Human ecdysoneless protein (ECD) plays an essential role in regulating cell cycle progression and cell survival. ECD has previously been implicated in RNA splicing through its association with spliceosomal proteins. Here, using electrophoretic mobility shift assay, fluorescence polarization assays, and mutational analysis, we demonstrate that ECD directly binds to RNA. Enhanced CLIP-seq analysis identified a broad repertoire of mRNAs bound to ECD in cells. RNA-seq analyses revealed that ECD depletion leads to widespread splicing aberrations and altered gene expression. ECD binding to RNAs was enriched near splice sites, and a substantial fraction of ECD-bound transcripts exhibited splicing defects upon ECD depletion. ECD associates with and stabilizes the U5 small nuclear ribonucleoprotein (snRNP) complex specific proteins. While depletion of ECD reduced the levels of key U5-specific proteins, these proteins exhibited an increased association with the R2TP complex in knockout cells. Notably, we found ECD to directly bind to U5 snRNA, and an RNA binding defective mutant of ECD (Δ135-148) failed to rescue the reduced levels of U5-specific proteins or the proliferation defect induced by ECD depletion. Collectively, these findings demonstrate that ECD binds to RNAs, including the U5 snRNA, and that RNA-binding is required for ECD to stabilize the U5 snRNP and for cellular functions.

G-quadruplexes (G4s) are non-canonical DNA secondary structures enriched at promoters, yet their regulatory role in transcription remains elusive. While G4-ligand-based studies suggest transcriptional repression, their prevalence at oncogene promoters and correlation with high expression suggest a positive regulatory role. Here, we provide direct genetic evidence that promoter G4s function as positive activators of gene expression through a novel mechanism. By selectively mutating endogenous promoter G4 motifs, we demonstrate that G4 loss significantly impairs oncogene expression. Using the endogenous CXCL1 promoter G4 as an example, we revealed that loss of a single promoter G4 motif led to a marked down-regulation of CXCL1 expression as well as inhibition of cellular functions such as cell migration and invasion. Mechanistically, we identified apurinic/apyrimidinic endonuclease (APE1), a multifunctional DNA repair and redox factor, as a G4-binding protein which was recruited to promoters via its unique N-terminus. Subsequently, the redox activity of APE1 enhances transcription factor binding at G4-containing promoters, driving a pro-metastatic gene expression program. Disruption of the G4-APE1 interaction, either genetically or pharmacologically, suppresses gene expression and impairs tumor cell malignant traits. Our findings establish a direct genetic link and mechanistic basis for promoter G4s as crucial drivers of oncogene expression and tumor progression.

Figure S3 shows USP1 knockdown or inhibition induces apoptotic cell death.

Prostate cancer (PC) is the most diagnosed cancer in males. Androgen receptor (AR) signaling is essential for PC progression. ECD (Ecdysoneless) protein participates in cell cycle progression and cell survival and is overexpressed in non-PC cancers. Current study explored the role and function of ECD in PC progression. TCGA Prostate Adenocarcinoma database was used to assess correlation between ECD and AR expression, and ECD’s expression in PC dataset. Bioinformatic analysis was performed to predict androgen response elements in the ECD promoter. Chromatin immunoprecipitation (ChIP) and Promoter-Luciferase assays were used to assess AR recruitment to the ECD promoter. ECD was overexpressed, as well as crispr knocked out in hormone-dependent LNCaP and hormone-independent C4-2B PC cell lines. RNA-seq analysis was performed to assess transcriptomics changes in ECD-overexpressing (ECD-OE) tumors. RNA immunoprecipitation (RIP) was performed to assess ECD protein association to RNAs. Glucose uptake, and glycolytic rates assays were carried out to assess glycolysis. ECD is overexpressed in PC patients’ tissues as compared to hyperplasia prostate samples, and ECD overexpression is associated with short patient survival. A significant positive correlation was observed between ECD and AR expression. Consensus androgen response elements in the ECD promoter showed androgen dependent recruitment of AR to the ECD promoter by ChIP assay. Luciferase assays showed androgen-dependent increase in ECD-luciferase activity. ECD overexpression and knockout in both LNCaP and C4-2B PC cell lines showed enhanced and decreased oncogenic traits, respectively. In vivo mice xenograft experiments showed ECD overexpression in LNCaP led to a significant increase in tumorigenesis. RNA-seq showed ECD overexpression led to increased levels of key glycolytic genes, which was confirmed by qRT-PCR. RIP assay revealed that ECD associates with the mRNA of these key glycolytic genes and enhances their stability. Functionally, ECD overexpressing PC cells showed higher glucose uptake and glycolytic rates, as compared to vector cells. Overall, our results demonstrate a novel role for androgen-regulated ECD overexpression in PC tumorigenesis through direct binding and stabilization of mRNAs of key glycolytic genes. Mohsin Raza, Asher Rajkumar Rajan, Benjamin B. Kennedy, Timothy E. Reznicek, Farshid Oruji, Sameer Mirza, M Jordan Rowley, Glen Kristiansen, Bhopal C. Mohapatra, Hamid Band, Vimla Band. Androgen regulated ECD overexpression promotes prostate cancer tumorigenesis through increased glycolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 266.

