Uncovering the Effect of SARS-CoV-2 on Liver Metabolism via Genome-Scale Metabolic Modeling for Reprogramming and Therapeutic Strategies.

Genome-scale metabolic models (GEMs) are promising computational tools that contribute to elucidating host-virus interactions at the system level and developing therapeutic strategies against viral infection. In this study, the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on liver metabolism was investigated using integrated GEMs of human hepatocytes and SARS-CoV-2. They were generated for uninfected and infected hepatocytes using transcriptome data. Reporter metabolite analysis resulted in significant transcriptional changes around several metabolites involved in xenobiotics, drugs, arachidonic acid, and leukotriene metabolisms due to SARS-CoV-2 infection. Flux balance analysis and minimization of metabolic adjustment approaches unraveled possible virus-induced hepatocellular reprogramming in fatty acid, glycerophospholipid, sphingolipid cholesterol, and folate metabolisms, bile acid biosynthesis, and carnitine shuttle among others. Reaction knockout analysis provided critical reactions in glycolysis, oxidative phosphorylation, purine metabolism, and reactive oxygen species detoxification subsystems. Computational analysis also showed that administration of dopamine, glucosamine, D-xylose, cysteine, and (R)-3-hydroxybutanoate contributes to alleviating viral infection. In essence, the reconstructed host-virus GEM helps us understand metabolic programming and develop therapeutic strategies to battle SARS-CoV-2.

Applications of 3D Bioprinting Technology to Brain Cells and Brain Tumor Models: Special Emphasis to Glioblastoma.

Primary brain tumor is one of the most fatal diseases. The most malignant type among them, glioblastoma (GBM), has low survival rates. Standard treatments reduce the life quality of patients due to serious side effects. Tumor aggressiveness and the unique structure of the brain render the removal of tumors and the development of new therapies challenging. To elucidate the characteristics of brain tumors and examine their response to drugs, realistic systems that mimic the tumor environment and cellular crosstalk are desperately needed. In the past decade, 3D GBM models have been presented as excellent platforms as they allowed the investigation of the phenotypes of GBM and testing innovative therapeutic strategies. In that scope, 3D bioprinting technology offers utilities such as fabricating realistic 3D bioprinted structures in a layer-by-layer manner and precisely controlled deposition of materials and cells, and they can be integrated with other technologies like the microfluidics approach. This Review covers studies that investigated 3D bioprinted brain tumor models, especially GBM using 3D bioprinting techniques and essential parameters that affect the result and quality of the study like frequently used cells, the type and physical characteristics of hydrogel, bioprinting conditions, cross-linking methods, and characterization techniques.

Identification of Therapeutic Targets for Medulloblastoma by Tissue-Specific Genome-Scale Metabolic Model.

Medulloblastoma (MB), occurring in the cerebellum, is the most common childhood brain tumor. Because conventional methods decline life quality and endanger children with detrimental side effects, computer models are needed to imitate the characteristics of cancer cells and uncover effective therapeutic targets with minimum toxic effects on healthy cells. In this study, metabolic changes specific to MB were captured by the genome-scale metabolic brain model integrated with transcriptome data. To determine the roles of sphingolipid metabolism in proliferation and metastasis in the cancer cell, 79 reactions were incorporated into the MB model. The pathways employed by MB without a carbon source and the link between metastasis and the Warburg effect were examined in detail. To reveal therapeutic targets for MB, biomass-coupled reactions, the essential genes/gene products, and the antimetabolites, which might deplete the use of metabolites in cells by triggering competitive inhibition, were determined. As a result, interfering with the enzymes associated with fatty acid synthesis (FAs) and the mevalonate pathway in cholesterol synthesis, suppressing cardiolipin production, and tumor-supporting sphingolipid metabolites might be effective therapeutic approaches for MB. Moreover, decreasing the activity of succinate synthesis and GABA-catalyzing enzymes concurrently might be a promising strategy for metastatic MB.

