Fold Enrichment Research Articles

Fatty acid synthesis promoted by PA1895-1897 operon delays quorum sensing activation in Pseudomonas aeruginosa

PA1895-1897 is a quorum sensing (QS) operon regulated by the anti-activator LuxR homologue QscR in Pseudomonas aeruginosa. We aimed to investigate its impact on bacterial metabolism, and whether it contributes to the delayed QS activation. We performed liquid chromatograph-mass spectrometer-based metabolomics using wildtype PAO1, PA1895-1897-knockout mutant, and mutant with pJN105.PA1895-1897 overexpression vector. The impact of metabolites on QS signaling molecule (3OC12-HSL and C4-HSL) concentrations, pyocyanin production, and QS gene (lasR, lasI, rhlR, and rhlI) expression was examined. Metabolomics analysis found that fatty acid biosynthesis had the highest fold enrichment among all metabolic pathways in PA1895-1897-overexpressed mutants. Among these enriched fatty acids, palmitoleic acid and acetic acid were the predominantly abundant ones that significantly affected by PA1895-1897 operon. When different doses of exogenous palmitoleic acid or acetic acid were added to the cultures of PA1895-1897 knockout mutants, their levels of 3OC12-HSL, C4-HSL, and pyocyanin were decreased in a dose-dependent manner. High doses of palmitoleic acid and acetic acid suppressed the mRNA expression of lasR, rhlR, and rhlI. Inhibition of fatty acid biosynthesis increased the production of 3OC12-HSL, C4-HSL, and pyocyanin in PA1895-1897-overexpressed cultures. Our data suggest that fatty acid synthesis is promoted by PA1895-1897 operon, and contributes the delayed expression of QS phenotypes, furthering the understanding about the regulation of bacterial QS activation.

Identification of MicroRNAs in children with increased asthma bronchodilator usage in genetics of asthma in Costa Rica Study.

Uncontrolled or severe asthma results in symptomatic usage of short-acting ß2-agonist (SABA) usage. MicroRNAs (miRNAs) are posttranslational regulators that can influence asthma biology. This study aims to identify miRNAs that are associated with increased SABA usage. Small RNA sequenced from blood serum of 1,132 children with asthma aged 6 to 14 years in the Genetics of Asthma in Costa Rica Study was used for this analysis. Logistic regression identified miRNAs in patients who required increased SABA usage. These miRNA were validated for association with SABA-induced bronchodilator responsiveness (BDR). Gene target pathway analysis was performed on validated miRNAs. Twenty-one miRNAs were significantly associated with increased SABA usage with OR ranging from 0.87 to 1.23. Two miRNAs, miR-378a-3p and miR-144-3p, had odds ratio 1.14 (1-1.29, p = .05) and 1.11 (1.01-1.22, p = .035), respectively, for increased SABA usage and were also significantly associated with bronchodilator response. Furthermore, a linear regression analysis involving these miRNA and bronchodilator response revealed that increased miR-378a-3p correlated with decreased BDR and increased expression of miR-144-3p correlated with improving pulmonary function with bronchodilators. In gene target Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the erythroblastosis viral oncogene signaling pathway had among one of the highest fold enrichment and p-value. Increased expression of miR-378a-3p and miR-144-3p was seen in this patient population who required increased SABA usage. There were different bronchodilatory effects seen in these two miRNAs, suggesting different potential mechanisms underlying increased SABA usage.

Exploring the anti-osteoporosis potential of Petroselinum crispum (Mill.) Fuss extract employing experimentally ovariectomized rat model and network pharmacology approach

One of the most prevalent secondary osteoporosis is ovariectomy-induced osteoporosis. Parsley (Petroselinum crispum) has potent estrogenic and antioxidant properties and was used traditionally in the treatment of amenorrhea and dysmenorrhea. The present study aimed to characterize parsley leaf extract (PLE) employing RP-HPLC-MS-MS/MS-based method and possible protective effect in ovariectomized (OVX)-induced osteoporosis in rats was assessed. Rats were randomly assigned into SHAM group, OVX group, PLE + OVX group (150 mg/kg/day, p.o), and estradiol benzoate (E2) + OVX group (30 μg/kg/day, s.c). After eight weeks following ovariectomy, biomarkers of bone strength, bone resorption, oxidative stress and histopathology were carried out. A network pharmacology approach investigated the key targets and potential mechanisms by of PLE metabolites against osteoporosis using databases: PubChem, BindingDB server, DisGeNET, ShinyGO, and KEGG Pathway. Moreover, FunRich 3.1.3, Cytoscape 3.10.0, and MOE 2019.0102 softwares were used for network pharmacology analysis and molecular docking studies.Flavones and hydroxycinnamic acid derivatives were predominant among 38 metabolites in PLE. It significantly restored bone strength and bone resorption biomarkers, osteocalcin (OST), oxidative stress biomarkers and histopathological alterations. The employed network pharmacology approach revealed that 14 primary target genes were associated with decreasing the severity of osteoporosis. Molecular docking revealed that cGMP-PKG signaling pathway has the highest fold enrichment and its downstream PDE5A. Luteolin, diosmetin, and isorhamnetin derivatives affected mostly osteoporosis targets. PLE exhibited protective action against ovariectomy-induced osteoporosis in rats and may be a promising therapy for premenopausal bone loss. cGMP-PKG signaling pathway could be a promising target for PLE in treating osteoporosis.