Figure S1 shows EWS::FLI1 and USP1 expression in EWS cell lines.

The ecdysoneless (ECD) mRNA and protein are overexpressed in breast cancer (BC), correlating with poor prognosis and shorter patient survival, particularly in ERBB2/HER2-positive BC. This study investigates the co-operative oncogenic mechanism of ECD and ERBB2 by deriving transgenic mice overexpressing ECD and/or ERBB2 (huHER2) in mammary epithelium under the MMTV promoter, as well as immortal human mammary epithelial cell lines (hMECs) overexpressing ECD and/or ERBB2. While the tumor latency and percentage of mice with tumors were similar between single and double-transgenic mice, we observed more and larger tumors in double transgenic mice in comparison to ECD or huHER2 single transgenic mice. Compared to huHER2Tg mice, which developed more homogenous solid nodular carcinomas, double transgenic mice (ECD;huHER2Tg) developed heterogenous and histologically aggressive mammary tumors with basal-like phenotype and epithelial mesenchymal transition (EMT) features, as seen in ECDTg mice and those reported in patients. ECD and ERBB2 overexpressing hMECs showed significant increase in oncogenic traits as compared to single gene expressing cells. Transcriptomic analysis revealed upregulation of two major oncogenic pathways, unfolded protein response (UPR) and glycolysis in ECD;huHER2Tg tumors as well as in ECD + ERBB2-overexpressing hMECs. ECD + ERBB2-overexpressing hMECs exhibited an increase in glucose uptake and enhanced glycolytic rate as compared to ECD or ERBB2-overexpressing hMECs. ECD as an RNA binding protein directly associated with mRNAs of three key glycolytic enzymes (LDHA, PKM2 and HK2) and mRNA of a major UPR regulated gene HSPA5, and increased mRNA stability. Knockdown of these genes resulted in decreased oncogenic traits of ECD + ERBB2 overexpressing hMECs. Taken together, our findings support a co-operative role of ECD and ERBB2 in oncogenesis by enhancing two major oncogenic pathways, UPR and glycolysis.

Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype characterized by poor therapeutic outcomes. Increased invasive capabilities expose tumor cells to higher plasma membrane stress resulting in frequent breaches that are incompatible with cancer cell survival and invasiveness. Upregulation of membrane repair mechanisms has been shown to counteract this inherent tumor cell vulnerability and targeting such mechanisms represents a novel potential therapeutic strategy. Previous studies have shown that plasma membrane repair involves multiple calcium-dependent protein complexes. We have previously demonstrated that EHD2, a member of the EHD (Eps15 homology domain-containing) protein family, functions in this role by sustaining the plasma membrane expression of Orai1, which is required to sustain the pro-oncogenic Store-Operated Calcium Entry (SOCE) pathway. EHD2 has been found to accumulate at plasma membrane repair sites in skeletal muscle models, suggesting the potential role of EHD2-SOCE axis in regulating plasma membrane integrity in breast cancer cells. We used CRISPR-Cas9 knockout of EHD2 or Orai1 as well as Orai1 inhibition in TNBC cell lines to examine the role of EHD2-Orai1 axis in the repair of mechanical or streptolysin O (SLO) toxin induced plasma membrane disruptions. The role of EHD2 as a mechano-transducer of external plasma membrane stress leading to Orai1 activation was investigated using micro-cantilevers to indent TNBC cells expressing genetic encoded calcium sensors. Functional linkage of EHD2 and Orai1 was assessed by measuring the impact of gene Knockout or complementation on SOCE and cell migration. EHD2 knockout impaired the ability of TNBC cells to efficiently repair the plasma membrane disruptions induced by mechanical injury or SLO. Orai1 knockout recapitulated the EHD2 knockout phenotype. A functional linkage of EHD2 and Orai1 was demonstrated by the ability of ectopic constitutively active Orai1, but not its dominant negative form, to rescue the cell migration defects of EHD2 knockout TNBC cells. Indenting the plasma membrane with micro-cantilevers induced rapid Ca2+ flux in TNBC cells and accumulation of EHD2 at the sites of deformation. Membrane deformation-induced Ca2+ entry was markedly reduced by EHD2 or Orai1 knockout. Orai1 inhibitors impaired the repair of mechanical or SLO induced plasma membrane disruption. Our results reveal an EHD2-Orai1 axis that is a critical regulator of mechanosensitive Ca2+ entry in TNBC cells and required for their plasma membrane repair. Our findings support a role of EHD2-Orai1 axis in mechano-sensing and plasma membrane integrity in TNBC cells under stress. These findings suggest that targeting the EHD2-SOCE axis could present a novel therapeutic strategy in TNBC. Haitao Luan, Azize Cerci, Bhopal Mohapatra, Timothy Bielecki, Matthew Storck, Jun-Yul Lim, Vimla Band, Subu Ramanathan, Hamid Band. The EHD2-Orai1 pro-oncogenic axis is required for calcium-dependent membrane repair in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6923.

Page 1: RT-qPCR primers used for DUB array; Page 2: Primers used for ChIP-qPCR analysis; Page 3: shRNA sequences used for EWS::FLI1 and USP1 knockdown cell line generation; Page 4: gRNA sequences used for USP1 knockout cell line generation; Page 5: List of working dilutions for antibodies used in this study; Page 6: List of reagents, with product information and RRIDs, used in this study; Page 7: List of biological resources, with product information and RRIDs, used in this study

Recurrence, therapy resistance and metastasis remain major challenges in cancer therapy, accounting for nearly all deaths from cancer. The inability of current therapies to eliminate cancer stem cells (CSCs) is widely believed to underlie these challenges. Targeting molecular pathways that help maintain the CSCs could open new avenues to devise durable cancer therapies. The CBL-family E3 ubiquitin ligases (CBL, CBL-B and CBL-C) attenuate tyrosine kinase signaling through ubiquitin-dependent degradation of tyrosine kinase-coupled receptors and signaling components. Gene deletion studies have shown that CBL-B, the predominant family member expressed in mature immune cells, mediates tumor-induced “immune editing” to generate a pro-tumor immune microenvironment. This mechanism has provided one line of evidence to develop chemical CBL-B inhibitors as cancer immune modulators, and candidate inhibitors exhibit anti-tumor activity in mouse implant models, supporting the general idea that CBL-family E3s are potential therapeutic targets. Using mouse genetic models, we have established that CBL and CBL-B are redundantly required for the maintenance of adult stem cell compartments, and that this role is dependent on the core function of CBL/CBL-B as negative regulators of the AKT-mTOR axis downstream of tyrosine kinase signaling. As normal organ stem cell programs are frequently co-opted by CSCs; we used a 3-pronged approach to ask if CBL/CBL-B are required to maintain CSC function. Methods/Results: 1. Based on higher CBL/CBL-B expression in TNBC cell lines, and TCGA data that CBL/CBL-B mRNA expression positively correlates with shorter patient survival, we engineered Dox-inducible shRNAs in TNBC cell lines and showed that CBL/CBL-B KD impaired cell migration and invasion, reduced the population with CSC markers and impaired orthotopic xenograft tumorigenesis and metastatic seeding. 2. We generated a unique mouse model in which the C3(1)-TAg transgene drives mammary tumors known to resemble human triple-negative breast cancer (TNBC) and floxed-Cbl and Cblb genes can be inducibly deleted using tamoxifen (via CreERT2). Tumors of these mice established as tumor-derived organoids (TDOs) exhibited significant impairment of growth and CSC signature gene expression upon Cbl/Cblb KO. 3. Since the developed “CBL-B” inhibitors target a mechanism shared between CBL/CBL-B, we used two active compounds (NTX-801, NTX-944) and one inactive compound (NTX-616) developed by Nimbus Therapeutics. The active inhibitors mimicked the genetic Cbl/Cblb KO to inhibit the mouse mammary TDOs growth, while the inactive analog was without effect. Our results establish a tumor-intrinsic requirement of CBL/CBL-B to maintain breast cancer CSCs and add a critical second rationale for the use of chemical inhibitors of CBL/CBL-B as targeted therapeutics to counter BC recurrence and metastasis. Bhopal C. Mohapatra, Aaqib M. Bhat, Mohsin Raza, Haitao Luan, Santosh Shrestha, Sowmya Kolluru, Matthew D. Storck, Lusheng Li, Fu-Shan Kuo, Silvana Leit, Fang Qiu, Subodh M. Lele, David Ciccone, Christine Loh, Shibiao Wan, Vimla Band, Hamid Band. Targeting CBL and CBLB ubiquitin ligases to exhaust cancer stem cells in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5697.

