Increase In Mitotic Index Research Articles

Abstract A024: Preclinical profile of novel and potent small molecule inhibitors of KIF18A inhibitors in chromosomally unstable solid tumor lines

Abstract Accurate alignment of chromosomes at the equator region and equal chromosome separation during cell division is essential for maintenance of genome integrity. KIF18A belongs to kinesin superfamily of microtubule associated motor proteins and is responsible for maintenance of bipolar spindle fiber integrity during mitotic phase. Cancer cells with chromosomally unstable (CIN+) DNA show synthetic lethal phenotype with genetic ablation of KIF18A. Thus, pharmacological inhibition of KIF18A represents a potential strategy aimed at attenuating growth of CIN +ve tumors. A microtubule dependent ADP Glo kinase assay was used to identify potent KIF18A inhibitors. Mitotic arrest, manifested by an increase in phosphoH3, was determined in cells using fluorescent microscopy. Anti-proliferative activity was confirmed in CIN+ve solid tumor cells with significant selectivity over CIN-ve cancer cells. Compounds inhibited KIF18A at with potencies ranging from 50-80 nM. A dose dependent increase in mitotic index in CIN+ve OVCAR-3 cells (via fluorescence microscopy) was observed in contrast to CIN-ve HCT-116 cells (10-100X selectivity). Translation was evident in an antiproliferation assay using CIN-ve cancer cells with IC50 <10 nM. Selected KIF18A inhibitors demonstrated favorable physicochemical, ADME and PK properties. In Vivo efficacy studies are planned. Citation Format: Sukanya Patra, Manoj Pothuganti, Navnath Karche, Ganesh Gudade, Venu Sankeshi, Githavani Kummari, Srikant Vishwanadha. Preclinical profile of novel and potent small molecule inhibitors of KIF18A inhibitors in chromosomally unstable solid tumor lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A024.

Abstract 4965: Targeting the mitotic kinesin, KIF18A, in chromosomally unstable cancers

Abstract Background: Chromosome instability (CIN), characterized by frequent and ongoing loss or gain of chromosome number, is commonly observed in tumor cells. Although long recognized as a vulnerability of cancer cells, potential CIN-selective therapeutic targets have only recently been discovered. Genetic studies from multiple groups have identified the mitotic kinesin, KIF18A, as selectively essential for the proliferation of CIN and aneuploid cells. By targeting KIF18A genetically and with novel small molecule inhibitors here we present data supporting KIF18A as therapeutic target in CIN tumors. Methods and Materials: To explore the therapeutic potential of KIF18A, we evaluated the effects of KIF18A genetic depletion and small molecule inhibition in both CIN-positive and CIN-negative cell lines. Biochemical, cell proliferation and phenotypic assays were used to characterize the potency and cellular activity of reference and novel KIF18A small molecule inhibitors. Anti-tumor activity of KIF18A inhibitors was assessed in CIN-positive cell line xenograft models. Results: In triple negative breast and colorectal CIN positive cancer cell lines siRNA mediated KIF18A knockdown and small molecule inhibition of the KIF18A ATPase activity both lead to a reduction in proliferation associated with an increase in mitotic index and multi-polar spindles. Consistent with KIF18A knockdown, KIF18A inhibition results in increased spindle length and chromosome alignment defects. Importantly, these effects are not observed with KIF18A knockdown or KIF18A inhibition in non-transformed, near diploid cells. In vivo, treatment of CIN-positive xenograft tumors with potent, novel KIF18A inhibitors results in robust anti-tumor activity with minimal impact on body weight. Conclusions: Collectively our data support KIF18A as a therapeutic target and provide rationale for the continued development of potent selective small molecule KIF18A inhibitors for the treatment of CIN positive cancers. Citation Format: James D. Joseph, Katherine Schutt, Kira Fisher, Katelyn Queen, Olivia Budington, Weifeng Mao, Wei Liu, Zhengqing Zhu, Xiangping Zhang, Yisong Xiao, Kunmin Lai, Xiaohui Gu, Jason Stumpff, Fred Aswad. Targeting the mitotic kinesin, KIF18A, in chromosomally unstable cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4965.

