Protein Levels Research Articles

7581 Periodic Administration of Quercetin and Dasatinib Alleviates Skeletal Fragility in the INS2AKITA Mouse Model of Type 1 Diabetes

Abstract Disclosure: R. Dresner-Pollak: Consulting Fee; Self; Opko Biologics LTD.. Speaker; Self; Medison Pharma. N. Lishinsky: None. I. Gurt: None. J. Stokar: None. V. Temkin: None. P. Shivam: None. G. Zatarah: None. J. Milgram: None. R. Shahar: None. Skeletal fragility is a recognized complication of type 1 diabetes (T1D). The risk of hip and non-vertebral fractures is significantly higher in people living with T1D compared to age- and sex-matched non-diabetic controls and those living with type 2 diabetes (T2D). Hip fracture risk is up to six-fold higher in T1D patients; hip fractures occur 10-15 years earlier and the outcome is poorer compared to non-diabetics. Alterations in bone quality are considered major contributors to T1D-related skeletal fragility. As people with T1D live longer, there is a growing need to discover new therapeutics to reduce fracture risk. TID is associated with accelerated biological aging. Cellular senescence is a fundamental mechanism of aging. Senescent cells stop dividing, are resistant to apoptosis and acquire a distinct phenotype that includes secretome changes termed “senescence-associated secretory phenotype” (SASP). Clearing senescent cells with senolytics is recognized as a promising therapeutic approach for multiple conditions in mice and humans We explored the ability of senolytics dasatinib (D), a tyrosine kinase inhibitor, and quercetin (Q), a flavanol, present in fruit and vegetables, drug combination, to alleviate femur fragility in mice with T1D. Ins2Akita mice show a sexual dimorphic effect of the mutant Ins2 allele causing severe hyperglycemia in males. We treated Ins2Akita male and female mice and C57BL/6 wild type (WT) male mice with D + Q administered once monthly for four months beginning at age 3 months. 3-month-old Ins2Akita male mice had higher blood levels of the SASPs components HMGB1, PAI-1, MMP12, and increased gene expression of p53 and senescence effectors p16, p15 and SASPs Il1b, Mmp3, Mmp9, Mmp13, Serpineb2 at the femur diaphysis and in bone marrow mesenchymal stem cells, respectively compared to age-matched WT male mice. In vitro, exposure to glucose levels equivalent to those observed in blood in Ins2Akita male mice induced cellular senescence in IDG-SW3 osteocyte-like cells including a 4-5-fold increase in gene expression of p16INK4a, p21Cip[1], p53, double percentage of SA-β-gal+ cells and P21 protein level. Ingenuity pathway analysis of differentially expressed proteins revealed enrichment for the senescence pathway (Z=1.213; P=1.11E-05). D+Q administration did not affect bone microarchitecture at the spine (L2), distal femur metaphysis and diaphysis assessed by µCT. D+Q administration significantly increased femur resistance to fracture assessed by the 3-point-bending test by increasing whole femur work-to-fracture, the ultimate parameter of bone resistance to failure, and post-yield displacement, an index of matrix composition, organization, and bone quality in Ins2Akita males. No effect was observed in Ins2Akita females or WT male mice, indicating that the intervention is specific for T1D. Senolyitcs D+Q show promise in improving bone health in T1D. Presentation: 6/1/2024

12234 Gα13 Promotes Clonogenic Growth By Increasing Tolerance To Oxidative Metabolic Stress In Prostate Cancer Cells

Abstract Disclosure: D. Wu: None. W. Lim: None. X. Chai: None. V.P. Seshachalam: None. S.A. Rasheed: None. S. Ghosh: None. P.J. Casey: None. Gα13 and Gα12 are members of the G12 family of Gα proteins that, along with their associated Gβγ subunits, mediate signaling from specific G protein-coupled receptors. Analyses of tumor specimens indicate that Gα13 expression correlates with patient survival in several solid cancers. We previously reported a role for Gα13 in cell migration and invasion in prostate cancer cell lines. Interestingly, patient data supports the notion that mitochondrial superoxide dismutase 2 (SOD2) correlates with prostate cancer risk and prognostic Gleason scores. Gα13 lower-expressing LNCaP cells also have lower SOD2 protein levels compared to the Gα13 higher-expressing PC3 cells. Hence, we explored the role of Gα13 in prostate tumorigenesis, and its effect on cellular processes such as cell survival and mitochondrial metabolism, in human prostate cancer cell lines PC3 and LNCaP. We stably knocked-down GNA13 expression in PC3 cells and overexpressed GNA13 in LNCaP cells. We found that Gα13 promoted anchorage-independent cell growth in PC3 and LNCaP cell lines, as assessed by soft agar colony formation, spheroid formation, and xenograft tumor growth. Whole-genome transcriptome analyses suggested that Gα13-regulated genes are functionally enriched in the mitochondria compartment and that GNA13 expression positively correlated to SOD2 transcript levels in PC3 cells. We found that silencing GNA13 sensitized PC3 cells to long-term oxidative metabolic stress when cultured in media containing non-glycolytic metabolites, namely galactose, glutamate plus malate, and succinate. Furthermore, Gα13 levels impacted the abundance of SOD2 protein in the mitochondria, as well as SOD2 promoter activity and mRNA expression. In addition, silencing GNA13 increased mitochondrial superoxide levels in PC3 cells when cultured in galactose medium, as assessed by MitoSOX staining and flow cytometry. Moreover, overexpression of SOD2 could rescue the effect of Gα13 loss on suppression of anchorage-independent cell growth in PC3 cells. Importantly, anchorage-independent cell growth was not rescued when a catalytically-inactive SOD2(Q167A) mutant was expressed. Likewise, stable knockdown of SOD2 suppressed the effect of overexpression of Gα13 on anchorage-independent cell growth in LNCaP cells. We propose a novel biological route of Gα13-mediated anchorage-independent growth and response to oxidative metabolic stress through regulation of SOD2 expression in prostate cancer cells. Given that SOD2 protein levels correlate with prostate cancer Gleason grade, identifying the upregulated GPCRs that signal through Gα13 in prostate cancer could lead to novel preventive or therapeutic strategies. Presentation: 6/1/2024

