Significant Reduction In Expression Research Articles

Abstract GMM-017: UPREGULATION OF MIR-328 CONTRIBUTES TO OVARIAN CANCER STEM CELL MAINTENANCE BY DOWNREGULATING DDB2

Abstract Cancer stem cells (CSCs) are a particular subpopulation of cells that are characterized by self renewal, differentiation and enhanced tumorigenicity. They are responsible for tumor metastasis, relapse and development of drug resistance. Thus, eradication of CSCs is essential for improved patient prognosis. Micro RNAs are a group of small non-coding, endogenous RNAs that are found to regulate cancer stem cell characteristics by binding to mRNA in a sequence specific manner. In ovarian cancers, a wide array of Micro RNAs have been found to show differential expression of which miR328-3p deserves special mention. In this study, a Micro RNA Nanostring profile analysis reveals a significant upregulation of miR-328-3p in ovarian cancer stem cells isolated from both ovarian cancer cell lines and primary ovarian tumors as compared to their corresponding bulk cells. Moreover, it was found that inhibition of miR-328 limited the CSC population in ovarian cancer cells whereas overexpression of miR-328 enriched the CSC population, thus accounting for miR-328 as an onco-miRNA. The upregulation of miR-328 not only increased the percentage of ALDH+ cells in ovarian cancer bulk cells, but also increased the tumorigenicity and sphere formation ability. This was supported by the orthotopic ovarian xenograft assay. Further investigation revealed that reduced phosphorylation of Erk in ovarian cancer stem cells owing to reduced levels of Reactive Oxygen species (ROS) could be a prospective mechanism behind elevated miR328 expression and maintenance of CSC characteristics. Inhibition of phosphorylated Erk expression in ovarian cancer bulk cells by use of commercially available Erk inhibitor, U0126, led to a significant increase in miR328 expression. Simultaneously, upregulation of phosphorylated Erk in ovarian cancer stem cells not only reduced miR328 expression, but also displayed a significant reduction in expression of cancer stem cell markers (Oct4, Sox2, Nanog), sphere formation ability and tumorigenesis. We obtained a similar trend of results on regulating the expression of pErk by use of Reactive Oxygen Species to ovarian cancer cells. These data further helped us confirm our speculation that reduced ROS promotes the maintenance of CSCs characteristics through inactivation of Erk signalling pathway. Besides, we also identified DDB2 as a direct target of miR328. Our previous findings demonstrate that DDB2 is able to limit ovarian CSC population by disrupting their self renewal capacity. Thus, we conclude that elevated miR328 in ovarian CSCs, resulting from inactivated Erk1/2 activity, is responsible for maintenance of stemness by inhibition of DDB2 expression. Targeting miR-328 could therefore be a novel therapeutic strategy to eradicate CSCs in ovarian cancer. Citation Format: Amit Kumar Srivastava, Tiantian Cui, Ananya Banerjee, Chunhua Han, Shurui Cai, Lu Liu, Dayong Wu1, Ri Cui, Zaibo Li, Xiaoli Zhang, Guozhen Xie, Selvendiran Karuppalyah, Adam Karpf, Jinsong Liu, David Cohn, Qi-En Wang. UPREGULATION OF MIR-328 CONTRIBUTES TO OVARIAN CANCER STEM CELL MAINTENANCE BY DOWNREGULATING DDB2 [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-017.

An innovative role for tenoxicam as a quorum sensing inhibitor in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen exhibiting higher resistance to commonly used antibiotics. Microbial resistance to antibiotics is a major problem that hinders attempts to control microbial infections. Quorum sensing inhibitors could help us solve such problem by repressing quorum sensing that controls the production of virulence factors in many pathogens including P. aeruginosa. In this study, the influence of tenoxicam, a non-steroidal anti-inflammatory drug, on quorum sensing in P. aeruginosa was characterized. Treatment of P. aeruginosa with tenoxicam decreased production of many virulence factors such as pyoverdin, rhamnolipids, pyocyanin, elastase, proteases, and hemolysins. Moreover, qRT-PCR revealed a significant reduction in expression of quorum sensing genes in tenoxicam-treated P. aeruginosa in comparison with untreated bacteria. Tenoxicam markedly reduced the capacity of P. aeruginosa to kill mice infection model. Mice injected with tenoxicam-treated P. aeruginosa exhibited higher survival rate as compared with those inoculated with untreated bacteria. Current data clearly demonstrate that tenoxicam has quorum sensing inhibitory effect on P. aeruginosa. Tenoxicam could play a role in reduction of Pseudomonas quorum sensing-dependant virulence factors production, and therefore affect its pathogenesis in the host. In summary, the current study suggests that tenoxicam could be used as adjuvant to antibiotics in the management of diseases caused by P. aeruginosa.

Virus-induced gene silencing for phenylalanine ammonia-lyase affects pepper adaption to low temperature

Reverse genetics approaches in plants rely on post-transcriptional gene silencing to study the function of genes. In particular, virus-induced gene silencing (VIGS) has been successfully applied to identify gene function in some crops. To date, it is unclear whether phenylalanine ammonia-lyase (PAL) is involved in low temperature tolerance in the pepper. Here, we used an agroinfiltration protocol with tobacco rattle virus (TRV) constructs containing partial sequences from CaPAL for VIGS to test its role in anthocyanin biosynthesis and response to low temperature in the pepper (Capsicum annuum). We found that accumulation of anthocyanins in the leaves of pepper plants transformed with the TRV2:CaPAL vector was significantly reduced compared with peppers transformed with the empty TRV2 vector (TRV2:00). A significant reduction in expression of genes related to anthocyanins synthesis was also detected in peppers transformed with TRV2:CaPAL. When silenced pepper plants were exposed to a low temperature, we found decreased antioxidant system, PAL activity, and photosynthesis in plants transformed with TRV2:CaPAL compared with peppers transformed with TRV2:00. Low transcriptions of cold stress-response genes demonstrated that pepper tolerance to low temperature decreased. Future studies focused on the interaction between CaPAL and other abiotic and biotic stressors will shed further light into the role of CaPAL in stress response.

