The purpose of this study was to evaluate the toxicity potential of cyanuric acid (CYA) and a combination of melamine (MEL) and CYA in broilers. A total of 1200 male COBB 500 broilers were randomly allocated into 1 of 10 treatment groups by a 5 × 2 factorial design in a 42-d experiment. The dietary treatments were as follows: T(1) to T(5): basal diets with 0, 10, 20, 33.3, and 50 mg CYA per kg diet; T(6) to T(10): basal diet with CYA regimens similar to T(1) to T(5) but with 100 mg MEL per kg diet. There were 6 replication pens with 20 chicks per pen. No differences were observed in alanine transaminase (ALT) and aspartate aminotransferase (AST) activities. But on d 22, uric acid (UA) and creatinine (Crea) concentrations were significantly greater when birds were fed CYA at 33.3 mg/kg with MEL 100 mg/kg, and Crea concentration was also higher when birds were fed CYA at 50 mg/kg. No crystals were found in kidneys but dilated renal tubules and small blood vessel expansion were found in kidneys of birds fed CYA at 50 mg/kg and CYA at 33.3 mg/kg with MEL 100 mg/kg. The apoptosis rate (AR) of kidneys of all birds fed CYA and MEL contaminated diets were higher than the control group. These results indicated that the dietary addition of CYA and MEL could induce kidney damage, and the effects were harmful when the ratio of CYA/MEL was 1:3.
Background: Leukemia inhibitory factor (LIF) and its receptor LIFR are over-expressed in multiple solid tumors and play a key role in tumor growth, progression, and resistance to standard anti-cancer treatments. Triple-negative breast cancer (TNBC) lacks targeted therapies and represents a disproportional share of breast cancer (BCa) mortality. TNBC exhibits autocrine stimulation of the LIF/LIFR axis and overexpression of LIF is associated with poorer relapse-free survival in BCa patients. LIF signaling also promotes maintenance of stem cells. Therefore, targeting the LIF/LIFR axis may have therapeutic utility in TNBC. Methods: We rationally designed a small organic molecule (EC359) that emulates the LIF/LIFR binding site and functions as a LIFR inhibitor from a library of compounds. In silico docking studies were used to identify the putative interaction of the EC359 and LIF/LIFR complex. Direct binding of EC359 to LIFR was confirmed using surface plasmon resonance (SPR) and microscale thermophoresis technique (MST) assays. In vitro activity was tested using Cell-Titer Glo, MTT, invasion, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays, and RNA-seq analysis. Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDEX) models were used for preclinical evaluation and toxicity. Results: Molecular docking studies showed that EC359 interacts at the LIF/LIFR binding interface. SPR and MST studies confirmed direct interaction of EC359 to LIFR. EC359 reduced the growth of TNBC cells with high potency (IC50 50-100nM) and promoted apoptosis. Further, EC359 treatment reduced invasion and stemness of TNBC cells. EC359 activity is dependent on the expression levels of LIFR and showed little or no activity on TNBC cells that have low levels of LIFR or ER+ve BCa cells. Further, EC359 significantly reduced the viability of cisplatin and taxane-resistant TNBC cells and enhanced the efficacy of HDAC inhibitors. Mechanistic and biochemical studies showed that EC359 interacts with LIFR and effectively blocking LIF/LIFR interactions. EC359 also blocked LIFR interactions with other LIFR ligands such as oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. EC359 treatment attenuated the activation of LIF/LIFR driven pathways including STAT3, mTOR, AKT, and MAPK. RNA-seq analysis identified regulation of apoptosis as one of the important pathway modulated by EC359. In TNBC xenograft and PDX assays, EC359 significantly reduced tumor progression. Further, using human primary BCa PDEX cultures, we demonstrated that EC359 has the potential to substantially reduce the proliferation of human BCa. Pharmacologically, EC359 exhibited high oral bioavailability and long half-life with a wide therapeutic window. Conclusions: EC359 is a novel targeted therapeutic agent that inhibits LIF/LIFR oncogenic signaling in TNBC via a unique mechanism of action. EC359 has the distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. (DOD BCRP grant #BC170312) Citation Format: Viswanadhapalli S, Luo Y, Sareddy GR, Santhamma B, Zhou M, Li M, Pratap UP, Altwegg KA, Li X, Srinivasan U, Ma S, Chang A, Riveros AC, Zhang KY, Dileep KV, Pan X, Murali R, Bajda M, Raj G, Brenner A, Manthati V, Rao M, Tekmal RR, Nair HB, Nickisch KJ, Vadlamudi RK. Development of a first-in-class small molecule inhibitor (EC359) targeting oncogenic LIF/LIFR signaling for the treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-02.
Background: Ongoing disease gene discoveries continue to drive our understanding of the molecular and cellular mechanisms underlying ALS. Causative genes from 60% of ALS families have been identified using modern genetic techniques, but the causal gene defect is yet to be identified in the remaining 40% of families. These remaining families often do not follow true Mendelian inheritance patterns and are challenging to solve using traditional genetic analysis alone. In vitro and in vivo studies have become critical in assessing and validating these ALS candidate genes. Objectives: In this study, we aim to develop and validate the utility of an in vitro functional pipeline for the discovery and validation of novel ALS candidate genes. Methods: A panel of cell based-assays were applied to candidate genes to examine the presence/absence of known ALS pathologies in cell lines as well as human autopsy tissues. These include immunofluorescence, flow cytometry and western blotting to study toxicity, neuronal inclusion formation, interaction with TDP-43, aberrant protein degradation and accumulation in detergent-insoluble cellular fractions. Immunohistochemistry and immunofluorescence were also used to examine if candidates were present in neuronal inclusions from ALS patient spinal cord tissues. Results: The in vitro pipeline was applied to five candidate genes from an ALS family that is negative for known ALS gene mutations. Two candidates were prioritized as top candidates based on their capacity to induce known ALS cellular pathologies. In transfected cells, the variants in these two genes caused a significantly higher toxicity than wild type, formed detergent insoluble inclusions and was able to co-aggregate with TDP-43 in neuronal cells. The variants have also led to protein degradation defects. One of the candidates also co-localised with TDP-43-positive neuronal inclusions in sporadic ALS patient post-mortem tissues, a signature pathology of ALS. Discussion and conclusions: We have demonstrated the utility of a functional prioritization pipeline and successfully prioritized two novel candidate ALS genes. These genes, and its associated pathways, will be further investigated through the development of animal models to establish if there is support for its role in ALS. New ALS genes offer fresh diagnostic and therapeutic targets and tools for the generation of novel animal models to better understand disease biology and offer preclinical testing of candidate treatments for ALS in the future.
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