (with at least one of increased dyspnoea, increased sputum volume and increased sputum purulence) of between 24 hours' and 21 days' duration. CRP POCTs to guide antibiotic prescribing decisions for AECOPD, compared with usual care (no CRP POCT), using remote online randomisation. Patient-reported antibiotic consumption for AECOPD within 4 weeks post randomisation and COPD health status as measured with the Clinical COPD Questionnaire (CCQ) at 2 weeks. For the economic evaluation, patient-reported resource use and the EuroQol-5 Dimensions were included. In total, 653 participants were randomised from 86 general practices. Three withdrew consent and one was randomised in error, leaving 324 participants in the usual-care arm and 325 participants in the CRP POCT arm. Antibiotics were consumed for AECOPD by 212 out of 274 participants (77.4%) and 150 out of 263 participants (57.0%) in the usual-care and CRP POCT arm, respectively [adjusted odds ratio 0.31, 95% confidence interval (CI) 0.20 to 0.47]. The CCQ analysis comprised 282 and 281 participants in the usual-care and CRP POCT arms, respectively, and the adjusted mean CCQ score difference at 2 weeks was 0.19 points (two-sided 90% CI -0.33 to -0.05 points). The upper limit of the CI did not contain the prespecified non-inferiority margin of 0.3. The total cost from a NHS perspective at 4 weeks was £17.59 per patient higher in the CRP POCT arm (95% CI -£34.80 to £69.98;  = 0.408). The mean incremental cost-effectiveness ratios were £222 per 1% reduction in antibiotic consumption compared with usual care at 4 weeks and £15,251 per quality-adjusted life-year gained at 6 months with no significant changes in sensitivity analyses. Patients and clinicians were generally supportive of including CRP POCT in the assessment of AECOPD. A CRP POCT diagnostic strategy achieved meaningful reductions in patient-reported antibiotic consumption without impairing COPD health status or increasing costs. There were no associated harms and both patients and clinicians valued the diagnostic strategy. Implementation studies that also build on our qualitative findings could help determine the effect of this intervention over the longer term. Current Controlled Trials ISRCTN24346473. This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in ; Vol. 24, No. 15. See the NIHR Journals Library website for further project information. People with chronic obstructive pulmonary disease (COPD) often experience flare-ups known as acute exacerbations of chronic obstructive pulmonary disease. Antibiotics are prescribed for most flare-ups, but they do not always benefit patients and may cause harm, such as side effects or subsequent infections that are resistant. Rapid point-of-care tests (POCTs) can be used to help determine when antibiotics are more likely to be needed. C-reactive protein (CRP) is a marker of inflammation that can be measured with a POCT. Patients with flare-ups and a low CRP value are less likely to benefit from antibiotics. The PACE trial asked whether or not measuring CRP with a POCT could lead to fewer antibiotics being consumed for flare-ups, without having negative effects for patients. We aimed to recruit 650 patients with a COPD flare-up from primary care. Patients were randomly assigned to either (1) usual care with the addition of a CRP POCT, or (2) usual care without the addition of the test. Antibiotic use over the first 4 weeks and patients’ self-assessment of their health 2 weeks after enrolment were measured in both groups. Patients in the CRP test group used fewer antibiotics than those managed as usual, and had improved patient-reported outcomes. Costs were a little higher in the CRP POCT group. Interviews with patients and clinicians found that they appreciated the CRP test being included in the decision-making process.This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessmentprogramme and will be published in full inHealth Technology Assessment; Vol. 24, No. 15. See the NIHRJournals Library website for further project informatio

Supramolecular behaviour and fluorescence of rhodamine-functionalised ROMP polymers

Inherently fluorescent polymers are of interest in materials and medicine. We report a ring-opening metathesis polymerisation (ROMP) platform for creation of amphiphilic block copolymers in which one block is formed from rhodamine B-containing monomers. The polymers self-assemble into well-defined micelles which are able to sequester molecular dyes and further interact with them by energy transfer. Despite incorporating a cationic dye known to bind DNA, the polymer micelles do not interact with DNA, indicating that they are potentially safe for use in bioanalytical applications

Associations with antibiotic prescribing for acute exacerbation of COPD in primary care: secondary analysis of a randomised controlled trial

Background C-reactive protein (CRP) point-of-care testing can reduce antibiotic use in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in primary care, without compromising patient care. Further safe reductions may be possible. Aim To investigate the associations between presenting features and antibiotic prescribing in patients with AECOPD in primary care. Design and setting Secondary analysis of a randomised controlled trial of participants presenting with AECOPD in primary care (the PACE trial). Method Clinicians collected participants’ demographic features, comorbid illnesses, clinical signs, and symptoms. Antibiotic prescribing decisions were made after participants were randomised to receive a point-of-care CRP measurement or usual care. Multivariable regression models were fitted to explore the association between patient and clinical features and antibiotic prescribing, and extended to further explore any interactions with CRP measurement category (CRP not measured, CRP <20 mg/l, or CRP ≥20 mg/l). Results A total of 649 participants from 86 general practices across England and Wales were included. Odds of antibiotic prescribing were higher in the presence of clinician-recorded crackles (adjusted odds ratio [AOR] = 5.22, 95% confidence interval [CI] = 3.24 to 8.41), wheeze (AOR = 1.64, 95% CI = 1.07 to 2.52), diminished vesicular breathing (AOR = 2.95, 95% CI = 1.70 to 5.10), or clinician-reported evidence of consolidation (AOR = 34.40, 95% CI = 2.84 to 417.27). Increased age was associated with lower odds of antibiotic prescribing (AOR per additional year increase = 0.98, 95% CI = 0.95 to 1.00), as was the presence of heart failure (AOR = 0.32, 95% CI = 0.12 to 0.85). Conclusion Several demographic features and clinical signs and symptoms are associated with antibiotic prescribing in AECOPD. Diagnostic and prognostic value of these features may help identify further safe reductions

Symbol Grounding Transfer with Hybrid Self-Organizing/Supervised Neural Networks

This paper reports new simulations on an extended neural network model for the transfer of symbol grounding. It uses a hybrid and modular connectionist model, consisting of an unsupervised, self-organizing map for stimulus classification and a supervised network for category acquisition and naming. The model is based on a psychologicallyplausible view of symbolic communication, where unsupervised concept formation precedes the supervised acquisition of category names. The simulation results demonstrate that grounding is transferred from symbols denoting object properties to newly acquired symbols denoting the object as a whole. The implications for cognitive models integrating neural networks and multi-agent systems are discussed. I