Significant Increase In mRNA Expression Research Articles

Ergosterol and its metabolites as agonists of Liver X receptor and their anticancer potential in colorectal cancer

Aberrant cholesterol homeostasis is a well-recognized hallmark of cancer and is implicated in metastasis as well as chemotherapeutic resistance, the two major causes of cancer associated mortality. Liver X receptors (LXRs) are the key transcription factors that induce cholesterol efflux via enhancing the expression of ABCA1 and ABCG1. Therefore, a comprehensive analysis of several novel sterols namely ergosta-7,22,24(28)-trien-3β-ol (Erg1), ergosta-5,22,25-trien-3-ol (Erg2), ergosta-5,7,22,24(28)-tetraen-3β-ol (Erg3), and ergosta-7,22-dien-3β-ol (Erg4) as LXR agonists has been performed. Molecular docking studies have shown that these sterols possess higher binding affinities for LXRs as compared to the reference ligands (GW3965 and TO901317) and also formed critical activating interactions. Molecular dynamic (MD) simulations further confirmed that docking complexes made of these sterols possess significant stability. To assess the extent of LXR activation, ABCA1 promoter was cloned into luciferase reporter plasmid and transfected into HCT116 cells. It was observed that treatment with Erg, Erg2 and Erg4 led to a significant LXR activation with an EC50 of 5.64 µM, 4.83 and 3.03 µM respectively. Furthermore, a significant increase in mRNA expression of NR1H2 and LXR target genes i.e. ABCA1, ABCG1 and ApoE was observed upon Erg treatment. Flow cytometric analysis have revealed a significant increase in the accumulation of ABCA1 upon Erg treatment. Cytotoxicity studies conducted on colorectal cancer cell and normal epithelial cell line showed that these sterols are selectively toxic towards cancer cells. Taken together, our findings suggests that ergosterol activates LXRs, have significant anticancer activity and could be a likely candidate to manage aberrant cholesterol homeostasis.

Lactuca sativa L. Extract Enhances Sleep Duration Through Upregulation of Adenosine A1 Receptor and GABAA Receptors Subunits in Pentobarbital-Injected Mice.

We investigated the effects of Lactuca sativa L. extracts (Lactuc) on pentobarbital-induced sleep in mice to elucidate the mechanisms underlying its impact on sleep quality. Mice were randomly assigned to five groups: control, positive control (diazepam 2 mg/kg b.w.), and three groups orally administered with Lactuc (50, 100, and 200 mg/kg b.w.). After 2 weeks of oral administration and intraperitoneal injections, the mice were killed. We found that the Lactuc-administered groups had significantly reduced sleep latency and increased sleep duration compared with the control group. Furthermore, the oral administration of Lactuc induced a significant increase in mRNA expression and protein expression of adenosine A1 receptor in the brains compared with the expressions in the control group. In addition, the Lactuc-administered groups exhibited significantly higher levels of mRNA expressions of GABAA receptors subunits α2, β2, γ1, and, γ2 in the brain tissue. Therefore, we suggest that Lactuc could be used to develop natural products that effectively improve sleep quality and duration.

Effect and Mechanism of Mycobacterium avium MAV-5183 on Apoptosis of Mouse Ana-1 Macrophages.

To investigate the effects and mechanisms of Mycobacterium avium MAV-5183 protein on apoptosis in mouse Ana-1 macrophages. A pET-21a-MAV-5183 recombinant plasmid was constructed. The recombinant MAV-5183 protein was cloned, expressed, purified, and identified using an anti-His-tagged antibody. Rabbits were immunized to obtain antiserum, and its potency and immunoreactivity were assessed through WB. Mouse Ana-1 macrophages were incubated with varying concentrations of MAV-5183 protein. Flow cytometry, following ANNEXIN V-FITC/PI double staining, detected apoptosis. Western Blot analysis was conducted to identify apoptosis-related molecules Caspase-9/8/3 and vesicle-related molecules ASC, NLRP3, and Cleaved-casp1. ELISA measured TNF-α and IL-6 levels in the culture supernatant. LDH activity and ROS levels were analyzed separately. RT-qPCR measured mRNA levels of Caspase-9/8/3, ASC, NLRP3, Caspase-1, IL-1β, Bax, MAPK-p38, Bcl-2, TNF-α, and IL-6. MAV-5183 protein was successfully cloned, purified, and identified. In in vitro studies on Ana-1 macrophages, MAV-5183 protein increased the expression of Caspase-9/8/3, ASC, NLRP3 (P < 0.01), induced ROS secretion (P < 0.05), and promoted inflammatory cytokine secretion (TNF-α, IL-6, P < 0.0001); however, it did not significantly affect LDH (P > 0.05). MAV-5183 also induced apoptosis in Ana-1 macrophages (P < 0.05). RT-qPCR results indicated a significant increase in mRNA expression of Caspase-9/8/3, ASC, NLRP3, TNF-α, IL-6, MAPK-p38, and pro-apoptotic factor Bax (P < 0.01), with no significant effect on Bcl-2 and IL-1β mRNA (P > 0.05). The data indicate that MAV-5183 induces macrophage apoptosis through a caspase-dependent pathway and promotes inflammatory cytokine secretion via ROS.