Understanding the Link between Inflammasome and Apoptosis through the Response of THP-1 Cells against Drugs Using Droplet-Based Microfluidics.

Droplet-based microfluidic devices are used to investigate monocytic THP-1 cells in response to drug administration. Consistent and reproducible droplets are created, each of which acts as a bioreactor to carry out single cell experiments with minimized contamination and live cell tracking under an inverted fluorescence microscope for more than 2 days. Here, the effects of three different drugs (temsirolimus, rifabutin, and BAY 11-7082) on THP-1 are examined and the results are analyzed in the context of the inflammasome and apoptosis relationship. The ASC adaptor gene tagged with GFP is monitored as the inflammasome reporter. Thus, a systematic way is presented for deciphering cell-to-cell heterogeneity, which is an important issue in cancer treatment. The drug temsirolimus, which has effects of disrupting the mTOR pathway and triggering apoptosis in tumor cells, causes THP-1 cells to express ASC and to be involved in apoptosis. Treatment with rifabutin, which inhibits proliferation and initiates apoptosis in cells, affects ASC expression by first increasing and then decreasing it. CASP-3, which has a role in apoptosis and is directly related to ASC, has an increasing level in inflammasome conditioning. Thus, the cell under the effect of rifabutin might be faced with programmed cell death faster. The drug BAY 11-7082, which is responsible for NFκB inhibition, shows similar results to temsirolimus with more than 60% of cells having high fluorescence intensity (ASC expression). The microfluidic platform presented here offers strong potential for studying newly developed small-molecule inhibitors for personalized/precision medicine.

The effects of high intensity-interval training on vaspin, adiponectin and leptin levels in women with polycystic ovary syndrome.

AIM: We aimed to investigate the metabolic effects of HIIT exercise on PCOS patients and how it affects adiponectin, vaspin and leptin. MATERIAL AND METHODS: Twenty women with PCOS were included in the study and were divided into two groups. HIIT program was applied for 10 PCOS and Medium Intensity Continuous Training (MICT) program was applied for other 10 PCOS. At the beginning and at the end of the study, total cholesterol, low-density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), triglyceride(TG), insulin, Adiponectin, Leptin, Vaspin levels of both PCOS groups were evaluated. RESULTS: When PCOS patients by performed HIIT exercise for 12 weeks, we found that the levels of leptin and vaspin did not change while adiponectin levels increased. Moreover serum levels of insulin, TG, total cholesterol, LDL-C decreased but levels of HDL-C increased. CONCLUSION: HIIT increased in the adiponectin levels in women with PCOS and provided more weight loss.

Real-Time Single-Cell Monitoring of Drug Effects Using Droplet-Based Microfluidic Technology: A Proof-of-Concept Study.

Drugs that act on ribosome biogenesis and cell proliferation play important roles in treatment of human diseases. Moreover, measurement of drug effects at a single-cell level would create vast opportunities for pharmaceutical innovation. We present in this study an original proof-of-concept study of single-cell measurement of drug effects with a focus on inhibition of ribosome biogenesis and cell proliferation, and using yeast (Saccharomyces cerevisiae) as a model eukaryotic organism. We employed a droplet-based microfluidic technology and nucleolar protein-tagged strain of the yeast for real-time monitoring of the cells. We report a comprehensive account of the ways in which interrelated pathways are impacted by drug treatment in a single-cell level. Self-organizing maps, transcription factor, and Gene Ontology enrichment analyses were utilized to these ends. This article makes a contribution to advance single-cell measurement of drug effects. We anticipate the microfluidic technology platform presented herein is well poised for future applications in personalized/precision medicine research as well as in industrial settings for drug discovery and clinical development. In addition, the study offers new insights on ribosome biogenesis and cell proliferation that should prove useful in cancer research and other complex human diseases impacted by these key cellular processes.

Thermoplastic microfluidic bioreactors with integrated electrodes to study tumor treating fields on yeast cells.

