Major Differences In Expression Research Articles

Importance of circulating monocyte subpopulations and Fcγ receptors in immune thrombocytopenia.

Monocytes have been linked to the pathogenesis of immune thrombocytopenia (ITP) because of their role in autoantibody-mediated platelet phagocytosis. However, monocytes constitute unique populations with major differences in expression for surface Fcγ receptors (FcγRs). Thus, we evaluated monocytes in whole blood samples from patients with newly diagnosed and chronic ITP. Monocyte subpopulations were identified phenotypically by flow cytometry and defined according to the surface expression of CD14 (lipopolysaccharide receptor) and of CD16 (low-affinity Fcγ receptor III) into classical (CLM), intermediate (INTM) and nonclassical (non-CLM) monocytes. We also examined the expression of FcγRI/CD64 and FcγRIII/CD16 by monocyte subpopulations. Newly diagnosed patients showed a decrease in non-CLM, expressed as a relative percentage of total monocytes compared with controls and chronic ITP patients. Both non-CLM and INTM of newly diagnosed patients closely correlated with platelet count. These monocyte subpopulations showed significantly enhanced CD64 expression in newly diagnosed patients. On the contrary, patients with chronic ITP presented higher non-CLM in percentage than controls and concomitant lower CLM and total monocytes, in percentage and number. The expression of CD64 was increased by all monocyte subpopulations, CLM, INTM, and non-CLM in chronic patients. In conclusion, differences in monocyte subpopulations, together with enhanced expression of FcγRI/CD64 are evident in patients with ITP.

Molecular taxonomy of nociceptors and pruriceptors.

Molecular taxonomy of nociceptors and pruriceptors.

Major differences in glycosylation and fucosyltransferase expression in low-grade versus high-grade bladder cancer cell lines.

Bladder cancer is the ninth most frequently diagnosed cancer worldwide, and there is a need to develop new biomarkers for staging and prognosis of this disease. Here we report that cell lines derived from low-grade and high-grade bladder cancers exhibit major differences in expression of glycans in surface glycoproteins. We analyzed protein glycosylation in three low-grade bladder cancer cell lines RT4 (grade-1-2), 5637 (grade-2), and SW780 (grade-1), and three high-grade bladder cancer cell lines J82COT (grade-3), T24 (grade-3) and TCCSUP (grade-4), with primary bladder epithelial cells, A/T/N, serving as a normal bladder cell control. Using a variety of approaches including flow cytometry, immunofluorescence, glycomics and gene expression analysis, we observed that the low-grade bladder cancer cell lines RT4, 5637 and SW780 express high levels of the fucosylated Lewis-X antigen (Lex, CD15) (Galβ1-4(Fucα1-3)GlcNAcβ1-R), while normal bladder epithelial A/T/N cells lack Lex expression. T24 and TCCSUP cells also lack Lex, whereas J82COT cells express low levels of Lex. Glycomics analyses revealed other major differences in fucosylation and sialylation of N-glycans between these cell types. O-glycans are highly differentiated, as RT4 cells synthesize core 2-based O-glycans that are lacking in the T24 cells. These differences in glycan expression correlated with differences in RNA expression levels of their cognate glycosyltransferases, including α1-3/4-fucosyltransferase genes. These major differences in glycan structures and gene expression profiles between low- and high-grade bladder cancer cells suggest that glycans and glycosyltransferases are candidate biomarkers for grading bladder cancers.

Major Differences in Expression of Inflammatory Pathways in Skin from Different Body Sites of Healthy Individuals

Major Differences in Expression of Inflammatory Pathways in Skin from Different Body Sites of Healthy Individuals

Comparative Transcriptomic Profiling of Yersinia enterocolitica O:3 and O:8 Reveals Major Expression Differences of Fitness- and Virulence-Relevant Genes Indicating Ecological Separation.

