Proteomic Analysis of Morphologically Changed Tissues after Prolonged Dexamethasone Treatment.

Prolonged dexamethasone (Dex) administration leads to serious adverse and decrease brain and heart size, muscular atrophy, hemorrhagic liver, and presence of kidney cysts. Herein, we used an untargeted proteomic approach using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneous identification of changes in proteomes of the major organs in Sprague-Dawley (SD rats post Dex treatment. The comparative and quantitative proteomic analysis of the brain, heart, muscle, liver, and kidney tissues revealed differential expression of proteins (n = 190, 193, 39, 230, and 53, respectively) between Dex-treated and control rats. Functional network analysis using ingenuity pathway analysis (IPA revealed significant differences in regulation of metabolic pathways within the morphologically changed organs that related to: (i) brain-cell morphology, nervous system development, and function and neurological disease; (ii) heart-cellular development, cellular function and maintenance, connective tissue development and function; (iii) skeletal muscle-nucleic acid metabolism, and small molecule biochemical pathways; (iv) liver-lipid metabolism, small molecular biochemistry, and nucleic acid metabolism; and (v) kidney-drug metabolism, organism injury and abnormalities, and renal damage. Our study provides a comprehensive description of the organ-specific proteomic profilesand differentially altered biochemical pathways, after prolonged Dex treatement to understand the molecular basis for development of side effects.

Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.

Very-long-chain fatty acids (VLCFAs) play important roles in membrane structure and cellular signaling, and their contribution to human health is increasingly recognized. Fatty acid elongases catalyze the first and rate-limiting step in VLCFA synthesis. Heterozygous mutations in ELOVL4, the gene encoding one of the elongases, are known to cause macular degeneration in humans and retinal abnormalities in mice. However, biallelic ELOVL4 mutations have not been observed in humans, and murine models with homozygous mutations die within hours of birth as a result of a defective epidermal water barrier. Here, we report on two human individuals with recessive ELOVL4 mutations revealed by a combination of autozygome analysis and exome sequencing. These individuals exhibit clinical features of ichthyosis, seizures, mental retardation, and spasticity-a constellation that resembles Sjögren-Larsson syndrome (SLS) but presents a more severe neurologic phenotype. Our findings identify recessive mutations in ELOVL4 as the cause of a neuro-ichthyotic disease and emphasize the importance of VLCFA synthesis in brain and cutaneous development.

Proteomics Profiling to Distinguish DOCK8 Deficiency From Atopic Dermatitis.

Dedicator of cytokinesis 8 deficiency is an autosomal recessive primary immune deficiency disease belonging to the group of hyperimmunoglobulinemia E syndrome (HIES). The clinical phenotype of dedicator of cytokinesis 8 (DOCK8) deficiency, characterized by allergic manifestations, increased infections, and increased IgE levels, overlaps with the clinical presentation of atopic dermatitis (AD). Despite the identification of metabolomics and cytokine biomarkers, distinguishing between the two conditions remains clinically challenging. The present study used a label-free untargeted proteomics approach using liquid-chromatography mass spectrometry with network pathway analysis to identify the differentially regulated serum proteins and the associated metabolic pathways altered between the groups. Serum samples from DOCK8 (n = 10), AD (n = 9) patients and healthy control (Ctrl) groups (n = 5) were analyzed. Based on the proteomics profile, the PLS-DA score plot between the three groups showed a clear group separation and sample clustering (R2 = 0.957, Q2 = 0.732). Significantly differentially abundant proteins (p < 0.05, FC cut off 2) were identified between DOCK8-deficient and AD groups relative to Ctrl (n = 105, and n = 109) and between DOCK8-deficient and AD groups (n = 85). Venn diagram analysis revealed a differential regulation of 24 distinct proteins from among the 85 between DOCK8-deficient and AD groups, including claspin, haptoglobin-related protein, immunoglobulins, complement proteins, fibulin, and others. Receiver-operating characteristic curve (ROC) analysis identified claspin and haptoglobin-related protein, as potential biomarkers with the highest sensitivity and specificity (AUC = 1), capable of distinguishing between patients with DOCK8 deficiency and AD. Network pathway analysis between DOCK8-deficiency and AD groups revealed that the identified proteins centered around the dysregulation of ERK1/2 signaling pathway. Herein, proteomic profiling of DOCK8-deficiency and AD groups was carried out to determine alterations in the proteomic profiles and identify a panel of the potential proteomics biomarker with possible diagnostic applications. Distinguishing between DOCK8-deficiency and AD will help in the early initiation of treatment and preventing complications.

A novel HAX1 gene mutation in severe congenital neutropenia (SCN) associated with neurological manifestations.