Abstract 517: Targeting chromosomally unstable tumors with a selective KIF18A inhibitor

Abstract Chromosome instability (CIN) is an attribute of cancer cells that has yet to be fully exploited therapeutically, despite its prevalence across many tumor types and lack of occurrence in normal cells. To identify genes uniquely essential to CINhigh cells, we mined the Cancer Dependency map (DepMap) for genes required for proliferation of tumor cell lines with high levels of copy number aberrations, an indicator of CIN. This analysis identified KIF18A, a mitotic kinesin also identified by others as a vulnerability of aneuploid and whole genome doubled cells. Genetic disruption of KIF18A with RNAi confirmed its necessity in CINhigh cells, and these findings translated to in vivo reduction in tumor growth, illustrating the potential of KIF18A as a therapeutic target. High-throughput screening of a chemical library for inhibitors of KIF18A motor domain’s ATPase activity identified an ATP-noncompetitive hit that was optimized for potency, selectivity, and pharmacokinetic properties. These optimized inhibitors are orally bioavailable, exhibit sub-nM potency, and are highly selective for KIF18A vs. other kinesins. They also display a long drug-target residence time, a unique property that allows cells to be trapped in mitosis long enough to result in cell death. Mechanistically, inhibition of KIF18A’s ATPase activity renders KIF18A unable to localize to the metaphase plate, instead becoming trapped at the spindle poles. This inability to translocate results in defects in chromosome congression, evidenced by a less compact metaphase plate, and an accumulation of cells in G2/M. Treatment of CIN cancer cells with these inhibitors results in a proliferative defect due in part to induction of programmed cell death. In contrast to the effect in cancer cells, KIF18A inhibition has no effect on rapidly proliferating non-transformed epithelial cells and primary T cells. The differential impact between normal and cancer cells sets KIF18A inhibitors apart from other anti-mitotic therapies, which have had limited clinical benefit due to their detrimental impact on both cancer and highly proliferative normal cells. The therapeutic potential of KIF18A inhibitors was assessed in human tumor xenograft models. Treatment of multiple tumor models resulted in substantial, dose-dependent inhibition of tumor growth. KIF18A inhibition in the tumor was confirmed through evaluation of KIF18A localization changes, chromosome congression and an increase in mitotic index. The strong biological rationale, robust preclinical data, and superior compound properties warrant clinical development of Volastra’s KIF18A inhibitor which is planned for development for the treatment of chromosomally unstable cancers. Citation Format: Aaron F. Phillips, Rumin Zhang, Akanksha Verma, Tamar Feinberg, Mia Jaffe, Marysol Chu Carty, Ryan Schulz, Sarah Bettigole, Celia Andreu, Scott Drutman, Michael Su, Derek Cogan, Christina H. Eng. Targeting chromosomally unstable tumors with a selective KIF18A inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 517.

Vicia faba seed: a bioindicator of phytotoxicity, genotoxicity, and cytotoxicity of light crude oil.

Crude oil contamination is a serious threat to the environment and human health as it can contaminate food chains. Therefore, it is necessary to find efficient tests to monitor soils for crude oil contamination. The present study investigates the efficacy of Vicia faba seeds for monitoring contaminated soils with light crude oil. Vicia faba seeds were planted in 0 (control), 1, 2, and 4 percentages (weight percentage) light crude oil-contaminated soils. The seed germination and root length were measured to evaluate phytotoxicity, while the mitotic index, chromosome aberrations, and micronucleus formation in the root tip cells were examined for cytotoxicity and genotoxicity tests. The results showed that light crude oil had toxic effects on Vicia faba growth characteristics, even at 1% contamination. The phytotoxicity assay showed that crude oil reduced seed germination and root length by 45% and 61.67%, respectively. In contrast, cellular observations indicated an increase in mitotic index, chromosome aberrations, and micronucleus formation up to 3, 3.59, and 5.6 times, respectively, compared to the control. The light crude oil at 4% induced the simultaneous occurrence of nuclear bud, polyploidy, and micronucleus that may be considered as severe clastogenic and aneugenic effects. Accordingly, Vicia faba can be considered a reliable living system for monitoring light crude oil pollution in soils, even at low concentrations.

Abstract 2981: Loss of the small GTPase RHOA enhances lymphomagenesis and impairs the activity of lenalidomide in mantle cell lymphoma