Yersinia enterocolitica is a zoonotic pathogen and an important cause of bacterial gastrointestinal infections in humans. Large-scale population genomic analyses revealed genetic and phenotypic diversity of this bacterial species, but little is known about the differences in the transcriptome organization, small RNA (sRNA) repertoire, and transcriptional output. Here, we present the first comparative high-resolution transcriptome analysis of Y. enterocolitica strains representing highly pathogenic phylogroup 2 (serotype O:8) and moderately pathogenic phylogroup 3 (serotype O:3) grown under four infection-relevant conditions. Our transcriptome sequencing (RNA-seq) approach revealed 1,299 and 1,076 transcriptional start sites and identified strain-specific sRNAs that could contribute to differential regulation among the phylogroups. Comparative transcriptomics further uncovered major gene expression differences, in particular, in the temperature-responsive regulon. Multiple virulence-relevant genes are differentially regulated between the two strains, supporting an ecological separation of phylogroups with certain niche-adapted properties. Strong upregulation of the ystA enterotoxin gene in combination with constitutive high expression of cell invasion factor InvA further showed that the toxicity of recent outbreak O:3 strains has increased. Overall, our report provides new insights into the specific transcriptome organization of phylogroups 2 and 3 and reveals gene expression differences contributing to the substantial phenotypic differences that exist between the lineages. IMPORTANCE Yersinia enterocolitica is a major diarrheal pathogen and is associated with a large range of gut-associated diseases. Members of this species have evolved into different phylogroups with genotypic variations. We performed the first characterization of the Y. enterocolitica transcriptional landscape and tracked the consequences of the genomic variations between two different pathogenic phylogroups by comparing their RNA repertoire, promoter usage, and expression profiles under four different virulence-relevant conditions. Our analysis revealed major differences in the transcriptional outputs of the closely related strains, pointing to an ecological separation in which one is more adapted to an environmental lifestyle and the other to a mostly mammal-associated lifestyle. Moreover, a variety of pathoadaptive alterations, including alterations in acid resistance genes, colonization factors, and toxins, were identified which affect virulence and host specificity. This illustrates that comparative transcriptomics is an excellent approach to discover differences in the functional output from closely related genomes affecting niche adaptation and virulence, which cannot be directly inferred from DNA sequences.

A motor neuron strategy to save time and energy in neurodegeneration: adaptive protein stoichiometry.

Neurofilament proteins (Nf) are a biomarker of disease progression in amyotrophic lateral sclerosis (ALS). This study investigated whether there are major differences in expression from in vivo measurements of neurofilament isoforms, from the light chain, NfL (68 kDa), compared with larger proteins, the medium chain (NfM, 150 kDa) and the heavy (NfH, 200‐210 kDa) chains in ALS patients and healthy controls. New immunological methods were combined with Nf subunit stoichiometry calculations and Monte Carlo simulations of a coarse‐grained Nf brush model. Based on a physiological Nf subunit stoichiometry of 7 : 3 : 2 (NfL:NfM:NfH), we found an ‘adaptive’ Nf subunit stoichiometry of 24 : 2.4 : 1.6 in ALS. Adaptive Nf stoichiometry preserved NfL gyration radius in the Nf brush model. The energy and time requirements for Nf translation were 56 ± 27k ATP (5.6 h) in control subjects compared to 123 ± 102k (12.3 h) in ALS with ‘adaptive’ (24:2.4:1.6) Nf stoichiometry (not significant) and increased significantly to 355 ± 330k (35.5 h) with ‘luxury’ (7:3:2) Nf subunit stoichiometry (p < 0.0001 for each comparison). Longitudinal disease progression‐related energy consumption was highest with a ‘luxury’ (7:3:2) Nf stoichiometry. Therefore, an energy and time‐saving option for motor neurons is to shift protein expression from larger to smaller (cheaper) subunits, at little or no costs on a protein structural level, to compensate for increased energy demands.

Differences in gene expression related to the outcomes of obesity treatment, peak oxygen uptake, and fatty acid metabolism measured in a cardiopulmonary exercise test.

