Natural Compounds for Bone Remodeling: A Computational and Experimental Approach Targeting Bone Metabolism-Related Proteins.

Osteoporosis, characterized by reduced bone density and increased fracture risk, affects over 200 million people worldwide, predominantly older adults and postmenopausal women. The disruption of the balance between bone-forming osteoblasts and bone-resorbing osteoclasts underlies osteoporosis pathophysiology. Standard treatment includes lifestyle modifications, calcium and vitamin D supplementation and specific drugs that either inhibit osteoclasts or stimulate osteoblasts. However, these treatments have limitations, including side effects and compliance issues. Natural products have emerged as potential osteoporosis therapeutics, but their mechanisms of action remain poorly understood. In this study, we investigate the efficacy of natural compounds in modulating molecular targets relevant to osteoporosis, focusing on the Mitogen-Activated Protein Kinase (MAPK) pathway and the gut microbiome's influence on bone homeostasis. Using an in silico and in vitro methodology, we have identified quercetin as a promising candidate in modulating MAPK activity, offering a potential therapeutic perspective for osteoporosis treatment.

Development of a multigram synthesis of the bradykinin receptor 2 agonist FR-190997 and analogs thereof.

Using Fujisawa's B2R agonist FR-190997, we recently demonstrated for the first time that agonism at the bradykinin receptor type 2 (B2R) produces substantial antiproliferative effects. FR-190997 elicited an EC50 of 80 nM in the triple-negative breast cancer cell line MDA-MB-231, a much superior performance to that exhibited by most approved breast cancer drugs. Consequently, we initiated a program aiming primarily at synthesizing adequate quantities of FR-190997 to support further in vitro and in vivo studies toward its repurposing for various cancers and, in parallel, enable the generation of novel FR-190997 analogs for an SAR study. Prerequisite for this endeavor was to address the synthetic challenges associated with the FR-190997 scaffold, which the Fujisawa chemists had constructed in 20 steps, 13 of which required chromatographic purification. We succeeded in developing a 17-step synthesis amenable to late-stage diversification that eliminated all chromatography and enabled access to multigram quantities of FR-190997 and novel derivatives thereof, supporting further anticancer research based on B2R agonists.

The clinical utility of polygenic risk scores in genomic medicine practices: a systematic review.

Genomic medicine aims to improve health using the individual genomic data of people to inform care. While clinical utility of genomic medicine in many monogenic, Mendelian disorders is amply demonstrated, clinical utility is less evident in polygenic traits, e.g., coronary artery disease or breast cancer. Polygenic risk scores (PRS) are subsets of individual genotypes designed to capture heritability of common traits, and hence to allow the stratification of risk of the trait in a population. We systematically reviewed the PubMed database for unequivocal evidence of clinical utility of polygenic risk scores, using stringent inclusion and exclusion criteria. While we identified studies demonstrating clinical validity in conditions where medical intervention based on a PRS is likely to benefit patient outcome, we did not identify a single study demonstrating unequivocally such a benefit, i.e. clinical utility. We conclude that while the routine use of PRSs hold great promise, translational research is still needed before they should enter mainstream clinical practice.

Drug-Induced Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis Call for Optimum Patient Stratification and Theranostics via Pharmacogenomics.

The Global Genomic Medicine Collaborative, a multinational coalition of genomic and policy experts working to implement genomics in clinical care, considers pharmacogenomics to be among the first areas in genomic medicine that can provide guidance in routine clinical practice, by linking genetic variation and drug response. Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe life-threatening reactions to medications with a high incidence worldwide. Genomic screening prior to drug administration is a key opportunity and potential paradigm for using genomic medicine to reduce morbidity and mortality and ultimately eliminate one of the most devastating adverse drug reactions. This review focuses on the current understanding of the surveillance, pathogenesis, and treatment of SJS/TEN, including the role of genomics and pharmacogenomics in the etiology, treatment, and eradication of preventable causes of drug-induced SJS/TEN. Gaps, unmet needs, and priorities for future research have been identified for the optimal management of drug-induced SJS/TEN in various ethnic populations. Pharmacogenomics holds great promise for optimal patient stratification and theranostics, yet its clinical implementation needs to be cost-effective and sustainable.

Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole-indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.

Documentation of clinically relevant genomic biomarker allele frequencies in the next-generation FINDbase worldwide database.

FINDbase (http://www.findbase.org) is a comprehensive data resource recording the prevalence of clinically relevant genomic variants in various populations worldwide, such as pathogenic variants underlying genetic disorders as well as pharmacogenomic biomarkers that can guide drug treatment. Here, we report significant new developments and technological advancements in the database architecture, leading to a completely revamped database structure, querying interface, accompanied with substantial extensions of data content and curation. In particular, the FINDbase upgrade further improves the user experience by introducing responsive features that support a wide variety of mobile and stationary devices, while enhancing computational runtime due to the use of a modern Javascript framework such as ReactJS. Data collection is significantly enriched, with the data records being divided in a Public and Private version, the latter being accessed on the basis of data contribution, according to the microattribution approach, while the front end was redesigned to support the new functionalities and querying tools. The abovementioned updates further enhance the impact of FINDbase, improve the overall user experience, facilitate further data sharing by microattribution, and strengthen the role of FINDbase as a key resource for personalized medicine applications and personalized public health.

Sensitive Monogenic Noninvasive Prenatal Diagnosis by Targeted Haplotyping.

During pregnancy, cell-free DNA (cfDNA) in maternal blood encompasses a small percentage of cell-free fetal DNA (cffDNA), an easily accessible source for determination of fetal disease status in risk families through non-invasive procedures. In case of monogenic heritable disease, background maternal cfDNA prohibits direct observation of the maternally inherited allele. Non-invasive prenatal diagnostics (NIPD) of monogenic diseases therefore relies on parental haplotyping and statistical assessment of inherited alleles from cffDNA, techniques currently unavailable for routine clinical practice. Here, we present monogenic NIPD (MG-NIPD), which requires a blood sample from both parents, for targeted locus amplification (TLA)-based phasing of heterozygous variants selectively at a gene of interest. Capture probes-based targeted sequencing of cfDNA from the pregnant mother and a tailored statistical analysis enables predicting fetal gene inheritance. MG-NIPD was validated for 18 pregnancies, focusing on CFTR, CYP21A2, and HBB. In all cases we could predict the inherited alleles with >98% confidence, even at relatively early stages (8 weeks) of pregnancy. This prediction and the accuracy of parental haplotyping was confirmed by sequencing of fetal material obtained by parallel invasive procedures. MG-NIPD is a robust method that requires standard instrumentation and can be implemented in any clinic to provide families carrying a severe monogenic disease with a prenatal diagnostic test based on a simple blood draw.

New molecular diagnostic trends and biomarkers for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rare and fatal neurodegenerative disorder. Two forms are recognized, familial (FALS) that accounts for 5-10% of ALS cases, and sporadic (SALS) that accounts for the rest. Early diagnosis of ALS is important because it improves their therapeutic efficacy. Current diagnosis is based on clinical assessment and requires approximately 12 months, leading to a significant delay in drug administration. Therefore, new methods are required for the earlier diagnosis of ALS. Screening for pathogenic variants in known ALS-associated genes is already exploited as a diagnostic tool in ALS but cannot be applied for population-based screening. New circulating biomarkers (proteins or small molecules) are needed for initial screening, whereas specific diagnostic methods can be applied to confirm the presence of pathogenic variants in the selected population subgroup. Lipids appear as promising biomarkers for population-based screening and for monitoring disease progression. Genetic analysis can also assist in the prediction of disease progression by analyzing disease-modifying genes, for example, EPHA4 and CHGB. Furthermore, molecular diagnosis will aid the stratification of ALS patients for improved pharmacological approaches. Here, we discuss current and novel diagnostic strategies and how they can be applied to revolutionize the field of ALS molecular diagnosis.