Effect of Ionizing Radiation from Computed Tomography on Differentiation of Human Embryonic Stem Cells into Neural Precursors.

We studied the effect of radiation from computed tomography (CT) scans on differentiation of human embryonic stem cells (hESCs) into neuronal lineage. hESCs were divided into three radiation exposure groups: 0-dose, low-dose, or high-dose exposure. Low dose was accomplished with a single 15 mGy CT dose index (CTDI) CT scan that approximated the dose for abdominal/pelvic CT examinations in adults while the high dose was achieved with several consecutive CT scans yielding a cumulative dose of 500 mGy CTDI. The neural induction was characterized by immunocytochemistry. Quantitative polymerase chain reaction (qPCR) and Western blots were used to measure expression of the neuronal markers PAX6 and NES and pluripotency marker OCT4. We did not find any visible morphological differences between neural precursors from irradiated and non-irradiated cells. However, quantitative analyses of neuronal markers showed that PAX6 expression was reduced following exposure to the high dose compared to 0-dose controls, while no such decrease in PAX6 expression was observed following exposure to the low dose. Similarly, a statistically significant reduction in expression of NES was observed following high-dose exposure, while after low-dose exposure, a modest but statistically significant reduction in NES expression was only observed on Day 8 of differentiation. Further studies are warranted to elucidate how lower or delayed expression of PAX6 and NES can impact human fetal brain development.

Abstract 937: Etv2 Regulates Endothelial Gene Expression With Its Novel Binding Partner Vezf1

Ets transcription factors function as important developmental regulators and are known to be modulators of cell fate. Previously, in conjunction with co-factors, we have described Ets variant 2 (Etv2) as an essential regulator of the hematopoietic and endothelial lineages. But the mechanism and the Etv2 interacting partners involved in achieving this critical function remains poorly understood. Through Yeast two-hybrid analysis we identified Vascular Endothelial Zinc Finger 1 (Vezf1) as an interacting factor with Etv2. Vezf1 is a conserved C2H2 zinc finger transcription factor known to regulate the formation, proliferation, and migration of endothelial cells through several gene targets. We verified Vezf1 as a binding partner of Etv2 through co-immunoprecipitation and GST-pull down studies. Bioinformatic analysis of ChIP-seq and Etv2-expressing single cell RNA sequencing was conducted to identify candidate genes containing both Etv2 and Vezf1 binding motifs in their regulatory regions. Histone deacetylase 7 (Hdac7) and angiomotin like protein 2 (Amotl2) were identified as genes of interest involved in endothelial development. Hdac7 is a conserved class II deacetylase that functions in endothelial cell adhesion and vascular integrity. Amotl2 is a Motin family protein that functions in angiogenesis, endothelial cell polarity and migration. RT-qPCR analysis showed upregulation of Hdac7 and Amotl2 in response to doxycycline inducible Etv2 and Vezf1; whereas significant reduction of expression of these two genes was observed in the Etv2 and Vezf1 knockout cells. Chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assays (EMSA) confirmed Etv2-Vezf1 adjacent binding sites in the promoters of Hdac7 and Amotl2. Histone Acetyl transferase (HAT) assays was performed to investigate Etv2-Vezf1 on global histone acetylation conditions in doxycycline inducible embryoid bodies. Vezf1 overexpression results in a significant reduction of acetylated histone. In summary, this study identifies Vezf1 as a novel binding partner of Etv2 and their combined role in regulating downstream target genes Amotl2 and Hdac7 in hematoendothelial development.

Abstract 2013: A carbonic anhydrase inhibitor methazolamide potentiates efficacy of gemcitabine by modulating anti-proliferative activity and cancer stem cell markers in pancreatic carcinoma

Abstract Pancreatic cancer is the fourth cause for cancer related deaths globally. By 2020, incidence of new cases is expected to be 420,000 with equal number of associated mortalities. Overall 5-year survival has marginally improved over past despite of our growing understanding of the disease. Surgery remains standard of care for resectable tumors followed by adjuvant chemotherapy which resulted in median survival of 26 months. Newer therapies targeting stroma and immunotherapies hold promise for the future. Noticeably, a high unmet medical need warrant novel therapy to extend survival over a year in metastatic settings. Drug repositioning holds promise over the conventional drug discovery, as it reduces development time and cost. Using this approach, we report promising anticancer activity of Methazolamide (MZM) in pancreatic cancer. MZM, a synthetic derivative of sulphonamide and a carbonic anhydrase IX (CA) inhibitor is used for the treatment of ocular conditions for lowering intraocular pressure. Overexpression of CA is implicated in acidification of tumor microenvironment leading to hypoxia which in turn promotes growth and metastasis of the tumor. Therefore, modulation of extracellular tumor pH via inhibition of CA represents a promising course to anticancer therapy. Here, we report anti-proliferative activity of MZM in 12 pancreatic cell lines including patient derived (PDX) in 2D models and anchorage independent growth inhibition in 3D models. Combination treatment with GEM yielded significant growth inhibition than drug alone in all tested cell lines. Additive effects were observed for the combination in PDX-1986, PDX-546, Capan-2, Mia-Pa-Ca and PANC-1 cells. Further, in vivo efficacy of MZM in combination with GEM was performed using PDX-546 in nude mice. Combination group showed significant tumor growth inhibition (72%, p<0.05) as against drug alone, MZM at 60 mg/kg, p.o. (50%, p<0.05) and GEM at 50 mg/kg i.v. (61%, p<0.05). There was no significant body weight loss. For PK/PD studies, MZM levels were measured in tumors and PD effect was evaluated using various markers in tumor samples. Expression of target genes was measured by qRT-PCR. Significant reduction in expression of stem cell markers viz. RAC1, OCT4, CD34, CD14, Nanog and proliferation marker (Ki67) was observed in combination group as compared to drug alone. In addition, anti-angiogenic potential viz. would healing, tube formation and expression of important anti-angiogenic markers like HIF1α, VEGF were profoundly modulated in MZM and combination group. Thus, our study indicates that the combination of MZM, a CA inhibitor with GEM is a promising novel approach for the treatment of pancreatic cancer especially in metastatic settings. Citation Format: Kalpana Joshi, Jeevan D. Ghosalkar, Vinay R. Sonawane, Siddhika R. Raut, Geena Malhotra. A carbonic anhydrase inhibitor methazolamide potentiates efficacy of gemcitabine by modulating anti-proliferative activity and cancer stem cell markers in pancreatic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2013.

