Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in-vitro enzyme activity with clinical phenotype in argininosuccinic aciduria

Background

The urea cycle defect argininosuccinate lyase (ASL) deficiency has a large spectrum of presentations from highly severe to asymptomatic. Enzyme activity assays in red blood cells or fibroblasts, although diagnostic of the deficiency, fail to discriminate between severe, mild or asymptomatic cases. Mutation/phenotype correlation studies are needed to characterize the effects of individual mutations on the activity of the enzyme.

Methods

Bacterial in-vitro expression studies allowed the enzyme analysis of purified mutant ASL proteins p.I100T (c.299?T?>?C), p.V178M (c.532?G?>?A), p.E189G (c.566A?>?G), p.Q286R (c.857A?>?G), p.K315E (c.943A?>?G), p.R379C (c.1135?C?>?T) and p.R385C (c.1153?C?>?T) in comparison to the wildtype protein.

Results

In the bacterial in-vitro expression system, ASL wild-type protein was successfully expressed. The known classical p.Q286R, the novel classical p.K315E and the known mutations p.I100T, p.E189G and p.R385C, which all have been linked to a mild phenotype, showed no significant residual activity. There was some enzyme activity detected with the p.V178M (5 % of wild-type) and p.R379C (10 % of wild-type) mutations in which K_m values for argininosuccinic acid differed significantly from the wild-type ASL protein.

Conclusion

The bacterially expressed enzymes proved that the mutations found in patients and studied here indeed are detrimental. However, as in the case of red cell ASL activity assays, some mutations found in genetically homozygous patients with mild presentations resulted in virtual loss of enzyme activity in the bacterial system, suggesting a more protective environment for the mutant enzyme in the liver than in the heterologous expression system and/or in the highly dilute assays utilized here.     

Post-subarachnoid Hemorrhage Vasospasm in Patients with Primary Headache Disorders

Background

Altered cerebral vasomotor reactivity leading to vasospasm can be seen both in patients with primary headache disorders (PHD) and in patients with subarachnoid hemorrhage (SAH). The pathogenesis of vasospasm in post-SAH patients and in headache disorder sufferers may be related. To address this hypothesis, we analyzed a large cohort of SAH patients to determine whether a diagnosis of PHD predisposes to vasospasm, delayed cerebral ischemia, or worsened clinical outcome.

Methods

Prospectively collected data from patients enrolled in the SAH Outcomes Project between 1996 and 2006 were analyzed. Patients were segregated based on whether they had a diagnosis of PHD or not and were subsequently compared for differences in clinical and radiographic outcome.

Results

A total of 921 SAH patients were analyzed, 265 of which had a diagnosis of PHD. In total, symptomatic vasospasm was seen in 17 %, while angiographic vasospasm was seen in 28 %. Vasospasm rates were similar among patients with a PHD and in those without a PHD (p > 0.05). However, on multivariate analysis new ischemic infarcts were more common in patients with a PHD as compared to patients without a PHD (p = 0.015). Functional outcomes at 3 months were similar among PHD and non-PHD patients (p > 0.05).

Conclusion

A history of PHD is associated with an increased rate of ischemic infarcts during admission for SAH. Increased rates of vasospasm within small cerebral blood vessels may be implicated. Further studies are warranted to more closely link the mechanisms of vasospasm in PHD and SAH patients.     

Cerebrovascular autoregulation in critically ill patients during continuous hemodialysis

Purpose

In chronic renal failure, intermittent hemodialysis decreases cerebral blood flow velocity (CBFV); however, in critically ill patients with acute renal failure, the effect of continuous venovenous hemodialysis (CVVHD) on CBFV and cerebrovascular autoregulation (AR) is unknown. Therefore, a study was undertaken to investigate the potential effect of CVVHD on CBFV and AR in patients with acute renal failure.

Methods

This cohort study investigated 20 patients with acute renal failure who required CVVHD. In these patients, the CBFV and index of AR (Mx) were measured using transcranial Doppler before and during CVVHD.

Results

The median Mx values at baseline were 0.33 [interquartile range (IQR): 0.02-0.55], and during CVVHD, they were 0.20 [0.07-0.40]. The differences in Mx (CVVHD – baseline) was (median [IQR]) ?0.015 [?0.19-0.05], 95% confidence interval (CI) ?0.16 to 0.05. The Mx was > 0.3 in 11/20 patients at baseline measurement. Six of these patients recovered to Mx < 0.3 during CVVHD. The CBFV was (median [IQR]) 47 [36-59] cm·sec^?1 at baseline and 49 [36-66] cm·sec^?1 during CVVHD. The difference of CBFV was 0.0 [?4 - 2.7], 95% CI ?2.5 to 4.2.

Conclusion

Compared with patients with intermittent hemodialysis, CVVHD did not influence CBFV and AR in critically ill patients with acute renal failure, possibly due to lower extracorporeal blood flow, slower change of plasma osmolarity, and a lower fluid extraction rate. In a subgroup of patients with sepsis, the AR was impaired at baseline in more than half of the patients, and this was reversed during CVVHD. The trial was registered at ClinicalTrials.gov ID: NCT01376531.     

Emergency Department Utilization of Adolescents and Young Adults with Autism Spectrum Disorder

Journal of Autism and Developmental Disorders - This study examined emergency department (ED) utilization by adolescents and young adults, 12–30&nbsp;years of age (AYA) with autism...     

25 Jahre Notfall+Rettungsmedizin – reine Männersache?

Notfall + Rettungsmedizin -     

Pitch Discrimination in Musicians and Non-Musicians: Effects of Harmonic Resolvability and Processing Effort

Musicians typically show enhanced pitch discrimination abilities compared to non-musicians. The present study investigated this perceptual enhancement behaviorally and objectively for resolved and unresolved complex tones to clarify whether the enhanced performance in musicians can be ascribed to increased peripheral frequency selectivity and/or to a different processing effort in performing the task. In a first experiment, pitch discrimination thresholds were obtained for harmonic complex tones with fundamental frequencies (F0s) between 100 and 500 Hz, filtered in either a low- or a high-frequency region, leading to variations in the resolvability of audible harmonics. The results showed that pitch discrimination performance in musicians was enhanced for resolved and unresolved complexes to a similar extent. Additionally, the harmonics became resolved at a similar F0 in musicians and non-musicians, suggesting similar peripheral frequency selectivity in the two groups of listeners. In a follow-up experiment, listeners’ pupil dilations were measured as an indicator of the required effort in performing the same pitch discrimination task for conditions of varying resolvability and task difficulty. Pupillometry responses indicated a lower processing effort in the musicians versus the non-musicians, although the processing demand imposed by the pitch discrimination task was individually adjusted according to the behavioral thresholds. Overall, these findings indicate that the enhanced pitch discrimination abilities in musicians are unlikely to be related to higher peripheral frequency selectivity and may suggest an enhanced pitch representation at more central stages of the auditory system in musically trained listeners.