8681 Preoteogenomic Identification Of Novel Translated And Regulatory Open Reading Frames In Human Beta-Cells

Abstract Disclosure: K. Walters: None. R.C. Guiterrez: None. S. Sakhar: None. A. Baldwin: None. E.S. Nakayasu: None. H. Russ: None. N. Mukherjee: None. Preoteogenomic Identification of Novel Translated and Regulatory Open Reading Frames in Human Beta-cells The discovery of novel autoreactive T cells that target β cells during type 1 diabetes (T1D) has been challenging due to our limited knowledge in autoreactive β cell antigens. Most of these antigens are derived from canonical open reading frames (ORFs) of protein coding genes, such as preproinsulin and chromogranin A. However, a defective ribosomal product (DRiP) was recently identified from the insulin mRNA (INS-DRiP). This antigen is the result of translation from a novel or unannotated open reading frames (nuORFs). nuORFs have a range of functions from regulating translation to encoding novel small bioactive peptides to producing neoantigens. In this study, we use a transcriptome-wide approach to identify nuORFs in human β cells in an unbiased manner. We performed ribosome profiling (ribo-seq) in stem cell derived β cells (sBCs). Human pluripotent stem cells were differentiated into sBCs expressing a flag-tagged ribosomal protein (RPL22-Flag) driven by the rat insulin promoter. We performed IP-ribo-seq on sBCs enriching for RPL22-Flag containing ribosomes. These data exhibited high-resolution 3-nucleotide periodicity consistent with translating codons and identified sequencing reads consistent with translation of the INS-DRiP. We performed de novo ORF-calling using two software using complementary statistical approaches (RiboCode and ORFQuant). We identified over 12,000 canonical annotated ORFs of which ∼85% were had protein level support from matched proteomics confirming the high specificity of our approach. Of nearly 1000 nuORFs identified in sBCs, over half had protein level support in sBCs and cadaveric human islets. We found that 149 nuORFs were associated with Type-1 Diabetes (T1D) genes, including a novel upstream ORF (uORF) in TYK2, which had the strongest association with T1D and is a promising therapeutic target to halt T1D progression. Using reporter assay, we show that the novel uORF in TYK2 is a translational activator and warrants further investigation. Finally, our data represents a valuable database for the search of novel autoreactive antigens in β cells and accelerate the discovery of novel autoreactive T cells implicated in T1D pathogenesis. Presentation: 6/2/2024

7274 Assessing The Interactome Of Hexose-6-Phosphate Dehydrogenase In Breast Cancer Cells And Identifying Anterior Gradient Protein 2 As Interacting Partner By Using A BioID Method

Abstract Disclosure: G. Sakalauskaite: None. M. Weingartner: None. T. Bock: None. J. Birk: None. M. Tsachaki: None. A. Odermatt: None. Hexose-6-phosphate dehydrogenase (H6PD) catalyzes the first two steps of the pentose-phosphate pathway in the endoplasmic reticulum (ER). It is so far the only enzyme shown to generate NADPH in the ER. H6PD is involved in physiological processes such as glucose and steroid metabolism, redox regulation, and response to ER-stress. So far, only one H6PD interacting partner, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), has been reported. This enzyme uses NADPH for its well-reported role in glucocorticoid activation. However, H6PD has 11β-HSD1-independent functions, such as the recently described modulation of breast cancer cell properties. Here, we adapted the BioID approach to identify novel H6PD interacting partners in the ER. First, we generated a triple negative breast cancer cell line MDA-MB 231 stably expressing a H6PD-biotin ligase fusion protein. Then, we validated the method to confirm the known interaction between H6PD and 11β-HSD1. Next, we analyzed enriched biotinylated proteins by mass spectrometry and further assessed potential candidates by co-immunoprecipitation and functional assays. The resulting interactome revealed a potential hit cluster of protein disulfide isomerase (PDI) family members, different ER-resident chaperones and luminal calcium binding proteins. Due to its association with breast cancer, we further examined the PDI Anterior gradient protein 2 (AGR2) as H6PD interacting partner. Physical interaction between AGR2 and H6PD was confirmed by co-immunoprecipitation assay in MCF7 cells. Downregulation of AGR2 by siRNA resulted in elevated H6PD protein levels but decreased activity. Furthermore, increased H6PD activity was observed when this protein was co-expressed with AGR2 in HEK-293 cells. In conclusion, the results validated the use of the BioID approach to identify protein interactions within the ER and they revealed that AGR2 physically interacts with H6PD, thereby controlling its expression and enzyme activity. Presentation: 6/3/2024

Apache is a neuronal player in autophagy required for retrograde axonal transport of autophagosomes

Neurons are dependent on efficient quality control mechanisms to maintain cellular homeostasis and function due to their polarization and long-life span. Autophagy is a lysosomal degradative pathway that provides nutrients during starvation and recycles damaged and/or aged proteins and organelles. In neurons, autophagosomes constitutively form in distal axons and at synapses and are trafficked retrogradely to the cell soma to fuse with lysosomes for cargo degradation. How the neuronal autophagy pathway is organized and controlled remains poorly understood. Several presynaptic endocytic proteins have been shown to regulate both synaptic vesicle recycling and autophagy. Here, by combining electron, fluorescence, and live imaging microscopy with biochemical analysis, we show that the neuron-specific protein APache, a presynaptic AP-2 interactor, functions in neurons as an important player in the autophagy process, regulating the retrograde transport of autophagosomes. We found that APache colocalizes and co-traffics with autophagosomes in primary cortical neurons and that induction of autophagy by mTOR inhibition increases LC3 and APache protein levels at synaptic boutons. APache silencing causes a blockade of autophagic flux preventing the clearance of p62/SQSTM1, leading to a severe accumulation of autophagosomes and amphisomes at synaptic terminals and along neurites due to defective retrograde transport of TrkB-containing signaling amphisomes along the axons. Together, our data identify APache as a regulator of the autophagic cycle, potentially in cooperation with AP-2, and hypothesize that its dysfunctions contribute to the early synaptic impairments in neurodegenerative conditions associated with impaired autophagy.

