Perturbation Of Signaling Pathways Research Articles

Perturbations in inflammatory pathways are associated with shortness of breath profiles in oncology patients receiving chemotherapy.

One plausible mechanistic hypothesis is the potential contribution of inflammatory mechanisms to shortness of breath. This study was aimed to evaluate for associations between the occurrence of shortness of breath and perturbations in inflammatory pathways. Patients with cancer reported the occurrence of shortness of breath six times over two cycles of chemotherapy. Latent class analysis was used to identify subgroups of patients with distinct shortness of breath occurrence profiles (i.e., none (70.5%), decreasing (8.2%), increasing (7.8%), high (13.5%)). Using an extreme phenotype approach, whole transcriptome differential gene expression and pathway impact analyses were performed to evaluate for perturbed signaling pathways associated with shortness of breath between the none and high classes. Two independent samples (RNA-sequencing (n = 293) and microarray (n = 295) methodologies) were evaluated. Fisher's combined probability method was used to combine these results to obtain a global test of the null hypothesis. In addition, an unweighted knowledge network was created using the specific pathway maps to evaluate for interconnections among these pathways. Twenty-nine Kyoto Encyclopedia of Genes and Genomes inflammatory signaling pathways were perturbed. The mitogen-activated protein kinase signaling pathway node had the highest closeness, betweenness, and degree scores. In addition, five common respiratory disease-related pathways, that may share mechanisms with cancer-related shortness of breath, were perturbed. Findings provide preliminary support for the hypothesis that inflammation contribute to the occurrence of shortness of breath in patients with cancer. In addition, the mechanisms that underlie shortness of breath in oncology patients may be similar to other respiratory diseases.

Protection of Ndrg2 deficiency on renal ischemia-reperfusion injury via activating PINK1/Parkin-mediated mitophagy.

Renal ischemia-reperfusion (R-I/R) injury is the most prevalent cause of acute kidney injury, with high mortality and poor prognosis. However, the underlying pathological mechanisms are not yet fully understood. Therefore, this study aimed to investigate the role of N-myc downstream-regulated gene 2 (Ndrg2) in R-I/R injury. We examined the expression of Ndrg2 in the kidney under normal physiological conditions and after R-I/R injury by immunofluorescence staining, real-time polymerase chain reaction, and western blotting. We then detected R-I/R injury in Ndrg2-deficient (Ndrg2-/-) mice and wild type (Ndrg2+/+) littermates in vivo, and detected oxygen and glucose deprivation and reperfusion injury (OGD-R) in HK-2 cells. We further conducted transcriptomic sequencing to investigate the role of Ndrg2 in R-I/R injury and detected levels of oxidative stress and mitochondrial damage by dihydroethidium staining, biochemical assays, and western blot. Finally, we measured the levels of mitophagy in Ndrg2+/+ and Ndrg2-/- mice after R-I/R injury or HK-2 cells in OGD-R injury. We found that Ndrg2 was primarily expressed in renal proximal tubules and significantly decreased its expression 24h after R-I/R injury. Ndrg2-/- mice exhibited significantly attenuated R-I/R injury compared to Ndrg2+/+ mice. Transcriptomics profiling showed that Ndrg2 deficiency induced perturbations of multiple signaling pathways, downregulated inflammatory responses and oxidative stress, and increased autophagy following R-I/R injury. Further studies revealed that Ndrg2 deficiency reduced oxidative stress and mitochondrial damage. Notably, Ndrg2 deficiency significantly activated phosphatase and tensin homologue on chromosome ten-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy. The downregulation of NDRG2 expression significantly increased cell viability after OGD-R injury, increased the expression of heme oxygenase-1, decreased the expression of nicotinamide adenine dinucleotide phosphate oxidase 4, and increased the expression of the PINK1/Parkin pathway. Ndrg2 deficiency might become a therapy target for R-I/R injury by decreasing oxidative stress, maintaining mitochondrial homeostasis, and activating PINK1/Parkin-mediated mitophagy.

Kinase signalling adaptation supports dysfunctional mitochondria in disease.