Tumor-treating fields (TTFields) are alternating electrical fields of intermediate frequency and low intensity that can slow or inhibit tumor growth by disrupting mitosis division of cancerous cells through cell cycle proteins. In this work, for the first time, an in-house fabricated cyclo-olefin polymer made microfluidic bioreactors are integrated with Cr/Au interdigitated electrodes to test TTFields on yeast cells with fluorescent protein:Nop56 gene. A small gap between electrodes (50 µm) allows small voltages (<150 mV) to be applied on the cells; hence, uninsulated gold electrodes are used in the non-faradaic region without causing any electrochemical reaction at the electrode-medium interface. Electrochemical modeling as well as impedance characterization and analysis of the electrodes are done using four different cell nutrient media. The experiments with yeast cells are done with 150 mV, 150 kHz and 30 mV, 200 kHz sinusoidal signals to generate electrical field magnitudes of 6.58 V/cm and 1.33 V/cm, respectively. In the high electrical field experiment, the cells go through electroporation. In the experiment with the low electrical field magnitude for TTFields, the cells have prolonged mitosis from typical 80-90 min to 200-300 min. Our results confirm the validity of the electrochemical model and the importance of applying a correct magnitude of the electrical field. Compared to the so far reported alternatives with insulated electrodes, the here developed thermoplastic microfluidic bioreactors with uninsulated electrodes provide a new, versatile, and durable platform for in vitro cell studies toward the improvement of anti-cancer therapies including personalized treatment.

Cell trapping microfluidic chip made of Cyclo olefin polymer enabling two concurrent cell biology experiments with long term durability.

Cyclo Olefin Polymer (COP) based microbioreactors on a microfluidic chip were produced in house by hot-embossing and thermo-compression bonding methods. The chip allows two different experiments to be performed on trapped cells at the same time. On one side of the chip, red fluorescent protein (RFP) tagged nucleolar Nop56 protein was used to track changes in cell cycle as well as protein synthesis within the yeast cells under the application of the anti-tumor agent hydroxyurea (HU). Simultaneously, on the other side of the chip, the response of yeast cells to the drug metformin, mTOR inhibitor, was investigated to reveal the role of TOR signaling in ribosome biogenesis and cell proliferation. The results of 20 h long experiments are captured by taking brightfield and fluorescent microscopy images of the trapped cells every 9 min. The expression of Nop56 protein of ribosome assembly and synthesis was densely observed during G1 phase of cell cycle, and later towards the end of cell cycle the ribosomal protein expression slowed down. Under HU treatment, the morphology of yeast cells changed, but after cessation of HU, the biomass synthesis rate was sustained as monitored by the cell perimeter. Under metformin treatment, the perimeters of single cells were observed to decrease, implying a decrease in biomass growth; however these cells continued their proliferation during and after the drug application. The relation between ribosome biogenesis and cell cycle was successfully investigated on single cell basis, capturing cell-to-cell variations, which cannot be tracked by regular macroscale bioreactors.

A Drug Repurposing and Protein-Protein Interaction Network Study of Ribosomopathies Using Yeast as a Model System.

Ribosomopathies result in various cancers, neurodegenerative and viral diseases, and other pathologies such as Diamond-Blackfan anemia and Shwachman-Diamond syndrome. Their pathophysiology at a proteome and functional level remains to be determined. Protein networks and highly connected hub proteins for ribosome biogenesis in Saccharomyces cerevisiae offer a potential as a model system to inform future therapeutic innovation in ribosomopathies. In this context, we report a ribosome biogenesis protein-protein interaction network in S. cerevisiae, created with 1772 proteins and 22,185 physical interactions connecting them. Moreover, by network decomposition analysis, we determined the linear pathways between the transcription factors and target proteins with a view to drug repurposing. While considering only the paths containing the three C/D box proteins (Nop56, Nop58, and Nop1), the most frequently encountered proteins were Aft1, Htz1, Ssa1, Ssb1, Ssb2, Gcn5, Cka1, Tef1, Nop1, Cdc28, Act1, Krr1, Rpl8B, and Tor1, which were then identified as potential drug targets. For drug repurposing, these candidate proteins were further searched in the DrugBank to find other diseases associated with them, as well as the drugs used to treat these diseases. To support the computational results, an experimental study was conducted using in-house manufactured microfluidic bioreactor platform, while the effect of the drug temsirolimus, Tor1 inhibitor, on yeast cells was investigated by following Nop56 protein expression. In conclusion, these results inform the ways in which ribosomopathies and associated common complex human diseases materialize and how drug repurposing might accelerate therapeutic innovation through bioinformatic studies of yeast.

