Treatment of Ionic Strength in Biomolecular Simulations of Charged Lipid Bilayers.

Biological membranes are complex systems that have recently attracted a significant scientific interest. Due to the presence of many different anionic lipids, these membranes are usually negatively charged and sensitive to pH. The protonation states of lipids and the ion distribution close to the bilayer are two of the main challenges in biomolecular simulations of these systems.

GRYFUN: a web application for GO term annotation visualization and analysis in protein sets.

Functional context for biological sequence is provided in the form of annotations. However, within a group of similar sequences there can be annotation heterogeneity in terms of coverage and specificity. This in turn can introduce issues regarding the interpretation of actual functional similarity and overall functional coherence of such a group.

New pharmacological approaches for cystic fibrosis: promises, progress, pitfalls.

With the discovery of the CFTR gene in 1989, the search for therapies to improve the basic defects of cystic fibrosis (CF) commenced. Pharmacological manipulation provides the opportunity to enhance CF transmembrane conductance regulator (CFTR) protein synthesis and/or function. CFTR modulators include potentiators to improve channel gating (class III mutations), correctors to improve abnormal CFTR protein folding and trafficking (class II mutations) and stop codon mutation read-through drugs relevant for patients with premature stop codons (most class I mutations).

Quality assessment and control of tissue specific RNA-seq libraries of Drosophila transgenic RNAi models.

RNA-sequencing (RNA-seq) is rapidly emerging as the technology of choice for whole-transcriptome studies. However, RNA-seq is not a bias free technique. It requires large amounts of RNA and library preparation can introduce multiple artifacts, compounded by problems from later stages in the process.

Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis.

The major purpose of the present study was to quantify correctly spliced CFTR transcripts in human nasal epithelial cells from cystic fibrosis (CF) patients carrying the splicing mutations c.580-1G>T (712-1G>T) and c.2657+5G>A (2789+5G>A) and to assess the applicability of this model in CFTR therapeutic approaches.

High-content siRNA screen reveals global ENaC regulators and potential cystic fibrosis therapy targets.

Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators.

Cardosin A contains two vacuolar sorting signals using different vacuolar routes in tobacco epidermal cells.

Several vacuolar sorting determinants (VSDs) have been described for protein trafficking to the vacuoles in plant cells. Because of the variety in plant models, cell types and experimental approaches used to decipher vacuolar targeting processes, it is not clear whether the three well-known groups of VSDs identified so far exhaust all the targeting mechanisms, nor if they reflect certain protein types or families. The vacuolar targeting mechanisms of the aspartic proteinases family, for instance, are not yet fully understood.

Control of cystic fibrosis transmembrane conductance regulator membrane trafficking: not just from the endoplasmic reticulum to the Golgi.

Biogenesis of cystic fibrosis transmembrane conductance regulator (CFTR) starts with its cotranslational insertion into the membrane of the endoplasmic reticulum (ER) and core glycosylation. These initial events are followed by a complex succession of steps with the main goal of checking the overall quality of CFTR conformation in order to promote its exit from the ER through the secretory pathway. Failure to pass the various checkpoints of the ER quality control targets the most frequent disease-causing mutant protein (F508del-CFTR) for premature degradation.

Changes in transcriptome of native nasal epithelium expressing F508del-CFTR and intersecting data from comparable studies.

Background: Microarray studies related to cystic fibrosis (CF) airway gene expression have gone some way in clarifying the complex molecular background of CF lung diseases, but have made little progress in defining a robust "molecular signature" associated with mutant CFTR expression. Disparate methodological and statistical analyses complicate comparisons between independent studies of the CF transcriptome, and although each study may be valid in isolation, the conclusions reached differ widely.

Methods: We carried out a small-scale whole genome microarray study of gene expression in human native nasal epithelial cells from F508del-CFTR homozygotes in comparison to non-CF controls.

Biochemical and biophysical characterization of recombinant yeast proteasome maturation factor ump1.

Protein degradation is essential for maintaining cellular homeostasis. The proteasome is the central enzyme responsible for non-lysosomal protein degradation in eukaryotic cells. Although proteasome assembly is not yet completely understood, a number of cofactors required for proper assembly and maturation have been identified.

Measurements of CFTR-mediated Cl- secretion in human rectal biopsies constitute a robust biomarker for Cystic Fibrosis diagnosis and prognosis.

Background: Cystic Fibrosis (CF) is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl(-)) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases.

Methodology/principal Findings: To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl(-) secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n=51), individuals with clinical CF suspicion (n=49) and age-matched non-CF controls (n=18).

Introduction to section III: biochemical methods to study CFTR protein.

This section of Cystic Fibrosis: Diagnosis and Protocols is focussed on methods aimed at detecting expression, localization, endocytic sorting and metabolism (biogenesis and turnover), as well as interacting partners of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein product of the gene mutated in cystic fibrosis (CF). An overview of the protocols to be found in subsequent chapters of this book section is provided here, as well as the rationale for utilizing these protocols (also as a workflow) explaining which scientific question(s) each of them helps to address. Protocols included in other sections of this book are also cross-referenced.

Quantification of CFTR transcripts.

Quantification and analysis of CFTR transcripts is of crucial importance not only for cystic fibrosis (CF) diagnosis and prognosis, but also in evaluating the efficiency of various therapeutic approaches to CF, including gene therapy. Reverse transcription (RT) followed by quantitative polymerase chain reaction (qPCR) is at present the most sensitive method for transcript abundance measurement. Classical RNA-based methods require significant expression levels in target samples for appropriate analysis, thus PCR-based methods have evolved towards reliable quantification.

Functional genomics assays to study CFTR traffic and ENaC function.

As several genomes have been sequenced, post-genomic approaches like transcriptomics and proteomics, identifying gene products differentially expressed in association with a given pathology, have held promise both of understanding the pathways associated with the respective disease and as a fast track to therapy. Notwithstanding, these approaches cannot distinguish genes and proteins with mere secondary pathological association from those primarily involved in the basic defect(s). New global strategies and tools identifying gene products responsible for the basic cellular defect(s) in CF pathophysiology currently being performed are presented here.

Targeting CFTR: how to treat cystic fibrosis by CFTR-repairing therapies.

Several novel compounds recently appeared as promising leads to develop effective drugs against the basic defect in Cystic fibrosis (CF) and the first rationale therapies for CF relying on the understanding of the basic defect started to hit the clinical setting. Most of these efforts are focused on correcting the F508del mutation (occurring in ≈90% of CF patients) which causes misfolding of the CF transmembrane conductance regulator (CFTR) protein, the intracellular retention of such abnormal conformation by the endoplasmic reticulum quality control and premature degradation, thus precluding CFTR from reaching the cell membrane where it normally functions as a cAMP-stimulated Cl- channel. Here, several rationale therapeutic strategies are briefly reviewed, namely, mutation-specific (or "CFTR-repairing") approaches (with a particular focus on the cellular defect associated with F508del-CFTR), manipulation of other ionic (non-CFTR) conductances and gene therapy.

Folding and rescue of a cystic fibrosis transmembrane conductance regulator trafficking mutant identified using human-murine chimeric proteins.

Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2.