Triple-negative breast cancer (TNBC) has a relatively high metastasis rate as compared to other breast cancer subtypes. Metastasis and tumor recurrence remain major challenges for TNBC patients, resulting in poor prognosis. Therefore, it is imperative to identify and elucidate the molecular mechanisms that drive metastasis to allow the development of new therapeutic agents to inhibit TNBC metastasis and improve patients’ outcomes. Herein, we uncovered a new mechanistic connection between a multifunctional DNA repair protein APE1 and G-quadruplex (G4), a secondary DNA structure present in many metastasis-related signature genes promoters in TNBC. We found that APE1 is highly enriched at the G4 regions on the promoters of metastasis related genes and APE1 knockout or knockdown impairs the tumorigenic and pro-metastatic traits of TNBC cell lines in vitro. Intriguingly, we found that G4 is required for APE1’s enrichment at these promoters and APE1 promotes transcription factors (TFs) binding to these promoters, thereby promoting the expression of these metastasis-related genes. Deletion of APE1 impeded TFs’ binding to promoters and downregulated expression of these genes. In addition, we found that G4 deletion in some target gene promoters exerted similar inhibitory effects as APE1 knockout on cell migration, cell invasion and tumor sphere formation. We discovered that a small molecule which disrupts APE1: G4 interaction significantly disrupted APE1’s enrichment to G4 on promoters, thereby blocking TFs binding, gene expression, cell migration, invasion and tumor sphere formation in vitro. We generated orthotopic TNBC metastasis mouse model using an aggressive TNBC cell line, MDA-MB-231 and found that disrupting G4-APE1 axis either by APE1 knockout or G4 deletion or small molecules significantly inhibited the primary tumor growth and impeded TNBC distant metastasis to lungs. Overall, our study uncovered a novel functional axis of G4-APE1 in regulating TNBC growth and metastasis and identified that this regulatory axis has the potential to serve as a new therapeutic target to prevent metastasis in TNBC patients. Yingling Chen, Suravi Pramanik, Mason Tarpley, Achyuth Kalluchi, Sutapa Ray, Bhopal Mohapatra, Kyle Hewitt, Jordan Rowley, Vimla Band, Kishor K. Bhakat. A new approach to suppress triple-negative breast cancer metastasis by blocking apurinic/apyrimidinic endonuclease 1 interaction with G-quadruplex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3866.