A low cost PS based microfluidic platform to investigate cell cycle towards developing a therapeutic strategy for cancer.

Inhibition of DNA damage response pathway in combination with DNA alkylating agents may enhance the selective killing of cancer cells leading to better therapeutic effects. MDM2 binding protein (MTBP) in human has a role in G1 phase (interphase of cell cycle) and its overexpression leads to breast and ovarian cancers. Sld7 is an uncharacterized protein in budding yeast and a potential functional homologue of MTBP. To investigate the role of Sld7 as a therapeutic target, the behavior of the wild-type cells and sld7∆ mutants were monitored in 0.5 nL microbioreactors. The brightfield microscopy images were used to analyze the change in the cell size and to determine the durations of G1 and S/G2/M phases of wild type cells and mutants. With the administration of the alkylating agent, the cell size decreased and the duration of cell cycle increased. The replacement of the medium with the fresh one enabled the cells to repair their DNA. The application of calorie restriction together with DNA alkylating agent to mutant cells resulted in smaller cell size and longer G1 phase compared to those in control environment. For therapeutic purposes, the potential of MTBP in humans or Sld7 in yeast as a drug target deserves further exploration. The fabrication simplicity, robustness and low-cost of this microfluidic bioreactor made of polystyrene allowed us to perform yeast culturing experiments and show a potential for further cell culturing studies. The device can successfully be used for therapeutic applications including the discovery of new anti-microbial, anti-inflammatory, anti-cancer drugs.

In Silico Identification of Novel Orthosteric Inhibitors of Sphingosine Kinase 1 (SK1).

BACKGROUND: Sphingosine kinase 1 (SK1) overexpression and elevated sphingosine-1-phosphate (S1P) levels have been correlated with many disease states from cancer to inflammatory diseases to diabetes. Even though SK1 inhibitors are of consideberable interest as effective chemotherapeutic agents, poor potency, lack of selectivity and poor pharmacokinetic properties have been major problems in the first generation SK1 inhibitors. OBJECTIVE: There is an urgent need for the discovery of novel in vivo, stable selective SK1 inhibitors with improved potency. The primary object of this study was to identify potential novel leads for orthosteric inhibition of SK1. METHODS: We propose a series of compounds from different chemotypes as potential selective SK1 inhibitors via virtual screening of the ZINC database using ligand-based and structure-based pharmacophore models, molecular docking, substructure search, selectivity calculations. Molecular dynamics (MD) simulations revealed key insights into the binding mode and the stability of the SK1-ligand complex. RESULTS: Ten ligands were proposed as potential SK1 inhibitors based on the high induced fit docking scores, BEI, LLE and %HOA. Ligands 2, 3, 5 and 9 were found to be selective toward SK1 with favorable binding free energy of - 95 ± 5 kcal/mol. MD simulation of ligand 5 showed that the ligand-SK1 complex reached equilibrium with favorable hydrogen bonding and hydrophobic interactions. The four selective compounds have less than 0.24 similarity with previously discovered potent inhibitors. CONCLUSION: The proposed compounds may serve as potential novel leads for orthosteric inhibition of SK1.

Endocan is markedly overexpressed in severe erectile dysfunction.