Cost-effectiveness analysis of pharmacogenomics-guided clopidogrel treatment in Spanish patients undergoing percutaneous coronary intervention.

Clopidogrel is an antiplatelet drug given to patients before and after having a percutaneous coronary intervention (PCI). Genomic variants in the CYP2C19 gene are associated with variable enzyme activities affecting drug metabolism and hence, patients with reduced or increased enzymatic function have increased risk of bleeding. We conducted a cost-effectiveness analysis to compare a pharmacogenomics versus a non-pharmacogenomics-guided clopidogrel treatment for coronary artery syndrome patients undergoing PCI in the Spanish healthcare setting. A total of 549 patients diagnosed with coronary artery disease followed by PCI were recruited. Dual antiplatelet therapy was administrated to all patients from 1 to 12 months after PCI. Patients were classified into two groups: the Retrospective group was treated with clopidogrel based on the clinical routine practice and the Prospective group were initially genotyped for the presence of CYP2C19 variant alleles before treatment with those carrying more than one CYP2C19 variant alleles given prasugrel treatment. We collected data on established clinical and health outcome measures, including, per treatment arm: the percentage of patients that suffered from (a) myocardial infraction, (b) major bleeding and minor bleeding, (c) stroke, (d) the number of hospitalization days, and (e) the number of days patients spent in Intensive Care Unit. Our primary outcome measure for the cost-effectiveness analysis was Quality Adjusted Life Years (QALYs). To estimate the treatment cost for each patient, individual data on its resource used were combined with unit price data, obtained from Spanish national sources. The analysis predicts a survival of 0.9446 QALYs in the pharmacogenomics arm and 0.9379 QALYs in the non-pharmacogenomics arm within a 1-year horizon. The cumulative costs per patient were €2971 and €3205 for the Prospective and Retrospective groups, respectively. The main cost driver of total cost in both arms was hospitalization costs. The incremental cost-effectiveness ratio (ICER) was negative indicating that the PGx was a dominant option. Our data show that pharmacogenomics-guided clopidogrel treatment strategy may represent a cost-effective choice compared with non-pharmacogenomics-guided strategy for patients undergoing PCI.

Expanded national database collection and data coverage in the FINDbase worldwide database for clinically relevant genomic variation allele frequencies.

FINDbase (http://www.findbase.org) is a comprehensive data repository that records the prevalence of clinically relevant genomic variants in various populations worldwide, such as pathogenic variants leading mostly to monogenic disorders and pharmacogenomics biomarkers. The database also records the incidence of rare genetic diseases in various populations, all in well-distinct data modules. Here, we report extensive data content updates in all data modules, with direct implications to clinical pharmacogenomics. Also, we report significant new developments in FINDbase, namely (i) the release of a new version of the ETHNOS software that catalyzes development curation of national/ethnic genetic databases, (ii) the migration of all FINDbase data content into 90 distinct national/ethnic mutation databases, all built around Microsoft's PivotViewer (http://www.getpivot.com) software (iii) new data visualization tools and (iv) the interrelation of FINDbase with DruGeVar database with direct implications in clinical pharmacogenomics. The abovementioned updates further enhance the impact of FINDbase, as a key resource for Genomic Medicine applications.

Role of Genomic Biomarkers in Increasing Fetal Hemoglobin Levels Upon Hydroxyurea Therapy and in ß-Thalassemia Intermedia: A Validation Cohort Study.