Breast cancer (BC) is a heterogenous disease, with estrogen receptor-positive (ER+) BC accounting for over 70% of cases. ER+ BC depend on estrogen signaling for oncogenesis and metastasis. Even though treatments targeting ER pathway have improved patient’s outcome, factors driving ER+ BC progression particularly in metastatic remain unclear. One of these factors is Ecdysoneless (ECD), a highly conserved protein from yeast to human. ECD expression correlates with ER expression, ECD overexpression is observed in ER+ BC and ECD overexpression correlates with poor patient survival. In this study, we assessed the relationship of ECD and ER, and the role of ECD in ER+ BC oncogenic traits. Bioinformatic analysis of publicly available datasets were explored for the relationship between ER and ECD and survival analysis was performed to examine the link between ECD, ER, and ER target MYC expression. ECD promoter analysis followed by chromatin immunoprecipitation (ChIP) and promoter-luciferase assays were used to assess estrogen-dependent ER recruitment to the ECD promoter. Estradiol (E2) treatments were performed to examine how estrogen signaling affects ECD levels. The effects of ECD overexpression were performed in ER+ breast cancer cells to evaluate changes in in vitro tumorigenic traits. ECD expression correlated with ER levels in publicly available datasets. Survival data showed patients with elevated levels of ECD in ER+ BC had worse outcome. The ECD promoter contained estrogen response elements, and ChIP assays confirmed E2 mediated recruitment of ER to the ECD promoter. E2 treatment enhanced ECD promoter-luciferase activity. Overexpression of ECD in ER+ cells enhanced oncogenic traits. We have identified ECD is a novel estrogen regulated protein that plays a critical role in ER-driven oncogenesis in ER+ BC. Current studies are exploring the role of overexpressed ECD in in vivo preclinical mice models and therapy resistance. Farshid Oruji, Asher Rajkumar Rajan, Mohsin Raza, Bhopal Mohapatra, Hamid Band, Vimla Band. Ecdysoneless (ECD) as a key estrogen-regulated protein driving progression in ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6727.

Androgen receptor (AR)-mediated signaling is essential for PC tumorigenesis. In the TCGA database we observed a positive correlation between ECD and AR expression. Consistently, Dihydrotestosterone (DHT) treatment of PC cell lines increased ECD mRNA and protein levels, and AR knockdown (KD) reduced ECD expression. Bioinformatic analysis predicted three consensus androgen response elements in the ECD promoter, and DHT treatment increased AR occupancy at the ECD promoter, and enhanced ECD promoter activity. Enzalutamide treatment decreased ECD levels, and ECD knockout (KO) in PC cells reduced oncogenic traits, suggesting a functional role of ECD to maintain PC oncogenesis. ECD mRNA and protein are overexpressed in PC patient tissues, and its overexpression predicts shorter survival. Overexpression of ECD in PC cell lines enhanced the oncogenic traits in vitro and developed faster and larger highly proliferative xenograft tumors. RNA-seq analysis of mouse tumors revealed an increase in mRNA levels of several glycolytic genes. ECD associates with mRNA of key glycolytic genes and is required for their stability, consistent with our recent demonstration of ECD is an RNA binding protein. Higher glucose uptake and glycolysis was seen upon ECD overexpression in PC cells. Together, we demonstrate the role of a novel AR target gene ECD in PC tumorigenesis.

Essential cations, such as alkali and alkaline earth metals, are crucial in aquatic ecosystems, but when present in water in concentrations that have been described above as toxic to the different classes of aquatic organisms, they become toxic. This work examines the effects of acute exposure to selected alkali and Alkaline earth metals on fish fry with both the lethal and the sub-lethal effects studied. Fish fry, being at a very developmental stage, is more sensitive to environmental stressors such as chemical toxicity and is more suitable for evaluating aquatic pollution. The present investigation deals with the acute toxicity of Sodium (Na), Potassium (K), Calcium (Ca) and Magnesium (Mg) on rohu, Labeo rohita (Ham.) fry. Different concentrations of sodium chloride, potassium chloride, calcium chloride and magnesium chloride were used as test salts and their toxicity tests (LC50 values) were studied in laboratory conditions by exposing the rohu fry with different durations like 6 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs and 96 hrs of exposure. The LC50 values of different metal ions ranged between 10001.87 ppm - 750.23ppm in the case of sodium chloride, 1096.98 ppm - 68.73 ppm in the case of potassium chloride, 9872.44 ppm to 6384.12 ppm in the case of calcium chloride and 12676.81 ppm to 11095.66 ppm in case of magnesium chloride. In the 96-hour fry toxicity test, potassium chloride is more toxic than other metals. The toxicities of the salts were observed in the order of KCl>NaCl >CaCl2 >MgCl2.