Mitochondria form a critical control nexus which are essential for maintaining correct tissue homeostasis. An increasing number of studies have identified dysregulation of mitochondria as a driver in cancer. However, which pathways support and promote this adapted mitochondrial function? A key hallmark of cancer is perturbation of kinase signalling pathways. These pathways include mitogen activated protein kinases (MAPK), lipid secondary messenger networks, cyclic-AMP-activated (cAMP)/AMP-activated kinases (AMPK), and Ca2+/calmodulin-dependent protein kinase (CaMK) networks. These signalling pathways have multiple substrates which support initiation and persistence of cancer. Many of these are involved in the regulation of mitochondrial morphology, mitochondrial apoptosis, mitochondrial calcium homeostasis, mitochondrial associated membranes (MAMs), and retrograde ROS signalling. This review will aim to both explore how kinase signalling integrates with these critical mitochondrial pathways and highlight how these systems can be usurped to support the development of disease. In addition, we will identify areas which require further investigation to fully understand the complexities of these regulatory interactions. Overall, this review will emphasize how studying the interaction between kinase signalling and mitochondria improves our understanding of mitochondrial homeostasis and can yield novel therapeutic targets to treat disease.

The Use of Fruit and Vegetable by-Products as Enhancers of Health Status of Piglets after Weaning: The Role of Bioactive Compounds from Apple and Carrot Industrial Wastes.

At weaning, piglets are exposed to a large variety of stressors, from environmental/behavioral factors to nutritional stress. Weaning transition affects the gastrointestinal tract especially, resulting in specific disturbances at the level of intestinal morphology, barrier function and integrity, mucosal immunity and gut microbiota. All these alterations are associated with intestinal inflammation, oxidative stress and perturbation of intracellular signaling pathways. The nutritional management of the weaning period aims to achieve the reinforcement of intestinal integrity and functioning to positively modulate the intestinal immunity and that of the gut microbiota and to enhance the health status of piglets. That is why the current research is focused on the raw materials rich in phytochemicals which could positively modulate animal health. The composition analysis of fruit, vegetable and their by-products showed that identified phytochemicals could act as bioactive compounds, which can be used as modulators of weaning-induced disturbances in piglets. This review describes nutritional studies which investigated the effects of bioactive compounds derived from fruit (apple) and vegetables (carrot) or their by-products on the intestinal architecture and function, inflammatory processes and oxidative stress at the intestinal level. Data on the associated signaling pathways and on the microbiota modulation by bioactive compounds from these by-products are also presented.

Anxiety in oncology outpatients is associated with perturbations in pathways identified in anxiety focused network pharmacology research

PurposeEvaluate for perturbed signaling pathways associated with subgroups of patients with low versus high levels of state anxiety. These pathways were compared to the pathways identified across eight network pharmacology studies of the anxiolytic effect(s) of a variety of compounds.MethodsAdult outpatients had a diagnosis of breast, gastrointestinal, gynecological, or lung cancer; had received chemotherapy within the preceding four weeks; and were scheduled to receive at least two additional cycles of chemotherapy. Latent profile analysis was used to identify subgroups of patients with distinct anxiety profiles based on Spielberger State Anxiety Inventory scores that were obtained six times over two cycles of chemotherapy. Blood samples were processed using RNA sequencing (i.e., RNA-seq sample, n = 244) and microarray (i.e., microarray sample; n = 256) technologies. Pathway perturbations were assessed using pathway impact analysis. Fisher’s combined probability method was used to combine test results using a false discovery rate of 0.01.ResultsIn the RNA-seq sample, 62.3% and 37.7% of the patients were in the low- and high-anxiety classes, respectively. In the microarray sample, 61.3% and 38.7% were in the low and high-anxiety classes, respectively. Forty-one perturbed signaling pathways were identified. Eight of these pathways were common to those identified in the network pharmacology studies.ConclusionsFindings increase our knowledge of the molecular mechanisms that underlie anxiety in patients receiving chemotherapy. This study provides initial insights into how anxiety in patients with cancer may share common mechanisms with anxiety in patients with other clinical conditions.

Targeting Esophageal Squamous Cell Carcinoma by Combining Copper Ionophore Disulfiram and JMJD3/UTX Inhibitor GSK J4.