Abstract The small GTPase Ras homolog family member A (RHOA) is one of the most extensively investigated members of the Rho GTPase family, that acts as a molecular switch controlling a wide variety of signal transduction pathways. Although RHOA has long been implicated in malignant transformation in solid tumors, recent evidences have demonstrated its tumor suppressor activity in different subgroups of B-cell non-Hodgkin lymphoma (B-NHL). Here, using a panel of 11 cell lines covering the most common and/or aggressive B-NHL subtypes, we observed that the lowest mRNA and protein levels of RHOA are found in mantle cell lymphoma (MCL), the most aggressive entity with a median overall survival of 5-7 years. Depletion of RHOA expression and activity was carried out in the two RHOA+ MCL cell lines, REC-1 and Z-138, by CRISPR/Cas9-mediated gene edition followed by RHOA pulldown activation assay. In RHOA knockout (KO) subclones, cell proliferation was increased by 40%, in association with a 10-fold increase in mitotic index and a 3-fold potentiation of cell migration, as assessed respectively by CellTitter-Glo assay, histone H3-pSer10 labeling and Transwell assay, thus suggesting a tumor suppressor role of RHOA in MCL malignancy. Comparative RNA-seq analysis of RHOAWT and RHOAKO MCL subclones then highlighted a crucial role of RHOA and its downstream signaling in the control of MCL cell proliferation, cell cycle and cell migration. Interestingly, the immunomodulatory drug lenalidomide has previously shown to regulate such processes and to be clinically active in patients with relapsed/refractory MCL. In a co-culture system associating RHOAWT MCL cells with peripheral blood-derived effector cells, we observed that lenalidomide treatment facilitated the formation of immunological synapse, as revealed by simultaneous immunodetection of effector T cells, target B cells and perforin, and by quantification of lactate dehydrogenase (LDH) release. Conversely, lenalidomide exhibited minimal effect in the RHOAKO MCL co-cultures, as shown by a 30-40% decrease in the formation of immune synapse and a 4-fold decrease in LDH secretion. Taken together, these data provide the first insight into the tumor suppressive activity of RHOA in MCL and into its requirement for the immune-mediated mechanism of action of lenalidomide in this disease. Ongoing in vivo validation studies will be presented. Citation Format: Juliana C. Santos, Marcelo L. Ribeiro, Gael Roue. Loss of the small GTPase RHOA enhances lymphomagenesis and impairs the activity of lenalidomide in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2981.

Modulation of cytotoxic effects of wastewater on barely seedlings by Aloe vera extract

Aim: This study was conducted to assess the mitigation effect of A. vera leaf ethanolic and water extracts on plant growth, MDA and protein content, and mitotic index of barley seedlings irrigated with sewage water. Methodology: The experiment was conducted in a green house in Al-Ahsa, Saudi Arabia, in plastic bags containing air-dried loam soil and compost (1:1). Wastewater samples were collected from Al-Ahsa Sewage Treatment Plant/Al-Ahsa/Saudi Arabia. Seeds of barley were exposed to the following treatments for three weeks: 100% treated wastewater, treated wastewater mixed with: 0.05 g ml-1 A. vera alcoholic extract (Alc Ext) or 0.05 g ml-1 A. vera water extract (Wat Ext), 0.15 g ml-1 Alc Ext or 0.15 g ml-1 Wat Ext. Similar test doses were prepared for untreated wastewater. A separate set of seeds was prepared using distilled water as a control. Germination rate, fresh and dry weight, protein content, MDA content and mitotic index were determined. Results: Untreated wastewater caused significant reduction in fresh weight while both treated and untreated wastewater showed insignificant decrease in dry weight of barley. No significant change in MDA contents were observed in all treatments, except for addition of 0.15 A. vera water extract to untreated wastewater. Protein content showed an increase in both treated wastewater (insignificant) and untreated wastewater (significant). Addition of A. vera extracts increased accumulation of protein in both shoot and root. A. vera alcoholic extract promoted significant increase in both root and shoot protein of treated wastewater, but only shoot protein of untreated wastewater. A significant reduction in mitotic index was induced by wastewater, while addition of A. vera extracts stimulated significant increase in mitotic index. Interpretation: A. vera possess reactive oxygen species scavenging ability and enzyme conservation activity in addition to stimulatory effects on cell proliferation and cell-cycle process that add a modulation credit on protein content and mitotic index of barley.

Gastrointestinal stromal tumors of the stomach: A 10-year experience of a single-center.

Gastrointestinal tract stromal tumors (GIST) are the most common mesenchymal tumors in the gastrointestinal tract (GIS). GISTs may cause significant morbidity and mortality rates. In this study, it was aimed to evaluate 10 years of gastric GIST cases followed in our hospital, and to analyze the prognostic factors. In this single-center retrospective study, a total of 64 patients who were operated between May 2010 and May 2020 due to gastric GIST tumor were reviewed. Clinical and pathological features, risk classifications, overall survival (OS) and disease-free survival (DFS) were evaluated. According to the risk classification, 18.8% of the patients were in the high-risk group. The overall 5-year OS and DFS rates were 85.7%. The mean OS of the patients was 47.9 SD36.2 months, and the duration of DFS was 45.5 months. Patients with a 5-year OS rate above 5 cm in diameter (P = 0.024), with a mitotic index above 5/50 high power field (HPF) (P = 0.038), and those with a high-risk group (P = 0.011) were significantly lower than the other group. In the correlation analysis, it was found that tumor diameter correlated significantly with OS (P = 0.034; r = -0.317). Tumor diameter and mitotic index were found to be inversely correlated with DFS duration (P = 0.004; r = -0.425 and P = 0.035; r = -0.316, respectively). Our findings showed that in gastric GIST cases, as the primary tumor diameter and mitotic index increase, correlate with survival rates and the mean overall and disease-free survival times decrease.