While better management of loco-regional prostate cancer (PC) has greatly improved survival, advanced PC remains a major cause of cancer deaths. Identification of novel targetable pathways that contribute to tumor progression in PC could open new therapeutic options. The di-ganglioside GD2 is a target of FDA-approved antibody therapies in neuroblastoma, but the role of GD2 in PC is unexplored. Here, we show that GD2 is expressed in a small subpopulation of PC cells in a subset of patients and a higher proportion of metastatic tumors. Variable levels of cell surface GD2 expression were seen on many PC cell lines, and the expression was highly upregulated by experimental induction of lineage progression or enzalutamide resistance in CRPC cell models. GD2high cell fraction was enriched upon growth of PC cells as tumorspheres and GD2high fraction was enriched in tumorsphere-forming ability. CRISPR-Cas9 knockout (KO) of the rate-limiting GD2 biosynthetic enzyme GD3 Synthase (GD3S) in GD2high CRPC cell models markedly impaired the in vitro oncogenic traits and growth as bone-implanted xenograft tumors and reduced the cancer stem cell and epithelial-mesenchymal transition marker expression. Our results support the potential role of GD3S and its product GD2 in promoting PC tumorigenesis by maintaining cancer stem cells and suggest the potential for GD2 targeting in advanced PC.

IntroductionAtherosclerosis is a lipid-driven inflammatory disease of the arterial wall, and the underlying cause of the majority of cardiovascular diseases. Recent advances in high-parametric immunophenotyping of immune cells indicate that T cells constitute the major leukocyte population in the atherosclerotic plaque. The E3 ubiquitin ligase Casitas B-lymphoma proto-oncogene-B (CBL-B) is a critical intracellular regulator that sets the threshold for T cell activation, making CBL-B a potential therapeutic target to modulate inflammation in atherosclerosis. We previously demonstrated that complete knock-out of CBL-B aggravated atherosclerosis in Apoe-/- mice, which was attributed to increased macrophage recruitment and increased CD8+ T cell activation in the plaque.MethodsTo further study the T cell specific role of CBL-B in atherosclerosis, Apoe-/- CD4creCblbfl/fl (Cbl-bcKO) mice and Apoe-/-CD4WTCblbfl/fl littermates (Cbl-bfl/fl) were fed a high cholesterol diet for ten weeks.ResultsCbl-bcKO mice had smaller atherosclerotic lesions in the aortic arch and root compared to Cbl-bfl/fl, and a substantial increase in CD3+ T cells in the plaque. Collagen content in the plaque was decreased, while other plaque characteristics including plaque necrotic core, macrophage content, and smooth muscle cell content, remained unchanged. Mice lacking T cell CBL-B had a 1.4-fold increase in CD8+ T cells and a 1.8-fold increase in regulatory T cells in the spleen. Splenic CD4+ and CD8+ T cells had increased expression of C-X-C Motif Chemokine Receptor 3 (CXCR3) and interferon-γ (IFN-γ), indicating a T helper 1 (Th1)-like/effector CD8+ T cell-like phenotype.ConclusionIn conclusion, Cbl-bcKO mice have reduced atherosclerosis but show increased T cell accumulation in the plaque accompanied by systemic T cell activation.