Hemoglobinopathies exhibit a remarkable phenotypic diversity in terms of disease severity, while individual genetic background plays a key role in differential response to drug treatment. In the last decade, genomic variants in genes located within, as well as outside the human ß-globin cluster have been shown to be significantly associated with Hb F increase, in relation to hydroxyurea (HU) therapy in patients with these diseases. Here, we aim to determine the effect of genomic variants located in genes, such as MAP3K5, ASS1, NOS2A, TOX, PDE7B, NOS1, FLT1 and ARG2, previously shown to modulate fetal hemoglobin (Hb F) levels in patients with ß type hemoglobinopathies and reflecting disease severity and response to HU therapy in an independent cohort of Greek patients with these diseases. We recruited and genotyped 45 ß-thalassemia patients (ß-thal), either transfusion-dependent (TDT) or non transfusion-dependent (NTDT), 42 Hb S (HBB: c.20A>T)-ß-thal compound heterozygotes, who were treated with HU, as well as 53 healthy individuals, all of Hellenic origin. Our study showed that genomic variants of the MAP3K5, NOS2A and ARG2 gene are associated with HU therapy efficacy in Hb S-ß-thal compound heterozygotes. We have also shown that FLT1 and ARG2 genomic variants are associated with the mild phenotype of NTDT patients. Our findings provide evidence that MAP3K5, NOS2A, ARG2 and FLT1 genomic variants could be considered as genomic biomarkers to predict HU therapy efficacy in Hb S-ß-thal compound heterozygotes and also to describe disease severity in patients with ß type hemoglobinopathies.

VEGF-A and ICAM-1 Gene Polymorphisms as Predictors of Clinical Outcome to First-Line Bevacizumab-Based Treatment in Metastatic Colorectal Cancer.

Bevacizumab is used to treat metastatic colorectal cancer (mCRC). However, there are still no available predictors of clinical outcomes. We investigated selected single nucleotide polymorphisms (SNPs) in the genes involved in VEGF-dependent and -independent angiogenesis pathways and other major intracellular signaling pathways involved in the pathogenesis of mCRC as an attempt to find predictors of clinical outcome. Forty-six patients treated with first-line bevacizumab-based chemotherapy were included in this study with a 5 year follow up. Genomic DNA was isolated from whole blood for the analysis of VEGF-A (rs2010963, 1570360, rs699947), ICAM-1 (rs5498, rs1799969) SNPs and from tumor tissue for the detection of genomic variants in KRAS, NRAS, BRAF genes. PCR and next generation sequencing were used for the analysis. The endpoints of the study were progression-free survival (PFS) and overall survival (OS). The VEGF-A rs699947 A/A allele was associated with increased PFS (p = 0.006) and OS (p = 0.043). The ICAM-1 rs1799969 G/A allele was associated with prolonged OS (p = 0.036). Finally, BRAF wild type was associated with increased OS (p = 0.027). We identified VEGF-A and ICAM-1 variants in angiogenesis and other major intracellular signaling pathways, such as BRAF, that can predict clinical outcome upon bevacizumab administration. These identified biomarkers could be used to select patients with mCRC who may achieve long-term responses and benefit from bevacizumab-based therapies.

Whole transcriptome analysis of human erythropoietic cells during ontogenesis suggests a role of VEGFA gene as modulator of fetal hemoglobin and pharmacogenomic biomarker of treatment response to hydroxyurea in ß-type hemoglobinopathy patients.

BACKGROUND: Human erythropoiesis is characterized by distinct gene expression profiles at various developmental stages. Previous studies suggest that fetal-to-adult hemoglobin switch is regulated by a complex mechanism, in which many key players still remain unknown. Here, we report our findings from whole transcriptome analysis of erythroid cells, isolated from erythroid tissues at various developmental stages in an effort to identify distinct molecular signatures of each erythroid tissue. RESULTS: From our in-depth data analysis, pathway analysis, and text mining, we opted to focus on the VEGFA gene, given its gene expression characteristics. Selected VEGFA genomic variants, identified through linkage disequilibrium analysis, were explored further for their association with elevated fetal hemoglobin levels in ß-type hemoglobinopathy patients. Our downstream analysis of non-transfusion-dependent ß-thalassemia patients, ß-thalassemia major patients, compound heterozygous sickle cell disease/ß-thalassemia patients receiving hydroxyurea as fetal hemoglobin augmentation treatment, and non-thalassemic individuals indicated that VEGFA genomic variants were associated with disease severity in ß-thalassemia patients and hydroxyurea treatment efficacy in SCD/ß-thalassemia compound heterozygous patients. CONCLUSIONS: Our findings suggest that VEGFA may act as a modifier gene of human globin gene expression and, at the same time, serve as a genomic biomarker in ß-type hemoglobinopathy disease severity and hydroxyurea treatment efficacy.