The Effect of Artemisia Dracunculus L. on Mitotic Index in Bone Marrow and Spleen Cells of Mice: In Vivo Study

Artemisia dracunculus L. (tarragon) is a rich source of herbal remedies with antioxidant and anti-inflammatoryproperties. In the present study, the proliferation of bone marrow and spleen cells of mice was evaluated afterextraction of Artemisia dracunculus with 80% of ethanol. Two doses of the extract (500mg/kg body weightand 1000mg/kg body weight) were given to the mice for seven successive days. On day eight, mice weresacrificed and cells from bone marrow and spleen were collected; mitotic index was calculated and resultswere compared with that of methotrexate at a dose 20mg/kg body weight ( positive control ) and distilledwater ( negative control). The results showed that the dose 500mg/kg body weight of Artemisia dracunculusextract caused significant increase in mitotic index in both bone marrow and spleen cells of mice whencompared with negative control. While, the dose 1000mg/kg body weight of Artemisia dracunculus extractcaused significant decrease in mitotic index in both bone marrow and spleen cells of mice when comparedwith negative control.

Genetic Study on the Effect of the Antidiabetic Drug ( Sitagliptin) on DNA and Chromosomes of Human Lymphocyte Culture

AbstractBackground: Diabetes mellitus is a worsening worldwide health problem. It constitutes a major global health concern. Sitagliptin selectively inhibits dipeptidyl peptidase-4 (DPP-4) to manage type 2 diabetes mellitus (T2DM). It regulates the blood glucose level without risk of hypoglycemia or increase in body weight. In our study we investigated the effects of Sitagliptin on DNA and chromosomes in cultured human lymphocytes. Aim: To assess the genotoxic and cytotoxic effects of different concentrations of Sitagliptin on cultured human lymphocytes.Material and methods: Cultures were divided into 6 groups: control, positive control (Cisplatin) at concentration of 10 μg/mL and 4 different concentrations of Sitagliptin (125,250,500,1000 μg/mL). Sitagliptin genotoxicity and cytotoxicity were determined by using chromosomal aberrations (CAs), mitotic index (MI), comet assay and nucleic acids electrophoresis.Results: There was high significant increase in total chromosomal aberrations (TCAs) at 500, 1000 μg/mL of Sitagliptin compared to control. Other studied concentrations of Sitagliptin exhibited an increase in TCAs without significant relation. Compared to control, there was a significant increase in mitotic index at 125 μg/mL of Sitagliptin but non-significant increase at 250 μg/mL of Sitagliptin. However, at 500, 1000 μg/mL of Sitagliptin, there was a significant decrease in MI. Regarding comet assay, there was significant and high significant increase in total DNA damage at 500,1000 μg/mL of Sitagliptin respectively. Nucleic acids electrophoresis not digested with RNase showed that optical density value of RNA was maximum at 125 μg/mL then gradually decreased till reach the minimum level at 1000 μg/mL of Sitagliptin indicating its toxicity. Genomic DNA fragmentation results indicated that Sitagliptin caused a slight damage of DNA in the form of necrosis in a concentration dependent manner. Conclusion: Sitagliptin induces significant genotoxic and cytotoxic effects on the cultured human lymphocytes at concentrations of (500, 1000 μg/mL).

CBIO-03. ATRX LOSS IN GLIOMA RESULTS IN EPIGENETIC DYSREGULATION OF CELL CYCLE PHASE TRANSITION

Abstract Gliomas are a leading cause of cancer mortality in children and adults, and new targeted therapies are desperately needed. ATRX is a chromatin remodeling protein that is recurrently mutated in H3F3A-mutant pediatric glioblastoma (GBM) and IDH-mutant grade 2/3 adult glioma. We previously showed that loss of ATRX in glioma results in tumor growth and additional tumor mutations. However, the mechanism driving these phenotypes has not been fully established. We found that in ChIP-Seq/ChIP-qPCR of mouse neuronal precursor cells (NPCs) and GBM cells with isogenic ATRX loss, ATRX binds regulatory elements for cell cycle phase transition gene sets, and ATRX loss subsequently results in reduced expression. Furthermore, human GBM cells with ATRX knock-out demonstrate higher rates of cells in S and G2/M phases, with clusters of cells demonstrating reduced expression of cell cycle regulatory gene sets by single-cell sequencing (scSeq) analysis. In human and mouse GBM in vitro models, ATRX-deficient cells exhibit a seven-fold increase in mitotic index at 16 hours after sub-lethal radiation and enhanced activation of the master cell cycle regulator ATM with radiation. Treatment of ATRX-deficient gliomas with ATM inhibitors results in a selective increase in dysfunctional cell cycling and increased radio-sensitization in ATRX-deficient glioma cells. Using an ATM-luciferase reporter in orthotopically-implanted human GBM cells, both AZD0156 and AZD1390 demonstrate in vivo pathway inhibition. Mice intra-cranially implanted with ATRX-deficient GBM cells demonstrate a doubling of median survival compared to radiated controls (p=0.0018) when treated with AZD0156 combined with radiation; this is not seen in ATRX-sufficient models. This study demonstrates that ATRX-deficient high-grade gliomas display epigenetic dysregulation of cell cycle phase transitions, which opens a new window for therapies targeting this unique phenotype.