Highlights Inclusion of the designed radial flow settler to the aquaponic system helps to remove more than 80% TSS, which is on par with or better in comparison to the TSS removal efficiency of the existing models. Removing excess suspended solids in traditional aquaponic setups could help prevent biofilter choking and hence, water contamination further down the line. The developed radial flow is simple in design, cost-effective, and yet, efficient. Abstract. One cylindrical settler of capacity 200 L (Ø 0.6×0.7 m) with a center-bottom inlet has been designed and developed in High-Density Poly Ethylene (HDPE) under the All-India Co-ordinated Research Project on Plastic Engineering in Agriculture Structures & Environment Management (AICRP on PEASEM) center at ICAR-CIFA, Bhubaneswar for fitting to the developed Nutrient Film Technique (NFT) aquaponics system. The water flow into the settling unit comes from a single-drain fish culture tank with the outlet at the bottom-center with a downward slope of 1:22. The experiment was carried out for the evaluation of the efficiency of Total Suspended Solid (TSS) removal in the developed radial-flow settling unit. The performance is assessed in terms of capturing the suspended solids from the water of aquaculture operation with varying specific gravity (1=SG>2) as well as the skimming of the floating organic surfactants before entering into the biofilter. During the 90 days of study, a total of 60 water samples were collected from the outlet of the fish culture tank and the overflow of the settler, which were filtered through the standard 11 µm pore paper filter for further processing in the laboratory. The average TSS concentration removed was calculated to be 128.55 mg/L on dry weight basis and a mean efficiency of 80.06 ± 9.36% was observed in the experiment with a surface loading rate of 0.00103 m3/min-m2 of the radial flow settler. The right-angle submerged overflow of the settler helped in complete surfactant removal. Keywords: Aquaponics, NFT, Radial-flow, Specific gravity, Suspended solid.

Among the signaling pathways that control the stem cell self-renewal and maintenance vs. acquisition of differentiated cell fates, those mediated by receptor tyrosine kinase (RTK) activation are well established as key players. CBL family ubiquitin ligases are negative regulators of RTKs but their physiological roles in regulating stem cell behaviors are unclear. While hematopoietic Cbl/Cblb knockout (KO) leads to a myeloproliferative disease due to expansion and reduced quiescence of hematopoietic stem cells, mammary epithelial KO led to stunted mammary gland development due to mammary stem cell depletion. Here, we examined the impact of inducible Cbl/Cblb double-KO (iDKO) selectively in the Lgr5-defined intestinal stem cell (ISC) compartment. Cbl/Cblb iDKO led to rapid loss of the Lgr5 Hi ISC pool with a concomitant transient expansion of the Lgr5 Lo transit amplifying population. LacZ reporter-based lineage tracing showed increased ISC commitment to differentiation, with propensity towards enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro , Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single cell RNAseq analysis of organoids revealed Akt-mTOR pathway hyperactivation in iDKO ISCs and progeny cells, and pharmacological inhibition of the Akt-mTOR axis rescued the organoid maintenance and propagation defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.

Summary: Cross-presentation of dead cell-associated antigens by conventional dendritic cells type 1 (cDC1s) is critical for CD8+ T cells response against many tumors and viral infections. It is facilitated by DNGR-1 (CLEC9A), an SYK-coupled cDC1 receptor that detects dead cell debris. Here, we report that DNGR-1 engagement leads to rapid activation of CBL and CBL-B E3 ligases to cause K63-linked ubiquitination of SYK and terminate signaling. Genetic deletion of CBL E3 ligases or charge-conserved mutation of target lysines within SYK abolishes SYK ubiquitination and results in enhanced DNGR-1-dependent antigen cross-presentation. We also find that cDC1 deficient in CBL E3 ligases are more efficient at cross-priming CD8+ T cells to dead cell-associated antigens and promoting host resistance to tumors. Our findings reveal a role for CBL-dependent ubiquitination in limiting cross-presentation of dead cell-associated antigens and highlight an axis of negative regulation of cDC1 activity that could be exploited to increase anti-tumor immunity.