This study aimed to measure the serum endocan level of patients with erectile dysfunction (ED) and to investigate the possible association between this and vasculogenic severe ED. We performed a prospective analysis of 86 consecutive patients affected by ED. Patients were divided into severe ED (IIEF-5 score < 7) and mild or moderate ED (IIEF-5 score > 7). A strong negative correlation was found between serum endocan levels and peak systolic velocity (p < .001 and r = -.665) in men with severe ED. Univariate logistic regression analysis demonstrated that tobacco consumption (p < .05), serum total 25-hydroxyvitamin D (p < .01), serum endocan levels (p < .01), peak systolic velocity (p < .01), hypertension (p < .001), dyslipidaemia (p < .001), metabolic syndrome (p = .026) and a history of a cardiovascular event (p < .001) significantly increase the risk of severe ED. In the multivariate logistic regression model, we also found that age, hypertension, metabolic syndrome, cardiovascular events and higher serum endocan levels were independently associated with severe ED. Circulating endocan may be used in daily practice as a new marker that correlates with cardiovascular risks and the severity of ED disease.

Multiomics Approach to Novel Therapeutic Targets for Cancer and Aging-Related Diseases: Role of Sld7 in Yeast Aging Network.

Genome instability has long been implicated as a salient causal factor in aging and age-related diseases such as cancer and neurodegeneration. However, the molecular mechanisms associated with genome instability remain unclear. Recent studies suggest growth signaling in the organism Saccharomyces cerevisiae and in higher eukaryotes might affect oxidative stress and aging/age-related diseases by activating DNA replication stress that causes DNA damage. In this broader integrative biology and clinical context, MDM two binding protein (MTBP) is a binding protein that has a role in activation of the tumor suppressor protein p53. Clinical studies suggest that the increase in expression of MTBP leads to reduction in survival of breast cancer patients. The functional homolog of MTBP in yeast, Sld7, is a hitherto uncharacterized protein that decreases the affinity of Sld3 (Treslin/Ticrr in humans) toward Cdc45 in the cell cycle process of S. cerevisiae. To investigate the putative function of Sld7 in chronological aging and replicative life span, we applied a network biology approach, integrating interactome and transcriptome data of budding yeast. The cell cycle and chronological aging networks of proteins in budding yeast were reconstructed. Furthermore, through meta-analysis of cell cycle and chronological aging-associated transcriptome datasets, we constructed coexpression networks and identified coexpressed gene clusters. In this study, we propose a dual role for Sld7: it participates in macromolecular complex binding in the cell cycle and has oxidoreductase activity in chronological aging in budding yeast. These potential roles of Sld7 in yeast can offer new insights on the role of MTBP in humans and consequently might lead to novel strategies for treatment of cancers and aging-related diseases.

A systematic methodology for large scale compound screening: A case study on the discovery of novel S1PL inhibitors.

Decrease in sphingosine 1-phosphate (S1P) concentration induces migration of pathogenic T cells to the blood stream, disrupts the CNS and it is implicated in multiple sclerosis (MS), a progressive inflammatory disorder of the central nervous system (CNS), and Alzheimer's disease (AD). A promising treatment alternative for MS and AD is inhibition of the activity of the microsomal enzyme sphingosine 1-phosphate lyase (S1PL), which degrades intracellular S1P. This report describes an integrated systematic approach comprising virtual screening, molecular docking, substructure search and molecular dynamics simulation to discover novel S1PL inhibitors. Virtual screening of the ZINC database via ligand-based and structure-based pharmacophore models yielded 10000 hits. After molecular docking, common substructures of the top ranking hits were identified. The ligand binding poses were optimized by induced fit docking. MD simulations were performed on the complex structures to determine the stability of the S1PL-ligand complex and to calculate the binding free energy. Selectivity of the selected molecules was examined by docking them to hERG and cytochrome P450 receptors. As a final outcome, 15 compounds from different chemotypes were proposed as potential S1PL inhibitors. These molecules may guide future medicinal chemistry efforts in the discovery of new compounds against the destructive action of pathogenic T cells.

Antifibrogenic role of valproic acid in streptozotocin induced diabetic rat penis.