Autosomal recessive severe congenital neutropenia (SCN) results from a maturation arrest of granulopoiesis at the level of promyelocytes and apoptosis of myeloid cells. In SCN patients, mutations have been described in the HAX1 gene. Most of the SCN patients who carry nonsense mutations that are common to both transcript variants of the HAX1 gene also exhibit neurological deficits. This study describes an SCN patient with neurological manifestations including daily episodes of atonic seizures, learning disabilities, and developmental delay. Sequencing of the HAX1 gene of this SCN patient identified a novel nonsense c.463_464insC homozygous mutation in exon 3, which is common to both transcript variants of the gene. This mutation encodes for a p.Gln155ProfsX14 change and causes premature truncation of the HAX1 protein. Neutrophils isolated from the patient exhibited spontaneous apoptosis and loss of inner mitochondrial membrane potential, which were further enhanced upon treatment with hydrogen peroxide. This study adds to the spectrum of novel HAX1 gene mutations and disease manifestations in ethnically distinct SCN patients. Our report describes the only nonsense mutation in the HAX1 gene present in SCN patients of Arab origin.

Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1.

BACKGROUND: A newly developed drug trastuzumab emtansine (T-DM1) has improved the survival of breast cancer (BC) patients. Despite an impressive initial clinical response, a subgroup of patient develop resistance and present therapeutic challenges. The underlying resistance mechanisms are not fully investigated. We report that T-DM1 treatment modulates the expression of ROR1 (type 1 receptor tyrosine kinase-like orphan receptor) and induces self-renewal of cancer stem cells (CSCs) leading to therapeutic resistance. METHODS: Using BC patient tumor samples, and BC cell lines we gained insight into the T-DM1 treatment induced ROR1 overexpression and resistance. In vitro sphere forming assays and in vivo extreme dilution assays were employed to analyze the stemness and self-renewal capacity of the cells. A series of molecular expression and protein assays including qRT-PCR, FACS-sorting, ELISA, immunostaining, Western blotting were used to provide evidence. FINDINGS: Exposure of cells to T-DM1 shifted ROR1 expression from low to high, enriched within the CSC subpopulation, coincident with increased Bmi1 and stemness factors. T-DM1 induced ROR1 cells showed high spheroid and tumor forming efficiency in vitro and in an animal model exhibiting shorter tumor-free time. Mechanistically, the overexpression of ROR1 is partly induced by the activation of YAP1 and its target genes. Silencing of ROR1 and YAP1 by pharmacologic inhibitors and/or sh/siRNA inhibited spheroid formation, the initiation of tumors and the capacity for self-renewal and ROR1 overexpression. INTERPRETATIONS: The results presented here indicate that simultaneous targeting of ROR1 and YAP1 may suppress CSC self-renewal efficacy and inhibit tumor progression in BC. In this manner such treatments may overcome the T-DM1 mediated therapeutic resistance and improve clinical outcome. FUND: This study was supported by Neurogen Technologies for interdisciplinary research.

Integrated Left Ventricular Global Transcriptome and Proteome Profiling in Human End-Stage Dilated Cardiomyopathy.

AIMS: The disease pathways leading to idiopathic dilated cardiomyopathy (DCM) are still elusive. The present study investigated integrated global transcriptional and translational changes in human DCM for disease biomarker discovery. METHODS: We used identical myocardial tissues from five DCM hearts compared to five non-failing (NF) donor hearts for both transcriptome profiling using the ABI high-density oligonucleotide microarrays and proteome expression with One-Dimensional Nano Acquity liquid chromatography coupled with tandem mass spectrometry on the Synapt G2 system. RESULTS: We identified 1262 differentially expressed genes (DEGs) and 269 proteins (DEPs) between DCM cases and healthy controls. Among the most significantly upregulated (>5-fold) proteins were GRK5, APOA2, IGHG3, ANXA6, HSP90AA1, and ATP5C1 (p< 0.01). On the other hand, the most significantly downregulated proteins were GSTM5, COX17, CAV1 and ANXA3. At least ten entities were concomitantly upregulated on the two analysis platforms: GOT1, ALDH4A1, PDHB, BDH1, SLC2A11, HSP90AA1, HSP90AB1, H2AFV, HSPA5 and NDUFV1. Gene ontology analyses of DEGs and DEPs revealed significant overlap with enrichment of genes/proteins related to metabolic process, biosynthetic process, cellular component organization, oxidative phosphorylation, alterations in glycolysis and ATP synthesis, Alzheimer's disease, chemokine-mediated inflammation and cytokine signalling pathways. CONCLUSION: The concomitant use of transcriptome and proteome expression to evaluate global changes in DCM has led to the identification of sixteen commonly altered entities as well as novel genes, proteins and pathways whose cardiac functions have yet to be deciphered. This data should contribute towards better management of the disease.

Protein signatures as potential surrogate biomarkers for stratification and prediction of treatment response in chronic myeloid leukemia patients.

