Targeting the PI3K/Akt/mTOR signalling pathway in Cystic Fibrosis.

Deletion of phenylalanine 508 of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) is a major cause of cystic fibrosis (CF), one of the most common inherited childhood diseases. ΔF508 CFTR is a trafficking mutant that is retained in the endoplasmic reticulum (ER) and unable to reach the plasma membrane. Efforts to enhance exit of ΔF508 CFTR from the ER and improve its trafficking are of utmost importance for the development of treatment strategies. Using protein interaction profiling and global bioinformatics analysis we revealed mammalian target of rapamycin (mTOR) signalling components to be associated with ∆F508 CFTR. Our results demonstrated upregulated mTOR activity in ΔF508 CF bronchial epithelial (CFBE41o-) cells. Inhibition of the Phosphatidylinositol 3-kinase/Akt/Mammalian Target of Rapamycin (PI3K/Akt/mTOR) pathway with 6 different inhibitors demonstrated an increase in CFTR stability and expression. Mechanistically, we discovered the most effective inhibitor, MK-2206 exerted a rescue effect by restoring autophagy in ΔF508 CFBE41o- cells. We identified Bcl-2-associated athanogene 3 (BAG3), a regulator of autophagy and aggresome clearance to be a potential mechanistic target of MK-2206. These data further link the CFTR defect to autophagy deficiency and demonstrate the potential of the PI3K/Akt/mTOR pathway for therapeutic targeting in CF.

Clinical and molecular characterization of individuals with recurrent genomic disorder at 10q22.3q23.2.

The identification of genomic imbalances in young patients can affect medical management by allowing early intervention for developmental delay and by identifying patients at risk for unexpected medical complications. Using a 105K-feature oligonucleotide array, we identified a 7.25 Mb deletion at 10q22.3q23.2 in six unrelated patients. Deletions of this region have been described in individuals with cognitive and behavioral abnormalities, including autistic features, and may represent a recurring genetic syndrome. All four patients in this study for whom clinical information was available had mild dysmorphic features and three had developmental delay. Of note is the emerging clinical phenotype in these individuals with similar dysmorphic features such as macrocephaly, hypertelorism, and arachnodactyly, and neurodevelopmental delay that includes failure to thrive, hypotonia, and feeding difficulties in the neonatal period, and receptive and expressive language delay with global neurodevelopmental delay after the neonatal period. However, there is no pattern of abnormalities, craniofacial, behavioral, or otherwise, that would have aroused clinical suspicion of a specific syndrome. Finally, the patients' deletions encompass BMPR1A but not PTEN, and these patients may be at risk for colon cancer and should be referred for appropriate prophylactic care and surveillance. Of the two patients in this study who had colonoscopy following the array results, neither had polyps. Therefore, the magnitude of the increased risk for colon cancer is currently unknown.

Insights into the platelet releasate.

The platelet releasate comprises of a multitude of inflammatory and vasoactive substances, which can attract atherogenic leukocytes from the circulation, activate endothelial cells and stimulate vessel growth and repair by triggering vascular cell proliferation, migration, and inflammation. Thus, platelets are believed central in the development and progression of atherosclerotic lesions and recent progress in uncovering more than 300 proteins in the thrombin-activated platelet releasate may advance our ability to understand the events involved and responses triggered in the progression of atherosclerosis. Furthermore, neutralisation of these platelet-derived pro-inflammatory factors may become an interesting means for therapeutic or preventative intervention in atherosclerosis.

Integration of proteomics and genomics in platelets: a profile of platelet proteins and platelet-specific genes.

Platelets, while anucleate, contain RNA, some of which is translated into protein upon activation. Hypothesising that the platelet proteome is reflected in the transcriptome, we identified 82 proteins secreted from activated platelets and compared these, as well as published proteomic data, to the transcriptional profile. We also compared the transcriptome of platelets to other tissues to identify platelet-specific genes and used ontology to determine gene categories over-represented in platelets. RNA was isolated from highly pure platelet preparations for hybridization to Affymetrix oligonucleotide arrays. We identified 2,928 distinct messages as being present in platelets. The platelet transcriptome was compared with the proteome by relating both to UniGene clusters. Platelet proteomic data correlated well with the transcriptome, with 69% of secreted proteins detectable at the mRNA level, and similar concordance was obtained using two published datasets. While many of the most abundant mRNAs are for known platelet proteins, messages were detected for proteins not previously reported in platelets. Some of these may represent residual megakaryocyte messages; however, proteomic analysis confirmed the expression of many previously unreported genes in platelets. Transcripts for well-described platelet proteins are among the most platelet-specific messages. Ontological categories related to signal transduction, receptors, ion channels, and membranes are over-represented in platelets, while categories involved in protein synthesis are depleted. Despite the absence of gene transcription, the platelet proteome is mirrored in the transcriptome. Conversely, transcriptional analysis predicts the presence of novel proteins in the platelet. Transcriptional analysis is relevant to platelet biology, providing insights into platelet function and the mechanisms of platelet disorders.

Altered distribution of mitochondria and actin fibers in 3T3 cells cultured on microcarriers.

The mitochondria and actin fibers of 3T3 fibroblasts cultured on microcarriers in spinner flasks were visualized using fluorescent stains. In contrast to cells grown on planar surfaces under static or steady laminar flow conditions, cells exposed to higher levels of turbulent agitation do not form actin stress fibers. Greater agitation also leads to a more diffuse appearance of the mitochondria and a wider distribution of them throughout the cytoplasm. This response may indicate damaged mitochondria, as similar results have been reported for chemical toxins.

Mae III positively detects the mitochondrial mutation associated with type I Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy is a blinding disease that usually causes acute or subacute central visual loss in adolescent and young adult males. In patients who lack a family history of a similar illness, Leber's disease has been a diagnosis of exclusion. The recent discovery of a specific mitochondrial mutation in many pedigrees affected with the disease has provided the basis for rapid molecular diagnosis of one genetic type of Leber's disease. We have developed a new method, based on a Mae III (Boehringer Mannheim Biochemicals, Indianapolis, Ind) restriction fragment length polymorphism, for detecting the Wallace-type Leber's mutation. The method has several advantages over the previously used SfaN I method that make it more suitable for use as a general laboratory test. We demonstrate the utility of this new test in the diagnosis of Leber's disease in a patient with no family history of visual loss.