A NADH-accepting imine reductase variant: Immobilization and cofactor regeneration by oxidative deamination.

Engineering cofactor specificity of enzymes is a promising approach that can expand the application of enzymes for biocatalytic production of industrially relevant chemicals. Until now, only NADPH-dependent imine reductases (IREDs) are known. This limits their applications to reactions employing whole cells as a cost-efficient cofactor regeneration system. For applications of IREDs as cell-free catalysts, (i) we created an IRED variant showing an improved activity for NADH. With rational design we were able to identify four residues in the (R)-selective IRED from Streptomyces GF3587 (IR-Sgf3587), which coordinate the 2'-phosphate moiety of the NADPH cofactor. From a set of 15 variants, the highest NADH activity was caused by the single amino acid exchange K40A resulting in a 3-fold increased acceptance of NADH. (ii) We showed its applicability using an immobilisate obtained either from purified enzyme or from lysate using the EziG(™) carriers. Applying the variant and NADH, we reached 88% conversion in a preparative scale biotransformation when employing 4% (w/v) 2-methylpyrroline. (iii) We demonstrated a one-enzyme cofactor regeneration approach using the achiral amine N-methyl-3-aminopentanone as a hydrogen donor co-substrate.

Embolic and Hemodynamic Transcranial Doppler Characteristics in Patients with Acute Ischemic Stroke due to Carotid Occlusive Disease: Contribution to the Different Infarct Patterns on MRI.

BACKGROUND AND PURPOSE: Whether hemodynamic and/or embolic transcranial Doppler (TCD) features of internal carotid artery (ICA) stenosis contribute to the classification of stroke patterns on MRI. PATIENTS AND METHODS: Consecutive patients presenting symptomatic ≥50% ICA stenosis were included. Microembolic signals (MES) detection and measurement of cerebral vasoreactivity (VR) were performed by TCD. Only acute MRI lesions, territorial (TT) and/or borderzone (BZ) were considered. RESULTS: A total of 72 ICA stenoses, 27 (38%) moderate (50-69%), and 45 (62%) high grade (70-99%) were included. MRI lesions showed 32 (44%) pure TT, 20 (28%) pure BZ, and 20 (28%) mixed TT and BZ. Impaired VR was found more frequently among patients with higher degrees of stenoses (P < .001) whereas MES were similarly encountered in both groups (P = NS). Impaired VR was more common in the BZ (10/20, 50%) than in the TT group (9/32, 28%, P < .1) while MES were present in 47% (15/32) of patients with TT and in 30% (6/20, P < .1) of those with BZ lesions, in particular in cortical BZ infarcts (P < .02). CONCLUSION: Our findings suggest that TCD characteristics of the ICA stenosis contribute to better define stroke patterns on MRI in about one-third of the patients presenting with pure TT or BZ lesions.

18FDG-PET-CT: an imaging biomarker of high-risk carotid plaques. Correlation to symptoms and microembolic signals.

BACKGROUND AND PURPOSE: We investigated whether uptake of (18)fluoro-2-deoxy-d-glucose (18FDG) positron emission tomography-computed tomography (PET-CT) correlated to clinical symptoms and presence of microembolic signals (MES) detected by transcranial Doppler in patients with carotid stenosis. METHODS: 18FDG-PET-CT and MES detection was performed in consecutive patients with 50% to 99% symptomatic or asymptomatic carotid stenoses. Uptake index was defined by a target to background ratio (TBR) between maximum standardized uptake value of the carotid plaque and the mean standardized uptake value of the jugular veins. End points for analysis were presence of symptoms and presence of MES. RESULTS: We included 123 stenosis derived from 110 patients, 60 symptomatic and 63 asymptomatic. MES positive (+) lesions were found in 16%. TBR values were higher in symptomatic compared with asymptomatic (median 2.07 versus 1.78; P<0.0018) and in MES+ compared with MES- plaques (median 2.14 versus 1.86; P<0.008). TBR values were also higher in asymptomatic MES+ compared with MES- plaques (median 1.97 versus 1.76; P<0.03). The best TBR threshold value for symptomatic versus asymptomatic, for MES+ versus MES-, for symptomatic MES+ versus symptomatic or asymptomatic MES-, and for asymptomatic MES+ versus asymptomatic MES- plaques was 1.9. Sensitivity/specificity were, respectively, 56/77%, 73/63%, 79/64%, and 80/77%. We found a strong correlation between number of MES and TBR values (ρ 0.26; P=0.0043). CONCLUSIONS: 18FDG-PET-CT accurately detected high-risk carotid plaques. Also given its strong correlation to MES, 18FDG-PET-CT may be a useful tool in clinical practice.

Contrast-enhanced ultrasound imaging of carotid plaque neo-vascularization: accuracy of visual analysis.

The aim of our study was to evaluate whether neo-vascularization of the carotid plaque can be accurately assessed by visual analysis of contrast-enhanced ultrasound images and whether these findings correlate with intensity-over-time curve analysis (ITC) and histopathology. Patients with ≥50% symptomatic or ≥60% asymptomatic stenosis according to European Carotid Surgery Trial criteria were included. Four investigators evaluated contrast enhancement visually (three grades), with positive agreement when three or more investigators were unanimous. ITC analysis of contrast enhancement was performed in the plaque and in the lumen. Histopathology (microvessel density with CD34 + staining) was completed when endarterectomy was performed. Visual grading (33 patients, inter-observer agreement = 94%) correlated significantly with ITC analysis (p = 0.03). Histopathology (n = 19) revealed a larger CD34 + area in patients with grade 1/2 versus grade 0 (p = 0.03). Visual analysis of neo-vascularization by means of contrast-enhanced ultrasound imaging is accurate and reproducible, with significant correlations with ITC and histopathology.

Rivastigmine for HIV-associated neurocognitive disorders: a randomized crossover pilot study.

OBJECTIVE: To assess the efficacy and safety of rivastigmine for the treatment of HIV-associated neurocognitive disorders (HAND) in a cohort of long-lasting aviremic HIV+ patients. METHODS: Seventeen aviremic HIV+ patients with HAND were enrolled in a randomized, double-blind, placebo-controlled, crossover study to receive either oral rivastigmine (up to 12 mg/day for 20 weeks) followed by placebo (20 weeks) or placebo followed by rivastigmine. Efficacy endpoints were improvement on rivastigmine in the Alzheimer's disease assessment scale-cognitive subscale (ADAS-Cog) and individual neuropsychological scores of information processing speed, attention/working memory, executive functioning, and motor skills. Measures of safety included frequency and nature of adverse events and abnormalities on laboratory tests and on plasma concentrations of antiretroviral drugs. Analyses of variance with repeated measures were computed to look for treatment effects. RESULTS: There was no change on the primary outcome ADAS-Cog on drug. For secondary outcomes, processing speed improved on rivastigmine (trail making test A: F(1,13) = 5.57, p = 0.03). One measure of executive functioning just failed to reach significance (CANTAB spatial working memory [strategy]: F(1,13) = 3.94, p = 0.069). No other change was observed. Adverse events were frequent, but not different from those observed in other populations treated with rivastigmine. No safety issues were recorded. CONCLUSIONS: Rivastigmine in aviremic HIV+ patients with HAND seemed to improve psychomotor speed. A larger trial with the better tolerated transdermal form of rivastigmine is warranted. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that rivastigmine is ineffective for improving ADAS-Cog scores, but is effective in improving some secondary outcome measures in aviremic HIV+ patients with HAND.