Abstract Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) among all the BC subtypes due to its exceptionally high metastasis rate. TNBC patients with metastasis have a short overall survival as the endocrine therapy and chemotherapy are inefficient in TNBC treatment. Hence, efforts are still needed to figure out the molecular mechanism driving TNBC metastasis and find promising therapeutic targets for TNBC treatment. A newly published study revealed that a secondary DNA structure G-quadruplex (G4) is highly enriched in TNBC signature genes’ promoters, and our lab was the first one to uncover that DNA repair protein Apurinic/apyrimidinic endonuclease 1 (APE1) binds to G4 and regulates its stability in cells, so we are aimed to investigate the interaction between APE1 and G4 in TNBC and their roles in regulating TNBC metastasis. We found that knocking out or knocking down APE1 in TNBC cell lines, MDA-MB-231 and BT-549, significantly impaired the cell migration ability. RNA-seq analysis and Gene Ontology analysis revealed that APE1 knockout downregulated expression of genes such as CXCL1, VEGFA, PIK3R3, BMI1, etc. which are mostly involved in promoting cell migration. Further, APE1 ChIP-seq analysis showed that APE1 was highly enriched in CXCL1, VEGFA, PIK3R3 and BMI1 gene promoters, and these regions contained potential G4 folding sequences, suggesting a potential interaction between APE1 and G4 in these gene promoters. Consisted with this, our study with the enzyme linked immunosorbent assays (ELISA) showed that recombinant APE1 bound with high affinity to CXCL1 and VEGFA G4 secondary structures as compared to double-strand DNA oligos in vitro, suggesting that APE1’s enrichment to the metastasis-related gene promoters attributes to the G4 structures in those promoter regions. Promoter-directed ChIP assay further revealed that downregulating APE1 inhibited transcription factor (TF) c-JUN’s occupancy to VEGFA promoters. Interestingly, we discovered that the G4-targeted small molecule TMPyP4 blocked APE1-G4 binding in vitro and reduced APE1’s occupancy at gene promoters in cells. Treatment of cells with TMPyP4 significantly downregulated CXCL1, VEGFA, PIK3R3, and BMI1 gene expression as well as inhibiting cell migration. In orthotopic TNBC metastasis mouse model, mice treating with TMPyP4 significantly reduced the frequency and volume of metastatic tumors in lung tissues. Overall, our study revealed that G4 in gene promoters recruited APE1 which further promoted transcription factors’ binding to promoters and enhanced metastasis-related gene expression. Blocking APE1-G4 interaction by TMPyP4 is a potential therapeutic approach to inhibit TNBC metastasis. Citation Format: Yingling Chen, Suravi Pramanik, Mason Tarpley, Achyuth Kalluchi, Bhopal Mohapatra, Kyle Hewitt, Michael J. Rowley, Vimla Band, Kishor Bhakat. The interplay between Apurinic/apyrimidinic Endonuclease 1 (APE1) and DNA G-quadruplex (G4) in regulating triple-negative breast cancer (TNBC) metastasis [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 2478.

One vertical aquaponics unit following the principle of Nutrient Film Technique (NFT) has been designed and developed at ICAR-Central Institute of Freshwater Aquaculture, Bhubaneswar centre of All India Coordinated Research Project on Plasticulture Engineering in Agriculture Structures and Environment Management (AICRP on PEASEM). The developed unit is constructed using three major components, viz., fish tank/ aquarium tank (0.127 m3), vertical hydroponic pipe (Ø0.16×1.5 m) and the water distribution system. The unit experimented with 30 numbers of Guppy fish (Poecilia reticulata) in 0.1 cubic meters of water and 6 numbers of Petunia plant (Petunia × hybrida) and 6 numbers of Zinnia plant (Zinnia angustifolia) in 780 CC hydroponics cups arranged at 20° from the vertical axis on a PVC pipe. The quad channel sprinkler is fixed inside the pipe at the top surface. The water dispersion efficiency concerning the availability of nutrient-rich water at the roots of the plants for its flowering has been evaluated in the present study. The optimum water quality and flower production from the plants were achieved at 200 LPH flow rate from the fish tank. At this hydrolic loading rate, maximum plant length & spread were observed among the three replications . The length & spread for Zinnia angustifolia and Petunia x hybrida were 37.17±4.27 cm & 39.54±7.45 cm, and 33.43±2.53 cm & 29.58±7.26 cm respectively. There was no significant difference (p>0.05) in the water quality parameters at 200 & 250 LPH flow rates in the vertical aquaponics system.The cost of one unit is calculated to be INR 7000, with approximately a 10-20% price reduction in case of mass production. The newly developed integrated system of aquarium is designed for peri-urban and urban hobby farming as well as home decoration.