7881 Using Selective Estrogen Receptor Modulators to Reveal Therapeutic Vulnerabilities in ESR1 Mutant Breast Cancer

Abstract Disclosure: S.W. Fanning: Grant Recipient; Self; Olema Oncology. Hotspot activating somatic mutations to ESR1, the gene for estrogen receptor alpha (ER), enable hormone therapy resistance and drive breast cancer metastasis. The Y537S missense mutation within the ER ligand binding domain is the most activating, therapy resistant, and increases metastatic potential. Next generation antiestrogens including lasofoxifene (laso) and elacestrant (RAD1901) show improved progression-free survival over fulvestrant (fulv), but further progress is needed to achieve durable response. To fully address Y537S ESR1, it is critically important to understand how the mutation boosts metastasis. We have developed a small molecule antiestrogen, T6I-29, that uniquely impacts transcriptional pathways related to cell-cell adhesions and morphology. Two allele-specific phenotypes identified by the Oesterreich Laboratory enhance the metastasis of Y537S ESR1 breast cancer cells. T6I-29-specific pathway effects were driven by a significant downregulation of dickkopf-1 (DKK1), which was also observed but to a lesser extent with fulv, laso, and RAD1901. DKK1 is an immunosuppressive, tumor-secreted glycoprotein and its overexpression correlates with poor outcomes in multiple cancers. However, DKK1 acts as a tumor-suppressor in colorectal cancer, which does not rely on ER. Our preliminary studies suggest that DKK1 plays a role in ER+ breast cancer progression. We measured significantly elevated DKK1 protein levels in the blood plasma of 108 diverse ER+ breast cancer patients compared to 100 matched healthy female donors and levels increase coincident with tumor stage. Unfortunately, we lack ESR1 mutation status and other critical details to make further insights on these patients. DKK1 gene expression and protein secretion is stimulated in WT cells by the pathogenic hormone 17β-estradiol (E2), while it is constitutively expressed in Y537S ESR1 cells. DKK1 is characterized as a WNT antagonist but can also induce PI3K/Akt signaling. Interestingly, DKK1 is elevated and ERα responsive in WT not mutant PI3KCA cell lines. DKK1 knockdown also reduced proliferation of Y537S ESR1 breast cancer cells where elevated DKK1 was observed. Presentation: 6/3/2024

8743 Regulation Of Tryptophan 2,3-Dioxygenase 2 And Androgen Receptor In Triple-Negative Breast Cancer

Abstract Disclosure: L. Kuo: None. K. Lacey: None. N. Spoelstra: None. J.K. Richer: None. Background: Triple Negative Breast Cancer (TNBC) is an aggressive subtype with a peak recurrence rate within 3-5 years after initial diagnosis. Androgen receptor (AR) and Tryptophan 2,3-dioxygenase 2 (TDO2) are elevated in TNBC cells grown under anchorage-independent conditions and facilitate anoikis resistance. Several studies reported that the luminal AR subset of TNBC showed a positive enrichment of the tryptophan (TRP) catabolism pathway gene signature. Recent studies have demonstrated that aryl hydrocarbon receptor (AhR) inhibition decreases AR promoter transcriptional activity in prostate cancer. We have observed that TNBC with high AR have high levels of genes/proteins in the tryptophan catabolism pathway including the rate limiting enzyme TDO2. Hypothesis: TDO2 activity regulates AR expression through the downstream tryptophan catabolite kynurenine (KYN), which binds and activates AhR and AhR and AR may work together to promote TNBC progression. Methods: Human TNBC cell lines: MDA-MB-453, BT549, SUM159PT ​ were used in this study and the expression of TDO2 and AR were examined by immunohistochemistry (IHC) and western blot. TDO2 was stably knocked down (KD) via shRNA or overexpressed (OE) using lentivirus transduction. Mass spectrometry-based metabolomics were used to measure changes in TRP catabolite levels, and a cytokine array (R&D) was utilized to analyze conditioned media. Results: TDO2 and AR protein are positively correlated in human TNBC cell lines. Although overexpression of TDO2 did not alter AR protein expression in SUM159PT, in the human MDA-MB-453, which have high levels of AR, knockdown of TDO2 (shTDO2) reduced AR protein by 40% and production of kynurenine (KYN) was reduced by 50% (p<0.0001). This suggests that TDO2 activity, specifically KYN binding to AhR, may influence AR protein levels. Conditioned media (CM) from TDO2 OE and KD respectively increased and decreased CXCL5 and IGFBP2. Lastly, TCGA database analysis of TNBC tumors with high levels of both AhR and AR showed significantly shorter patient survival (HR=1.47, log rank p=0.046) compared to those with tumors with lower amounts of both genes. Conclusion: TDO2 and AR are positively correlated in human TNBC cell lines. Tumors with high levels of both AR and AhR result in significantly shorter survival time for TNBC patients. Knockdown of TDO2 significantly reduced KYN production and AR expression in luminal AR TNBC cell lines and future experiments will focus on the underlying mechanism behind this and whether it is dependent on KYN-mediated activation of AhR and if AhR stabilizes AR in TNBC. Presentation: 6/3/2024