We investigated the therapeutic effects of valproic acid (VPA) on erectile dysfunction and reducing penile fibrosis in streptozocin (STZ)-induced diabetic rats. Eighteen male rats were divided into three experimental groups (Control, STZ-DM, STZ-DM plus VPA) and diabetes was induced by transperitoneal single dose STZ. Eight weeks after, VPA and placebo treatments were given according to groups for 15 days. All rats were anesthetised for the measurement of in vivo erectile response to cavernous nerve stimulation. Afterward penes were evaluated histologically in terms of immune labelling scores of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and transforming growth factor-ß1 (TGF-ß1). Slides were also evaluated in terms of collagen/smooth muscle ratio and penile apoptosis. After the treatment with VPA, erectile responses were found as improved when compared with STZ-DM rats but not statistically meaningful. eNOS and VEGF immune expressions diminished in penile corpora of STZ-DM rats and improved with VPA treatment. VPA led to decrease in TGF-ß1 expression and collagen content of diabetic rats' penes. Penile apoptosis was not diminished with VPA. In conclusion, VPA treatment seems to be effective for reducing penile fibrosis in diabetic rats and more prolonged treatment period may enhance erectile functions.

Targeting the Akt1 allosteric site to identify novel scaffolds through virtual screening.

Preclinical data and tumor specimen studies report that AKT kinases are related to many human cancers. Therefore, identification and development of small molecule inhibitors targeting AKT and its signaling pathway can be therapeutic in treatment of cancer. Numerous studies report inhibitors that target the ATP-binding pocket in the kinase domains, but the similarity of this site, within the kinase family makes selectivity a major problem. The sequence identity amongst PH domains is significantly lower than that in kinase domains and developing more selective inhibitors is possible if PH domain is targeted. This in silico screening study is the first time report toward the identification of potential allosteric inhibitors expected to bind the cavity between kinase and PH domains of Akt1. Structural information of Akt1 was used to develop structure-based pharmacophore models comprising hydrophobic, acceptor, donor and ring features. The 3D structural information of previously identified allosteric Akt inhibitors obtained from literature was employed to develop a ligand-based pharmacophore model. Database was generated with drug like subset of ZINC and screening was performed based on 3D similarity to the selected pharmacophore hypotheses. Binding modes and affinities of the ligands were predicted by Glide software. Top scoring hits were further analyzed considering 2D similarity between the compounds, interactions with Akt1, fitness to pharmacophore models, ADME, druglikeness criteria and Induced-Fit docking. Using virtual screening methodologies, derivatives of 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol were proposed as potential leads for allosteric inhibition of Akt1.

Serum carbohydrate antigen 19-9 levels in patients with unilateral hydronephrosis due to urinary lithiasis.

AIM: We aimed to investigate that the association between serum carbohydrate antigen 19-9 (CA 19-9) levels and unilateral hydronephrosis due to urinary lithiasis in homogeneous groups who had no urinary tract infection and renal impairment. METHODS: A total of 50 patients with ureteral stone enrolled in this study prospectively. The patients were divided into two groups according to degree of hydronephrosis. Patients without hydronephrosis were taken into Group I and who had hydronephrosis were taken into Group II. All patients were underwent treatment of shock wave lithotripsy (SWL). Serum CA 19-9 was measured using immunassay method pre-treatment of SWL. After complete stone clearence and recovery of hydronephrosis in all patients, patiens were re-evaluated and CA 19-9 levels were measured. RESULTS: There were no significant differences for age, gender, body mass index, stone volume, shock wave number, number of SWL seasons, and serum creatinin between the groups (P>0.05). Pre-SWL serum mean Ca 19-9 levels were 14.82±14.64 U/mL. in Group I and 13.89±13.03 U/mL. in Group II (P=0.8686). After complete stone clearance and recovery of hydronephosis with SWL, the mean CA 19-9 values were measured as 14.14±10.67 U/mL. in Group II and there were not found significant change as compared pre-SWL values for Group II (P=0.7334). CONCLUSION: The relationship between CA 19-9 values and hydronephrosis was not found to be statistically meaningful on the contrary to previous reports and it does not appear as a useful parameter to predict of urinary obstruction non-invasively.