Genomic variants in the FTO gene are associated with sporadic amyotrophic lateral sclerosis in Greek patients.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devastating disease whose complex pathology has been associated with a strong genetic component in the context of both familial and sporadic disease. Herein, we adopted a next-generation sequencing approach to Greek patients suffering from sporadic ALS (together with their healthy counterparts) in order to explore further the genetic basis of sporadic ALS (sALS). RESULTS: Whole-genome sequencing analysis of Greek sALS patients revealed a positive association between FTO and TBC1D1 gene variants and sALS. Further, linkage disequilibrium analyses were suggestive of a specific disease-associated haplotype for FTO gene variants. Genotyping for these variants was performed in Greek, Sardinian, and Turkish sALS patients. A lack of association between FTO and TBC1D1 variants and sALS in patients of Sardinian and Turkish descent may suggest a founder effect in the Greek population. FTO was found to be highly expressed in motor neurons, while in silico analyses predicted an impact on FTO and TBC1D1 mRNA splicing for the genomic variants in question. CONCLUSIONS: To our knowledge, this is the first study to present a possible association between FTO gene variants and the genetic etiology of sALS. In addition, the next-generation sequencing-based genomics approach coupled with the two-step validation strategy described herein has the potential to be applied to other types of human complex genetic disorders in order to identify variants of clinical significance.

Novel genetic risk variants for pediatric celiac disease.

BACKGROUND: Celiac disease is a complex chronic immune-mediated disorder of the small intestine. Today, the pathobiology of the disease is unclear, perplexing differential diagnosis, patient stratification, and decision-making in the clinic. METHODS: Herein, we adopted a next-generation sequencing approach in a celiac disease trio of Greek descent to identify all genomic variants with the potential of celiac disease predisposition. RESULTS: Analysis revealed six genomic variants of prime interest: SLC9A4 c.1919G>A, KIAA1109 c.2933T>C and c.4268_4269delCCinsTA, HoxB6 c.668C>A, HoxD12 c.418G>A, and NCK2 c.745_746delAAinsG, from which NCK2 c.745_746delAAinsG is novel. Data validation in pediatric celiac disease patients of Greek (n = 109) and Serbian (n = 73) descent and their healthy counterparts (n = 111 and n = 32, respectively) indicated that HoxD12 c.418G>A is more prevalent in celiac disease patients in the Serbian population (P < 0.01), while NCK2 c.745_746delAAinsG is less prevalent in celiac disease patients rather than healthy individuals of Greek descent (P = 0.03). SLC9A4 c.1919G>A and KIAA1109 c.2933T>C and c.4268_4269delCCinsTA were more abundant in patients; nevertheless, they failed to show statistical significance. CONCLUSIONS: The next-generation sequencing-based family genomics approach described herein may serve as a paradigm towards the identification of novel functional variants with the aim of understanding complex disease pathobiology.

Identification of cancer predisposition variants in apparently healthy individuals using a next-generation sequencing-based family genomics approach.