A200 EXPOSURE TO CANNABIS SMOKE IN UTERO AFFECTS THE DEVELOPMENT OF THE ENDOCANNABINOID SYSTEM IN THE GASTROINTESTINAL TRACT

Abstract Background Recreational use of cannabis has been legalized in Canada since 2018. Increasing numbers of women are using cannabis while pregnant, with reported rates ampersand:003E10%. There is a disconnect with societal perceptions, with up to one third of women believing that cannabis is safe for use in pregnancy, but with preclinical and clinical studies demonstrating negative neurodevelopmental outcomes. A key component of gut-brain axis regulation is found in the endocannabinoid system (ECS), of which receptors, ligands and enzymes are extensively distributed throughout the gastrointestinal (GI) tract and are actively involved in GI motility and sensation. Aims We tested the hypothesis that exposure to cannabis smoke in utero could influence the expression of ECS components in the developing GI tract. Methods We developed a “real world” cannabis smoke inhalation model in which CD-1 pregnant mouse dams were exposed daily to a delta-9-tetrahydrocannabinol (THC)-dominant strain of cannabis in a smoking chamber unit, from embryonic days 6 (E6) to E18. Control mice were placed in a restraining cage and exposed to room air for the equivalent time-period. Dams were sacrificed at E18, fetuses removed and the fetal GI tract (from stomach to distal colon) collected for RNA extraction. Key components of the ECS were investigated by developing a NanoString Custom CodeSet, including receptors (cannabinoid receptors 1 and 2; CB1 and CB2, transient receptor potential vanilloid type 1; TRVP1, GPR55) and enzymes (diacylglycerol lipase; DAGL, fatty acid amide hydrolase; FAAH, monoacylglycerol lipase; MAGL, N-acylphosphatidyletanolamine phospholipase D; NAPE-PLD). Samples (n=per litter; 7 cannabis-exposed and 8 control) were processed in the Farncombe Metagenomics Facility and NanoString data analyzed using nSolver Analysis Software. Results We found a significant increase in the expression of ECS receptors in the cannabis-exposed compared to control GI tract at E18, including CB2 (pampersand:003C0.05), TRPV1 (pampersand:003C0.05) and GPR55 (pampersand:003C0.0005). Interestingly, no significant change in mRNA expression was detected for CB1. We also found a significant increase in mRNA encoding the full panel of ECS enzymes, including DAGL (pampersand:003C0.005), FAAH (pampersand:003C0.05), MAGL (pampersand:003C0.05) and NAPE-PLD (pampersand:003C0.005) in cannabis-exposed compared to control GI tract. Conclusions In conclusion, we have found a significant increase in mRNA expression in a panel of key ECS receptors and enzymes in the GI tract at a point of maximal exposure to cannabis in utero. Future research will be needed to further characterize potential impacts on postnatal GI development and function. Funding Agencies CIHRThe Azrieli Foundation

A12 ROLE OF INTESTINAL EPITHELIAL HNF4A IN PROTECTING AGAINST ACUTE INFLAMMATORY STIMULI

Abstract Background Inflammatory bowel disease is a group of chronic resulting from complex interactions between host susceptibility genes, the host microbiome, the immune system, and environmental triggers. The gastrointestinal epithelium acts as a barrier against harmful stimuli, and defects in its integrity are a precursor for the development of IBD. Our laboratory has shown that the conditional deletion of Hnf4a in the mouse intestinal epithelium leads to chronic intestinal inflammation. However, the functional role of HNF4A on epithelial barrier integrity is still controversial. Aims To evaluate the impact of Hnf4a deletion on barrier integrity during acute inflammation. Methods We used a tamoxifen-inducible CreER-loxP system to delete Hnf4a in the adult intestinal epithelium (Hnf4aΔIEC-ind). Intestinal permeability was assessed in Hnf4aΔIEC-ind and control mice in the collected serum after oral gavage of FITC-dextran. Mice were infected with an invasion-attenuated strain of Salmonella Typhimurium (SB103). S. Typhimurium loads were scored in the feces, liver, and spleen tissue. Histological analysis was carried out by various staining. Also, we used a model of T-cell-induced enteropathy triggered by a T-cell activating monoclonal anti-CD3ε Ab (145-2C11). Mice were assessed for clinical signs of enteropathy and sacrificed 1-, 3-, and 5 days post-injection. Gene expression of selected targets was studied by RT-qPCR. Results FITC-dextran assay showed a significant increase of the intestinal permeability in uninfected mutant mice compared to controls. In contrast, an oral infection with an invasion-deficient S. Typhimurium strain did not differentially impact in tissue bacterial loads or epithelial damage between groups. Likewise, the loss of epithelial HNF4A led to increased Muc2 transcripts expression in the ileum of infected mutant mice but not in the colon. Histological examinations at four days post-infection showed an elevated number and size of goblet cells in the ileal crypts of mutant mice. Bacterial localization using the general bacterial probe EUB388-cy3 (red) staining showed that infected mutant mice tended to enhance barrier mucus layer thickness compared to the controls. On the contrary, acute T cell activation with anti-CD3 mAb induced a significant increase of mRNA expression of Cxcl1 in the small intestine of mutant mice. H&amp;E staining showed elongated ileal crypts in mutant mice compared to controls after anti-CD3 injection. Conclusions Altogether, these results support that HNF4A could play a crucial adaptive role in modulating the small intestine epithelial barrier function and controlling the immune system during acute inflammatory stress. Funding Agencies CCC, CIHR