8743 Regulation of Tryptophan 2,3-Dioxygenase 2 and Androgen Receptor in Triple-Negative Breast Cancer

Abstract Disclosure: L. Kuo: None. K. Lacey: None. N. Spoelstra: None. J.K. Richer: None. Background: Triple Negative Breast Cancer (TNBC) is an aggressive subtype with a peak recurrence rate within 3-5 years after initial diagnosis. Androgen receptor (AR) and Tryptophan 2,3-dioxygenase 2 (TDO2) are elevated in TNBC cells grown under anchorage-independent conditions and facilitate anoikis resistance. Several studies reported that the luminal AR subset of TNBC showed a positive enrichment of the tryptophan (TRP) catabolism pathway gene signature. Recent studies have demonstrated that aryl hydrocarbon receptor (AhR) inhibition decreases AR promoter transcriptional activity in prostate cancer. We have observed that TNBC with high AR have high levels of genes/proteins in the tryptophan catabolism pathway including the rate limiting enzyme TDO2. Hypothesis: TDO2 activity regulates AR expression through the downstream tryptophan catabolite kynurenine (KYN), which binds and activates AhR and AhR and AR may work together to promote TNBC progression. Methods: Human TNBC cell lines: MDA-MB-453, BT549, SUM159PT ​ were used in this study and the expression of TDO2 and AR were examined by immunohistochemistry (IHC) and western blot. TDO2 was stably knocked down (KD) via shRNA or overexpressed (OE) using lentivirus transduction. Mass spectrometry-based metabolomics were used to measure changes in TRP catabolite levels, and a cytokine array (R&D) was utilized to analyze conditioned media. Results: TDO2 and AR protein are positively correlated in human TNBC cell lines. Although overexpression of TDO2 did not alter AR protein expression in SUM159PT, in the human MDA-MB-453, which have high levels of AR, knockdown of TDO2 (shTDO2) reduced AR protein by 40% and production of kynurenine (KYN) was reduced by 50% (p<0.0001). This suggests that TDO2 activity, specifically KYN binding to AhR, may influence AR protein levels. Conditioned media (CM) from TDO2 OE and KD respectively increased and decreased CXCL5 and IGFBP2. Lastly, TCGA database analysis of TNBC tumors with high levels of both AhR and AR showed significantly shorter patient survival (HR=1.47, log rank p=0.046) compared to those with tumors with lower amounts of both genes. Conclusion: TDO2 and AR are positively correlated in human TNBC cell lines. Tumors with high levels of both AR and AhR result in significantly shorter survival time for TNBC patients. Knockdown of TDO2 significantly reduced KYN production and AR expression in luminal AR TNBC cell lines and future experiments will focus on the underlying mechanism behind this and whether it is dependent on KYN-mediated activation of AhR and if AhR stabilizes AR in TNBC. Presentation: 6/3/2024

8672 Disrupted Glucocorticoid Receptor Mediated Signalling Causes a Primary Cilia Defect in the Fetal Mouse Renal Tubule

Abstract Disclosure: J. Lao: None. K.L. Short: None. J. Ng: None. D.L. Cottle: None. T.J. Cole: None. Primary cilia are microtubule-based organelles that protrude from cell membranes to mediate diverse developmental signalling pathways and senses extracellular stimuli to maintain tissue homeostasis. We show that glucocorticoid (GC) signalling via the glucocorticoid receptor (GR) is a regulator of normal primary cilia formation in kidney renal tubules. RNA sequencing of whole E18.5 fetal kidney from mice with global deletion of the GR (GR-null) compared to littermate wild-type controls identified significantly reduced mRNA levels of key ciliogenesis-related genes. Further analysis of whole fetal kidney RNA by real-time-qPCR confirmed reduced mRNA levels for the cilia-associated proteins Ccp110 (fold -2.18), Cep97 (fold -1.78), Cep290 (fold -2.91), Kif3a (fold -1.82) and Rpgr (fold -1.90). Confocal microscopy revealed abnormal, stunted primary cilia in renal proximal tubules, collecting ducts and podocytes in GR-null or in renal tubule conditional GR-deleted mice, created using the HoxB7 Cre-driver allele. Primary cilia length was significantly decreased in kidney proximal tubule cells in GR-null mice (5.01 ± 0.11μm) compared with wildtype controls (6.20 ± 0.15μm). Furthermore, scanning electron microscopy of E18.5 GR-null kidney further confirmed aberrant primary cilia morphology in the proximal renal tubules and the absence of an apical brush border. Activation of GR signalling with the synthetic GC dexamethasone in cultured mouse IMCD3 kidney tubule cells significantly increased primary cilia length (2.89 ± 0.04μm) compared to vehicle controls (2.46 ± 0.28μm), an effect blocked by the GR antagonist RU486 (vehicle + RU486: 2.06 ± 0.02μm, dexamethasone + RU486: 2.00 ± 0.02μm). Aurora kinase A (AURKA) is a known regulator of primary cilia formation via the AKT cell signalling pathway and distinct proteins at different stages of the mitotic cell cycle. AURKA protein levels were downregulated in GR-null kidney and in IMCD3 cells which suggest GR regulates AURKA to control the assembly and disassembly of primary cilia. Together, these results demonstrate that GC signalling via the GR is required for normal primary ciliogenesis in the developing renal tubule and suggests that synthetic GR agonists may provide a novel therapeutic option for human ciliopathies such as those observed in forms of polycystic kidney disease. Presentation: 6/2/2024

6603 Impact of the Monocarbonyl Analogs of Curcumin, C66 and B2BrBC, on the Protein Expression of Epithelial and Mesenchymal Markers in MCF-7 Breast Cancer Cells

Abstract Disclosure: R. Stojchevski: None. A. Lavi: None. J. Bogdanov: None. N. Hadzi-Petrushev: None. M. Mladenov: None. L. Poretsky: None. D.B. Avtanski: None. The synthesis of monocarbonyl analogs of curcumin (MACs) presents a solution to the challenges posed by curcumin, a natural polyphenol extracted from the roots of the turmeric plant (Curcuma longa), which is known for its antioxidant and anti-inflammatory properties. The limitations of curcumin, including poor stability, low bioavailability, and rapid metabolism, hinder its application, and MACs, designed without a β-diketone moiety, address these drawbacks and exhibit enhanced stability and bioavailability. This study aimed to assess the effects of two monocarbonyl analogs of curcumin, (2E,6E)-2,6-bis[(2-trifluoromethyl)benzylidene]cyclohexanone (C66) and (2E,6E)-2,6-bis(2-bromobenzylidene)cyclohexanone (B2BrBC), on epithelial-to-mesenchymal transition (EMT) in MCF-7 breast cancer cells using western blot analysis. Our previous findings indicated that these analogs can potentially affect EMT by modulating the epithelial and mesenchymal gene expression. MCF-7 cells were cultured in complete medium supplemented with EMT-inducing factors for two days, followed by C66 and B2BrBC administration (100 µM) for three additional days. After the experiments, proteins were extracted, and western blot analyses were performed to measure the expression levels of various epithelial and mesenchymal markers (E-cadherin, N-cadherin, SNAI1, and Claudin-1). The results revealed that both C66 and B2BrBC increased the protein levels of the epithelial marker E-cadherin and suppressed those of the mesenchymal marker N-cadherin. Moreover, C66 and B2BrBC decreased the protein expression of the transcription factor SNAI1 and transmembrane protein Claudin-1. Between the two MACs, B2BrBC showed a more pronounced effect on N-cadherin expression, while C66 was more potent in suppressing SNAI1 protein expression. Our findings demonstrate that C66 and B2BrBC counteract EMT, contributing to the potential therapeutic applications of MACs in managing breast cancer progression. Presentation: 6/3/2024