Cancer, like many common disorders, has a complex etiology, often with a strong genetic component and with multiple environmental factors contributing to susceptibility. A considerable number of genomic variants have been previously reported to be causative of, or associated with, an increased risk for various types of cancer. Here, we adopted a next-generation sequencing approach in 11 members of two families of Greek descent to identify all genomic variants with the potential to predispose family members to cancer. Cross-comparison with data from the Human Gene Mutation Database identified a total of 571 variants, from which 47 % were disease-associated polymorphisms, 26 % disease-associated polymorphisms with additional supporting functional evidence, 19 % functional polymorphisms with in vitro/laboratory or in vivo supporting evidence but no known disease association, 4 % putative disease-causing mutations but with some residual doubt as to their pathological significance, and 3 % disease-causing mutations. Subsequent analysis, focused on the latter variant class most likely to be involved in cancer predisposition, revealed two variants of prime interest, namely MSH2 c.2732T>A (p.L911R) and BRCA1 c.2955delC, the first of which is novel. KMT2D c.13895delC and c.1940C>A variants are additionally reported as incidental findings. The next-generation sequencing-based family genomics approach described herein has the potential to be applied to other types of complex genetic disorder in order to identify variants of potential pathological significance.

Development and validation of a UHPLC-UV method for the determination of a prostate secretory protein 94-derived synthetic peptide (PCK3145) in human plasma and assessment of its stability in human plasma.

PCK3145 is a synthetic peptide, derived from the Prostate Secreted Protein 94 (PSP94), with promising in vitro and animal in vivo results in prostate cancer. The aim of the present study was to develop and validate a fast and robust ultra-high-performance liquid chromatography with ultraviolet detection for the determination of PCK3145 in human plasma which would be suitable for the assessment of PCK3145 stability to proteolytic degradation. Following protein precipitation, chromatographic separation was carried out on an Aeris Peptide C18 column with mobile phase consisting of acetonitrile-water at a flow-rate of 0.50 mL/min. The calibration curve was linear over the range 0.50-20.00 µg/mL. Intra- and inter-day percentage relative standard deviation and relative error were ≤10%. The limit of detection and the lower limit of quantification were 0.15 and 0.50 µg/mL, respectively. Recovery of PCK3145 from human plasma was ≥96%. The peptide presented high stability in whole blood and in human plasma (>98% intact peptide after 24 h incubation at 37°C in human plasma), which represents a distinctive advantage in the therapeutic use of the compound. This is the first validated UHPLC method for the determination of PCK3145 reported, and it was successfully applied in the study of the proteolytic stability of PCK3145 in human plasma ex vivo. Copyright © 2016 John Wiley & Sons, Ltd.

Functional Analysis of an (A)γ-Globin Gene Promoter Variant (HBG1: g.-225_-222delAGCA) Underlines Its Role in Increasing Fetal Hemoglobin Levels Under Erythropoietic Stress.

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by persistent γ-globin gene expression and synthesis of high levels of fetal hemoglobin (Hb F; α2γ2) during adult life. It is usually caused by promoter variants or large deletions affecting the human fetal globin (HBG1 and HBG2) genes. Some of these HPFH-causing variants, such as HBG2: g.-158 C > T, exert their effect only under conditions of erythropoietic stress, typical for ß-thalassemia (ß-thal) patients. Namely, the presence of HBG2: g.-158 C > T favors a higher Hb F response, while it has little effect in healthy individuals. We analyzed a previously reported deletion residing in the promoter region of the HBG1 gene (HBG1: g.-225_-222delAGCA), both in normal conditions and under conditions of erythropoietic stress. Our results indicate that this deletion is responsible for decreased HBG1 gene expression. Specifically, this deletion was shown to result in drastically reduced reporter gene expression in K562 cells, compared to the wild-type sequence but only under conditions of erythropoietic stress, mimicked by introduction of erythropoietin (EPO) into the cell culture. Also, electrophoretic mobility shift analysis showed that the HBG1: g.-225_-222delAGCA deletion creates additional transcriptional factors' binding sites, which, we propose, bind a transcriptional repressor, thus decreasing the HBG1 gene promoter activity. These results are consistent with in silico analysis, which indicated that this deletion creates a binding site for GATA1, known to be a repressor of the γ-globin gene expression. These data confirm the regulatory role of the HBG1: g.-225_-222 region that exerts its effect under conditions of erythropoietic stress characteristic for ß-thal patients.