The alcohol-averse drug disulfiram has been reported to have anti-tumor effects and is well suited for drug combinations. In order to identify potential drug combinations in esophageal squamous cell carcinoma (ESCC), we screened a bioactive compound library with the disulfiram copper chelation product CuET. The Jumonji domain-containing protein 3 (JMJD3) and the ubiquitously transcribed tetratricopeptide repeat protein X-linked (UTX) inhibitor GSK J4 were identified. To further understand the molecular mechanism underlying the efficient drug combination, we applied quantitative mass spectrometry to analyze the signaling pathway perturbation after drug treatment. The data revealed that the synergistic effect of GSK J4 and CuET was due to the interaction among JMJD3 and UTX, which may play important roles in maintaining endoplasmic reticulum (ER) homeostasis in tumor cells. Interestingly, our clinical data analysis showed that high expression of JMJD3 and UTX was associated with T stage and worse prognosis of ESCC patients, further supporting the importance of the above findings. In conclusion, our findings suggest that the combination of CuET and targeting JMJD3/UTX may be a safe, effective, and available treatment for ESCC.

Mechanistic insights into hormesis induced by erythromycin in the marine alga Thalassiosira weissflogii

Erythromycin (ERY) is a typical macrolide antibiotic with large production and extensive use on a global scale. Detection of ERY in both freshwaters and coaster seawaters, as well as relatively high ecotoxicity of ERY have been documented. Notably, hormesis has been reported on several freshwater algae under ERY stress, where growth was promoted at relatively lower exposures but inhibited at higher treatment levels. On the contrary, there is limited information of ERY toxicity in marine algae, hampering the risk assessment on ERY in the coaster waters. The presence of hormesis may challenge the current concept of dose-response adopted in chemical risk assessment. Whether and how exposure to ERY can induce dose-dependent toxicity in marine algae remain virtually unknown, especially at environmentally relevant concentrations. The present study used a model marine diatom Thalassiosira weissflogii (T. weissflogii) to reveal its toxicological responses to ERY at different biological levels and decipher the underlying mechanisms. Assessment of multiple apical endpoints shows an evident growth promotion following ERY exposure at an environmentally relevant concentration (1 µg/L), associated with increased contents reactive oxygen species (ROS) and chlorophyll-a (Chl-a), activated signaling pathways related to ribosome biosynthesis and translation, and production of total soluble protein. By contrast, growth inhibition in the 750 and 2500 µg/L treatments was attributed to reduced viability, increased ROS formation, reduced content of total soluble protein, inhibited photosynthesis, and perturbed signaling pathways involved in xenobiotic metabolism, ribosome, metabolism of amino acid, and nitrogen metabolism. Measurements of multiple apical endpoints coupled with de novo transcriptomics analysis applied in the present study, a systems biology approach, can generate detailed mechanistic information of chemical toxicity including dose-response and species sensitivity difference used in environmental risk assessment.

Retinoic acid signaling pathway perturbation impacts mesodermal-tissue development in the zebrafish embryo: Biomarker candidate identification using transcriptomics.

The zebrafish embryo (ZE) model provides a developmental model well conserved throughout vertebrate embryogenesis, with relevance for early human embryo development. It was employed to search for gene expression biomarkers of compound-induced disruption of mesodermal development. We were particularly interested in the expression of genes related to the retinoic acid signaling pathway (RA-SP), as a major morphogenetic regulating mechanism. We exposed ZE to teratogenic concentrations of valproic acid (VPA) and all-trans retinoic acid (ATRA), using folic acid (FA) as a non-teratogenic control compound shortly after fertilization for 4 h, and performed gene expression analysis by RNA sequencing. We identified 248 genes specifically regulated by both teratogens but not by FA. Further analysis of this gene set revealed 54 GO-terms related to the development of mesodermal tissues, distributed along the paraxial, intermediate, and lateral plate sections of the mesoderm. Gene expression regulation was specific to tissues and was observed for somites, striated muscle, bone, kidney, circulatory system, and blood. Stitch analysis revealed 47 regulated genes related to the RA-SP, which were differentially expressed in the various mesodermal tissues. These genes provide potential molecular biomarkers of mesodermal tissue and organ (mal)formation in the early vertebrate embryo.

The Latest Developments in Using Proteomic Biomarkers from Urine and Serum for Non-Invasive Disease Diagnosis and Prognosis.