Transcriptomic and Metabolomic Analyses Reveal the Attenuating Role of Cordycepin and Cordyceps militaris Extract on Acute Liver Injury Induced by LPS in Piglets

Cordyceps militaris extract (CME) contains many bioactive compounds, mainly cordycepin (CPN). This study aimed to investigate the possible mechanisms underlying the amelioration of LPS-induced acute liver injury in piglets by CME or CPN supplementation using multi-omics analysis. Twenty-four weaned piglets were randomly distributed into 4 groups (n = 6): the control and LPS groups were fed basal diets; the CPN + LPS (CPN-LPS) and CME + LPS (CME-LPS) groups were fed the basal diets supplemented with CME or CPN. The results showed that CPN or CME supplementation significantly decreased the C-reactive protein level (p < 0.05) and improved liver tissue pathology to prevent acute liver injury after LPS treatment. Compared with LPS, the transcriptomic analysis indicated that CPN supplementation significantly downregulated cell adhesion molecules, while CME supplementation significantly downregulated inflammatory mediator regulation of TRP channels, complement and coagulation cascades and cytokine-cytokine receptor interaction. The metabolomic results showed that CPN or CME supplementation significantly reduced disease biomarker of bicyclo-prostaglandin E2, and increased levels of deoxyinosine and 3-hydroxyanthranilic acid (p < 0.05). The combined transcriptome and metabolome helped identify two metabolites PC 34:2 and PC 36:0, which may be associated with the restoration of liver cell morphology. In conclusion, CPN and CME could attenuate LPS-induced acute liver injury by regulating immune-related genes and metabolites. This study elucidates the potential protective mechanism of CPN or CME supplementation against acute liver injury.

7447 Rare KSR2 Variants: The Effect of Glycine and N-acetylcysteine (GlyNAC) Supplementation

Abstract Disclosure: J. Youn: None. Y. Carcamo-Bahena: None. E. Lartigue: None. S. Sisley: None. Kinase suppressor of ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Of note, disruption of the kinase domain of KSR2 in humans and mice causes obesity by reducing metabolic rate, suggesting its important role in energy homeostasis. In the present study, we identified two variants (P189L, T290A) with mutations located in the proximal CA2 region from children with severe, early-onset obesity. Since variants in this region have not been confirmed to cause obesity, we generated plasmid constructs containing these KSR2 mutations by site directed mutagenesis. The HEK293 cells transfected with KSR2 plasmid construct containing P189L or T290A had reduced binding affinity with AMPK and phosphorylation of AMPK without affecting KSR2 protein integrity. These variants also significantly reduced cellular metabolic rates measured by Seahorse analysis in C2C12 cells, suggesting that KSR2 variants in the proximal CA2 region can result in impaired KSR2 function, subsequently leading to a decrease in cellular metabolic rate. In addition, these KSR2 variants decreased protein levels of NRF-2, a transcription factor of cytoprotective genes regulating the antioxidant glutathione (GSH) pathway compared to wild type KSR2 which augmented NRF2 levels. We also observed lower levels of total GSH and GSH/Glutathione disulfide (GSSG) ratio and higher levels of thiobarbituric acid reactive substances (TBARS), an indicator of oxidative stress, indicating an imbalance of antioxidant and oxidant with proximal KSR2 variants. However, treatment of cells with Glycine and N-acetylcysteine (GlyNAC) reversed the detrimental effects of KSR2 variants by normalizing protein expression levels of NRF-2, GSH/GSSG ratios and TBARS levels. Taken together, we show strong evidence that two new KSR2 variants, P189L and T290A, cause disruption of KSR2 function. Additionally, we also report a novel role of KSR2 in maintaining redox homeostasis suggesting that increased oxidative stress following GSH deficiency may play an important role in the development of obesity from KSR2 variants. Presentation: 6/1/2024

8624 Maternal ZIKV Infection Causes Oxidative Damage in Placenta of Both Female and Male Mice

Abstract Disclosure: V.M. Nascimento: None. I.S. Andrade: None. C.B. Andrade: None. R.S. Alves: None. R.P. Sousa: None. A.C. Valim: None. L.S. Dias: None. K.N. Fontes: None. I.F. Miranda: None. S.A. Coelho: None. L.B. Arruda: None. R.S. Fortunato: None. T.M. Ortiga-Carvalho: None. The placenta is a maternal-fetal organ with nutritional, exchanging, hormonal and protecting functions that are vital for fetal growth and development during gestation. However, these functions can be disturbed by vertically transmitted pathogens such as Zika virus (ZIKV). In 2015, ZIKV was responsible for an increasing number of newborns with congenital abnormalities in Brazil, known as Congenital Zika Syndrome (CZS). Nonetheless, the extension of ZIKV infection in the placenta, leading to CZS, is still unclear. It is known that ZIKV causes oxidative stress in trophoblasts. However, no one has ever studied placental oxidative stress caused by ZIKV infection in vivo. Here, we investigated whether maternal ZIKV infection could cause sex-specific oxidative stress in murine placenta. At gestational day (GD)12.5, pregnant mice (C57BL/6; 8-12 weeks old) were infected with ZIKV-BRPE243 (5x107 PFU; ZIKV group, n=22) or Mock (supernatants of noninfected C6/36 cells; control group, n=28). At GD18.5, c-section was performed and maternal spleens, placentas, fetuses and fetal brains were collected. We measured placental carbonylated protein levels (n=7), by 2D OxyBlot, nitrotyrosine residues (n=5), by immunohistochemistry, reduced thiol content (n=9), total superoxide dismutase (SOD; n=9) and total glutathione peroxidase (GPx; n=9) activities, by spectrophotometry. Data were analyzed by unpaired Student’s t test and Two-Way ANOVA with Tukey’s post test and represented as mean±SEM. ZIKV infection increased maternal spleen weight (p<0.0001) and decreased both female (p=0.0429) and male (p=0.0171) placental weight. Although placental weights were affected by the infection, female and male fetal weight and fetal crown-rump length were not changed. Conversely, maternal ZIKV infection increased only female fetal brain weight (p=0.0028). Moreover, we found that ZIKV infection increased nitrotyrosine residues in the placental labyrinth region of both female (p<0.0001) and male (p=0.0007) fetuses. Meanwhile, nitrotyrosine residues were increased by the infection only in the placental junctional zone of female fetuses (p=0.0131). Although ZIKV increased this oxidative damage biomarker, placental carbonylated protein levels were not changed by the infection in both sexes. Also, reduced thiol content and total SOD and GPx activities were not affected. Together, our data show that maternal ZIKV infection caused oxidative stress in both placental regions of female and male fetuses, as we saw that ZIKV increased oxidative damage, although antioxidant defenses were not changed by the infection, at least at term (GD18.5). ZIKV infection may have disturbed placental growth, as we found that placental weight was decreased in both sexes. Moreover, we believe that the increase in the female fetal brain weight could be a suggestive feature of cerebral edema or hydrocephalus. Presentation: 6/2/2024

