Electrophysiological evaluation of Cystic Fibrosis Conductance Transmembrane Regulator (CFTR) expression in human monocytes

Background

Cystic fibrosis is caused by mutations of CFTR gene, a protein kinase A-activated anion channel, and is associated to a persistent and excessive chronic lung inflammation, suggesting functional alterations of immune cells. Leukocytes express detectable levels of CFTR but the molecule has not been fully characterized in these cells.

Methods

Freshly isolated monocytes from healthy individuals and CF patients were assessed by protein expression, single cell electrophysiological and membrane depolarization assays.

Results

We recorded chloride currents by patch clamp in healthy monocytes, after the administration of a CFTR stimulus. Currents were sensitive to a specific blocker of the CFTR channel, CFTR_inh-172 and were absent in CF monocytes. Next, we evaluated the effects of ex vivo exposure of monocytes from cystic fibrosis patients carrying the F508del mutation to a chemical corrector, Vertex-325. We found an increase in CFTR expression by confocal microscopy and a recovery of CFTR function by both patch clamp and single cell fluorescence analysis.

Conclusions

We confirm the expression of functional CFTR in human monocytes and demonstrate that blood monocytes can represent an adequate source of primary cells to assess new therapies and define diagnosis of CF.

General significance

Tests to evaluate CFTR functional abnormalities in CF disease might greatly benefit from the availability of a convenient source of primary cells. This electrophysiological study promotes the use of monocytes as a minimally invasive tool to study and monitor CFTR function in individual patients.     

Assembly and annotation of full mitochondrial genomes for the corn rootworm species, Diabrotica virgifera virgifera and Diabroticabarberi (Insecta: Coleoptera: Chrysomelidae), using Next Generation Sequence data

Complete mitochondrial genomes for two corn rootworm species, Diabrotica virgifera virgifera (16,747 bp) and Diabrotica barberi (16,632; Insecta: Coleoptera: Chrysomelidae), were assembled from Illumina HiSeq2000 read data. Annotation indicated that the order and orientation of 13 protein coding genes (PCGs), and 22 tRNA and 2 rRNA sequences were in typical of insect mitochondrial genomes. Non-standard nad4 and cox3 stop codons were composed of single T nucleotides and likely completed by adenylation, and atypical TTT start codons was predicted for both D. v. virgifera and D. barberinad1 genes. The D. v. virgifera and D. barberi haplotypes showed 819 variable nucleotide positions within PCG regions (7.36% divergence), which suggest that speciation may have occurred ~ 3.68 million years ago assuming a linear rate of short-term substitution. Phylogenetic analyses of Coleopteran MtD genome show clustering based on family level, and may have the capacity to resolve the evolutionary history within this Order of insects.     

Characterization of western corn rootworm (Coleoptera: Chrysomelidae) population dynamics in relation to landscape attributes

Abstract 1 The western corn rootworm (WCR), Diabrotica virgifera virgifera Leconte (Coleoptera: Chrysomelidae), creates economic and environmental concerns in the Corn Belt region of the U.S.A. To supplement the population control tactics of the Areawide Pest Management Program in Brookings, South Dakota, Geographical Information Systems (GIS) were used to examine the spatial relationships from 1997 to 2001 between WCR population dynamics, habitat structure, soil texture and elevation. 2 Using the inverse distance weighted interpolation technique, WCR population density maps were created from georeferenced emergence and post‐emergence traps placed in maize fields. For each year, these maps were overlaid with vegetation, soil and elevation maps to search for quantitative relationships. 3 Through visual interpretation and correlation analysis, shifts in landscape structure, such as size, number and arrangement of patches, were shown to associate with WCR population abundance and distribution in varying degrees. Contingency analysis showed that WCR population abundance is associated with soil texture and elevation. 4 An understanding of the interactions between WCR population dynamics and landscape variables provides information to pest managers, and this can be used to identify patterns in the landscape that promote high insect population density patches to improve pest management strategies.     

Marginal analyses of clustered data when cluster size is informative

We propose a new approach to fitting marginal models to clustered data when cluster size is informative. This approach uses a generalized estimating equation (GEE) that is weighted inversely with the cluster size. We show that our approach is asymptotically equivalent to within-cluster resampling (Hoffman, Sen, and Weinberg, 2001, Biometrika 73, 13-22), a computationally intensive approach in which replicate data sets containing a randomly selected observation from each cluster are analyzed, and the resulting estimates averaged. Using simulated data and an example involving dental health, we show the superior performance of our approach compared to unweighted GEE, the equivalence of our approach with WCR for large sample sizes, and the superior performance of our approach compared with WCR when sample sizes are small.