Pesticide induced visual abnormalities in Asian honey bees (Apis cerana L.) in intensive agricultural landscapes

Pesticide stress is one of the important factors for global bee declines. Apart from physiological and developmental anomalies, pesticides also impose cognitive damages on bees. The present study investigates the visual acuity of wild populations of honey bees, in an agricultural intensification landscape, and corroborates the findings with controlled laboratory experiments. Even though overall morphometric examinations revealed no significant differences between the populations, correct color choices by bees in pesticide exposed populations were significantly reduced. The study reports, for the first time, the significant reduction in ommatidia facet diameter in these populations, as viewed under scanning electron microscope, along with the molecular underpinnings to these findings. Western blot studies revealed a significant reduction in expression of two visual proteins – blue-sensitive opsin and rhodopsin – in the pesticide exposed populations in both field and laboratory conditions. The novel findings from this study form the basis for further investigations into the effects of field realistic doses of multiple pesticide exposures on wild populations of honey bees.

351 Phenotypic Modulation of Adipose-Derived Stem Cells Within 3D Scaffolds; Applications in Skin Bioengineering

Large burn injuries and wounds contribute to increased patient morbidity and mortality and impose a significant financial burden on healthcare systems. The main goal of wound treatment is to achieve a rapid closure of the lesion and promote healing with minimal scarring. Extracellular matrix- based biomaterials such as acellular dermal matrices (ADM) or in situ-forming scaffolds are advantageous for treatment of large wounds due to their mechanical strength and retained biological activity when compared with synthetic polymer materials. Further, recellularization of ADM with adipose-derived stem cells (ASCs) significantly increases the healing capacity. The aim of this study was to develop an ASC-populated ADM and assess its characteristics in vitro with the ultimate goal of engineering an optimized wound healing composite. To this end, we developed a novel method for de-cellularization of skin and used the acquired ADM as a 3D scaffold to seed human ASCs. We compared this 3D model with traditional 2D ASC cultures at different time points post-culture. A panel of cell surface markers was used for phenotypic characterization of ASCs, post-culture. Combinations of positive (CD146, CD44, CD90, and CD73) and negative (CD31, CD34, CD45) cell surface proteins were used as stem cell markers. Morphology and myofibroblast differentiation capacity of ASCs were also evaluated. The results showed a significant reduction in expression of CD73 and CD44 markers in ASCs when embedded within a 3D ADM, compared to the cells that were cultured in a 2D culture condition. We found that ASCs cultured under regular 2D conditions mainly differentiated towards a myofibroblastic phenotype with increased myofibroblast marker α-smooth muscle actin (α-SMA) and type I pro-collagen. In contrast, ASCs cultured on ADM showed a more balanced differentiation pattern with maintenance of important stem cell markers such as CD146. Taken together, these findings suggest that ASC differentiation can be regulated by a 3D scaffold. Embedding ASCs within 3D scaffolds prevents universal differentiation to stromal cells and maintain stemness features associated with ASCs. The ASC-ADM combination shows a promising potential as a novel therapeutic approach for treatment of large burn injuries and wounds. This bioengineered composite can reduce risk of fibrosis while maintaining the regenerative capacity of stem cells.

Christmas disease in a Hovawart family resembling human hemophilia B Leyden is caused by a single nucleotide deletion in a highly conserved transcription factor binding site of the F9 gene promoter.

Hemophilia B is a classical monogenic, X-chromosomal, recessively transmitted bleeding disorder caused by genetic variants within the coagulation factor IX gene (F9). Although hemophilia B has been described in dogs, it has not yet been reported in the Hovawart breed. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males were reported to suffer from recurrent hemorrhagic episodes. A blood sample from one of these males with only 2% of the normal concentration of plasma factor IX together with samples from seven relatives were provided. Next-generation sequencing of the mother and grandmother revealed a single nucleotide deletion in the F9 promoter. Genotyping of the deletion in 1,298 dog specimens including 720 Hovawarts revealed that the mutant allele was only present in the aforementioned Hovawart family. The deletion is located 73 bp upstream of the F9 start codon in the conserved overlapping DNA binding sites of hepatocyte nuclear factor 4α (HNF-4α) and androgen receptor (AR). The deletion only abolished binding of HNF-4α, while AR binding was unaffected as demonstrated by electrophoretic mobility shift assay using human HNF-4α and AR with double-stranded DNA probes encompassing the mutant promoter region. Luciferase reporter assays using wildtype and mutated promoter fragment constructs transfected into Hep G2 cells showed a significant reduction in expression from the mutant promoter. The data provide evidence that the deletion in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.