7872 Adiponectin Enhances Hepatic Insulin Sensitivity in a Rat Model of Polycystic Ovary Syndrome: Role of PI3K/Akt/mTORC2 Pathway

Abstract Disclosure: A. Abdelhameed: None. S. Rezq: None. R.M. Vinson: None. J. Flaherty: None. B. Kim: None. D.G. Romero: None. L.L. Yanes Cardozo: None. Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women and is associated with a high prevalence of obesity and insulin resistance (IR). Phosphoinositide-3-kinase (PI3K)/Akt/ the mammalian target of rapamycin complex 2 (mTORC2) pathway is one major insulin signaling pathway and its impairment is involved in multiple metabolic disorders by inducing IR. Phosphorylation of protein kinase B (Akt) at (Ser)-473 by mTORC2 leads to its full activation and subsequently enhances insulin sensitivity. Adiponectin is a peptide secreted by adipocytes that increases insulin sensitivity. PEG-BHD1028 is a novel potent peptide adiponectin receptor agonist that enhances insulin sensitivity in different models of diabetes. However, its potential to attenuate androgen-mediated hepatic IR by modulating PI3K/Akt/mTORC2 pathway remains unknown in PCOS. Hypothesis: We tested the hypothesis that PEG-BHD1028 attenuates hepatic IR via modulating PI3K/Akt/mTORC2 pathway in a rat model of PCOS. Methods: To induce PCOS, four-week-old female Sprague Dawley rats were implanted with dihydrotestosterone (DHT) silastic tubes (7.5mg) or placebo (PBO) for 90 days (n=4-8/group). During the final three weeks of DHT treatment, rats received PEG-BHD1028 (25μg/kg/day) subcutaneously. Body weight (BW) by gravimetry, fat and lean mass were measured by Echo-MRI. Fasting plasma insulin was measured by ELISA while fasting plasma glucose was measured with a clinical chemistry analyzer. HOMA-IR, an index of systemic IR, was calculated. Protein levels of insulin signaling markers including IRS1, p-IRS1 (Ser1101), PI3 kinase p110, Akt, p-Akt (Ser473), mTOR, p-mTOR (Ser2481) and mTORC2 regulatory protein Rictor were quantified in the liver by Western-blot. Results: DHT significantly (p<0.05) increased BW, fat mass and lean mass by 1.4-, 2.2-, 1.4- folds, respectively compared to PBO group. DHT significantly (p<0.05) increased HOMA-IR by 3.8- fold, compared to PBO group. DHT downregulated hepatic p-Akt, Akt and mTOR protein expression by 20% to 40% while upregulating IRS-1 by 1.6- fold with no impact on PI3 kinase, p-mTOR, Rictor and p-IRS1 compared to the PBO group. PEG-BHD1028 significantly decreased BW, fat mass and lean mass while attenuating IR (34.7% reduction in HOMA-IR) in PCOS rats. Interestingly, PEG-BHD1028 significantly (p<0.05) increased PI3K, active Akt (pAKT), mTOR and Rictor levels by 1.6-, 2.2-, 1.5-, 1.8- folds, respectively with no effect on the other insulin signaling markers compared to vehicle-treated group. Conclusion: Our findings suggest that PEG-BHD1028 is a novel and effective therapeutic agent to attenuate hepatic IR in PCOS via activating PI3K/Akt/mTORC2 pathway. Significance: Targeting adiponectin signaling could be a novel and effective therapeutic approach to mitigate metabolic dysfunction in females with excess androgens. Presentation: 6/2/2024

6603 Impact Of The Monocarbonyl Analogs Of Curcumin, C66 And B2BrBC, On The Protein Expression Of Epithelial And Mesenchymal Markers In MCF-7 Breast Cancer Cells

Abstract Disclosure: R. Stojchevski: None. A. Lavi: None. J. Bogdanov: None. N. Hadzi-Petrushev: None. M. Mladenov: None. L. Poretsky: None. D.B. Avtanski: None. The synthesis of monocarbonyl analogs of curcumin (MACs) presents a solution to the challenges posed by curcumin, a natural polyphenol extracted from the roots of the turmeric plant (Curcuma longa), which is known for its antioxidant and anti-inflammatory properties. The limitations of curcumin, including poor stability, low bioavailability, and rapid metabolism, hinder its application, and MACs, designed without a β-diketone moiety, address these drawbacks and exhibit enhanced stability and bioavailability. This study aimed to assess the effects of two monocarbonyl analogs of curcumin, (2E,6E)-2,6-bis[(2-trifluoromethyl)benzylidene]cyclohexanone (C66) and (2E,6E)-2,6-bis(2-bromobenzylidene)cyclohexanone (B2BrBC), on epithelial-to-mesenchymal transition (EMT) in MCF-7 breast cancer cells using western blot analysis. Our previous findings indicated that these analogs can potentially affect EMT by modulating the epithelial and mesenchymal gene expression. MCF-7 cells were cultured in complete medium supplemented with EMT-inducing factors for two days, followed by C66 and B2BrBC administration (100 µM) for three additional days. After the experiments, proteins were extracted, and western blot analyses were performed to measure the expression levels of various epithelial and mesenchymal markers (E-cadherin, N-cadherin, SNAI1, and Claudin-1). The results revealed that both C66 and B2BrBC increased the protein levels of the epithelial marker E-cadherin and suppressed those of the mesenchymal marker N-cadherin. Moreover, C66 and B2BrBC decreased the protein expression of the transcription factor SNAI1 and transmembrane protein Claudin-1. Between the two MACs, B2BrBC showed a more pronounced effect on N-cadherin expression, while C66 was more potent in suppressing SNAI1 protein expression. Our findings demonstrate that C66 and B2BrBC counteract EMT, contributing to the potential therapeutic applications of MACs in managing breast cancer progression. Presentation: 6/3/2024