Due to diagnostic improvements, medical diagnostics is demanding non-invasive or minimally invasive methods. Non-invasively obtained body fluids (eg., Urine, serum) can replace cerebral fluid, amniotic fluid, synovial fluid, bronchoalveolar lavage fluid, and others for diagnostic reasons. Many illnesses are induced by perturbations of cellular signaling pathways and associated pathway networks as a result of genetic abnormalities. These disturbances are represented by a shift in the protein composition of the fluids surrounding the tissues and organs that is, tissue interstitial fluid (TIF). These variant proteins may serve as diagnostic "signatures" for a variety of disorders. This review provides a concise summary of urine and serum biomarkers that may be used for the diagnosis and prognosis of a variety of disorders, including cancer, brain diseases, kidney diseases, and other system diseases. The studies reviewed in this article suggest that serum and urine biomarkers of various illnesses may be therapeutically useful for future diagnostics. Correct illness management is crucial for disease prognosis, hence non-invasive serum and urine biomarkers have been extensively studied for diagnosis, subclassification, monitoring disease activity, and predicting treatment results and consequences.

The Cyclin-G Associated Kinase (GAK) Is a Critical Dependency for Cell-Cycle Progression in Diffuse Large B-Cell Lymphoma Associated with Deregulation of the Retinoblastoma Tumor Suppressor

Background: Recurrent genomic alterations in specific cancers reveal tumor drivers that inform the development of rational therapeutic approaches. In diffuse large B-cell lymphoma (DLBCL), such oncoprotein-focused drug development has achieved only limited success. Phenotypic screening, less explored in DLBCL drug discovery, is an emerging alternative that can be leveraged especially through the application of machine-learning analyses to raw screening data. Under this paradigm, we reveal strong therapeutic potential in DLBCL of inhibiting the cyclin-G associated kinase (GAK). GAK participates in clathrin-mediated endocytosis and regulates mitotic-spindle alignment during cell division. It has been studied as an antiviral target that interrupts internalization at cell membranes but is minimally explored in cancer therapeutics and is a novel target in DLBCL. Methods and Results: We performed a first-of-its-kind screen of kinase inhibitors in DLBCL followed by machine learning-based analysis using our in-house target identification platform, idTRAX. The screen revealed GAK as a strong target for both activated B-cell (ABC) and germinal center B-cell (GCB) derived cell lines with complete sparing of peripheral-blood mononuclear cells (PBMCs), included as non-malignant controls. We analyzed gene-expression data available for 414 previously untreated DLBCL patients (Lenz cohort) and found tumors with high GAK expression had dramatically worse overall survival (OS) compared to those with low expression (hazard ratio=1.84 (95% conf. int. 1.33-2.53), p=0.0002). Mechanistically, we explored GAK's role in endocytosis but found no change in cell-surface B-cell receptor (BCR) expression after treatment with the GAK tool compound SGC-GAK-1. In addition, there was no evident perturbation of signaling pathways downstream of BCR by immunoblotting. We found instead that GAK inhibition in DLBCL results in profound disruption of progression through mitosis, potently triggering the spindle assembly checkpoint (SAC) and causing accumulation in G2 before onset of apoptosis. We explored whether GAK dependency is induced due to cell cycle deregulation. Analysis of gene-expression data in the Lenz cohort showed a striking correlation between high GAK expression and low expression of RB1, encoding the tumor suppressive master cell-cycle regulator retinoblastoma-associated protein (RB, p<0.0001). The ABC-DLBCL cell lines RIVA and U2932 completely lack RB expression and show high sensitivity and accumulation in G2 in response to SGC-GAK-1 treatment. Immunofluorescent confocal microscopy revealed profound disruption of mitotic spindles and shattering of chromosomes in these cells and, to a lesser extent, in RB-competent lines. SGC-GAK-1 is not optimized for use as a chemical probe in vivo, but target validation through shRNA knockdown in DLBCL patient-derived xenograft (PDX) models is underway and expected at time of presentation. Conclusions: GAK is a novel kinase therapeutic target in DLBCL revealed by our target identification platform, idTRAX. GAK's role in mitotic-spindle alignment represents a critical kinase dependency for DLBCL tumors, in particular those with cell-cycle deregulation due to loss of RB function. Our ongoing efforts to synthesize novel GAK inhibitors and reveal synergistic targets may reveal new therapeutic strategies for translation to clinical trials for DLBCL patients with unmet clinical needs.