Omega-3 fatty acids and docosahexaenoic acid oxymetabolites modulate the inflammatory response of equine recombinant interleukin1β-stimulated equine synoviocytes

Omega-3 fatty acid (n-3 PUFA) supplementation may have beneficial effects in certain chronic diseases, potentially including osteoarthritis. Favorable effects are attributed, in part, to downstream pro-resolving oxylipid metabolites. We investigated the role of n-3 PUFA and docosahexaenoic acid (DHA)-derived oxylipids (docosanoids) on equine synoviocyte metabolism. We hypothesized that n-3 PUFA and selected docosanoids would modulate inflammatory mediator gene expression by recombinant equine (re)IL-1β-stimulated synovial fibroblasts. Synoviocyte monolayer cultures were prepared from grossly normal equine carpal synovium. Cellular incorporation of eicosapentaenoic acid (EPA) and DHA was determined using LC–MS and docosanoid biosynthesis by LC–MS-MS. The influence of n-3 PUFA and docosanoids on osteoarthritis marker gene expression was determined by quantitative real time polymerase chain reaction (qPCR). Synoviocytes incorporated EPA and DHA in significant amounts and DHA treatment augmented the synthesis of several docosanoids. Synoviocyte cultures pre-treated with EPA or DHA followed by reIL-1β stimulation had significant reductions in expression of ADAMTS4, MMP-1, MMP-13, IL-1β, IL-6 and COX-2. The docosanoids resolvin D1 and D2, maresin 1 and protectin DX, alone and in combination, abrogated ADAMTS4, MMP-1, MMP-13, and IL-6 gene expression in reIL-1β-stimulated synoviocytes. Similarly, both resolvins and maresin 1 stifled COX-2 expression. Our results demonstrate that synoviocytes readily incorporate n-3 PUFA. DHA incorporation was sufficient for biosynthesis of significant concentrations of several docosanoids which modulated the synovial inflammatory response in vitro. These data indicate n-3 PUFA supplementation may prove useful in the prevention or treatment of osteoarthritis.

Oncogenic STRAP Supports Hepatocellular Carcinoma Growth by Enhancing Wnt/β-Catenin Signaling.

Aberrant activation of Wnt/β-catenin signaling plays a key role in the onset and development of hepatocellular carcinomas (HCC), with about half of them acquiring mutations in either CTNNB1 or AXIN1. The serine/threonine kinase receptor-associated protein (STRAP), a scaffold protein, was recently shown to facilitate the aberrant activation of Wnt/β-catenin signaling in colorectal cancers. However, the function of STRAP in HCC remains completely unknown. Here, increased levels of STRAP were observed in human and mouse HCCs. RNA sequencing of STRAP knockout clones generated by gene editing of Huh6 and Huh7 HCC cells revealed a significant reduction in expression of various metabolic and cell-cycle-related transcripts, in line with their general slower growth observed during culture. Importantly, Wnt/β-catenin signaling was impaired in all STRAP knockout/down cell lines tested, regardless of the underlying CTNNB1 or AXIN1 mutation. In accordance with β-catenin's role in (cancer) stem cell maintenance, the expressions of various stem cell markers, such as AXIN2 and LGR5, were reduced and concomitantly differentiation-associated genes were increased. Together, these results show that the increased STRAP protein levels observed in HCC provide growth advantage among others by enhancing Wnt/β-catenin signaling. These observations also identify STRAP as a new player in regulating β-catenin signaling in hepatocellular cancers. IMPLICATIONS: Elevated STRAP levels in hepatocellular cancers provide a growth advantage by enhancing Wnt/β-catenin signaling.

Dietary Fish Oil and Vitamin E Reduce Cryoinjuries and Apoptosis in Vitrified Laying Hens' Ovarian Tissue.

The occurrence of cryoinjuries during ovarian tissue freezing necessitates development of methods that can overcome these challenges in cell and tissue cryopreservation. It has been hypothesized that omega-3 and vitamin E diet supplementation to the animal could be an appropriate strategy to preserve post-thaw ovarian quality. The laying hen is an appropriate animal model that can be used to study the effects of providing dietary supplements on the outcomes of ovary cryopreservation. The purpose of the study was to evaluate the dietary effects of fish oil, sunflower oil, and vitamin E on 68 laying hens according to the following treatments: basal diet + 1.5% sunflower oil (control; C); basal diet + 1.5% sunflower oil + 1.1 IU alpha-tocopherol/hen/day (E); basal diet + 1.5% fish oil + 1.1 IU alphatocopherol/hen/day (n-3+E); and basal diet + 1.5% fish oil (n-3). The effects on ovarian structure and preservation, apoptosis-related gene expression, and the fatty acid profiles in ovarian laying hen (n = 7 in each group) were studied. The number of intact primordial follicles in n-3+E group was significantly higher than other groups (85% vs. 71%, 72%, and 77% for n-3+E, C, E, and n-3, respectively; p < 0.01). There was a significant reduction in expression of Cas3, as well as Cas8, in n-3 and n-3+E than C and E (p ≤ 0.05). A trend to decrease in Bak (p = 0.089) and Bak/Bcl2 ratio (p = 0.095) in the mRNA was observed in n-3+E. Ovarian eicosapentaenoic acid (C20:5n3) concentration in n-3 was the highest among C, E, and n-3+E (p < 0.01). Docosahexaenoic acid (C22:6 n-3) concentrations in ovaries of the n-3 group were elevated five times more than control. The n-3: n-6 ratio in groups receiving omega-3 (n-3+E and n-3) was higher than other groups (p < 0.01). In conclusion, consumption of dietary omega-3 fatty acids with vitamin E improves the results of vitrification of ovarian tissues in laying hens.