Deletion of a conserved genomic region associated with adolescent idiopathic scoliosis leads to vertebral rotation in mice.

Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis, in which spinal curvature develops in adolescence, and 90% of patients are female. Scoliosis is a debilitating disease that often requires bracing or surgery in severe cases. AIS affects 2%-5.2% of the population; however, the biological origin of the disease remains poorly understood. In this study, we aimed to determine the function of a highly conserved genomic region previously linked to AIS using a mouse model generated by CRISPR-CAS9 gene editing to knockout this area of the genome to understand better its contribution to AIS, which we named AIS_CRMΔ. We also investigated the upstream factors that regulate the activity of this enhancer in vivo, whether the spatial expression of the LBX1 protein would change with the loss of AIS-CRM function, and whether any phenotype would arise after deletion of this region. We found a significant increase in mRNA expression in the developing neural tube at E10.5, and E12.5, for not only Lbx1 but also other neighboring genes. Adult knockout mice showed vertebral rotation and proprioceptive deficits, also observed in human AIS patients. In conclusion, our study sheds light on the elusive biological origins of AIS, by targeting and investigating a highly conserved genomic region linked to AIS in humans. These findings provide valuable insights into the function of the investigated region and contribute to our understanding of the underlying causes of this debilitating disease.

A polyamino acid-based phosphatidyl polymer library for in vivo mRNA delivery with spleen targeting ability.

A qualified delivery system is crucial for the successful application of messenger RNA (mRNA) technology. While lipid nanoparticles (LNPs) are currently the predominant platform for mRNA delivery, they encounter challenges such as high inflammation and difficulties in targeting non-liver tissues. Polymers offer a promising delivery solution, albeit with limitations including low transfection efficiency and potential high toxicity. Herein, we present a poly(L-glutamic acid)-based phosphatidyl polymeric carrier (PLG-PPs) for mRNA delivery that combines the dual advantages of phospholipids and polymers. The PLGs grafted with epoxy groups were firstly modified with different amines and then with alkylated dioxaphospholane oxides, which provided a library of PLG polymers grafted with various phosphatidyl groups. In vitro studies proved that PLG-PPs/mRNA polyplexes exhibited a significant increase in mRNA expression, peaking 14 716 times compared to their non-phosphatidyl parent polymer. Impressively, the subset PA8-PL3 not only facilitated efficient mRNA transfection but also selectively delivered mRNA to the spleen instead of the liver (resulting in 69.73% protein expression in the spleen) once intravenously administered. This type of phosphatidyl PLG polymer library provides a novel approach to the construction of mRNA delivery systems especially for spleen-targeted mRNA therapeutic delivery.

Citrullinated and malondialdehyde-acetaldehyde modified fibrinogen activates macrophages and promotes an aggressive synovial fibroblast phenotype in patients with rheumatoid arthritis.

Post-translational protein modifications with malondialdehyde-acetaldehyde (MAA) and citrulline (CIT) are implicated in the pathogenesis of rheumatoid arthritis (RA). Although precise mechanisms have not been elucidated, macrophage-fibroblast interactions have been proposed to play a central role in the development and progression of RA. The purpose of our study was to evaluate the downstream effects of macrophage released soluble mediators, following stimulation with fibrinogen (FIB) modified antigens, on human fibroblast-like synoviocytes (HFLS). PMA-treated U-937 monocytes (Mϕ) and macrophage-differentiated peripheral blood mononuclear cells (MP) were stimulated with FIB, FIB-MAA, FIB-CIT, or FIB-MAA-CIT. HFLS-RA cells were stimulated directly with FIB antigens or with supernatants (SN) from macrophages (Mϕ-SN or MP-SN) stimulated with FIB antigens. Genes associated with an aggressive HFLS phenotype, extracellular matrix proteins, and activated signaling pathways were evaluated. HFLS-RA cells treated with Mϕ-SNFIB-CIT and Mϕ-SNFIB-MAA-CIT demonstrated significant increases in mRNA expression of genes associated with an aggressive phenotype at 24-h as compared to direct stimulation with the same antigens. Similar results were obtained using MP-SN. Cellular morphology was altered and protein expression of vimentin (p<0.0001 vs. Mϕ-SNFIB) and type II collagen (p<0.0001) were significantly increased in HFLS-RA cells treated with any of the Mϕ-SN generated following stimulation with modified antigens. Phosphorylation of JNK, Erk1/2, and Akt were increased most substantially in HFLS-RA treated with Mϕ-SNFIB-MAA-CIT (p<0.05 vs Mϕ-SNFIB). These and other data suggested the presence of PDGF-BB in Mϕ-SN. Mϕ-SNFIB-MAA-CIT contained the highest concentration of PDGF-BB (p<0.0001 vs. Mϕ-SNFIB) followed by Mϕ-SNFIB-CIT then Mϕ-SNFIB-MAA. HFLS-RA cells treated with PDGF-BB showed similar cellular morphology to the Mϕ-SN generated following stimulation with modified FIB, as well as the increased expression of vimentin, type II collagen, and the phosphorylation of JNK, Erk1/2 and Akt signaling molecules. Together, these findings support the hypothesis that in response to MAA-modified and/or citrullinated fibrinogen, macrophages release soluble factors including PDGF-BB that induce fibroblast activation and promote an aggressive fibroblast phenotype. These cellular responses were most robust following macrophage activation with dually modified fibrinogen, compared to single modification alone, providing novel insights into the combined role of multiple post-translational protein modifications in the development of RA.