Abstract 1808: Loss of BAP1/BRCA1 counteracts spindle assembly checkpoint activation by the anti-mesothelin antibody drug-conjugate anetumab ravtansine

Abstract Mesothelioma is a rapidly lethal cancer with limited treatment options. Spindle poisons have demonstrated clinical usefulness in mesothelioma, however, predictive biomarkers are lacking. Anetumab ravtansine (ARav) is an antibody drug-conjugate (ADC) comprising a mesothelin targeting antibody coupled to the payload, DM4, a microtubule destabilizer. We have recently identified BRCA1 as a direct regulator of the spindle assembly checkpoint (SAC), which is activated by spindle poisons and mediates mitotic arrest. BAP1 is one of the most frequently inactivated tumor suppressors in mesothelioma and it interacts with and regulates expression of BRCA1. We therefore hypothesized that BAP1 inactivation may confer resistance to ARav, highlighting a potential opportunity to refine personalization with this ADC. Three primary cell lines were derived from mesotheliomas resected during pleurectomy decortication expressed mesothelin (MSLN) and further characterized via whole exome sequencing. Two of the three primary cell lines have BAP1 mutation (R417 frameshift (fs); E631fs) and show reduced BRCA1/MAD2 protein expression. MSLN expression was found to be independent of BAP1 inactivation status or BRCA1 expression level. BAP1 mutant primary cell lines exhibited resistance to ARav by overriding the mitotic arrest with continued cell cycle progression, indicating a defective SAC whereas the cell line with wt BAP1 underwent mitotic arrest followed by cell death. Increase in mitotic index was also observed in primary cell line with wt BAP1 following ARav treatment without any significant change in BAP1 mutant cell lines. Resistance to ARav in BAP1 deleted cell line NCI-H226 was reversed by stable expression of wildtype BAP1 in NCI-H226 indicating critical regulation of SAC by BAP1. Vinorelbine resistant cell line RVR with a defective SAC also exhibited resistance to ARav compared to the parental REN with no change in MSLN expression suggesting that an active SAC is required for prolonged mitotic arrest and subsequent cell death. Wildtype BAP1 and MSLN is critical for efficacy of ARav. Inactivation of BAP1 correlates with loss of BRCA1 expression and dysregulation of the SAC. Citation Format: Anita Singh, Jin-li Luo, Sara Busacca, Alan Dawson, Krishna Kalyan Kolluri, Samuel Janes, Andrew Fry, Anette Sommer, Dean Fennell. Loss of BAP1/BRCA1 counteracts spindle assembly checkpoint activation by the anti-mesothelin antibody drug-conjugate anetumab ravtansine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1808.

Phosphodiesterase Type 5 Inhibitors Synergize Vincristine in Killing Castration-Resistant Prostate Cancer Through Amplifying Mitotic Arrest Signaling.

Combination therapies that display cancer-killing activities through either coexistent targeting of several cellular factors or more efficient suppression of a specific pathway are generally used in cancer treatment. Sildenafil, a specific phosphodiesterase type 5 (PDE5) inhibitor, has been suggested to display both cardioprotective and neuroprotective activities that provide a rationale for the combination with vincristine on the treatment against castration-resistant prostate cancer (CRPC). In the present work, vincristine arrested cells in the metaphase stage of mitosis. Vincristine-induced mitotic arrest was identified by Cdk1 activation (i.e., increased Cdk1Thr161 phosphorylation and decreased Cdk1Tyr15 phosphorylation), cyclin B1 upregulation, and increased phosphorylation of multiple mitotic proteins and stathmin. Sildenafil synergistically potentiated vincristine-induced mitotic arrest and a dramatic increase of mitotic index. Furthermore, sildenafil potentiated vincristine-induced mitochondrial damage, including Mcl-1 downregulation, Bcl-2 phosphorylation and downregulation, Bak upregulation and loss of mitochondrial membrane potential, and sensitized caspase-dependent apoptotic cell death. Sildenafil-mediated synergistic effects were mimicked by other PDE5 inhibitors including vardenafil and tadalafil, and also by PDE5A knockdown in cells, suggesting PDE5-involved mechanism. Notably, sildenafil amplified vincristine-induced phosphorylation and cleavage of BUBR1, a protein kinase in spindle assembly checkpoint (SAC) function and chromosome segregation. Sildenafil also significantly decreased kinetochore tension during SAC activation. Moreover, sildenafil synergized with vincristine on suppressing tumor growth in an in vivo model. In conclusion, the data suggest that sildenafil, in a PDE5-dependent manner, potentiates vincristine-induced mitotic arrest signaling, and sensitizes mitochondria damage–involved apoptosis in CRPC. Both in vitro and in vivo data suggest the combination potential of PDE5 inhibitors and vincristine on CRPC treatment.