8624 Maternal ZIKV Infection Causes Oxidative Damage in Placenta of Both Female and Male Mice

Abstract Disclosure: V.M. Nascimento: None. I.S. Andrade: None. C.B. Andrade: None. R.S. Alves: None. R.P. Sousa: None. A.C. Valim: None. L.S. Dias: None. K.N. Fontes: None. I.F. Miranda: None. S.A. Coelho: None. L.B. Arruda: None. R.S. Fortunato: None. T.M. Ortiga-Carvalho: None. The placenta is a maternal-fetal organ with nutritional, exchanging, hormonal and protecting functions that are vital for fetal growth and development during gestation. However, these functions can be disturbed by vertically transmitted pathogens such as Zika virus (ZIKV). In 2015, ZIKV was responsible for an increasing number of newborns with congenital abnormalities in Brazil, known as Congenital Zika Syndrome (CZS). Nonetheless, the extension of ZIKV infection in the placenta, leading to CZS, is still unclear. It is known that ZIKV causes oxidative stress in trophoblasts. However, no one has ever studied placental oxidative stress caused by ZIKV infection in vivo. Here, we investigated whether maternal ZIKV infection could cause sex-specific oxidative stress in murine placenta. At gestational day (GD)12.5, pregnant mice (C57BL/6; 8-12 weeks old) were infected with ZIKV-BRPE243 (5x107 PFU; ZIKV group, n=22) or Mock (supernatants of noninfected C6/36 cells; control group, n=28). At GD18.5, c-section was performed and maternal spleens, placentas, fetuses and fetal brains were collected. We measured placental carbonylated protein levels (n=7), by 2D OxyBlot, nitrotyrosine residues (n=5), by immunohistochemistry, reduced thiol content (n=9), total superoxide dismutase (SOD; n=9) and total glutathione peroxidase (GPx; n=9) activities, by spectrophotometry. Data were analyzed by unpaired Student’s t test and Two-Way ANOVA with Tukey’s post test and represented as mean±SEM. ZIKV infection increased maternal spleen weight (p<0.0001) and decreased both female (p=0.0429) and male (p=0.0171) placental weight. Although placental weights were affected by the infection, female and male fetal weight and fetal crown-rump length were not changed. Conversely, maternal ZIKV infection increased only female fetal brain weight (p=0.0028). Moreover, we found that ZIKV infection increased nitrotyrosine residues in the placental labyrinth region of both female (p<0.0001) and male (p=0.0007) fetuses. Meanwhile, nitrotyrosine residues were increased by the infection only in the placental junctional zone of female fetuses (p=0.0131). Although ZIKV increased this oxidative damage biomarker, placental carbonylated protein levels were not changed by the infection in both sexes. Also, reduced thiol content and total SOD and GPx activities were not affected. Together, our data show that maternal ZIKV infection caused oxidative stress in both placental regions of female and male fetuses, as we saw that ZIKV increased oxidative damage, although antioxidant defenses were not changed by the infection, at least at term (GD18.5). ZIKV infection may have disturbed placental growth, as we found that placental weight was decreased in both sexes. Moreover, we believe that the increase in the female fetal brain weight could be a suggestive feature of cerebral edema or hydrocephalus. Presentation: 6/2/2024

6652 Precision Diagnostic Approaches for Assessing Clinical Vitamin D Status

Abstract Disclosure: Z. Shahidzadeh Yazdi: None. E.A. Streeten: None. H.B. Whitlatch: Grant Recipient; Self; Regeneron Pharmaceuticals. M.E. Montasser: Grant Recipient; Self; Regeneron Pharmaceuticals. A.L. Beitelshees: None. S.I. Taylor: Consulting Fee; Self; Ionis Pharmaceuticals Inc.. Background . The body has evolved effective homeostatic mechanisms to maintain free 1,25-dihydroxyvitamin D [1,25(OH)2D] within narrow physiological ranges. Using a mathematical model to quantitate 24-hydroxylase activity, we concluded that mild-moderate vitamin D [VitD] deficiency suppresses 24-hydroxylase activity (the first line of defense), thereby suppressing clearance of 1,25(OH)2D. Severe VitD deficiency triggers secondary hyperparathyroidism (the second line of defense) to sustain physiological levels of 1,25(OH)2D. Objective. To compare performance of various indices of VitD status in predicting whether homeostatic mechanisms have been triggered. Methods. We studied 11 otherwise healthy VitD deficient (25-hydroxyvitamin D [25(OH)D] ≤20 ng/mL) participants before and after VitD3 supplementation (50,000 IU once or twice a week depending on BMI for 4-6 weeks). We measured 25(OH)D, 1,25(OH)2D, 24,25-dihydroxyvitamin D [24,25(OH)2D], PTH, and FGF23 before and after VitD3 supplementation. We calculated three VitD metabolite ratios and also a PTH index (i.e., PTH level before divided by PTH level after VitD3 supplementation) to normalize PTH levels relative to each individual’s setpoint. We then compared the correlation between the PTH index and various indices of VitD status. Results. VitD3 supplements induced a 2.7-fold increase (p=0.0006) in mean 25(OH)D and a 4.3-fold increase (p=0.0002) in 24,25(OH)2D, but did not change 1,25(OH)2D, PTH, or FGF23. While relative 24-hydroxylase activity (h) was highly correlated with the 1,25(OH)2D/24,25(OH)2D ratio (ρ2=0.94; p=3E-13), it was less well correlated with the 24,25(OH)2D/25(OH) ratio (ρ2=0.67; p=3E-06) and baseline total 25(OH)D (ρ2=0.72; p=7E-07). Furthermore, we investigated the correlation between the PTH index and baseline 25(OH)D and three VitD metabolite ratios. The correlation coefficient was higher when we plotted the PTH index as a function of baseline 25(OH)D before VitD3 supplementation (R2=0.23; p=2E-06) compared to the correlation of baseline PTH with baseline 25(OH)D (R2=0.08; p=5E-05). While the correlation coefficient for the correlation of PTH index with 1,25(OH)2D/24,25(OH)2D was 0.60 (R2=0.36; p=0.0002), it was 0.44 for 25(OH)D/1,25(OH)2D ratio (R2=0.20; p=0.0001) and 0.35 for the 24,25(OH)2D/25(OH)D ratio (R2=0.12; p=2E-06). Conclusions. Measuring total 25(OH)D level did not provide optimal assessment of clinical VitD status – probably because it does not account for the threefold inter-individual variation in VitD binding protein level. Of all the indices, the 1,25(OH)2D/24,25(OH)2D ratio best predicted triggering of two homeostatic mechanisms: suppression of 24-hydroxylase and induction of secondary hyperparathyroidism due to VitD deficiency. Presentation: 6/2/2024