INTRODUCTION Theobesity pandemic requires development of methods that could be used on alarge scale, such as thecardiopulmonary exercise test (CPET). Gene expression may explain CPET results on themolecular level. OBJECTIVES The aim of this study was to compare gene expression in obesity, depending on CPET results. PATIENTS AND METHODS Thestudy group consisted of 9 obese patients and 7 controls. Thetreatment encompassed diet, rehabilitation, and behavioral therapy. Diet was based on the body composition analyzed by bioelectrical impedance, resting metabolic rate, and subjective patient preferences. Therehabilitation depended on theCPET results: maximal oxygen uptake and fatty acid metabolism. Behavioral intervention focused on thediagnosis of health problems leading to obesity, lifestyle modification, training in self‑assessment, and development of healthy habits. The intensive treatment lasted for 12 weeks and consisted of consultations with a physician, dietitian, and medical rehabilitation specialist. RNA was isolated from the whole blood. A total of 47 323 transcripts were analyzed, of which 32 379 entities were confirmed to havehigh quality of RNA. RESULTS We observed differences in gene expression related to the CPET results indicating abnormalities in fat oxidation and maximal oxygen uptake. The genes with major differences in expression were: CLEC12A, HLA‑DRB1, HLA‑DRB4, HLA‑A29.1, IFIT1, and LOC100133662. CONCLUSIONS Thedifferences in gene expression may account for theoutcomes of treatment related to inflammation caused by obesity, which affects the muscles, fat tissue, and fatty acid metabolism.

Pyruvate dehydrogenase kinase 1 is essential for transplantable mouse bone marrow hematopoietic stem cell and progenitor function.

BackgroundAccumulating evidence suggests that hypoxic areas in the bone marrow are crucial for maintenance of hematopoietic stem cells (HSCs) by supporting a quiescent state of cell cycle and regulating the transplantation capacity of long-term (LT)-HSCs. In addition, HSCs seem to express a metabolic profile of energy production away from mitochondrial oxidative phosphorylation in favor of glycolysis. At oxygen deprivation, hypoxia inducible factor 1α (HIF-1α) is known to induce glycolytic enzymes as well as suppressing mitochondrial energy production by inducing pyruvate dehydrogenase kinase 1 (Pdk1) in most cell types. It has not been established whether PDK1 is essential for HSC function and mediates hypoxia-adapting functions in HSCs. While the Pdk gene family contains four members (Pdk1-4), it was recently shown that Pdk2 and Pdk4 have an important role in regulating LT-HSCs.Principle findingsHere we demonstrate that PDK1 activity is crucial for transplantable HSC function. Whereas Pdkl, Pdk2, and Pdk3 transcripts were expressed at higher levels in different subtypes of HSCs compared to differentiated cells, we could not detect any major differences in expression between LT-HSCs and more short-term HSCs and multipotent progenitors. When studying HIF-1α-mediated regulation of Pdk activity in vitro, Pdk1 was the most robust target regulated by hypoxia, whereas Pdk2, Pdk3, and Pdk4 were not affected. Contrary, genetic ablation in a cre-inducible Hif-1α knockout mouse did not support a link between HIF-1α and Pdk1. Silencing of Pdk1 by shRNA lentiviral gene transfer partially impaired progenitor colony formation in vitro and had a strong negative effect on both long-term and short-term engraftment in mice.ConclusionsOur study demonstrates that PDK1 has broad effects in hematopoiesis and is a critical factor for engraftment of both HSCs and multipotent progenitors upon transplantation to recipient mice. While Pdk1 was a robust hypoxia-inducible gene mediated by HIF-1α in vitro, we could not find evidence of any in vivo links between Pdk1 and HIF-1α.