Abstract 568: Activation of the RXR nuclear receptor regulates CD4 T cell activity in a murine model of HER2+ breast cancer

Abstract The role of CD4 T cells for anti-tumor responses is less recognized than CD8 cells. Evidence suggests that CD4 T cells can act directly as antitumor cells or direct the maturation and activation of dendritic cells (DCs). The retinoid X receptor (RXR) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors. RXR and its heterodimers have profound effects on the function of myeloid cells, but effects on T cells are less known. Treatment with the RXR agonist MSU42011 (100 mg/Kg diet) reduced tumor burden in a HER2+ model of breast cancer (p=0.0068) and in a KRAS-driven model of lung cancer (p=0.0015). However, when human lung cancer cells were implanted into immunodeficient mice, MSU42011 failed to reduce tumor burden, suggesting that the anti-tumor properties of MSU42011 are dependent on a functional immune system. After 10 days of treatment, the percentage of CD4, CD62L+ T cells in HER2+ mammary tumors treated with MSU42011 was significantly higher compared to the control group (2.41 vs 3.78, p=0.05), as analysed by flow cytometry. Additionally, tumor antigen presenting cells (CD11c+, CD11b+, IA-IE+) and DCs (CD11c+, IA-IE+) had increased expression of PDL1 in the MSU42011 group when compared with controls (MFI=4016 vs 5225, p=0.069; 5484 vs 7140, p=0.045, respectively). RT-PCR analysis of whole tumors showed a significant increase in mRNA expression of interferon (IFN; p=0.0026) and IL12a (p=0.0011) in treated mice. Similar increases in IFN and IL12a were found in the murine model of lung cancer. Next, T cells were depleted to determine if required for the anti-tumor effects of MSU42011. CD8 cells depletion did not reverse the anti-tumor efficacy of MSU42011. CD4 depletion in mice treated with MSU42011 rescued tumor growth (p=0.016 MSU42011 vs MSU42011+anti-CD4), as tumors of mice treated with MSU42011 and anti-CD4 antibodies for 10 days grew at the same rate as tumors treated with control plus or minus anti-CD4 antibodies. Tumors treated with MSU42011 and anti-CD4 antibodies also showed reduced levels of IL12a compared with MSU42011 alone (2.28 vs 0.87, p&amp;gt;0.05), as determined by RT-PCR.To dissect the effects of RXR activation on CD4 and DC cells, primary DCs, alone or co-cultured with T cells and conditioned media from cancer cells, were treated with MSU42011 (100 nM). This treatment increased PDL1 and IL12 mRNA expression in DCs. T cells, co-cultured with DC and conditioned media and treated with MSU42011, expressed higher IL2 mRNA levels. MSU42011 also increased expression of IFN in CD3 T cells. In conclusion, MSU42011 increased recruitment and activation of CD4 T cells in vitro and in HER2+ mammary tumors. These data, in combination with our previous work showing that RXR agonists reduce expression of tumor-promoting FOXP3 T cells in vitro and in vivo, confirm a strong correlation between RXR pharmacologic activation and CD4 T cell activation within the tumor microenvironment. Citation Format: Ana Sofia Leal, Karen T. Liby. Activation of the RXR nuclear receptor regulates CD4 T cell activity in a murine model of HER2+ breast cancer [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 568.