There is unmet need for prediction of treatment response for chronic myeloid leukemia (CML) patients. The present study aims to identify disease-specific/disease-associated protein biomarkers detectable in bone marrow and peripheral blood for objective prediction of individual's best treatment options and prognostic monitoring of CML patients. Bone marrow plasma (BMP) and peripheral blood plasma (PBP) samples from newly-diagnosed chronic-phase CML patients were subjected to expression-proteomics using quantitative two-dimensional gel electrophoresis (2-DE) and label-free liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of 2-DE protein fingerprints preceding therapy commencement accurately predicts 13 individuals that achieved major molecular response (MMR) at 6 months from 12 subjects without MMR (No-MMR). Results were independently validated using LC-MS/MS analysis of BMP and PBP from patients that have more than 24 months followed-up. One hundred and sixty-four and 138 proteins with significant differential expression profiles were identified from PBP and BMP, respectively and only 54 proteins overlap between the two datasets. The protein panels also discriminates accurately patients that stay on imatinib treatment from patients ultimately needing alternative treatment. Among the identified proteins are TYRO3, a member of TAM family of receptor tyrosine kinases (RTKs), the S100A8, and MYC and all of which have been implicated in CML. Our findings indicate analyses of a panel of protein signatures is capable of objective prediction of molecular response and therapy choice for CML patients at diagnosis as 'personalized-medicine-model'.

Proteome analysis of skin distinguishes acute guttate from chronic plaque psoriasis.

Psoriasis is a disease with considerable heterogeneity in clinical presentation. This is the first study using two-dimensional gel electrophoresis to compare global protein expression patterns in lesional and non-lesional skin from subjects with acute guttate psoriasis associated with streptococcal throat infection and chronic plaque psoriasis. Samples from experimentally induced contact eczema and normal skin from healthy controls were also included. Proteins with statistically significant differences in expression were used in hierarchical cluster analyses resulting in separation of the different samples into groups. Chronic plaque and guttate psoriasis samples were distinctly separated, indicating that they represent discrete phenotypes at the protein expression level. Interestingly, there was a trend in which guttate psoriasis lesions clustered closer to eczema than to chronic plaque psoriasis lesions, indicating that the duration of the inflammatory reaction may affect clustering. Several of the differentially expressed proteins were identified by mass spectrometry.

Proteomics-based signature for human benign prostate hyperplasia and prostate adenocarcinoma.

Prostate adenocarcinoma often presents at a late stage, due to a lack of early clinical symptoms and lack of accurate objective markers. This study aimed to identify and validate proteomics-based biomarkers useful for prostate cancer diagnosis and to establish a marker-panel for prostate cancer and benign prostate hyperplasia (BPH). Global protein expression patterns in fresh tissue specimens from 8 patients with prostate carcinoma and 16 with BPH were analyzed by two-dimensional gel electrophoresis. Differentially expressed proteins were identified by MALDI-TOF mass spectrometry. We compared our results with those of published studies and defined a set of common biomarkers. We identified 22 differentially expressed proteins between BPH and prostate carcinomas. The up-regulated proteins in cancer compared to BPH included protein disulfide-isomerase, 14-3-3-protein, Enoyl CoA-hydrase, prohibitin and B-tubulin ß-2. Keratin-II, desmin, HSP71, ATP-synthase-ß-chain and creatine kinase-ß-chain were down-regulated. Survey of the literature showed that 15 of our 22 identified proteins have been previously reported to differ in their expression levels between BPH and prostate cancer by other laboratories. The expression patterns of these biomarkers could successfully cluster BPH and adenocarcinomas as well as prostate cancer of low and high Gleason scores. This study validates protein-biomarkers that can be useful for accurate diagnosis and prognostic monitoring of prostate adenocarcinoma. Despite varied prevalence of the disease between different ethnic populations (i.e., high in Sweden, low in Saudi Arabia); the biomarkers indicate that BPH and prostate cancers are biologically 'homogeneous' in their protein expression patterns across wide geographical regions.

Molecular classification of borderline ovarian tumors using hierarchical cluster analysis of protein expression profiles.

Ovarian tumors range from benign to aggressive malignant tumors, including an intermediate class referred to as borderline carcinoma. The prognosis of the disease is strongly dependent on tumor classification, where patients with borderline tumors have much better prognosis than patients with carcinomas. We here describe the use of hierarchical clustering analysis of quantitative protein expression data for classification of this type of tumor. An accurate classification was not achieved using an unselected set of 1,584 protein spots for clustering analysis. Different approaches were used to select spots that were differentially expressed between tumors of different malignant potential and to use these sets of spots for classification. When sets of proteins were selected that differentiated benign and malignant tumors, borderline tumors clustered in the benign group. This is consistent with the biologic properties of these tumors. Our results indicate that hierarchical clustering analysis is a useful approach for analysis of protein profiles and show that this approach can be used for differential diagnosis of ovarian carcinomas and borderline tumors.