Ethanol exposure perturbs sea urchin development and disrupts developmental timing

Ethanol is a known vertebrate teratogen that causes craniofacial defects as a component of fetal alcohol syndrome (FAS). Our results show that sea urchin embryos treated with ethanol similarly show broad skeletal patterning defects, potentially analogous to the defects associated with FAS. The sea urchin larval skeleton is a simple patterning system that involves only two cell types: the primary mesenchymal cells (PMCs) that secrete the calcium carbonate skeleton and the ectodermal cells that provide migratory, positional, and differentiation cues for the PMCs. Perturbations in RA biosynthesis and Hh signaling pathways are thought to be causal for the FAS phenotype in vertebrates. Surprisingly, our results indicate that these pathways are not functionally relevant for the teratogenic effects of ethanol in developing sea urchins. We found that developmental morphology as well as the expression of some ectodermal and PMC genes was delayed by ethanol exposure. Temporal transcriptome analysis revealed significant impacts of ethanol on signaling and metabolic gene expression, and a disruption in the timing of GRN gene expression that includes both delayed and precocious gene expression throughout the specification network. We conclude that the skeletal patterning perturbations in ethanol-treated embryos likely arise from a loss of temporal synchrony within and between the instructive and responsive tissues.

Detect the early-warning signals of diseases based on signaling pathway perturbations on a single sample

BackgroundDuring the pathogenesisof complex diseases, a sudden health deterioration will occur as results of the cumulative effect of various internal or external factors. The prediction of an early warning signal (pre-disease state) before such deterioration is very important in clinical practice, especially for a single sample. The single-sample landscape entropy (SLE) was proposed to tackle this issue. However, the PPI used in SLE was lack of definite biological meanings. Besides, the calculation of multiple correlations based on limited reference samples in SLE is time-consuming and suspect.ResultsAbnormal signals generally exert their effect through the static definite biological functions in signaling pathways across the development of diseases. Thus, it is a natural way to study the propagation of the early-warning signals based on the signaling pathways in the KEGG database. In this paper, we propose a signaling perturbation method named SSP, to study the early-warning signal in signaling pathways for single dynamic time-series data. Results in three real datasets including the influenza virus infection, lung adenocarcinoma, and acute lung injury show that the proposed SSP outperformed the SLE. Moreover, the early-warning signal can be detected by one important signaling pathway PI3K-Akt.ConclusionsThese results all indicate that the static model in pathways could simplify the detection of the early-warning signals.

Y1 receptors modulate taste-related behavioral responsiveness in male mice to prototypical gustatory stimuli

Mammalian taste bud cells express receptors for numerous peptides implicated elsewhere in the body in the regulation of metabolism, nutrient assimilation, and satiety. The perturbation of several peptide signaling pathways in the gustatory periphery results in changes in behavioral and/or physiological responsiveness to subsets of taste stimuli. We previously showed that Peptide YY (PYY) – which is present in both saliva and in subsets of taste cells – can affect behavioral taste responsiveness and reduce food intake and body weight. Here, we investigated the contributions of taste bud-localized receptors for PYY and the related Neuropeptide Y (NPY) on behavioral taste responsiveness. Y1R, but not Y2R, null mice show reduced responsiveness to sweet, bitter, and salty taste stimuli in brief-access taste tests; similar results were seen when wildtype mice were exposed to Y receptor antagonists in the taste stimuli. Finally, mice in which the gene encoding the NPY propeptide was deleted also showed reduced taste responsiveness to sweet and bitter taste stimuli. Collectively, these results suggest that Y1R signaling, likely through its interactions with NPY, can modulate peripheral taste responsiveness in mice.

Perturbed transcriptional profiles after chronic low dose rate radiation in mice.