Cortical expression of AMPA receptors during postnatal development in a genetic model of absence epilepsy

Childhood absence epilepsy has been associated with poor academic performance, behavioural difficulties, as well as increased risk of physical injury in some affected children. The frequent episodes of ‘absence’ arise from corticothalamocortical network dysfunction, with multifactorial mechanisms potentially involved in genetically different patients. Aberrations in glutamatergic neurotransmission has been implicated in some seizure models, and we have recently reported that reduced cortical AMPA receptor (AMPAR) expression (predominantly GluA4- containing AMPARs) in parvalbumin-containing (PV+) inhibitory interneurons, could underlie seizure generation in the stargazer mutant mouse. In the present study, we investigate AMPA receptor subunit changes occurring during postnatal development in the stargazer mouse, to determine when these changes occur relative to seizure onset and thus could be contributory to seizure generation. Using quantitative western blotting, we analysed the expression of AMPAR GluA1-4 subunits in the somatosensory cortex at three critical time points; two before seizure onset (postnatal days (PN) 7–9 and 13–15), and one at seizure onset (PN17–18) in stargazers. We report that compared to their non-epileptic littermates, in the stargazer somatosensory cortex, there was a significant reduction in expression of AMPARs containing GluA1, 3 and 4 subunits prior to seizure onset, whereas reduction in expression of GluA2-AMPARs appears to be a post-seizure event. Thus, while loss of GluA4-containing AMPARs (likely GluA1/4 and GluA3/4) may be linked to seizure induction, the loss of GluA2-containing AMPARs is a secondary post-seizure mechanism, which is most likely involved in seizure maintenance.

Stimulatory effects of fish oil and vitamin E on ovarian function of laying hen

In recent years, there has been an increase in the consumption of omega-6 fatty acids (FAs) over omega-3. Moreover, the antioxidant nature of oil supplemented diet in human and animal studies is not clear. In this work, the n-3 effects on hen ovarian cells, ovarian dynamics, serum FA profiles and expression level of apoptosis genes in the presence or absence of vitamin E were studied. Sixty-eight laying hens were randomly divided into four groups and the groups were fed the following diets: basal diet +1.5% sunflower oil (control; C); basal diet +1.5% sunflower oil +1.1 U alpha-tocopherol/hen/day (E); basal diet +1.5% fish oil +1.1 U alpha-tocopherol/hen/day (n-3 + E); and basal diet +1.5% fish oil (n-3). In E, n-3 + E and n-3, small yellow follicle numbers were higher than control (p < .05). Large yellow follicle numbers as well as total numbers of follicles of n-3 + E and n-3 were higher (p < .05) than control and E. The rate of ovulation was significantly (p < .05) higher in n-3 (96%) compared to the control (89%). The proportion of intact primordial follicles in n-3 and n-3 + E was significantly more than control and E (p < .01). The expression of Bak (Bcl2 antagonist/killer) was significantly (p < .05) higher in control than the other groups. There was significant reduction in expression of Cas9 in n-3 and E compared to the control and n-3 + E (p < .05). While omega-3 and vitamin E can protect ovarian cells from apoptotic death, omega-3 source could have promoting roles on follicular development and ovulation rate, being independent of dietary vitamin E.HighlightsDespite the perceived importance of supplementing antioxidant with fatty acids, our results indicated that alone low level of n-3 has a great influence on ovary performance.

Inhibition of PTEN activates bovine non-growing follicles in vitro but increases DNA damage and reduces DNA repair response.

STUDY QUESTIONDoes ovarian follicle activation by phosphatase homologue of chromosome-10 (PTEN) inhibition affect DNA damage and repair in bovine oocytes and granulosa cells?SUMMARY ANSWERPTEN inhibition promotes bovine non-growing follicle activation but results in increased DNA damage and impaired DNA repair capacity in ovarian follicles in vitro.WHAT IS KNOWN ALREADYInhibition of PTEN is known to activate primordial follicles but may compromise further developmental potential. In breast cancer cells, PTEN inhibition represses nuclear translocation of breast cancer susceptibility 1 (BRCA1) and Rad51; this impairs DNA repair resulting in an accumulation of damaged DNA, which contributes to cell senescence.STUDY DESIGN, SIZE, DURATIONBovine ovarian tissue fragments were exposed to control medium alone or containing either 1 or 10 μM bpv(HOpic), a pharmacological inhibitor of PTEN, in vitro for 24 h. A sub-group of tissue fragments were collected for Western blot analysis after bpv(HOpic) exposure. The remainder were incubated in control medium for a further 5 days and then analysed histologically and by immunohistochemistry to detect DNA damage and repair pathways.PARTICIPANTS/MATERIALS, SETTING, METHODSBovine ovaries were obtained from abattoir-slaughtered heifers. Tissue fragments were exposed to either control medium alone or medium containing either 1 μM or 10 μM bpv(HOpic) for 24 h. Tissue fragments collected after 24 h were subjected to Akt quantification by Western blotting (six to nine fragments per group per experiment). Follicle stage and morphology were classified in remaining fragments. Immunohistochemical analysis included nuclear exclusion of FOXO3 as a marker of follicle activation, γH2AX as a marker of DNA damage, meiotic recombination 11 (MRE11), ataxia telangiectasia mutated (ATM), Rad51, breast cancer susceptibility 1 (BRCA1) and breast cancer susceptibility 2 (BRCA2) as DNA repair factors. A total of 29 550 follicles from three independent experiments were analysed.MAIN RESULTS AND THE ROLE OF CHANCETissue fragments exposed to bpv(HOpic) had increased Akt phosphorylation at serine 473 (pAkt/Akt ratio, 2.25- and 6.23-fold higher in 1 and 10 μM bpv(HOpic) respectively compared to control, P < 0.05). These tissue fragments contained a significantly higher proportion of growing follicles compared to control (78.6% in 1 μM and 88.7% in 10 μM versus 70.5% in control; P < 0.001). The proportion of morphologically healthy follicles did not differ significantly between 1 μM bpv(HOpic) and control (P < 0.001) but follicle health was lower in 10 μM compared to 1 μM and control in all follicle types (P < 0.05). DNA damage in oocytes, indicated by expression of γH2AX, increased following exposure to 1 μM bpv(HOpic) (non-growing, 83%; primary follicles, 76%) and 10 μM (non-growing, 77%; primary, 84%) compared to control (non-growing, 30% and primary, 59%) (P < 0.05 for all groups). A significant reduction in expression of DNA repair proteins MRE11, ATM and Rad51 was observed in oocytes of non-growing and primary follicles of treatment groups (primary follicles in controls versus 10 μM bpv(HOpic): MRE, 68% versus 47%; ATM, 47% versus 18%; Rad51, 48% versus 24%), P < 0.05 for all groups. Higher dose bpv(HOpic) also resulted in lower expression of BRCA1 compared to control and 1 μM bpv(HOpic) (P < 0.001) in non-growing and primary follicles. BRCA2 expression was increased in oocytes of primary follicles in 1 μM bpv(HOpic) (36%) compared to control (20%, P = 0.010) with a marked decrease in 10 μM (1%, P ≤ 0.001). Granulosa cells of primary and secondary follicles in bpv(HOpic) groups showed more DNA damage compared to control (P < 0.05). However, bpv(HOpic) did not impact granulosa cell DNA repair capacity in secondary follicles, but BRCA1 declined significantly in higher dose bpv(HOpic).LARGE-SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis study focuses on non-growing follicle activation after 6 days culture and may not reflect DNA damage and repair capacity in later stages of oocyte and follicle growth.WIDER IMPLICATIONS OF THE FINDINGS In vitro activation of follicle growth may compromise the bidirectional signalling between oocyte and granulosa cells necessary for optimal oocyte and follicle health. This large animal model may be useful in optimising follicle activation protocols with a view to transfer for clinical application.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by Indonesia endowment fund for education. No competing interest.TRIAL REGISTRATION NUMBERNot applicable.