Astrocytic CREB in nucleus accumbens promotes susceptibility to chronic stress

BackgroundIncreasing evidence implicates astrocytes in stress and depression in both rodent models and human Major Depressive Disorder (MDD). Despite this, little is known about the transcriptional responses to stress of astrocytes within the nucleus accumbens (NAc), a key brain reward region, and their influence on behavioral outcomes. MethodsWe used whole cell sorting, RNA-sequencing, and bioinformatic analyses to investigate the NAc astrocyte transcriptome in male mice in response to chronic social defeat stress (CSDS). Immunohistochemistry was used to determine stress-induced changes in astrocytic CREB within the NAc. Finally, astrocytic regulation of depression-like behavior was investigated using viral-mediated manipulation of CREB in combination with CSDS. ResultsWe found a robust transcriptional response in NAc astrocytes to CSDS in stressed mice, with changes seen in both stress-susceptible and stress-resilient animals. Bioinformatic analysis revealed CREB, a transcription factor widely studied in neurons, as one of the top-predicted upstream regulators of the NAc astrocyte transcriptome, with opposite activation states implicated in resilient vs. susceptible mice. This bioinformatic deduction was confirmed at the protein level with immunohistochemistry. Moreover, NAc astrocyte morphological complexity correlated with CREB activation and was reduced selectively in astrocytes of resilient mice. Viral overexpression of CREB selectively in NAc astrocytes promoted susceptibility to chronic stress. ConclusionsTogether, our data demonstrate that the astrocyte transcriptome responds robustly to CSDS and that transcriptional regulation in astrocytes contributes to depressive-like behaviors. A better understanding of transcriptional regulation in astrocytes may reveal unknown molecular mechanisms underlying neuropsychiatric disorders.

Evaluating the effects of dietary patterns on circulating C-reactive protein levels in the general adult population: an umbrella review of meta-analyses of interventional and observational studies.

Adopting a healthy dietary pattern may be an initial step in combating inflammation-related chronic diseases; however, a comprehensive synthesis evaluating current evidence is lacking. This umbrella review aimed to summarise the current evidence on the effects of dietary patterns on circulating C-reactive protein (CRP) levels in adults. We conducted an exhaustive search of the Pubmed, Scopus and Epistemonikos databases, spanning from their inception to November 2023, to identify systematic reviews and meta-analyses across all study designs. Subsequently, we employed a random-effects model to recompute the pooled mean difference. Methodological quality was assessed using the A Measurement Tool to Assess Systematic Reviews 2 (AMSTAR 2) checklist, and evidence certainty was categorised as non-significant, weak, suggestive, highly suggestive or convincing (PROSPERO: CRD42023484917). We included twenty-seven articles with thirty meta-analyses of seven dietary patterns, fifteen of which (50 %) exhibited high methodological quality. The summary effects of randomised controlled trials (RCT) found that the Mediterranean diet was the most effective in reducing circulating CRP levels, followed by Vegetarian/Vegan and Energy-restricted diets, though the evidence was of weak quality. In contrast, Intermittent Fasting, Ketogenic, Nordic and Paleolithic diets did not show an inverse correlation with circulating CRP levels. Some results from combined interventional and observational studies, as well as solely observational studies, also agreed with these findings. These dietary patterns show the potential in reducing CRP levels in adults, yet the lack of high-quality evidence suggests future studies may alter the summary estimates. Therefore, further well-conducted studies are warranted.

NAT10/ac4C/JunB facilitates TNBC malignant progression and immunosuppression by driving glycolysis addiction

BackgroundN4-Acetylcytidine (ac4C), a highly conserved post-transcriptional mechanism, plays a pivotal role in RNA modification and tumor progression. However, the molecular mechanism by which ac4C modification mediates tumor immunosuppression remains elusive in triple-negative breast cancer (TNBC).MethodsNAT10 expression was analyzed in TNBC samples in the level of mRNA and protein, and compared with the corresponding normal tissues. ac4C modification levels also measured in the TNBC samples. The effects of NAT10 on immune microenvironment and tumor metabolism were investigated. NAT10-mediated ac4C and its downstream regulatory mechanisms were determined in vitro and in vivo. The combination therapy of targeting NAT10 in TNBC was further explored.ResultsThe results revealed that the loss of NAT10 inhibited TNBC development and promoted T cell activation. Mechanistically, NAT10 upregulated JunB expression by increasing ac4C modification levels on its mRNA. Moreover, JunB further up-regulated LDHA expression and facilitated glycolysis. By deeply digging, remodelin, a NAT10 inhibitor, elevated the surface expression of CTLA-4 on T cells. The combination of remodelin and CTLA-4 mAb can further activate T cells and inhibite tumor progression.ConclusionTaken together, our study demonstrated that the NAT10-ac4C-JunB-LDHA pathway increases glycolysis levels and creates an immunosuppressive tumor microenvironment (TME). Consequently, targeting this pathway may assist in the identification of novel therapeutic strategies to improve the efficacy of cancer immunotherapy.