OctylPhenol (OP) Alone and in Combination with NonylPhenol (NP) Alters the Structure and the Function of Thyroid Gland of the Lizard Podarcis siculus

Abstract: Different environmental contaminants disturb the thyroid system at many levels. AlkylPhenols (APs), by-products of microbial degradation of AlkylPhenol Polyethoxylates (APEOs), constitute an important class of Endocrine Disrupting Chemicals (EDCs), the two most often used environmental APs being 4-nonylphenol (4-NP) and 4-tert-octylphenol (4-t-OP). The purpose of the present study was to investigate the effects on the thyroid gland of the bioindicator Podarcis siculus of OP alone and in combination with NP. We used radioimmunoassay to determine their effects on plasma 3,3′,5-triiodo-L-thyronine (T3), 3,3′,5,5′-L-thyroxine (T4), thyroid-stimulating hormone (TSH), and thyrotropin-releasing hormone (TRH) levels in adult male lizards. We also investigated the impacts of AP treatments on hepatic 5′ORD (type II) deiodinase and hepatic content of T3 and T4. After OP and OP + NP administration, TRH levels increased, whereas TSH, T3, and T4 levels decreased. Lizards treated with OP and OP + NP had a higher concentration of T3 in the liver and 5′ORD (type II) activity, whereas T4 concentrations were lower than that observed in the control group. Moreover, histological examination showed that the volume of the thyroid follicles became smaller in treated lizards suggesting that that thyroid follicular epithelial cells were not functionally active following treatment. This data collectively suggest a severe interference with hypothalamus–pituitary–thyroid axis and a systemic imbalance of thyroid hormones. Graphic Abstract: [Figure not available: see fulltext.

Immunohistochemical localization of NPY, VIP and 5-HT in the thyroid gland of the lizard, Podarcis sicula.

The thyroid gland of the lizard Podarcis sicula was immunohistochemically studied in adult male specimens using specific antibodies against NPY, VIP and 5-HT and the avidin-biotin peroxidase complex (ABC) procedure to localize the three peptides. Fine beaded VIP-immunoreactive nerve fibers ran between the follicles, and VIP-immunoreactivity was evenly distributed in the apical cytoplasm of follicular cells. NPY-immunoreactive fibers were found around the follicles, and, in the cells, immunoreactivity was localizated only in the cellular apices. Immunoreactivity to 5-HT was observed in the colloid, with a concentration in the follicular lumen exceeding that in the follicular cells. In fact, most follicles showed immunoreactivity in the cytoplasmic bridges formed between the apical portion of the follicular cells and the colloid

Let’s Take A Walk: Exploring the Impact of an Inclusive Walking Program on the Physical and Mental Health of Adults with Intellectual Disability

Background: People with intellectual disabilities experience health disparities and poorer health outcomes than people without disabilities. Increased physical activity has been found to reduce the impact of chronic health conditions among people with intellectual disabilities. Method: The current study explored the impact of an inclusive walking program on the physical and mental health of adults with intellectual disabilities. Let’s Take A Walk paired adults with intellectual disabilities, hereafter referred to as Community Walkers (n = 27), with college students to walk around a college campus twice a week for 45 minutes across 10 weeks. Data on mental health outcomes, specifically depression and anxiety, were collected from 24 Community Walkers across four-time points (pre-, mid-, post-, and 3-months following intervention), and data on physical health outcomes were collected across two-time points (pre- and post-intervention). Results: Community Walkers reported significant decreases in both depression and anxiety from pre to post-implementation. Particularly promising was clinically significant decreases in anxiety symptoms over the 10-week program. No differences were noted on Community Walkers’ measures of physical health. Conclusion: Inclusive walking programs are a valuable and promising mechanism for building social connections and inclusion and improving mental health for adults with intellectual disabilities

Electricity Generation using Sulfolobus solfataricus in a High-Temperature Microbial Fuel Cell