Enhancing the Biological Relevance of Secretome-Based Proteomics by Linking Tumor Cell Proliferation and Protein Secretion.

Secretome profiling has become a methodology of choice for the identification of tumor biomarkers. We hypothesized that due to the dynamic nature of secretomes cellular perturbations could affect their composition but also change the global amount of protein secreted per cell. We confirmed our hypothesis by measuring the levels of secreted proteins taking into account the amount of proteome produced per cell. Then, we established a correlation between cell proliferation and protein secretion that explained the observed changes in global protein secretion. Next, we implemented a normalization correcting the statistical results of secretome studies by the global protein secretion of cells into a generalized linear model (GLM). The application of the normalization to two biological perturbations on tumor cells resulted in drastic changes in the list of statistically significant proteins. Furthermore, we found that known epithelial-to-mesenchymal transition (EMT) effectors were only statistically significant when the normalization was applied. Therefore, the normalization proposed here increases the sensitivity of statistical tests by increasing the number of true-positives. From an oncology perspective, the correlation between protein secretion and cellular proliferation suggests that slow-growing tumors could have high-protein secretion rates and consequently contribute strongly to tumor paracrine signaling.

Comprehensive characterization of the neurogenic and neuroprotective action of a novel TrkB agonist using mouse and human stem cell models of Alzheimer's disease.

BACKGROUND: Neural stem cell (NSC) proliferation and differentiation in the mammalian brain decreases to minimal levels postnatally. Nevertheless, neurogenic niches persist in the adult cortex and hippocampus in rodents, primates and humans, with adult NSC differentiation sharing key regulatory mechanisms with development. Adult neurogenesis impairments have been linked to Alzheimer's disease (AD) pathology. Addressing these impairments by using neurotrophic factors is a promising new avenue for therapeutic intervention based on neurogenesis. However, this possibility has been hindered by technical difficulties of using in-vivo models to conduct screens, including working with scarce NSCs in the adult brain and differences between human and mouse models or ethical limitations. METHODS: Here, we use a combination of mouse and human stem cell models for comprehensive in-vitro characterization of a novel neurogenic compound, focusing on the brain-derived neurotrophic factor (BDNF) pathway. The ability of ENT-A011, a steroidal dehydroepiandrosterone derivative, to activate the tyrosine receptor kinase B (TrkB) receptor was tested through western blotting in NIH-3T3 cells and its neurogenic and neuroprotective action were assessed through proliferation, cell death and Amyloid-ß (Aß) toxicity assays in mouse primary adult hippocampal NSCs, mouse embryonic cortical NSCs and neural progenitor cells (NPCs) differentiated from three human induced pluripotent stem cell lines from healthy and AD donors. RNA-seq profiling was used to assess if the compound acts through the same gene network as BDNF in human NPCs. RESULTS: ENT-A011 was able to increase proliferation of mouse primary adult hippocampal NSCs and embryonic cortical NSCs, in the absence of EGF/FGF, while reducing Aß-induced cell death, acting selectively through TrkB activation. The compound was able to increase astrocytic gene markers involved in NSC maintenance, protect hippocampal neurons from Αß toxicity and prevent synapse loss after Aß treatment. ENT-A011 successfully induces proliferation and prevents cell death after Aß toxicity in human NPCs, acting through a core gene network shared with BDNF as shown through RNA-seq. CONCLUSIONS: Our work characterizes a novel BDNF mimetic with preferable pharmacological properties and neurogenic and neuroprotective actions in Alzheimer's disease via stem cell-based screening, demonstrating the promise of stem cell systems for short-listing competitive candidates for further testing.