Transgenic Drosophila lines for LexA-dependent gene and growth regulation.

Conditional expression of short hairpin RNAs with binary genetic systems is an indispensable tool for studying gene function. Addressing mechanisms underlying cell-cell communication in vivo benefits from simultaneous use of 2 independent gene expression systems. To complement the abundance of existing Gal4/UAS-based resources in Drosophila, we and others have developed LexA/LexAop-based genetic tools. Here, we describe experimental and pedagogical advances that promote the efficient conversion of Drosophila Gal4 lines to LexA lines, and the generation of LexAop-short hairpin RNA lines to suppress gene function. We developed a CRISPR/Cas9-based knock-in system to replace Gal4 coding sequences with LexA, and a LexAop-based short hairpin RNA expression vector to achieve short hairpin RNA-mediated gene silencing. We demonstrate the use of these approaches to achieve targeted genetic loss-of-function in multiple tissues. We also detail our development of secondary school curricula that enable students to create transgenic flies, thereby magnifying the production of well-characterized LexA/LexAop lines for the scientific community. The genetic tools and teaching methods presented here provide LexA/LexAop resources that complement existing resources to study intercellular communication coordinating metazoan physiology and development.

Optimizing the risk estimation after a transient ischaemic attack - the ABCDE⊕ score.

BACKGROUND AND PURPOSE: The risk of stroke after a transient ischaemic attack (TIA) can be predicted by scores incorporating age, blood pressure, clinical features, duration (ABCD-score), and diabetes (ABCD2-score). However, some patients have strokes despite a low predicted risk according to these scores. We designed the ABCDE+ score by adding the variables 'etiology' and ischaemic lesion visible on diffusion-weighted imaging (DWI) -'DWI-positivity'- to the ABCD-score. We hypothesized that this refinement increases the predictability of recurrent ischaemic events. METHODS: We performed a prospective cohort study amongst all consecutive TIA patients in a university hospital emergency department. Area under the computed receiver-operating curves (AUCs) were used to compare the predictive values of the scores with regard to the outcome stroke or recurrent TIA within 90 days. RESULTS: Amongst 248 patients, 33 (13.3%, 95%-CI 9.3-18.2%) had a stroke (n = 13) or a recurrent TIA (n = 20). Patients with recurrent ischaemic events more often had large-artery atherosclerosis as the cause for TIA (46% vs. 14%, P < 0.001) and positive DWI (61% vs. 35%; P = 0.01) compared with patients without recurrent events. Patients with and those without events did not differ with regard to age, clinical symptoms, duration, blood pressure, risk factors, and stroke preventive treatment. The comparison of AUCs [95%CI] showed superiority of the ABCDE+ score (0.67[0.55-0.75]) compared to the ABCD(2) -score (0.48[0.37-0.58]; P = 0.04) and a trend toward superiority compared to the ABCD-score (0.50[0.40-0.61]; P = 0.07). CONCLUSION: In TIA patients, the addition of the variables 'etiology' and 'DWI-positivity' to the ABCD-score seems to enhance the predictability of subsequent cerebral ischaemic events.

Significance of microbleeds in patients with transient ischaemic attack.

BACKGROUND AND PURPOSE: The aim of this study was to determine the prognostic significance of microbleeds in TIA-patients. In patients with a transient ischaemic attack (TIA), the prognostic value of microbleeds is unknown. METHODS: In 176 consecutive TIA patients, the number, size, and location of microbleeds with or without acute ischaemic lesions were assessed. We compared microbleed-positive and microbleed-negative patients with regard to the end-point stroke within 3 months. RESULTS: Four of the seven patients with subsequent stroke had microbleeds. Microbleed-positive patients had a higher risk for stroke [odds ratios (OR) 8.91, 95% CI 1.87-42.51, P<0.01] than those without microbleeds. Microbleed-positive patients with accompanying acute ischaemic lesions had a higher stroke risk than those with neither an acute ischaemia nor a microbleed (OR 6.20, 95% CI 1.10-35.12; P=0.04). CONCLUSION: Microbleeds alone or in combination with acute ischaemic lesions may increase the risk for subsequent ischaemic stroke after TIA within 3 months.

Stress hormones predict cerebrovascular re-events after transient ischemic attacks.

BACKGROUND: TIA is a strong predictor of subsequent stroke. The hypothalamic stress hormone copeptin is an accurate prognostic marker in acute ischemic stroke. This study assessed prognostic reliability of 2 distinct stress hormones, copeptin and cortisol, for the risk stratification of re-events in patients with TIA. METHODS: We conducted a prospective study in patients admitted to the emergency department with a TIA. Clinical risk scoring using the ABCD2 score was determined and both hormones were measured in plasma on admission. The primary endpoint was a cerebrovascular re-event within 90 days. RESULTS: We included 107 consecutive patients with TIA. Re-events occurred in 10 patients (9%). Copeptin levels were higher in patients with a re-event compared with patients without re-event (p = 0.02), in contrast to cortisol (p = 0.53). Copeptin revealed a higher area under the receiver operating characteristics curve (AUC) to predict re-events compared to the ABCD2 score (AUC of 0.73 vs 0.43; p < 0.01) and improved its prognostic accuracy (AUC of combined model of 0.77; p = 0.002). CONCLUSION: Measurement of plasma copeptin but not cortisol levels in patients with TIA provides additional prognostic information beyond the ABCD2 clinical risk score alone. If confirmed in future studies, routine copeptin measurement may be an additional tool for risk stratification and targeted resource allocation after TIA.