Microbial fuel cells (MFCs) are a developing technology that breaks down organic materials in liquids while generating electricity. They come in several forms and applications, including: micro-sized for medical implants, sediment for remote sensing and communications, and large-scale for industrial or environmental remediation. Few studies have looked at MFCs operating over 45ºC. Use of extremophiles as the fuel cell culture allows for high-temperature applications including industry, deserts, and alien space environments. This project includes the construction and operation of a membrane-less single chamber microbial fuel cell (ML-SCMFC), using the hyperthermophilic archaeon Sulfolobus solfataricus at about 80°C. The volcanic spring native S. solfataricus was used within a MFC to demonstrate feasibility of an extremely high temperature MFC and characterize the electrical power parameters from this device. A maximum power density of 25.26 mWm-3 was obtained using a carbon cloth anode and cellobiose as the substrate. Maximum sustained current densities ranging from 5.63 and 39.9 mAm-2 persisted for 15-30 hour durations. Continued modifications can potentially improve observed values, including new substrates, inclusion of separators and new anode materials.https://ecommons.udayton.edu/stander_posters/1013/thumbnail.jp

PO-042 Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2

Introduction Pancreatic ductal adenocarcinoma (PDAC) is an abysmal disease with a 5 year survival rate of merely 8%. The tumour microenvironment is one of the factors contributing to PDAC chemoresistance. More specifically, the hypoxic tumour cores and the metabolic switch to aerobic glycolysis (e.g. the Warburg effect), contribute to the lack of drug response. Interestingly, two glycolysis components glucose transporter 1 (GLUT-1) and lactate dehydrogenase A (LDH-A) are overexpressed in PDAC. The latter, LDH-A, is also correlated with prognosis in metastatic PDAC. N-Hydroxyindole-based LDH-A inhibitors (NHI-1 and NHI-2) have shown a synergistic effect in hypoxic PDAC cells when combined with gemcitabine. A glucose conjugated NHI-Glc-2 was designed to exploit the GLUT-1 overexpression in PDAC cells and in the present study we evaluated whether this novel compound further improved the pharmacological effect of LDH-A inhibitors. Material and methods The effect of NHI-Glc-2 on cell growth is tested in our primary PDAC cancer cell cultures, characterised for their hypoxic signature and LDH-A/GLUT-1 expression levels by next-generation sequencing. Inhibition of cell and tumour growth was evaluated by the SRB assay, 3D spheroid-cultures and with an orthotopic bioluminescent in vivo model. Additionally, LDH-A enzyme activity inhibition and the effect on the glycolytic rate by NHI-Glc-2 were assessed by spectrophotometry and with the Seahorse XF analyzer, respectively. Results and discussions NHI-Glc-2 is capable of inhibiting PDAC cell growth in, especially in hypoxia, in nanomolar range and shows a synergistic effect with gemcitabine. In 3D cultures NHI-Glc-2 disrupts spheroid integrity, and preliminary in vivo studies show promising results. Conclusion Lactate dehydrogenase A is a viable target in PDAC, and the novel LDH-A inhibitor showed improved pharmacological effect in normoxic and hypoxic PDAC cells compared to NHI-1 and NHI-2. Moreover, this compound displays a synergistic cytotoxic activity with gemcitabine, offering an innovative tool in hypoxic tumours

Impact of hypoxia on chemoresistance of mesothelioma mediated by the proton-coupled folate transporter, and preclinical activity of new anti-LDH-A compounds