Adverse health outcomes of ionizing radiation given chronically at low dose rates are highly debated, a controversy also relevant for other stressors. Increased knowledge is needed for a more comprehensive understanding of the damaging potential of ionizing radiation from all dose rates and doses. There is a lack of relevant low dose rate data that is partly ascribed to the rarity of exposure facilities allowing chronic low dose rate exposures. Using the FIGARO facility, we assessed early (one day post-radiation) and late (recovery time of 100-200 days) hepatic genome-wide transcriptional profiles in male mice of two strains (CBA/CaOlaHsd and C57BL/6NHsd) exposed chronically to a low dose rate (2.5 mGy/h; 1200h, LDR), a mid-dose rate (10 mGy/h; 300h, MDR) and acutely to a high dose rate (100 mGy/h; 30h, HDR) of gamma irradiation, given to an equivalent total dose of 3 Gy. Dose-rate and strain-specific transcriptional responses were identified. Differently modulated transcriptional responses across all dose rate exposure groups were evident by the representation of functional biological pathways. Evidence of changed epigenetic regulation (global DNA methylation) was not detected. A period of recovery markedly reduced the number of differentially expressed genes. Using enrichment analysis to identify the functional significance of the modulated genes, perturbed signaling pathways associated with both cancer and non-cancer effects were observed, such as lipid metabolism and inflammation. These pathways were seen after chronic low dose rate and were not restricted to the acute high dose rate exposure. The transcriptional response induced by chronic low dose rate ionizing radiation suggests contribution to conditions such as cardiovascular diseases. We contribute with novel genome wide transcriptional data highlighting dose-rate-specific radiation responses and emphasize the importance of considering both dose rate, duration of exposure, and variability in susceptibility when assessing risks from ionizing radiation.

Signaling pathway perturbation analysis for assessment of biological impact of cigarette smoke on lung cells

Exposure to cigarette smoke (CS) results in injury to the epithelial cells of the human respiratory tract and has been implicated as a causative factor in the development of chronic obstructive pulmonary disease and lung cancers. The application of omics-scale methodologies has improved the capacity to understand cellular signaling processes underlying response to CS exposure. We report here the development of an algorithm based on quantitative assessment of transcriptomic profiles and signaling pathway perturbation analysis (SPPA) of human bronchial epithelial cells (HBEC) exposed to the toxic components present in CS. HBEC were exposed to CS of different compositions and for different durations using an ISO3308 smoking regime and the impact of exposure was monitored in 2263 signaling pathways in the cell to generate a total effect score that reflects the quantitative degree of impact of external stimuli on the cells. These findings support the conclusion that the SPPA algorithm provides an objective, systematic, sensitive means to evaluate the biological impact of exposures to CS of different compositions making a powerful comparative tool for commercial product evaluation and potentially for other known or potentially toxic environmental smoke substances.

Heterodimerization of TPO and IFNγ Impairs Human Hematopoietic Stem/Progenitor Cell Signaling and Survival in Chronic Inflammation