Gochujang Consumption Prevents Metabolic Syndrome in a High-Fat Diet Induced Obese Mouse Model

High-fat and high-salt diets are risk factors for metabolic syndrome development. However, gochujang, which has a high salt content, possesses antiobesity properties in cell and animal models. We aimed to evaluate the effects of Sunchang traditional and modern factory produced gochujang on metabolic syndrome factors in high-fat diet (HFD)-induced obese mice. For 14 weeks, 4-week-old C57BL/6J male mice were separated into five groups and fed a normal diet (ND), a high-fat diet only (HD), a HD with salt (SALT), a HD with traditional Sunchang gochujang (TS), and HD with modern factory made Sunchang gochujang (FS). Compared to HD and SALT groups, the gochujang groups had lower body weight, blood leptin, and insulin levels with reduced Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index and improved serum and liver lipid profiles. In addition, gochujang supplemented groups exhibited a significant reduction in mRNA expression of anabolic lipid metabolism related factors; PPARγ, CEBPα, and FABP4, and a significant increase in mRNA expression of energy expenditure-related factors; PPARα and CPT1. Protein expressions of SREBP1 were downregulated in the gochujang fed groups. TS and FS intakes improved obesity in HFD-induced obese mice. Compared to the gochujang groups, the SALT group did not exhibit any of those benefits suggesting that the high salt content of gochujang has different effects compared with added salt alone. Our findings provide evidence that gochujang could be a functional food to attenuate metabolic syndrome.

Enterobacter asburiae E7, a Novel Potential Probiotic, Enhances Resistance to Aeromonas veronii Infection via Stimulating the Immune Response in Common Carp (Cyprinus carpio).

Probiotics are an alternative strategy for antibiotics, but most probiotics are Gram-positive bacteria suitable for terrestrial animals. Therefore, it is imperative to develop dedicated probiotics for the common carp industry to be ecologically efficient and environmentally friendly. A novel Enterobacter asburiae named E7 was isolated from the intestine of healthy common carp and displayed an extensive antibacterial spectrum against Aeromonas hydrophila, A. veronii, A. caviae, A. media, A. jandaei, A. enteropelogenes, A. schubertii, A. salmonicida, Pseudomonas aeruginosa, Ps. putida, Plesiomonas shigelloides, and Shewanella. E7 was nonpathogenic to the host and susceptible to the majority of antibiotics used in human clinical practice. E7 could grow between 10 and 45°C and between pH 4 and 7 and was extremely resistant to 4% (wt/vol) bile salts. Diets were supplemented with 1 × 107 CFU/g E. asburiae E7 for 28 days. No significant difference in the growth of fish was observed. Expression of immune-related genes IL-10, IL-8, and lysozyme in common carp kidney was significantly upregulated at weeks 1, 2, and 4 (P < 0.01). A significant upregulation of IL-1β, IFN, and TNF-α expression was observed after week 4 (P < 0.01). There was a significant increase in mRNA expression of TGF-β at week 3 (P < 0.01). Following challenge by Aeromonas veronii, the survival rate (91.05%) was significantly higher than observed in the controls (54%; P < 0.01). Collectively, E. asburiae E7 is a promising new Gram-negative probiotic that can enhance health and bacterial resistance of aquatic animals and could thus be developed as an exclusive aquatic probiotic. IMPORTANCE In the present study, we evaluated for the first time the efficiency of Enterobacter asburiae as a prospective probiotic for aquaculture applications. The E7 strain showed extensive resistance to Aeromonas, no pathogenicity to the host, and stronger environmental tolerance. We observed that the resistance of common carp to A. veronii was enhanced by feeding a diet containing 1 × 107 CFU/g E. asburiae E7 for 28 days, but growth was not improved. Strain E7 can act as an immunostimulant to induce the upregulation of some innate cellular and humoral immune responses, resulting in enhanced resistance to A. veronii. Hence, the continuous activation of immune cells can be maintained by adding suitable fresh probiotics to the diet. E7 has the potential to act as a probiotic agent for green, sustainable aquaculture and aquatic product safety.

Effect of polycyclic aromatic hydrocarbons on homeobox gene expression during embryonic development of cuttlefish, Sepia officinalis

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POPs) commonly found in marine environments. Their bioaccumulation can cause harm to aquatic organisms, including invertebrates, particularly during the early stages of embryonic development. In this study, we evaluated, for the first time, the patterns of PAH accumulation in both capsule and embryo of common cuttlefish (Sepia officinalis). In addition, we explored the effects of PAHs by analysing the expression profiles of seven homeobox genes [i.e., gastrulation brain homeobox (GBX), paralogy group labial/Hox1 (HOX1), paralogy group Hox3 (HOX3), dorsal root ganglia homeobox (DRGX), visual system homeobox (VSX), aristaless-like homeobox (ARX) and LIM-homeodomain transcription factor (LHX3/4)]. We found that PAH levels in egg capsules were higher than those observed in chorion membranes (35.1 ± 13.3 ng/g vs 16.4 ± 5.9 ng/g). Furthermore, PAHs were also found in perivitellin fluid (11.5 ± 5.0 ng/ml). Naphthalene and acenaphthene were the congeners present at highest concentrations in each analysed egg component suggesting higher bioaccumulation rates. Embryos with high concentrations of PAHs also showed a significant increase in mRNA expression for each of the analysed homeobox genes. In particular, we observed a 15-fold increase in the ARX expression levels. Additionally, the statistically significant variation in homeobox gene expression patterns was accompanied by a concomitant increase in mRNA levels of both aryl hydrocarbon receptor (AhR) and estrogen receptor (ER). These findings suggest that bioaccumulation of PAHs may modulate developmental processes of cuttlefish embryos by targeting homeobox gene-mediated transcriptional outcomes. Mechanisms underlying the upregulation of homeobox genes could be related to the ability of PAHs to directly activate AhR- or ER-related signaling pathways.