Background: Expression of proton-coupled folate transporter (PCFT) is associated with survival of mesothelioma patients treated with pemetrexed, and is reduced by hypoxia, prompting studies to elucidate their correlation. Methods: Modulation of glycolytic gene expression was evaluated by PCR arrays in tumour cells and primary cultures growing under hypoxia, in spheroids and after PCFT silencing. Inhibitors of lactate dehydrogenase (LDH-A) were tested in vitro and in vivo. LDH-A expression was determined in tissue microarrays of radically resected malignant pleural mesothelioma (MPM, N = 33) and diffuse peritoneal mesothelioma (DMPM, N = 56) patients. Results: Overexpression of hypoxia marker CAIX was associated with low PCFT expression and decreased MPM cell growth inhibition by pemetrexed. Through integration of PCR arrays in hypoxic cells and spheroids and following PCFT silencing, we identified the upregulation of LDH-A, which correlated with shorter survival of MPM and DMPM patients. Novel LDH-A inhibitors enhanced spheroid disintegration and displayed synergistic effects with pemetrexed in MPM and gemcitabine in DMPM cells. Studies with bioluminescent hypoxic orthotopic and subcutaneous DMPM athymic-mice models revealed the marked antitumour activity of the LDH-A inhibitor NHI-Glc-2, alone or combined with gemcitabine. Conclusions: This study provides novel insights into hypoxia/PCFT-dependent chemoresistance, unravelling the potential prognostic value of LDH-A, and demonstrating the preclinical activity of LDH-A inhibitors

Impact of hypoxia on chemoresistance of mesothelioma mediated by the proton-coupled folate transporter, and preclinical activity of new anti-LDH-A compounds

BACKGROUND: Expression of proton-coupled folate transporter (PCFT) is associated with survival of mesothelioma patients treated with pemetrexed, and is reduced by hypoxia, prompting studies to elucidate their correlation. METHODS: Modulation of glycolytic gene expression was evaluated by PCR arrays in tumour cells and primary cultures growing under hypoxia, in spheroids and after PCFT silencing. Inhibitors of lactate dehydrogenase (LDH-A) were tested in vitro and in vivo. LDH-A expression was determined in tissue microarrays of radically resected malignant pleural mesothelioma (MPM, N = 33) and diffuse peritoneal mesothelioma (DMPM, N = 56) patients. RESULTS: Overexpression of hypoxia marker CAIX was associated with low PCFT expression and decreased MPM cell growth inhibition by pemetrexed. Through integration of PCR arrays in hypoxic cells and spheroids and following PCFT silencing, we identified the upregulation of LDH-A, which correlated with shorter survival of MPM and DMPM patients. Novel LDH-A inhibitors enhanced spheroid disintegration and displayed synergistic effects with pemetrexed in MPM and gemcitabine in DMPM cells. Studies with bioluminescent hypoxic orthotopic and subcutaneous DMPM athymic-mice models revealed the marked antitumour activity of the LDH-A inhibitor NHI-Glc-2, alone or combined with gemcitabine. CONCLUSIONS: This study provides novel insights into hypoxia/PCFT-dependent chemoresistance, unravelling the potential prognostic value of LDH-A, and demonstrating the preclinical activity of LDH-A inhibitors

Splicing modulation as novel therapeutic strategy against diffuse malignant peritoneal mesothelioma

Introduction: Therapeutic options for diffuse malignant peritoneal mesothelioma (DMPM) are limited to surgery and locoregional chemotherapy. Despite improvements in survival rates, patients eventually succumb to disease progression. We investigated splicing deregulation both as molecular prognostic factor and potential novel target in DMPM, while we tested modulators of SF3b complex for antitumor activity. Methods: Tissue-microarrays of 64 DMPM specimens were subjected to immunohistochemical assessment of SF3B1 expression and correlation to clinical outcome. Two primary cell cultures were used for gene expression profiling and in vitro screening of SF3b modulators. Drug-induced splicing alterations affecting downstream cellular pathways were detected through RNA sequencing. Ultimately, we established bioluminescent orthotopic mouse models to test the efficacy of splicing modulation in vivo. Results: Spliceosomal genes are differentially upregulated in DMPM cells compared to normal tissues and high expression of SF3B1 correlated with poor clinical outcome in univariate and multivariate analysis. SF3b modulators (Pladienolide-B, E7107, Meayamycin-B) showed potent cytotoxic activity in vitro with IC50 values in the low nanomolar range. Differential splicing analysis of Pladienolide-B-treated cells revealed abundant alterations of transcripts involved in cell cycle, apoptosis and other oncogenic pathways. This was validated by RT-PCR and functional assays. E7107 demonstrated remarkable in vivo antitumor efficacy, with significant improvement of survival rates compared to vehicle-treated controls. Conclusions: SF3B1 emerged as a novel potential prognostic factor in DMPM. Splicing modulators markedly impair cancer cell viability, resulting also in potent antitumor activity in vivo. Our data designate splicing as a promising therapeutic target in DMPM