Biomonitoring of urban wastewaters treated by an integrated system combining microalgae and constructed wetlands

The objectives of the present study were to apply different, toxicological assays for monitoring the toxicity of treated and untreated urban effluents produced at a university campus. The research was conducted at the wastewater treatment plant of the University of Santa Cruz do Sul, (UNISC), from october 2018 to april 2019. An integrated system with, anaerobic reactor (AR), microalgae (MA) and constructed wetlands (CWs) was, proposed for detoxification of the wastewaters produced at the university campus with a hydraulic detention time of 17 days. Daphnia, magna (ecotoxicity) and Allium cepa (phytotoxicity, cytotoxicity, and, genotoxicity) were used as tools to monitor the efficiency of the integrated system. Obtained results showed that the integrated system (MA, + CWs) presented good COD and BOD5 reductions, besides removal rates of, almost 98% for N-NH3, being much more efficient than the UNISC wastewater, treatment plant (UWTTP). The results of ecotoxicity presented the raw wastewaters (RW) as slightly toxic and an absence of ecotoxicity in all the treatments steps. Regarding phytotoxicity, the results showed no significant differences between the treatments. The cytogenetic assays indicated a significant increase in mitotic index (MI) (p < 0.001) after treatment by CWs compared to the final treatment UWTTP while the results, regarding binucleated cells (BNC) did not present significant differences, among the treatments. Micronucleus (MN) indexes were significantly different between the UWWPT and the integrated system (p < 0.01). In relation to chromosome aberrations (CA) the results indicate a significant difference between the CWs and UWWTP treatments (p < 0.01) and, RW and CWs (p < 0.001), confirming the detoxifying potential of the integrated system when compared to UWWPT. Thus, the results of the present research highlight the relevance in the proposition of the integrated system as an alternative of cleaner technology to the detriment of conventional technologies applied in wastewater treatment.

GENE-17. ATRX LOSS IN GLIOMA RESULTS IN EPIGENETIC DYSREGULATION OF THE G2/M CHECKPOINT AND SENSITIVITY TO ATM INHIBITION

Abstract Gliomas are a leading cause of cancer mortality in children and adults and new targeted therapies are desperately needed. ATRX is a chromatin remodeling protein that is recurrently mutated in H3F3A-mutant pediatric GBM and IDH-mutant grade 2/3 adult glioma. We previously showed that loss of ATRX in glioma results in tumor growth and additional tumor mutations. However, the mechanism driving these phenotypes has not been fully established. We found that in ChIP-Seq datasets of mouse neuronal precursor cells (NPCs) and experimental models of human glioma cells, ATRX binds and regulates the chromatin state of promoters and enhancers for gene sets associated with regulation of the cell cycle G2/M checkpoint. In line with this, analysis of single-cell seq (sc-seq) data from IDH-mutant gliomas (n=16) shows that ATRX-mutant tumors (IDH-A) demonstrate a population of cycling cells with dysregulated cell cycle phase gene set expression when compared to ATRX-wildtype tumors (IDH-O). In glioma models, ATRX-deficient cells exhibit a seven-fold increase in mitotic index at 16 hours after sub-lethal radiation and enhanced activation of the master cell cycle regulator ATM with radiation. Treatment of ATRX-deficient gliomas with ATM inhibitors results in a selective increase in dysfunctional cell cycling and increased radio-sensitization in ATRX-deficient glioma cells. Using an ATM-luciferase reporter in orthotopically-implanted human GBM cells, both AZD0156 and AZD1390 demonstrate in vivo pathway inhibition. Mice intra-cranially implanted with ATRX-deficient GBM cells demonstrate a doubling of median survival compared to radiated controls (p=0.0018) when treated with AZD0156 combined with radiation. This study demonstrates that ATRX-deficient glioma display epigenetic dysregulation of the G2/M checkpoint, which opens a new window for therapies targeting this unique phenotype.