Myeloid Cells Differentiated in the Presence of Interferon-γ (INF-γ) Compared to Mature Cells Exposed to This Cytokine Exhibit Different Phox Protein Expression and Nox2 Activity

Introduction: Most information about the immunoregulatory functions of INF-γ has focused on the interaction with the adaptive immune system and less is reported about neutrophils. The majority of studies with neutrophils evaluate the effects of INF-γ on differentiated cells. Since an important issue INF-γ treatment is the function of neutrophils developed under the influence of this cytokine, we investigated the effects of INF-γ on a myeloid cell line that can be differentiated into neutrophil-like cells.Methods: PLB-985 cells were grown under standard culture conditions (5% CO2, 37oC) with RPMI and 10% bovine serum and were matured with the addition of 1.3% DMSO in the presence and absence of various concentrations of INF-γ-1b (Vidara Therapeutics Ltd.). After 72 and 96 hours, the cells exhibited neutrophil-like phenotype. As controls, cells were incubated with media or media plus INF-γ alone.Superoxide anion (O2-) production was measured as S00 inhibitable cytochrome c reduction after exposure to PMA (200 ng/mL) or FMLP (1 µM). Cell lysates were prepared in the presence of 0.1% SDS, 1% Triton X 100 and a protease inhibitor mix. Proteins from the lysate were separated on 10-15% SDS PAGE, blotted onto PVDF or nitrocellulose, exposed to primary antibodies for phox proteins overnight at 4oC and then appropriate peroxidase labeled secondary antibodies. Protein/antibody complexes were detected with an ECL detection system and were quantitated by densitometry with Image J software.Results: PLB-985 cells cultured for 4 days with 30ng/ml ( 600 IU/ml) INF-γ, DMSO or DMSO+ INF-γ were stimulated with PMA or fMLF and O2- production was measured over 5 minutes (for fMLF) or 10 minutes (for PMA). INF-γ alone resulted in minimal O2- generation. For fMLF a significant (p<0.05) increase in O2- was seen when DMSO was included in the culture (2.4+/- 0.3 nmol O2-, mean+/-SEM) and for PMA a significant (p<0.005) increase (17.9+/-1.3) was seen. For both agonists an additional increase (p<0.005) was documented when INF-γ was included with DMSO during the culture, fMLF, 8.5+/-0.53 and PMA 45.8+/-2.9.When a range of INF-γ concentrations (5-60 ng/ml) were co-applied during DMSO mediated PLB-985 differentiation over 3 days, the increase in O2-production was concentration dependent and reached a plateau at 30ng/ml for both stimuli.Culture of cells with INF-γ alone resulted in dramatic increases in p22phox and gp91phox but failed to cause more than minimal increases in the initially low levels of p40phox, p47phox and p67phox accounting for the inability of INF-γ alone to induce oxidase activity. Differentiation of the cells with DMSO caused a small decrease in p22phox and a small increase in gp91phox but also resulted in large increases in p40phox, p47phox and p67phox which would explain the ability of DMSO to induce Nox2 activity. Inclusion of INF-γ with DMSO further increased p47phox, and increased p22phox and gp91phox relative to DMSO alone, potentially explaining the enhanced Nox2 activity noted above.In striking contrast, when INF-γ was applied to cells first differentiated with DMSO, a minimal increase in fMLF induced O2- production was seen and only a small increase in PMA induced O2-generation was determined. Consistent with this, INF-γ applied to pre-differentiated PLB-985 cells did not enhance p40phox, and gp91phox, minimally increased p22phox, and decreased p47phox and p67phox slightly.Conclusions: These studies demonstrate that INF-γ induces very different phenotypes in myeloid cells depending on the differentiation level of the cell. Dramatic differences were noted whether the INF-γ was present during maturation of these myeloid cells as opposed to after differentiation and include major differences in expression of phox proteins as well as activity of the Nox2 enzyme system. These data not only support the administration of INF-γ for specific neutrophil dysfunction disorders, but also suggest, in theory, expanded uses of this cytokine. DisclosuresNo relevant conflicts of interest to declare.

Stage-specific reprogramming of gene expression characterizes Lr48-mediated adult plant leaf rust resistance in wheat.