Thrombopoietin (TPO) is the main regulator of hematopoietic stem and progenitor cell (HSPC) self-renewal and survival. Upon binding to its receptor, c-MPL, TPO activates cell signaling through JAK-STAT and other pathways. In recent clinical trials, eltrombopag, a small molecule c-MPL agonist, improved trilineage hematopoiesis in subjects with severe aplastic anemia (SAA). Paradoxically, TPO levels are already markedly elevated in these patients. To explain this paradox, we previously showed that IFNγ, a key proinflammatory cytokine implicated in the destruction of HSPCs in SAA, inhibited TPO signaling in human CD34+ HSPCs cultured in the presence of both cytokines. In contrast, eltrombopag could evade this inhibition in vitro, resulting in improved maintenance of progenitors in clonogenic CFU assays, and long-term repopulating cells in NSG transplantation models compared with TPO-containing cultures (Cheng et al., ASH abstract 2016). In this study, we sought to characterize the mechanisms by which eltrombopag evades IFNγ blockade of c-MPL signaling. Because activation of both c-MPL and IFNγ receptor by their respective ligands induces negative regulatory feedback mechanisms from the SOCS family, we first measured expression of SOCS proteins in CD34+ cells exposed to IFNγ and TPO or eltrombopag. SOCS expression was equally upregulated by TPO and eltrombopag in the presence of IFNγ, suggesting an alternative explanation for eltrombopag's ability to escape IFNγ-induced perturbation of TPO signaling. Because TPO and eltrombopag distinctively bind to the extracellular or juxtamembrane domain of c-MPL, respectively, we hypothesized that IFNγ may decrease binding affinity of TPO, but not eltrombopag, to c-MPL. We used microscale thermophoresis (MST) to assess the impact of IFNγ on TPO binding to its receptor. As previously reported, we showed that TPO and c-MPL interact via both a high affinity (KD, app<0.11 ± 0.04 nM) and a low affinity (KD, app=1100 ± 130 nM) binding site. In contrast, IFNγ had no interaction with c-MPL. Remarkably, addition of 100-molar excess IFNγ prevented binding of TPO to c-MPL at the low affinity site, but had no impact on the high affinity binding site (KD, app<0.20 ± 0.04 nM) (Panel A). To explain this result, we hypothesized that TPO and IFNγ may directly interact and form heteromeric complexes that hinder binding to c-MPL, whereas the non-peptide small molecule eltrombopag could conceivably evade that process. Strikingly, MST assays revealed a specific, one-site heterodimeric interaction between TPO and IFNγ (KD, app=540 ± 30 nM), while no heterodimer formation was observed between either TPO or IFNγ and other early-acting cytokines (SCF and Flt3L) (Panel B). These data suggest that TPO:IFNγ heterodimers may be responsible for the hindered TPO:c-MPL low-affinity interaction observed in the presence of IFNγ. To confirm this finding, we investigated whether TPO-induced c-MPL dimerization was interrupted in the presence of IFNγ. Using raster image correlation spectroscopy (RICS) to determine average diffusion coefficients of GFP-tagged c-MPL in live cells, we have preliminarily found that IFNγ negatively affects c-MPL dimerization in cells cultured with TPO, but not with eltrombopag. Further studies are underway. Taken together, our data provide a new and provocative paradigm to explain the observed negative impact of the proinflammatory cytokine IFNγ on human HSPC maintenance in bone marrow failure syndromes, and the ability of a small molecule (eltrombopag) to evade this inhibition. We propose that, under chronic inflammatory conditions, IFNγ specifically heterodimerizes with TPO, resulting in (i) occlusion of the low-affinity binding site of TPO to c-MPL, (ii) impaired receptor dimerization, (iii) perturbation of TPO-induced signaling pathways, and (iv) decreased survival of human HSPCs (Panel C). This new understanding could have far-reaching clinical implications for other disorders of chronic inflammation. [Display omitted] DisclosuresHuntsman:Novartis: Research Funding. Cheng:Novartis: Research Funding. Larochelle:Novartis: Research Funding; Novartis: Research Funding; StemCell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit.

Microarray analysis reveals ONC201 mediated differential mechanisms of CHOP gene regulation in metastatic and nonmetastatic colorectal cancer cells

The imipramine ONC201 has antiproliferative effects in several cancer cell types and activates integrated stress response pathway associated with the induction of Damage Inducible Transcript 3 (DDIT3, also known as C/EBP homologous protein or CHOP). We investigated the signaling pathways through which ONC201/CHOP crosstalk is regulated in ONC201-treated nonmetastatic and metastatic cancer cell lines (Dukes' type B colorectal adenocarcinoma nonmetastatic SW480 and metastatic LS-174T cells, respectively). Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry, gene expression was assessed by Affymetrix microarray, signaling pathway perturbations were assessed in silico, and key regulatory proteins were validated by Western blotting. Unlike LS-174T cells, SW480 cells were resistant to ONC201 treatment; Gene Ontology analysis of differentially expressed genes showed that cellular responsiveness to ONC201 treatment also differed substantially. In both ONC201-treated cell lines, CHOP expression was upregulated; however, its upstream regulatory mechanisms were perturbed. Although, PERK, ATF6 and IRE1 ER-stress pathways upregulated CHOP in both cell types, the Bak/Bax pathway regulated CHOP only LS-174T cells. Additionally, CHOP RNA splicing profiles varied between cell lines; these were further modified by ONC201 treatment. In conclusion, we delineated the signaling mechanisms by which CHOP expression is regulated in ONC201-treated non-metastatic and metastatic colorectal cell lines. The observed differences could be related to cellular plasticity and metabolic reprogramming, nevertheless, detailed mechanistic studies are required for further validations.

Variation in phenotypes from a Bmp-Gata3 genetic pathway is modulated by Shh signaling.