Nanopriming technology enhances vigor and mitotic index of aged Vicia faba seeds using chemically synthesized silver nanoparticles

This investigation was planned to utilize chemically synthesized silver nanoparticles (AgNPs), as a priming agent, for improving the germination of aged Vicia faba seeds. Application of AgNPs (0, 10, 50 and 100 ppm) to Vicia faba seeds led to distinctive endpoints indicating a reduction of aging-related genotoxic impacts at both cytological and physiological levels. Thus, priming with AgNPs induced a significant increase only in length of radicles, whereas the other determined growth parameters elicited non-significant changes in relation to control. In all treatments, total phenols increased slightly above control levels. Meanwhile, the highest value for catalase (CAT) activity was recorded with 100 ppm AgNPs. Ascorbate peroxidase (APX) decreased in the radicles but increased in the plumules with all concentrations used. Peroxidase (POX) and polyphenol oxidase (PPO) activities showed an opposite pattern compared with that of APX. For cyto-genotoxicity and mutagenicity assays, all of the treatments induced a significant increase in mitotic indices above those of control. Concerning genotoxicity assays, treated V. faba seeds significantly showed lower percentages of micronuclei and chromosomal abnormalities below those of control, in both interphasic and mitotic cells. Furthermore, treatment with silver nanoparticles elicited lower level of genotoxic and mutagenic aberrations than those maintained in the control. In conclusion, with increasing of AgNPs concentrations, the mitotic cell cycle and seedling growth were increased as compared with control. To our knowledge, this is the first report for seven-year-old seeds using nanoparticles. Thus, utilization of silver nanoparticles to enhance plant vigor under unfavorable environments appears to be conceivable.

Effect of GINGER AGAINST THE TOXICITY OF BRODIFACOUM ON ALBINO MALE MICE

Brodifacoum is a 4-hydroxycoumarin anticoagulant rodenticide of Rodents. This denotes a high possibility that contaminated the diet of man and his farm animals this fact alarmed some scientists to study their biological influence. Ginger (Zingiber officinale) has been used as an antioxidant and preventing agent against numbers of diseases. The study deals with effects of brodifacoum on the chromosome and on the liver and testis of male mice as well as and the protective effects of ginger extract. 130 Adult albino male mice were used in this study. Group (G1) served as control. Groups (G2&3) was received ginger extract doses 1.5% and 3%. Groups (G4&5) received single dose (1/10 LD50) of brodifacoum for group (G4) and (1/20 LD50) for group (G5). Groups (G6, 7, 8 & 9) received brodifacoum then ginger extract. Groups (G10, 11, 12&13) received ginger extract then brodifacoum. Animals from all the groups were sacrificed and their bone marrow cells were analyzed for chromosomal damage. Liver and testis samples are collected for histopathological examination. The animals of the groups that treated with brodifacoum showed significant increase of chromosomal aberrations and the value of mitotic index were reduced. Treated male mice with brodifacoum induced various histological changes in liver and testis. Treated animals with brodifacoum and ginger lead to reduction in chromosomal aberrations, significant increase in mitotic index and slight improvement in the histological change in liver and testis. Results obtained from this study demonstrated that brodifacoum has a mutagenic effect on chromosome and histopathological change in different organ (liver and testis) and the use of ginger may be a potent protective against the toxicity of brodifacoum anticoagulant rodenticide.

4-(3,4-dihydroxybenzoyloxymethyl)phenyl-O-β-D-glucopyranoside effect in liver regeneration.

Following an injury or resection, the mammalian liver has the capacity to regain its former volume and functioning by restoring itself. Studies have demonstrated that antioxidants play a role in hepatic regeneration. This study investigated the effect of 4-(3,4-dihydroxybenzoyloxymethyl) phenyl-O-β-D-glucopyranoside (PG) obtained from Origanum micranthum on liver regeneration. Sixty Wistar Albino rats were used. In the sham-operated group, a midline abdominal laparotomy was performed without hepatectomy. In the partial hepatectomy (PHx) group, the median and left lateral lobes were removed. Rats in the PHx group received 20 mg/kg/day PG intraperitoneally before being sacrificed at 24, 48, and 72 hrs, and 7 days later. Liver tissues were collected for immunohistochemical analysis and electron microscopic evaluation. We found an increase in mitotic index, and the numbers of Ki-67 stained hepatocytes in all PHx early stage groups (24 hr, 48hr, 72 hr), but not in 7-day groups. The regeneration mediators eNOS, iNOS, TNF-α and NF-κB were shown to increase in PHx groups. This increase was more prominent dependening on time. In the PHx treatment (PHx+PG) groups, while eNOS was still high, iNOS, TNF-α and NF-κB had decreased. The apoptotic index was markedly high in the PHx groups; this was prevented by PG treatment. These findings were supported by the ultrastructural results. Our findings indicate that PG supports liver regeneration, hepatocyte proliferation, reduced liver damage, and inflammatory mediators following PHx.