Exploring alternative splicing in cancer: from insights in drug resistance to new therapies

Genomic characterization of cancer subtypes became a major research goal worldwide after the release of the complete human genome sequence in 2001. International consortia (e.g. The Cancer Genome Atlas - TCGA) have profiled and analyzed large numbers of human tumors matched to non-malignant tissues and created databases of molecular aberrations at the DNA, RNA, protein and epigenetic level. Research efforts aimed at understanding the molecular determinants of cancer prompted the rapid development of molecularly targeted chemotherapeutics which significantly improved clinical outcomes. Unfortunately, most cancer patients experience disease relapse manifested as recurrence of residual malignant cells which apparently did not respond to treatment regimes. Such cells either already carried or acquired genetic alterations (e.g. mutation, gene amplification, gene deletion or chromosomal translocation) that provide a clonal advantage to survive under selective therapeutic pressure. DNA aberrations have largely been documented to activate and drive oncogenic transformation as well as modulate response to therapy. Recent evidence highlighted the connection between alternative pre-mRNA splicing, cancer development and response to therapy. Alternative splicing is the essential process in eukaryotic gene expression by which non-coding intron sequences are removed from the pre-mRNA transcripts and specific exons are included or excluded from mature mRNAs. The aim of this thesis is to unravel the role of altered splicing in drug-resistant pediatric leukemia and evaluate the efficacy of small molecule splicing modulators in rare and aggressive tumors which pose major therapeutic challenges in the adult population. Chapter 2 is an extensive overview of the latest findings in the field of alternative splicing regulation in cancer. We describe the molecular mechanisms by which cancer cells harness alternative splicing to drive oncogenesis and evade anticancer drug treatment. Moreover, we discuss current challenges in the domain of bioinformatic analyses arising from genome-wide detection of aberrant splicing, which form the basis for the research presented in chapters 4 and 5. Finally, we identify splicing-based vulnerabilities that can provide novel treatment opportunities, as illustrated in chapters 6, 7 and 8. In Chapter 3 we studied the association of splicing alterations affecting the gene folylpolyglutamate synthetase (FPGS) with ex vivo methotrexate (MTX) resistance as well as clinical response in pediatric acute lymphoblastic leukemia (ALL) patients. In Chapter 4 we present an experimental protocol for genome-wide detection and validation of alternative splicing associated with in vitro drug resistance in solid tumors and hematological malignancies. We analyzed the transcriptomic profiles of several drug resistant in vitro models through RNA-seq and bioinformatic tools and established a qRT-PCR based method to validate the detected splice variants of candidate genes. This method is applied in chapter 5 to ex vivo leukemic specimens. In Chapter 5 we characterize whole-genome splicing profiles associated with glucocorticoid (GC) resistance in pediatric ALL samples by using RNA-seq-based differential splicing analysis, as established in chapter 3. Our analyses revealed markedly distinct splicing landscapes in ALL samples of B-cell precursor and T-cell lineages associated with GC-resistant phenotype. To determine whether splicing modulation could serve as a novel therapeutic option for GC-resistant patients, we tested the efficacy of splicing modulator Pladienolide-B in GC-resistant ALL cell lines and primary leukemic specimens as single agent or in combination with GCs. Chapter 6 and 7 illustrate the rationale of targeting spliceosome in malignant peritoneal mesothelioma and pancreatic ductal adenocarcinoma. Here we tested the antitumor effects of splicing modulators in relevant in vitro and in vivo disease models. Finally, Chapter 8 provides a novel splicing-based strategy to specifically target non-small cell lung cancer by silencing core spliceosomal Sm proteins as an alternative to SF3b modulation. In Chapter 9 we discuss the relevance of the findings presented in this thesis