The Non-Canonical, R-Loop Regulatory Function of PIWIL4 Maintains Genomic Integrity and Leukemic Potential of AML Cells

During DNA replication and transcription, RNA:DNA hybrids are formed as part of three stranded nucleic acid structures known as R-loops. R-loops occur frequently in the genome at highly transcribed regions, ribosomal genes, mitochondria and intergenic regions, and are predominantly resolved by Ribonuclease (RNase) H family of enzymes. However, unscheduled and unresolved R-loops represent a potent source of DNA damage, especially in rapidly dividing cells such as cancer cells. It is imperative for cancer cells to prevent accumulation of unresolved R-loops in order to limit DNA damage. So far, the mechanism how leukemic cells prevent accumulation of R-loops is not well understood. In this study, we show that an RNase H-like protein, PIWIL4, is aberrantly and highly expressed in AML patients, prevents R-loop accumulation via its RNase H activity and thereby acts as important regulator of leukemic growth. In our initial analysis, we observed that the recombinant human PIWIL4 protein digested radiolabeled-RNA-containing R-loops in vitro, exhibiting an RNase H-like activity with increasing efficiency, in incremental concentrations and time durations. Moreover, immunoprecipitation of PIWIL4 followed by liquid chromatography mass spectrometry (LC/MS) in HEK cells showed that PIWIL4 was bound with multiple nuclear and nucleolar RNA processing factors that are associated with formation of R-loops. Published RNA-seq and microarray datasets revealed that, among all cancers, PIWIL4 was significantly highest expressed in myeloid leukemia. Quantitative real time PCR (qRT-PCR) of acute myeloid leukemia (AML) patients revealed that PIWIL4 showed an average of 21.6 ± 5.0-fold higher expression in AML patients (n=68; p<0.0001), compared to healthy CD34+ bone marrow (BM) and BM mononuclear cells (n=3). Western blot of AML patient samples and intracellular (IC) staining confirmed higher PIWIL4 protein expression levels in AML cells compared to cord blood CD34+ HSPCs. Piwil4 expression increased by 6-8 fold in murine BM healthy HPSCs within 48h after transduction with MLL-AF9, AML1-ETO9A and CDX2 oncogenes compared to empty vector (n=3, p<0.0001). Stable knockdown of PIWIL4 in AML cell lines and primary AML samples using shRNA, followed by IC staining and confocal microscopy using an antibody against R-loops (S9.6) revealed a marked increase in accumulation of R-loops within 72h post-transduction in PIWIL4 depleted cells, in contrast to healthy cord blood HSPCs which remained unaffected (n=3). PIWIL4 depleted AML cells exhibited an accumulation of DNA damage associated gH2AX foci, replication stress associated BrdU foci, higher levels of phosphorylated ATR (p-ATR), a marked increase in apoptosis and block in the G2M phase of the cell cycle. Depletion of PIWIL4 significantly impaired clonogenic potential of AML patient samples in vitro (avg. 4.9 ± 0.9-fold reduction, p<0.0001, n=3). In vivo, PIWIL4 depletion in cell lines delayed onset of leukemia (n=8, p<0.001) and in AML patient cells reduced leukemic engraftment in xenografts 12 weeks post-transplantation (avg. scr - 50.6±21% vs avg. shRNA-14.6±10, n=6). Of note, PIWIL4 depletion in cord blood CD34+ HSPCs had no impact on colony formation or differentiation in vitro. RNA-seq of PIWIL4 depleted THP-1 cell line followed by GSEA revealed a significant reduction in expression of ribosomal genes and increased expression of G2M checkpoint repair pathway (n=2, p<0.05, FDR<0.05). qRT-PCR of pre-rRNA (45S rRNA) showed a significant reduction in rRNA transcription in shRNA transduced cell lines (avg. 2.5 ± 0.3-fold reduction, n=3, p<0.01). Overexpression of PIWIL4 or RNase H1 in PIWIL4 depleted AML cell lines rescued R-loop and gH2AX signals, induced a decrease in p-ATR and gH2AX protein levels, and rescued the impact on apoptosis and growth phenotype in colony assays. RNA polymerase I inhibitor CX-5461, known to stabilize R-loop associated secondary structures, acted synergistically with PIWIL4 depletion and induced complete cell death of PIWIL4 depleted AML cells compared to scrambled control at IC50 concentrations. Thus, collectively, we could show for the first time that PIWIL4 is a functional RNase H like enzyme in AML cells, suppresses formation of R-loops, thereby preventing DNA damage and apoptosis of AML cells. Our data also suggest that impairing resolution of R-loops is a powerful therapeutic tool in AML. DisclosuresBuske:Roche: Honoraria, Research Funding; Bayer: Research Funding; Janssen: Honoraria, Research Funding.