Characterization of a Stemness-Optimized Purification Method for Human Dental-Pulp Stem Cells: An Approach to Standardization.

Human dental pulp stem cells (hDPSCs) are promising for oral/craniofacial regeneration, but their purification and characterization is not yet standardized. hDPSCs from three donors were purified by magnetic activated cell sorting (MACS)-assisted STRO-1-positive cell enrichment (+), colony derivation (c), or a combination of both (c/+). Immunophenotype, clonogenicity, stemness marker expression, senescence, and proliferation were analyzed. Multilineage differentiation was assessed by qPCR, immunohistochemistry, and extracellular matrix mineralization. To confirm the credibility of the results, repeated measures analysis and post hoc p-value adjustment were applied. All hDPSC fractions expressed STRO-1 and were similar for several surface markers, while their clonogenicity and expression of CD10/44/105/146, and 166 varied with the purification method. (+) cells proliferated significantly faster than (c/+), while (c) showed the highest increase in metabolic activity. Colony formation was most efficient in (+) cells, which also exhibited the lowest cellular senescence. All hDPSCs produced mineralized extracellular matrix. Regarding osteogenic induction, (c/+) revealed a significant increase in mRNA expression of COL5A1 and COL6A1, while osteogenic marker genes were detected at varying levels. (c/+) were the only population missing BDNF gene transcription increase during neurogenic induction. All hDPSCs were able to differentiate into chondrocytes. In summary, the three hDPSCs populations showed differences in phenotype, stemness, proliferation, and differentiation capacity. The data suggest that STRO-1-positive cell enrichment is the optimal choice for hDPSCs purification to maintain hDPSCs stemness. Furthermore, an (immuno) phenotypic characterization is the minimum requirement for quality control in hDPSCs studies.

S-acylthioalkyl ester (SATE)-based prodrugs of deoxyribose cyclic dinucleotides (dCDNs) as the STING agonist for antitumor immunotherapy

Cancer immunotherapy is a powerful weapon in the fight against cancers. Cyclic dinucleotides (CDNs) have demonstrated the great potential by evoking the immune system to fight cancers. There are still a lot of unmet needs for highly active CDNs in clinical applications due to low cell permeation and serum stability. Here we reported S-acylthioalkyl ester (SATE)-based prodrugs of deoxyribose cyclic dinucleotides (dCDNs) with three different types of internucleotide linkages (3′,3′:11a; 2′,3′:11b; 2′,2′:11c). The parent dCDNs could be efficiently released from SATE-dCDNs by cellular esterases. Compared to 2′,3′-cGAMP and ADU-S100, 11a exhibited much higher potency of activating STING pathway and higher serum stability. In a CT26-Luc tumor-bearing animal model, 11a showed the efficient antitumor activity in eliminating the established tumor and induced significant increase of mRNA expression of IFN-β and other related inflammatory cytokines. Hence, SATE-dCDN prodrugs demonstrated their benefits in promoting cell penetration, improving serum stability, and thus enhancing bioactivity, suggesting their potential application as immunotherapy in a variety of malignancies.

Renin-Angiotensin System in Huntington's Disease: Evidence from Animal Models and Human Patients.

The Renin-Angiotensin System (RAS) is expressed in the central nervous system and has important functions that go beyond blood pressure regulation. Clinical and experimental studies have suggested that alterations in the brain RAS contribute to the development and progression of neurodegenerative diseases. However, there is limited information regarding the involvement of RAS components in Huntington’s disease (HD). Herein, we used the HD murine model, (BACHD), as well as samples from patients with HD to investigate the role of both the classical and alternative axes of RAS in HD pathophysiology. BACHD mice displayed worse motor performance in different behavioral tests alongside a decrease in the levels and activity of the components of the RAS alternative axis ACE2, Ang-(1-7), and Mas receptors in the striatum, prefrontal cortex, and hippocampus. BACHD mice also displayed a significant increase in mRNA expression of the AT1 receptor, a component of the RAS classical arm, in these key brain regions. Moreover, patients with manifest HD presented higher plasma levels of Ang-(1-7). No significant changes were found in the levels of ACE, ACE2, and Ang II. Our findings provided the first evidence that an imbalance in the RAS classical and counter-regulatory arms may play a role in HD pathophysiology.