Increased expression of nestin in the major pelvic ganglion following cavernous nerve injury.

In an effort to identify neuronal repair mechanisms of the major pelvic ganglion (MPG), we evaluated changes in the expression of nestin, an intermediate filament protein and neural stem cell marker following cavernous nerve crush injury (CNI). We utilized two groups of Sprague Dawley rats: (i) sham and (ii) bilateral CNI. Erectile responses to cavernous nerve stimulation (CNS) were determined at 48 h in a subset of rats. The MPG was isolated and removed at 48 h after CNI, and nestin immunolocalization, protein levels and RNA expression were evaluated. At 48 h, erectile responses to CNS in CNI rats were substantially reduced (P<0.05; ∼70% decrease in intracavernous pressure/mean arterial pressure) compared with sham surgery controls. This coincided with a dramatic 10-fold increase (P<0.05) in nestin messenger RNA expression and protein levels in the MPG of rats with CNI. Immunoflourescence microscopy demonstrated that nestin upregulation after CNI occurred within the ganglion cell bodies and nerve fibers of the MPG. In conclusion, CNI induces nestin in the MPG. These data suggest that nestin may be involved in the regenerative process of the cavernous nerve following crush injury.

Reconstruction and crosstalk of protein-protein interaction networks of Wnt and Hedgehog signaling in Drosophila melanogaster.

In the last few years, researchers have an intense interest in the evolutionarily conserved signaling pathways which have crucial roles during embryonic development. The most intriguing factor of this interest is that malfunctioning of these signaling pathways (Hedgehog, Notch, Wnt etc.) leads to several human diseases, especially to cancer. This study deals with the ß-catenin dependent branch of Wnt signaling and the Hedgehog signaling pathways which offer potential targeting points for cancer drug development. The identification of all proteins functioning in these signaling networks is crucial for the efforts of preventing tumor formation. Here, through integration of protein-protein interaction data and Gene Ontology annotations, Wnt/ß-catenin and Hedgehog signaling networks consisting of proteins that have statistically high probability of being biologically related to these signaling pathways were reconstructed in Drosophila melanogaster. Next, by the structural network analyses, the crucial components functioning in these pathways were identified. The proteins Arm, Frizzled receptors (Fz and Fz2), Arr, Apc, Axn, Ci and Ptc were detected as the key proteins in these networks. Futhermore, the hub protein Mer having tumor suppressor function may be proposed as a putative drug target for cancer and deserves further investigation via experimental methods. Finally, the crosstalk analysis between the reconstructed networks reveals that these two signaling networks crosstalk to each other.

Stoichiometric network reconstruction and analysis of yeast sphingolipid metabolism incorporating different states of hydroxylation.

The first elaborate metabolic model of Saccharomyces cerevisiae sphingolipid metabolism was reconstructed in silico. The model considers five different states of sphingolipid hydroxylation, rendering it unique among other models. It is aimed to clarify the significance of hydroxylation on sphingolipids and hence to interpret the preferences of the cell between different metabolic pathway branches under different stress conditions. The newly constructed model was validated by single, double and triple gene deletions with experimentally verified phenotypes. Calcium sensitivity and deletion mutations that may suppress calcium sensitivity were examined by CSG1 and CSG2 related deletions. The model enabled the analysis of complex sphingolipid content of the plasma membrane coupled with diacylglycerol and phosphatidic acid biosynthesis and ATP consumption in in silico cell. The flux data belonging to these critically important key metabolites are integrated with the fact of phytoceramide induced cell death to propose novel potential drug targets for cancer therapeutics. In conclusion, we propose that IPT1, GDA1, CSG and AUR1 gene deletions may be novel candidates of drug targets for cancer therapy according to the results of flux balance and variability analyses coupled with robustness analysis.