Mesenchymal Stem Cells Gene Signature in High-Risk Myeloma Bone Marrow Linked to Suppression of Distinct IGFBP2-Expressing Small Adipocytes

Introduction:Certain studies suggest that multiple myeloma (MM) induces expansion of bone marrow (BM) mesenchymal stem cells (MSCs), but others showed induction of MSC senescence. MM cells suppress MSC lineages such as osteoblasts, while their effects on adipocytes remain to be elucidated. Recent studies identified “regulated” adipocytes that are smaller in size than large adipocytes that are constitutively present (Scheller et al, Nature Commun 2015), and that function as an endocrine tissue and regulators of hematopoiesis (Cawthorn et al, Cell Metab 2014), suggesting that BM adipocytes are functionally heterogeneous.We established a MSC gene signature in whole bone biopsies and showed that it gradually changes in different disease stages and is associated with outcome (Schinke et al, CCR 2018).The aim of the study was to identify changes in expression of MSC genes in BM of patients with high risk MM and in focal lesions (FLs), and to elucidate whether these changes reflect altered proportion and function of MSCs and their lineages.Methods:MSC gene expression in whole bone biopsies from normal donors (n=68), and patients with MGUS/SMM (n=90) and MM (n=531) was analyzed using global gene expression profiles. Unexpanded single MSCs from normal donors (n=3; 175 single MSCs) and MM patients (n=3; 162 single MSCs) were sorted by FACSAria and expression of mesenchymal cell, proliferation and senescence markers were analyzed by qRT-PCR using Fluidigm Biomark HD. Functionally, single MSCs were tested for their ability to multiply using supportive serum and MSC-conditioned media. Cell senescence was analyzed by SA-βGal staining. IGFBP2 and adiponectin protein were detected using immunohistochemistry (IHC). Numbers of IGFBP2+ cells were analyzed in biopsies from patients with MGUS/SMM, low risk (LR) and high risk (HR) MM (10 biopsies/group). Cultured MSCs were differentiated to adipocytes by treatment with dexamethasone, insulin and indomethacin for 3-4 weeks. The effects of recombinant IGF1 and IGFBP2 on MM cell growth were performed on BM-dependent MM lines (n=3) cultured in serum-free conditions for 48 hrs.Results:We compared MSC gene expression levels in random interstitial BM biopsies of patients with LR and HR MM, and in paired biopsy samples from random BM and FLs; 41 of the 345 MSC genes were differentially expressed in both comparisons. Most overexpressed genes were related to angiogenesis and ECM, including several collagen genes (e.g., COL4A1, POSTN, and HSPG2). Several underexpressed genes were associated with adipocytes, including IGFBP2 and aldo/keto reductases.To unravel whether these differences in gene expression reflect changes in the proportion of MSCs we tested expression of genes associated with MSCs, osteoblastogenesis, adipogenesis, proliferation and senescence in unexpanded singleMSCs. MM single MSCs had significant reduction in expression of KI67 and increased expression of the senescence marker, CDKN2A/p16, whereas expression of other tested genes were modestly differentially expressed between the two groups. Functionally, unexpanded MM MSCs had increased SA-βGal expression, and only 65±8% of single MSCs divided at least once compared to 90±4% of their normal donor counterparts (p<0.03), suggesting that the in vivo MSC gene expression in MM reflects modifications in mesenchymal cell lineages that encompass most of the mesenchymal compartment in bone.Since IGFBP2 is involved in adipogenesis and bone homeostasis we traced the source of this factor using IHC. Double staining for IGFBP2 and adiponectin revealed that IGFBP2 was mainly expressed by small adipocytes. The number of IGFBP2+ cells was higher in BM biopsies from patients with MGUS/SMM than in those from patients with MM (p<0.02), and the number was lower in biopsies from patients with HR MM than with LR MM (p<0.01).Normal MSCs differentiated to adipocytes produced a high level of IGFBP2, while co-culturing MSCs with MM cells inhibits their differentiation to adipocytes by 6 folds (p<0.001) and reduced expression of IGFBP2 and adiponectin. Recombinant IGFBP2 effectively blocked IGF1-mediated growth of MM cells, indicating its role in controlling IGF1 bioavailability.Conclusions:Our data demonstrate that MM MSCs are less proliferative and that IGFBP2+ small adipocytes are a distinct mesenchymal cell population suppressed by MM, and their depletion may contribute to disease progression. DisclosuresEpstein:University of Arkansas for Medical Sciences: Employment. Davies:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; ASH: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; TRM Oncology: Honoraria. Morgan:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding.

Transcriptomic Analysis Reveals Erythroferrone Modulates BMP6 Signaling Pathways Involved in Iron Homeostasis and Metabolism