Wheat genotype CSP44 carrying a recessive gene Lr48 exhibits adult plant resistance (APR; incompatible reaction) but gives a compatible reaction (susceptibility) at the seedling stage against leaf rust. A comparative gene expression analysis involving cDNA-amplified fragment length polymorphism (cDNA-AFLP) and quantitative PCR (qPCR) was carried out for incompatible and compatible reactions in the genotype CSP44. cDNA-AFLP analysis was conducted using RNA samples that were isolated from flag leaves following inoculation with leaf rust race 77-5 (the most virulent race) and also after mock inoculation. As many as 298 of a total of 493 expressed transcript-derived fragments (TDFs) exhibited differential expression (262 upregulated and 36 downregulated). Of these 298 TDFs, 48 TDFs were eluted from gels, re-amplified, cloned, and sequenced. Forty two of these 48 TDFs had homology with known genes involved in the following biological processes: energy production, metabolism, transport, signaling, defense response, plant-pathogen interaction, transcriptional regulation, translation, and proteolysis. The functions of the remaining six TDFs could not be determined; apparently, these represented some novel genes. The qPCR analysis for 18 TDFs (with known and unknown functions, but showing major differences in expression) was conducted using RNA isolated from the seedlings as well as from the adult plants. The expression of at least 11 TDFs was induced and that of 4 other TDFs attenuated or remained near normal in adult plants following leaf rust inoculations. The remaining three TDFs had non-specific/developmental stage-specific expression. Functional annotation of TDFs that were upregulated suggest that the APR was supported by transient recruitment and reprogramming of processes like perception and recognition of pathogen effector by receptors, followed by CDPK and MAPK signaling, transport, metabolism, and energy release.

Reduced expression of TLR3, TLR10 and TREM1 by human macrophages in Chronic cavitary pulmonary aspergillosis, and novel associations of VEGFA, DENND1B and PLAT

Chronic cavitary pulmonary aspergillosis (CCPA) is an uncommon but serious pulmonary disease of humans, with an annual mortality rate of 10–30%. It is caused by the fungus Aspergillus fumigatus. Patients are overtly immunocompetent; however, some immunogenetic defect is likely. To investigate this, we performed a genetic association study analysing biologically plausible candidate genes in 112 CCPA patients and 279 healthy controls, and investigated gene expression in monocyte-derived macrophages from patients and controls at baseline and during stimulation with A. fumigatus. Single-nucleotide polymorphisms (SNPs) associated with CCPA were found in TLR1, CLEC7A (dectin-1), PLAT (n = 2), VEGFA, and DENND1B. Macrophages from CCPA patients showed low TLR3 and TLR10 expression and high TREM1 expression at baseline, as compared with macrophages from healthy subjects, with major expression differences being seen in most Toll-like receptors (TLRs) during 9 h of co-culture with A. fumigatus. The differences in baseline expression between the healthy and CCPA groups suggest roles for TLR3 and TLR10 in susceptibility to CCPA, and the association of SNPs in PLAT (n = 2), VEGFA and DENND1B supports novel roles for plasminogen activation and angiogenesis and of these genes specifically in susceptibility to CCPA.

Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures.

Tissue Distribution and Dependence of Responsiveness of Human Antigen-Specific Memory B Cells

Memory B cells (mBCs) are a key to immunologic memory, yet their distribution within lymphoid organs and the individual role of these for mBC functionality remain largely unknown. This study characterized the distribution and phenotype of human (Ag-specific) mBCs in peripheral blood (PB), spleen, tonsil, and bone marrow. We found that the spleen harbors most mBCs, followed by tonsils, BM, and PB, and we detected no major differences in expression of markers associated with higher maturity. Testing the distribution of tetanus toxoid-specific (TT(+)) mBCs revealed their presence in PB during steady state, yet absolute numbers suggested their largest reservoir in the spleen, followed by tonsils. To explore the role of both tissues in the maintenance of reactive B cell memory, we revaccinated controls and splenectomized and tonsillectomized individuals with TT. All donor groups exhibited comparable emergence of anti-TT IgG, TT(+) plasma cells, and TT(+) mBCs in the PB, together with similar molecular characteristics of TT(+) plasma cells. In summary, human mBCs recirculate through PB and reside in different lymphoid organs that do not reflect different mBC maturity stages. The spleen and tonsil, although harboring the largest number of overall and TT(+) mBCs, appear to be dispensable to preserve adequate responsiveness to secondary antigenic challenge.

Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: Implications for population susceptibility to diseases

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

Proteomic Profiling of Smad Proteins Expression in AML: Pan Expression with High Phosphorylation Is Prognostically Adverse

Abstract 891The Transforming growth factor β (TGF-β) signaling pathway has been previously known to play a tumor suppressor role in hematologic malignancies. Smad proteins and their phosphorylation play a vital role in TGF-β signaling pathway. There are three class of Smads; Receptor-regulated Smads (1, 2, 3, 5, 8), Common mediator Smad (4) and Inhibitory Smads (6, 7). However, little is known about the expression and activation of Smad proteins in AML and nothing has been reported about correlation with clinical features or outcomes. Interestingly, Tabe et.al, (ASH 2012) recently identified pro-survival effect of TGF-β in leukemia cells via upregulation of MMP-1 and that the anti-apoptotic effect of TGF-β was associated with G0/G1 cell cycle arrestWe performed proteomic profiling of Smad expression, measuring the level of total Smad 1, 2, 3, 4, 5, 6, and phosphorylated Smad 2 (p245, p465) and 5 (p463) using reverse phase protein array (RPPA) technology. All antibodies were strictly validated. Analysis was performed on a cohort of 511 newly diagnosed AML (non APL) cases randomly divided into training and test sets. Normal bone marrow derived CD34+ cells (n=11) served as expression controls. Hierarchical clustering with Wald linkage rules and Euclidean distance matrix were used to define signatures. Cox model and long rank test were used to assess the survival outcome with different sample signatures.When comparing expression of individual Smad proteins with control CD34+ cells, most cases had expression within the normal CD34+ cell range, but levels of Smad 2, 2p465 and 4 had higher percentages of cases with expression below normal, while levels of Smad 3, 5, 5 (p463) and 6 were more frequently expressed at levels above normal. There were no major differences in expression between bone marrow and blood, and diagnosis and relapse samples. When unbiased hierarchical clustering was performed on the training set, four distinct Smad protein expression signatures were identified (Figure 1). Group 1 is characterized by pan-low Smad expression; Group 2 by high expression in Smad 2, 5, 5 (p463); Group 3 by high expression of phosphorylated Smad 2 (p245, p465) and 5 (p463); and Group 4, by pan-high Smad expression. Group 2 was associated with Ras mutation (28% vs. 9% for the other 3, p=0.03) and FLT3 ITD (p=0.009) mutation frequency was significantly lower in group 1. Smad group was not associated with FAB classification, demographics, prior treatment history, cytogenetics, and other molecular mutations. Higher Smad expression was statistically significantly correlated with higher counts of WBC (p=0.04), bone marrow and peripheral blast % (p=0.009, 0.004), CD33 and 34 counts (p=0.006, 0.002). Intriguingly, among 210 other proteins assayed in RPPA, expression of Integrin/Adhesion proteins IGFBP2, CD49B, CD11A and Fibronectin were inversely correlated with Smad expression consistent with the above observation. Pan Smad expression was strongly correlated with AKT pathway activation and high expression of several proliferation promoting proteins. Pan-high Smad expression (group 4) was associated with inferior overall survival (OS) (Figure 2) and event free survival (EFS) whereas the OS and EFS of Group 1, 2, and 3 were similar (log-rank test OS p=0.017; EFS p=0.03). Median OS, EFS were 36.3 and 19.3 weeks in Group 4 versus 56.1 and 26.9 weeks in other groups, respectively. Patients in group 4 had a lower remission rate (51% vs. 66%). When validated with the test set, similar results were observed and group 4 again had inferior survival (median 26.7 vs. 58 weeks, p = 0.0047) compared to the other groups.In conclusion, we observed that Smad expression in AML segregates into four distinct heterogeneous expression and activation patterns. Pan-high Smad expression was linked with significantly worse OS, EFS, and trends for inferior CR rates. The clinical features (high WBC and % PB and BM blasts) and inferior clinical outcome associated with pan-high Smad expression suggest that dominant TGF-β signaling is adverse in AML and that these patients may benefit from TGF-β blockade. Our finding suggest a tumor promoting, rather than tumor suppressing role, for TGF- β in AML, possibly mediated via MMP-1 activation. Further studies are required to investigate the mechanism of TGF-β pathway activation possibly inducing chemotherapy resistance leading to poor survival. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Molecular characterization of PRR13 and its tissue-specific expression in rainbow trout (Oncorhynchus mykiss)