We sought to understand how perturbation of signaling pathways and their targets generates variable phenotypes. In humans, GATA3 associates with highly variable defects, such as HDR syndrome, microsomia and choanal atresia. We previously characterized a zebrafish point mutation in gata3 with highly variable craniofacial defects to the posterior palate. This variability could be due to residual Gata3 function, however, we observe the same phenotypic variability in gata3 null mutants. Using hsp:GATA3-GFP transgenics, we demonstrate that Gata3 function is required between 24 and 30 hpf. At this time maxillary neural crest cells fated to generate the palate express gata3. Transplantation experiments show that neural crest cells require Gata3 function for palatal development. Via a candidate approach, we determined if Bmp signaling was upstream of gata3 and if this pathway explained the mutant's phenotypic variation. Using BRE:d2EGFP transgenics, we demonstrate that maxillary neural crest cells are Bmp responsive by 24 hpf. We find that gata3 expression in maxillary neural crest requires Bmp signaling and that blocking Bmp signaling, in hsp:DN-Bmpr1a-GFP embryos, can phenocopy gata3 mutants. Palatal defects are rescued in hsp:DN-Bmpr1a-GFP;hsp:GATA3-GFP double transgenic embryos, collectively demonstrating that gata3 is downstream of Bmp signaling. However, Bmp attenuation does not alter phenotypic variability in gata3 loss-of-function embryos, implicating a different pathway. Due to phenotypes observed in hypomorphic shha mutants, the Sonic Hedgehog (Shh) pathway was a promising candidate for this pathway. Small molecule activators and inhibitors of the Shh pathway lessen and exacerbate, respectively, the phenotypic severity of gata3 mutants. Importantly, inhibition of Shh can cause gata3 haploinsufficiency, as observed in humans. We find that gata3 mutants in a less expressive genetic background have a compensatory upregulation of Shh signaling. These results demonstrate that the level of Shh signaling can modulate the phenotypes observed in gata3 mutants.

Intense terahertz pulses inhibit Ras signaling and other cancer-associated signaling pathways in human skin tissue models

Terahertz (THz) radiation has shown unique advantages in biomedical applications for novel diagnostic technologies due to the high sensitivity to molecular structure and chemical concentration. However, emerging evidence shows that intense pulses of THz radiation can induce significant non-thermal biological effects that must be characterized. In human skin exposed to intense THz pulses, relatively large responses characterized by differential gene expression profiles are observed. These data are analyzed by signaling pathway perturbation analysis to predict phenotypic endpoints and dysregulatory effects on cancer-related processes. The activities of several important pathways that drive the initiation, development, and progression of many human cancers are predicted to be suppressed, and this effect is intensity-dependent. Some affected pathways are targets for current and emerging anti-cancer therapies. In particular, the activity of the Ras signaling and Calcium signaling pathways is predicted to be significantly inhibited. These results indicate the possibility of an additional therapeutic mechanism of intense THz pulses, due to the potential for targeted suppression of pro-mitotic activity in diseased tissue.

Analysis of astaxanthin molecular targets based on network pharmacological strategies.

In order to further explore the potential pharmacological activity of astaxanthin (AST), network pharmacological approaches were employed in this work to systematically investigate its affinity targets, perturbed signaling pathways, and related disease applications. First, potential targets were captured based on AST chemical structure information. Enrichment analysis was then performed using bioinformatics tools to predict the biological processes and diseases in which AST targets are involved. The results suggest that AST is involved in steroid hormone metabolism, and the regulation of glucocorticoids may be one of the potential mechanisms of its known therapeutic effects on depression and insulin resistance. Molecular docking experiments confirmed that AST can form stable binding to several key nodes (SRD5A2, STS, AKR1C2, HSD11B1, and CYP17A1) in steroid hormone biosynthesis. More importantly, the molecular targets of AST were the most significantly associated with endometriosis. Functionally, grouped analysis of key therapeutic nodes was carried out by establishing the interaction network between drug targets and disease targets. While exerting inflammatory effects, the regulation of estrogen and other semiochemicals by targeting steroid metabolism may be the biological basis for the potential treatment of endometriosis with AST. This work provides a theoretical basis for further exploring the pharmacological mechanisms of AST and development of new therapeutic applications. PRACTICAL APPLICATIONS: In this study, systematic pharmacological methods were used to identify the potential therapeutic effects and associated mechanisms of astaxanthin, providing a bioinformatics basis for further exploration of astaxanthin's new pharmacological properties in foods.