Abstract LB022: A hybrid epithelial to mesenchymal transition in ex-vivo cutaneous squamous cell carcinoma tissues

Abstract While most cases of cutaneous squamous cell carcinoma (cSCC) are benign, invasive cSCC is associated with higher mortality and is often more difficult to treat. As such, understanding the factors that influence the progression of cSCC are important. Aggressive cancers metastasize through a series of evolutionary changes, collectively called the epithelial-to-mesenchymal transition (EMT). During EMT, epithelial cells transition to a highly mobile mesenchymal cell type with metastatic capacities. While changes in expression of TGF-β, vimentin, and E-cadherin are hallmarks, EMT is not an “all or none” process. Using patient-derived cSCC and adjacent normal tissues (ANT), we observed a significant increase in mRNA expression of TGF-β (p&amp;lt;0.05), TGF-βR2 (p&amp;lt;0.001), and E-cadherin (p&amp;lt;0.005) in cSCC tissues by qPCR. Immunoblots performed on homogenized tissues yielded significant and parallel increases in protein expression of TGF-β (p&amp;lt;0.05) and E-cadherin (p&amp;lt;0.05). We also observed increased expression of the EMT-associated transcription factors Zeb1 and Snai1 in cSCC tissue sections using immunohistochemistry. Interestingly, we observed a significant increase in expression of vimentin (p&amp;lt;0.01) by immunoblot and differential expression of four oncogenic and EMT-associated miRNAs; miR-31 expression was significantly higher in cSCC tissues (p&amp;lt;0.001), while we observed a significant decrease in expression of miR-34a (p&amp;lt;0.05), miR-200b, and miR-200c in cSCC tissues. Examination of ANT and cSCC tissue sections using immunofluorescence and immunohistochemistry showed differential, and reverse, expression of E-cadherin and vimentin among cells within an individual tumor. Collectively, our data support a hypothesis that cSCC tumors are undergoing a hybrid EMT program, where there is variation in expression of EMT markers by cells within a tumor mass that may be facilitating invasion. This hybrid EMT program may be promoting both survival and invasiveness of the tumors. A better understanding of this hybrid EMT process may influence therapeutic strategies in more invasive disease. Citation Format: Christopher S. Pulford, Chandana K. Uppalapati, McKale R. Montgomery, Elizabeth E. Hull, Kathryn J. Leyva. A hybrid epithelial to mesenchymal transition in ex-vivo cutaneous squamous cell carcinoma tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB022.

Systemic Ketone Replacement Does Not Improve Survival or Cancer Cachexia in Mice With Lung Cancer.

Cachexia is a debilitating comorbidity affecting many lung cancer patients. We have previously found that cachectic mice with lung cancer have reduced serum ketone body levels due to low PPARα activity in the liver. Restoring hepatic PPARα activity with fenofibrate increased circulating ketones and delayed muscle and white adipose tissue wasting. We hypothesized that the loss of circulating ketones plays a pathophysiologic role in cachexia and performed two dietary intervention studies to test this hypothesis. In the first study, male and female mice were randomized to consume either a very low carbohydrate, ketogenic diet (KD) or normal chow (NC) after undergoing tumor induction. The KD successfully restored serum ketone levels and decreased blood glucose in cachectic mice but did not improve body weight maintenance or survival. In fact, there was a trend for the KD to worsen survival in male but not in female mice. In the second study, we compounded a ketone ester supplement into the NC diet (KE) and randomized tumor-bearing mice to KE or NC after tumor induction. We confirmed that KE was able to acutely and chronically increase ketone body abundance in the serum compared to NC. However, the restoration of ketones in the circulation was not able to improve body weight maintenance or survival in male or female mice with lung cancer. Finally, we investigated PPARα activity in the liver of mice fed KE and NC and found that animals fed a ketone ester supplement showed a significant increase in mRNA expression of several PPARα targets. These data negate our initial hypothesis and suggest that restoring ketone body availability in the circulation of mice with lung cancer does not alter cachexia development or improve survival, despite increasing hepatic PPARα activity.

Ginger Root Extract Mitigates Neuropathic Pain via Suppressing Neuroinflammation: Gut-Brain Connection

ObjectivesEmerging evidence suggests an important role of the gut-brain-axis in the development of neuropathic pain (NP). We investigated the effects of gingerol-enriched ginger (GEG) on pain sensitivity and mRNA expression of inflammation and tight junction protein in GI tissues (colon and ileum) and nervous tissues (amygdala and spinal cord) of animals with NP.MethodsTwenty-eight male rats were randomly divided into 5 groups: Naïve control, sham control, spinal nerve ligation (SNL, pain model), SNL + 0.375% (w/w in diet) GEG, and SNL + 0.75% (w/w in diet) GEG for 4 weeks. Pain sensitivity was assessed by von Frey filament tests, evoked audible vocalizations, and grimace tests in subjects. Intestinal permeability was assessed by lactulose/mannitol ratio in urine. The levels of mRNA expression of neuroinflammation (NF-κB) and tight junction (Claudin-1, Zonulin) in colon, ileum, amygdala, and spinal cord were determined by qRT-PCR. Data was analyzed by one-way ANOVA or one-way repeated measures ANOVA followed by post-hoc analysis.ResultsCompared to the sham group, the SNL group had significantly greater hypersensitivity (von Frey test), emotional responses (vocalizations), and spontaneous pain (grimace test). GEG supplementation significantly reduced hypersensitivity, emotional responses, and spontaneous pain (nose bulge, whisker change, and ear position) in SNL rats. GEG supplementation tended to decrease intestinal permeability of SNL-operated rats. The SNL group showed a significant increase in mRNA expression of Zonulin in amygdala and Claudin-1 in colon compared to the sham control group. GEG supplementation significantly suppressed the gene expression levels of Zonulin and Claudin-1 in amygdala and colon of SNL animals. GEG addition into the diet suppressed NF-κB gene expression in the amygdala, colon, and ileum of SNL rats.ConclusionsThis study suggests GEG supplementation mitigated pain behaviors in a preclinical NP animal model. GEG also decreased SNL-induced neuroinflammation, intestinal permeability, and possible blood-brain barrier breakdown, which may explain the behavioral effects of GEG.Funding SourcesUSDA-NIFA 2021-67,017-34,026.