The proline-rich protein 13 (PRR13) is reported to be a key regulator of the resistance to cytostatica by decreasing the copy number of the proapoptotic gene thrombospondin-1. We isolated and characterized the complete PRR13 gene sequence of rainbow trout (Oncorhynchus mykiss). The gene comprises four exons and three introns, the latter of comparatively short lengths (100-811bp). The full-length PRR13 cDNA consists of 1,101 nucleotides, including an open reading frame of 563bp, which is predicted to encode a 187 amino acid protein with a molecular mass of 18.8kDa. A continuous stretch of ten serine residues at the C-terminus is highly conserved and characteristic for vertebrate PRR13, but not for other known proline-rich proteins. Phylogenetic analyses suggest a clear separation of teleostean PRR13 proteins and those from mammalian and reptilian species. Comparison of the tissue-specific PRR13 mRNA abundance in two strains of the rainbow trout coastal form (TCO Steelhead II-WA vs. BORN Steelhead II-Germany) revealed an increased expression in the BORN trout in nearly all examined tissues. The major expression differences were detected in gill (2.29-fold) and in liver tissue (2.16-fold). Hence, the increased PRR13 expression in BORN trout might cause improved protection from natural cytostatica and therefore support our assumption that PRR13 is a candidate gene possibly involved in the varying ability of the two rainbow trout strains to handle environmental stress under local conditions of the Southern Baltic.

Injury Is a Major Inducer of Epidermal Innate Immune Responses during Wound Healing

We examined the importance of injury for the epidermal innate immune response in human skin wounds. We found that injury, independent of infiltrating inflammatory cells, generated prominent chemotactic activity toward neutrophils in injured skin because of IL-8 production. Furthermore, injury was a major inducer of the expression of antimicrobial (poly)peptides (AMPs) in skin wounds. In human skin, these injury-induced innate immune responses were mediated by activation of the epidermal growth factor receptor (EGFR). Consequently, inhibition of the EGFR blocked both the chemotactic activity generated in injured skin and the expression of the majority of the AMPs. The importance of injury was confirmed in mouse experiments in vivo, in which injury independent of infection was a potent inducer of AMPs in skin wounds. To our knowledge, these data thereby provide a previously unreported molecular link between injury and neutrophil accumulation and identify the molecular background for the vast expression of IL-8 and AMPs in wounded epidermis. Conceptually, these data show that the growth factor response elicited by injury is important for the recruitment of neutrophils in skin wounds.