Introduction: Erythroferrone (ERFE), the recently identified erythroid regulator of iron absorption, is a member of the C1q/TNF-related protein (CTRP) family. It is produced in erythroblasts in response to an increased erythropoietic drive, downregulates hepcidin expression and thereby promotes intestinal iron absorption and mobilization. The levels of Erfe are inappropriately high under conditions of ineffective erythropoiesis in inherited anemias such as thalassemia and congenital dyserythropoietic anemias. However, the mechanism of Erfe mediated Hepcidin inhibition remains unknown. Here, we use transcriptomic analysis of the human hepatoma cell line, Huh7 to identify genes and pathways involved in hepatocyte response to Erfe.Methods: Huh7 cells were seeded in a 6-well cell culture plate. Twenty four hours later, the cells were washed with PBS, and treated with a recombinant murine ERFE monomeric Fc (mEFRE-FC, 10µg/ml) protein or a control IgG (10 ug/ml) for 1h, 6h or 24h in growth media. RNA was isolated, followed by RNA quantification and quality assessment using a 2100 Agilent Bioanalyzer. ERFE is known to regulate Hepcidin antimicrobial peptide (HAMP) transcription and a treatment effect on HAMP expression was demonstrated by qPCR prior to sequencing. A total of 27 mRNA sequencing libraries were constructed from 1ug of human total RNA with the Illumina TruSeq Stranded mRNA Sample Prep protocol and single-end 75 bp reads were generated on an Illumina NextSeq 500. DESeq2 statistical package was used for differential expression testing. Multiple comparisons were adjusted for using a false discovery rate of 5%. Additionally, results were filtered to consider only those genes that demonstrated a fold-change point estimate >2 in either direction. Pathway analysis was performed using the Biological Process gene sets from the Gene Ontology annotation.Results: Compared to control treated Huh7 cells, 3 transcripts were differentially regulated following a one hour treatment with mERFE-Fc, ID1 (Log2 fold-change -1.55, q-value 4.99E-09), BPIFB2 (Log2 fold-change -1.88, q-value 0.036) and ANO1 (Log2 fold-change -1.43, q-value 0.019). A larger number of genes were differentially regulated with longer treatments, 32 genes at 6h and 828 genes at 24h. Among selected genes differentially expressed at 24h, between control treated and Erfe treated cells, we observed a significant reduction in expression of genes known to be directly upregulated by bone morphogenetic proteins (BMPs), including DNA binding protein inhibitor ID1(ID1), DNA binding protein inhibitor ID2 (ID2), DNA binding protein inhibitor ID3 (ID3) and HAMP. BMP6 has been demonstrated to regulate several biological processes such as iron metabolism in the liver, adipogenesis, and insulin sensitization. Interestingly, we also observed upregulation of SLC27A1 (or FATP1, fatty acid transporter, Log2 fold-change 2.27, q-value 0.003), GDNF (Log2 fold-change 2.57, q-value 2.4E-05), GIPR (Log2 fold-change 2.53, q-value 7.9E-07) upon treatment with Erfe at 24h. Previous work has demonstrated that all of these genes are downregulated by BMP6 treatment 1-3. Pathway analysis indicated a number of genes differentially regulated in the GOBP (gene ontology biological processes) iron ion homeostasis pathway, BMP signaling or TGF-β receptor signaling pathway. This assessment further indicated downregulation of cellular hormone levels and metabolic processes and ion homeostasis as biological processes impacted by erythroferrone.Conclusions: Significant differences in gene expression occur in hepatocytes upon interaction with erythroferrone. Many genes in the iron homeostasis, BMP6 signaling metabolic processes were also differentially regulated. This further supports the mechanism of ERFE on iron homeostasis. DisclosuresJasuja:Pfizer: Employment. Sawant:Pfizer: Employment. Pittman:Pfizer: Employment. Quan:Pfizer: Employment.

Costimulatory Blockade with CTLA4-Ig Abrogates Prolonged Graft Survival in Old Recipients

Introduction Although they represent a rapidly growing population, elderly organ transplant recipients are underrepresented in clinical trials. Age-specific aspects of established immunosuppressants are therefore poorly understood. Methods Here, we assessed the impact of immunosuppressive treatment with CTLA4-Ig, a fusion protein blocking costimulatory signaling between APCs and T cells through CD28, on alloimmune responses in old and young recipients (2-3 months vs. 16 months) in a fully MHC-mismatched murine transplantation models. Results While treatment with CTLA4-Ig prolonged skin graft survival in young recipients, the same treatment was unable to prolong graft survival in old recipients. Conversely, cardiac allografts in young mice treated with CTLA4-Ig survived indefinitely, while 80% of old recipients treated with CTLA4-Ig had lost their graft after 100 days (log-rank test, p<0.001; n=5/group; Fig. 1).{{AbstractFigure.1}} CTLA4-Ig reduced the in-vivo proliferation of CD4+ and CD8+ T cells (as assessed by BrdU incorporation) uniformly in both young and old recipients; in contrast, CTLA4-Ig reduced CD4+ central-memory and effector-memory T cells only in young but not old recipients. Moreover, systemic frequencies of CD4+IFN-γ+ T cells and systemic levels of IFN-γ cytokine production were reduced in young recipients, but remained unchanged in old. CTLA4-Ig caused significant perturbation in the Treg compartments with reduced frequencies and compromised proliferation of CD4+CD25+Foxp3+ cells. These differences correlated with a significant reduction in expression of CD28 on T cells in old mice, while levels of CTLA4 remained stable.Conclusion Immunosuppressive effects of costimulatory blockade with CTLA4-Ig showed distinct age-dependent effects in both skin and cardiac transplantation. Reduced expression of CD28 with aging may represent an escape mechanism for old alloreactive T cells, with unique clinical consequences for immunosuppression in the growing population of elderly transplant recipients.

Cytotoxic activity of Cardia draba leaves extracted on some cancer cell line

Cardaria draba is considered as one of the most fundamental plants that have wide diversity in medical uses, other researches had shown that this plant has a strong antioxidant activity for this reason the idea came to look for the cytotoxicity of this plant extract against different carcinogenic cell lines after subjection of this plant to methanolic extraction method.Result declared strong cytotoxicity against those carcinogenic cell line with a variable values of IC50 and the best effect was seen against HepG2 cell line, then we looked for the reason behind this reduction in viability of the cells via performing Caspase 9 assay which is used to check the induction of apoptosis and the expression of the TNF-α as a strong transcription factor which is activated in tumor cell lines.&#x0D; Interestingly, our finding suggested that the plant extract produced a positive effect in the induction of Caspase 9 which led to induce early apoptosis, regarding TNF-α expression; the result explained a significant reduction in expression of those genes which were correlated with induction of tumor. This meant that the reduction in cell viability resulted in the induction of apoptosis and reduction of TNF-α transcription genes.