Regulation of Extracellular Matrix Gene Expression by Desmosomal Cadherins

Desmosomes are protein complexes crucial for maintaining cell‐cell adhesion and integrity of tissues. These complexes are made up of proteins from three families: transmembrane cadherins (Desmoglein and Desmocollin) link adjacent cells in the extracellular space, armadillo proteins (Plakophilin and Plakoglobin) stabilize the intracellular plaque, and the cytolinker Desmoplakin (DP) connects the plaque to the intermediate filament network. Desmosomal proteins have also been shown to coordinate gene expression pathways required for processes such as proliferation, differentiation and cell migration. In particular, several lines of evidence have linked desmosomal proteins to gene expression of extracellular matrix (ECM) proteins. Loss of Plakophilin‐2 or Desmoplakin causes increases in expression of fibronectin and collagen, while in contrast, loss of plakoglobin results in a significant decrease in expression of fibronectin. These data indicate that individual components of the desmosome control ECM gene expression via distinct cellular signaling networks. In our study, we sought to investigate the role of desmosomal cadherins in ECM gene expression, via use of A431 cells lacking Desmoglein‐2, generated via CRISPR‐mediated knock‐out. Compared to control cells, Dsg2 KO cells demonstrated a dramatic increase in expression of Fibronectin (Fn1) mRNA, and relatively minor changes in expression of Collagen 1 (Col1a1) and Collagen 2 (Col2a1) mRNA. An increase in expression of Fibronectin protein levels in Dsg2‐deficient cells was also observed via western blot. We show that loss of Dsg2 KO also caused a significant increase in mRNA expression of the pro‐fibrotic signaling molecule transforming growth factor beta 2 (Tgfb2), but not Tgfb1 or Tgfb3. Increased expression of Fn1, Col1a1, Col2a1 and Tgfb2 was also observed upon siRNA‐mediated knockdown of Dsg2 in both A431 cells and HaCaT keratinocytes, verifying that these changes are not clone‐specific or due to off‐target CRISPR effects. siRNA‐mediated knockdown of other desmosomal proteins could not rescue Fn1 or Tgfb2 increases in Dsg2 KO cells, indicating that changes are not due to mislocalization of other desmosomal components. An extensive analysis of signaling pathways known to be involved in regulation of ECM gene expression uncovered an important role for PI3‐Kinase and Akt signaling, as inhibition of either signaling protein resulted in a rescue of Dg2KO‐mediated increases in Fn1 gene expression. Moreover, we have documented a significant increase in phosphorylation of Akt in Dsg2‐deficient cells. Taken together, our study highlights a novel role for Dsg2 in mediating ECM gene expression via PI3‐K/Akt signaling, adding significant insight into the mechanisms by which desmosomal cadherins control the adhesive behavior of cancer cells.

Identification of a novel gene signature in second-trimester amniotic fluid for the prediction of preterm birth

Preterm birth affects approximately 5% to 7% of live births worldwide and is the leading cause of neonatal morbidity and mortality. Amniotic fluid supernatant (AFS) contains abundant cell-free nucleic acids (cfNAs) that can provide genetic information associated with pregnancy complications. In the current study, cfNAs of AFS in the early second-trimester before the onset of symptoms of preterm birth were analyzed, and we compared gene expression levels between spontaneous preterm birth (n = 5) and term birth (n = 5) groups using sequencing analysis. Differential expression analyses detected 24 genes with increased and 6 genes with decreased expression in the preterm birth group compared to term birth. Upregulated expressions of RDH14, ZNF572, VOPP1, SERPINA12, and TCF15 were validated in an extended AFS sample by quantitative PCR (preterm birth group, n = 21; term birth group, n = 40). Five candidate genes displayed a significant increase in mRNA expression in immortalized trophoblast HTR-8/SVneo cell with H2O2 treatment. Moreover, the expression of five candidate genes was increased to more than twofold by pretreatment with lipopolysaccharide in HTR-8/SVneo cells. Changes in gene expression between preterm birth and term birth is strongly correlated with oxidative stress and infection during pregnancy. Specific expression patterns of genes could be used as potential markers for the early identification of women at risk of having a spontaneous preterm birth.