Time-Warped Comparison of Gene Expression in Adaptive and Maladaptive Cardiac Hypertrophy

Cardiac hypertrophy is classically regarded as a compensatory response, yet the active tissue remodeling processes triggered by various types of mechanical stress can enhance or diminish the function of the heart. Despite the disparity in outcomes, there are similarities in the hypertrophic responses. We hypothesized that a generic genetic response that is not dependent on the particular nature of the hypertrophic stimulus exists. To test our hypothesis, we compared the temporal evolution of transcriptomes measured in hearts subjected to either adaptive (exercise-induced) or maladaptive (aortic banding-induced) hypertrophy. Generic hypertrophy-associated genes were identified and distinguished from stimulus-dependent transcripts by coupling a metric of cardiac growth with a dynamic time-warping algorithm to align transcriptome changes with respect to the hypertrophy response. The major differences in expression between the adaptive and maladaptive hypertrophy models were centered around the genes involved in metabolism, fibrosis, and immune response. Conversely, transcripts with common expression patterns in both hypertrophy models were associated with signal transduction, cytoskeletal development, and muscle contraction. Thus, despite the apparent differences in the expression response of the heart to either athletic conditioning or pressure overload, there is a set of genes that displays similar expression profiles. This finding lends support to the notion of a generalized cardiac growth mechanism that is activated in response to mechanical perturbation. The common and unique genetic signatures of adaptive and maladaptive hypertrophy may be useful in the diagnosis and treatment of pathological myocardial remodeling.

Increased oxidative stress and antioxidant expression in mouse keratinocytes following exposure to paraquat

Paraquat (1,1′-dimethyl-4,4′-bipyridinium) is a widely used herbicide known to induce skin toxicity. This is thought to be due to oxidative stress resulting from the generation of cytotoxic reactive oxygen intermediates (ROI) during paraquat redox cycling. The skin contains a diverse array of antioxidant enzymes which protect against oxidative stress including superoxide dismutase (SOD), catalase, glutathione peroxidase-1 (GPx-1), heme oxygenase-1 (HO-1), metallothionein-2 (MT-2), and glutathione- S-transferases (GST). In the present studies we compared paraquat redox cycling in primary cultures of undifferentiated and differentiated mouse keratinocytes and determined if this was associated with oxidative stress and altered expression of antioxidant enzymes. We found that paraquat readily undergoes redox cycling in both undifferentiated and differentiated keratinocytes, generating superoxide anion and hydrogen peroxide as well as increased protein oxidation which was greater in differentiated cells. Paraquat treatment also resulted in increased expression of HO-1, Cu,Zn-SOD, catalase, GSTP1, GSTA3 and GSTA4. However, no major differences in expression of these enzymes were evident between undifferentiated and differentiated cells. In contrast, expression of GSTA1-2 was significantly greater in differentiated relative to undifferentiated cells after paraquat treatment. No changes in expression of MT-2, Mn-SOD, GPx-1, GSTM1 or the microsomal GST's mGST1, mGST2 and mGST3, were observed in response to paraquat. These data demonstrate that paraquat induces oxidative stress in keratinocytes leading to increased expression of antioxidant genes. These intracellular proteins may be important in protecting the skin from paraquat-mediated cytotoxicity.

Expression of Bax, Bcl‐xL and Bcl‐2 proteins in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C

To determine the expression of regulators of apoptosis in chronic hepatitis C. Expression of Bax, Bcl-xL and Bcl-2 proteins was assessed immunohistochemically in liver biopsy specimens obtained from 89 adults with chronic hepatitis C. Expression of Bax in hepatocytes correlated inversely with grade of inflammation (P < 0.001) and stage of fibrosis (P = 0.011), classified according to the Scheuer score; expression of Bcl-xL in hepatocytes did not correlate with grade of inflammation (P = 0.106) or stage of fibrosis (P = 0.078); maximum Bcl-xL expression was observed in grade 3 inflammation and stage 4 fibrosis. Expression of Bcl-2 protein in hepatocytes was present in only two cases (both with advanced disease); the expression of Bcl-2 protein in interlobular bile duct epithelial cells correlated with the grade of inflammation (P = 0.018), but not with stage of fibrosis (P = 0.154). The expression of Bcl-2 protein in lymphoid cells infiltrating portal zones and lobules did not correlate with grade of inflammation (P = 0.113) or stage of fibrosis (P = 0.815). Major differences in expression of studied proteins were observed in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C.