Rescue of Murine F508del CFTR Activity in Native Intestine by Low Temperature and Proteasome Inhibitors

Most patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cells. New therapies are therefore aimed at improving the folding and trafficking of F508del CFTR, (CFTR correctors) or at enhancing the open probability of the CFTR chloride channel (CFTR potentiators). Preventing premature breakdown of F508del CFTR is an alternative or additional strategy, which is investigated in this study. We established an ex vivo assay for murine F508del CFTR rescue in native intestinal epithelium that can be used as a pre-clinical test for candidate therapeutics. Overnight incubation of muscle stripped ileum in modified William''s E medium at low temperature (26°C), and 4 h or 6 h incubation at 37°C with different proteasome inhibitors (PI: ALLN, MG-132, epoxomicin, PS341/bortezomib) resulted in fifty to hundred percent respectively of the wild type CFTR mediated chloride secretion (forskolin induced short-circuit current). The functional rescue was accompanied by enhanced expression of the murine F508del CFTR protein at the apical surface of intestinal crypts and a gain in the amount of complex-glycosylated CFTR (band C) up to 20% of WT levels. Sustained rescue in the presence of brefeldin A shows the involvement of a post-Golgi compartment in murine F508del CFTR degradation, as was shown earlier for its human counterpart. Our data show that proteasome inhibitors are promising candidate compounds for improving rescue of human F508del CFTR function, in combination with available correctors and potentiators.     

Rapid kinetics of glucose uptake inSaccharomyces cerevisiae

In a multiple deletion mutanthxt1Δhxt2Δhxt3Δ hxt4Δsnf3Δ ofSaccharomyces cerevisiae growing on 2 % glucose, high-affinity glucose-uptake (lowK _m) was exhibited throughout growth on glucose in contrast to the wild-type, which exhibited the usual low-affinity to high-affinity transition as the glucose in the medium was consumed. elevated levels of invertase activity throughout growth on glucose, in this mutant as compared to the wild-type, indicate that glucose repression may be impaired. Howver, in a mutant containing only theHXT2 gene (hxt1Δhxt3Δhxt4Δ snf3Δ), invertase levels were similar to those in the wild-type. It is likely, therefore, that some of these putative glucose transporters, such asHXT2, also have regulatory roles in cellular metabolism. In triple hexose-kinase mutants, rapid (200-ms) measurements of initial glucose-uptake revealed high-affinity glucose uptake (K _m approx. 2 mmol/L) while measurements on the slower 5-s scale clearly demonstrate that uptake is not linear over this longer period. These results suggest that this high-affinity component does not require a functional hexose-kinase.     

Efficacy of carnitine in the treatment of children with attention-deficit hyperactivity disorder

To determine safety and the efficacy of carnitine treatment in children with attention-deficit hyperactivity disorder (ADHD). The ADHD behavior was observed by parents completing the Child Behavior Checklist (CBCL) and by teachers completing the Conners teacher-rating score, in a randomized, double-blind, placebo-controlled double-crossover trial. In 13/24 boys receiving carnitine, home behavior improved as assessed with the CBCL total score (P < 0.02). In 13/24 boys, school behavior improved as assessed with the Conners teacher-rating score (P < 0.05). Before treatment, the CBCL total and sub-scores were significantly different from those of normal Dutch boys (P < 0.0001). Responders showed a significant improvement of the CBCL total scores compared to baseline (P < 0.0001). In the majority of boys no side effects were seen. At baseline and after carnitine treatment, responders showed higher levels of plasma-free carnitine (P < 0.03) and acetylcarnitine (P < 0.05). Compared to baseline, the carnitine treatment caused in the responsive patients a decrease of 20-65% (8-48 points) as assessed by the CBCL total problem rating scale. Treatment with carnitine significantly decreased the attention problems and aggressive behavior in boys with ADHD.     

Coupling between MRI-assessed regional aortic pulse wave velocity and diameters in patients with thoracic aortic aneurysm: a feasibility study

Aims

Thoracic aortic aneurysm (TAA) is potentially life-threatening and requires close follow-up to prevent aortic dissection. Aortic stiffness and size are considered to be coupled. Regional aortic stiffness in patients with TAA is unknown. We aimed to evaluate coupling between regional pulse wave velocity (PWV), a marker of vascular stiffness, and aortic diameter in TAA patients.

Methods

In 40 TAA patients (59 ± 13 years, 28 male), regional aortic diameters and regional PWV were assessed by 1.5 T MRI. The incidence of increased diameter and PWV were determined for five aortic segments (S1, ascending aorta; S2, aortic arch; S3, thoracic descending aorta; S4, suprarenal and S5, infrarenal abdominal aorta). In addition, coupling between regional PWV testing and aortic dilatation was evaluated and specificity and sensitivity were assessed.

Results

Aortic diameter was 44 ± 5 mm for the aortic root and 39 ± 5 mm for the ascending aorta. PWV was increased in 36 (19 %) aortic segments. Aortic diameter was increased in 28 (14 %) segments. Specificity of regional PWV testing for the prediction of increased regional diameter was ≥ 84 % in the descending thoracic to abdominal aorta and ≥ 68 % in the ascending aorta and aortic arch.

Conclusion

Normal regional PWV is related to absence of increased diameter, with high specificity in the descending thoracic to abdominal aorta and moderate results in the ascending aorta and aortic arch.     

Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established.     

Loneliness and Hypervigilance to Social Cues in Females: An Eye-Tracking Study

The goal of the present study was to examine whether lonely individuals differ from nonlonely individuals in their overt visual attention to social cues. Previous studies showed that loneliness was related to biased post-attentive processing of social cues (e.g., negative interpretation bias), but research on whether lonely and nonlonely individuals also show differences in an earlier information processing stage (gazing behavior) is very limited. A sample of 25 lonely and 25 nonlonely students took part in an eye-tracking study consisting of four tasks. We measured gazing (duration, number of fixations and first fixation) at the eyes, nose and mouth region of faces expressing emotions (Task 1), at emotion quadrants (anger, fear, happiness and neutral expression) (Task 2), at quadrants with positive and negative social and nonsocial images (Task 3), and at the facial area of actors in video clips with positive and negative content (Task 4). In general, participants tended to gaze most often and longest at areas that conveyed most social information, such as the eye region of the face (T1), and social images (T3). Participants gazed most often and longest at happy faces (T2) in still images, and more often and longer at the facial area in negative than in positive video clips (T4). No differences occurred between lonely and nonlonely participants in their gazing times and frequencies, nor at first fixations at social cues in the four different tasks. Based on this study, we found no evidence that overt visual attention to social cues differs between lonely and nonlonely individuals. This implies that biases in social information processing of lonely individuals may be limited to other phases of social information processing. Alternatively, biased overt attention to social cues may only occur under specific conditions, for specific stimuli or for specific lonely individuals.     

Testing for Starch,Respiring Tissues,and Vascular Bundles: Inquiry-Based Seed and Stem Anatomy Labs

In this article, the authors present lab exercises in which students use the microscope to study cells. After learning how to recognize the parts of a cell, differentiate between types of cells, and identify the nucleus, the students study blood samples from both familiar and “unknown” animals. The activities allow students to work independently and to become more proficient at microscope use. Students also gain a deeper understanding of the more “invisible” world of science.