Evaluation of phytotoxicity, cytotoxicity, and genotoxicity of ZnO nanoparticles in Vicia faba

Due to the accelerating use of manufactured nanomaterials, more research is needed to define their impact on plants. The present investigation aimed at evaluating the effect of different levels (0.0, 10, 25, 50, and 100mg/L) of ZnO nanoparticles (NPs) on Vicia faba during seed germination and seedling establishment. Additionally, V. faba root meristems were used as a model to monitor the cytotoxic and genotoxic effects resulting from exposure to ZnO NPs. The influence of ZnO NPs on three isoenzyme systems, peroxidase, α, and β esterase, was also evaluated using native-PAGE. Our results showed that lower concentrations of ZnO NPs (especially 10 and 25mg/L) enhanced seed germination and improved seedling growth, while higher concentrations (100 and 200mg/L) resulted in phytotoxicity. Cytological investigations of ZnO NPs-treated V. faba root cells denoted the clastogenic and aneugenic nature of ZnO NPs. Differential increase in mitotic index and significant alterations in cell cycle were observed upon exposure to ZnO NPs. High concentrations of ZnO NPs markedly induced chromosomal aberration, micronuclei, and vacuolated nuclei formation. Chromosomal breakage, chromosomal bridges, ring chromosomes, laggard chromosomes, and stickiness were also observed at a higher rate. The PAGE analysis showed that ZnO NPs treatments altered the expression patterns of all studied enzyme systems. Collectively, results from this work will help to further understand the phytotoxic effects of nanomaterials.

Fluoxetine or Sox2 reactivate proliferation-defective stem and progenitor cells of the adult and aged dentate gyrus

The dentate gyrus of the hippocampus and the subventricular zone are neurogenic niches where the production of new neurons from glia-like stem cells continues throughout adult life. It is not clear whether the pool of stem cells is fated to be exhausted or is conserved until old age. We observed that the antiproliferative gene Btg1 maintains the quiescence of stem cells, and its ablation causes an increase of stem/progenitor cells proliferation in neonatal mice followed by progressive loss of proliferation during adulthood. Fluoxetine is an antidepressant, which exerts a powerful neurogenic effect on dentate gyrus progenitor cells, but is ineffective on stem cells.Here we show that adult dentate gyrus stem cells in the Btg1 knockout mice, with reduced self-renewal and proliferative capability, can be reactivated by fluoxetine, which increases their number greatly above the level of control or fluoxetine-treated wild-type mice. The increase of mitotic index above wild-type in Btg1 knockout fluoxetine-treated stem cells indicates that fluoxetine forces quiescent stem cells to enter the cycle. Stem cell proliferation undergoes continuous reactivation until fluoxetine is administered. Remarkably, fluoxetine reactivates proliferation-defective stem cells also in aged Btg1 knockout mice (15-month-old), an effect absent in wild-type aged mice. Moreover, overexpression of Sox2 retrovirally transduced in Btg1 knockout dentate gyrus cells significantly increases the number of neuroblasts, indicating that Sox2 is able to promote the self-renewal of proliferation-defective stem cells.Overall, the deletion of an antiproliferative gene, such as Btg1, reveals that dentate gyrus stem cells retain a hidden plasticity for self-renewal also in old age, in agreement with a model of permanent self-renewal.

Antigenotoxic potential of plant leaf extracts of Parkinsonia aculeata L. using Allium cepa assay

The aim of the present study was to evaluate the antigenotoxic potential of P. aculeata L. leaf extract/fractions against maleic hydrazide (MH) using Allium cepa root chromosomal aberration assay. The excessive reduction in root growth and mitotic index value was observed after 3 h treatment of MH as compared to negative control (water). In case of MH treatment, frequency of aberrated cells significantly (p ≤ 0.05) raised from 129 to 337 at 0.1 ppm and 2.0 ppm concentrations respectively. From root growth inhibition test with MH treatment, EC50 value i.e. 0.5 ppm was selected to study the antigenotoxic effect of different extract/fractions of P. aculeata L. leaves. All the extract/fractions showed increase in mitotic index and great reduction in chromosomal aberrations with rise in concentration against the genotoxicity of MH. Among all the extract/fractions, butanol and ethyl acetate fractions showed significant reduction in chromosomal aberrations in A. cepa cells and indicates the chemo preventive activity. Antigenotoxic property of this plant is due to the presence of various phytochemicals in leaf such as epi-orientin, Parkinsonin-A, Parkinsonin-B, orientin, iso-orientin, vitexin, iso-vitexin, C-glycosylflavone, parkintin, rotenoids, terpenoids, flavonoids, saponins, alkaloids, glycosides and anthraquinone etc. Our result showed that among all the treatments, simultaneous treatment showed best result followed by pre and post treatment. Further studies in animal model are suggested for further evaluation of the use of P. aculeata leaf extract in human welfare.