Abstract 271: The effects of B-cell acute lymphoblastic leukemia cells on the adipose tissue microenvironment

Abstract Obesity increases the risk of developing many cancers, including breast, colon, kidney, and acute lymphoblastic leukemia (ALL). We have previously shown that ALL cells are recruited to migrate into adipose tissue, where they are protected from chemotherapy. In vitro, ALL cells induce significant changes in adipocytes which may contribute to these protective effects. However, how ALL cells affect the complex cell populations found in adipose tissue in vivo remains unknown. We therefore conducted single-cell RNA sequencing (scRNAseq) of the non-adipocyte stromal vascular fraction (SVF) from adipose tissue in obese mice with and without implanted syngeneic BCR-ABL positive ALL. Differential expression analysis was performed on individual SVF cell types to compare ALL vs. control adipose tissue and pathway enrichment analysis was used in identifying altered molecular processes between conditions. SVF from leukemic mice showed substantial differences in gene expression compared to control mice, suggesting that the ALL potentially induces changes in the microenvironment. Some of the largest differences were found in adipose tissue macrophages, and pathway enrichment analysis identified neutrophil recruitment and regulation as one of the top ten pathways upregulated in adipose macrophages from leukemic mice (4.10 fold enrichment, p<1 × 10-17 by Fisher’s exact test). Selected macrophage genes of interest in this pathway included CYBB (Cytochrome B-245 Beta), a neutrophil interaction gene that has been associated with macrophage infiltration in gastric cancer, was expressed 2.28 log2-fold higher in macrophages from leukemic compared to nonleukemic mice (p<1 × 10−99 by Wilcoxon test). ITGAL (Integrin subunit alpha L), which has been shown to be involved in neutrophil recruitment and implicated in cancer cell growth and transformation was expressed 2.85 log2-fold higher in macrophages from leukemic mice (p<1 × 10−99). Interestingly, adipose from leukemic mice showed a population of neutrophils (1.9% of SVF cells), while neutrophils were largely absent in adipose from nonleukemic mice (<0.1% of SVF cells), which is consistent with macrophage involvement in neutrophil recruitment. Our findings highlight the importance of adipose tissue in the leukemia microenvironment and show that ALL cells can influence this microenvironment in complex ways. Further work will confirm and investigate these cell-cell interactions, potentially improving our understanding of how adipose tissue contributes to cancer mortality. Citation Format: Jia Tan, Michael Cohen, Jessica Ding, In Sook Ahn, Xia Yang, Steven D. Mittelman. The effects of B-cell acute lymphoblastic leukemia cells on the adipose tissue microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 271.

Abstract 523: Aronia berry and oligomeric proanthocyanidins synergistically exhibit anticancer effects in colorectal cancer by inhibiting LMNB1

Abstract Background: Colorectal cancer (CRC) is one of the most common and recurrent diseases globally, ranking as the second leading cause of mortality. The high mortality rates associated with this disease are largely due to the ineffectiveness of current treatments and the development of chemotherapeutic resistance to conventional therapies. In recent years, several natural agents, such as Aronia berry extract (ABE) and oligomeric proanthocyanidins (OPCs), have shown promise in providing a safe, cost-effective, and synergistically multi-targeted treatment approach across various cancers. Herein, we hypothesized that the combined treatment with ABE and OPCs may synergistically regulate multiple oncogenic pathways, thereby presenting a more potent anti-cancer activity, particularly targeting CRC. Methods: We performed a series of cell culture experiments to evaluate the synergistic anticancer effect of the combination of ABE and OPCs in CRC cell lines. By performing genome-wide transcriptomic profiling, we identified specific growth-signaling pathways targeted by the combined treatment and subsequently validated these findings in cell lines and patient-derived 3D organoids. Results: The combined treatment with ABE and OPCs exhibited superior synergistic anti-tumor potential, affecting cell viability, proliferation, migration, and invasion in CRC cells. The transcriptomic analysis identified that LMNB1, associated with the cell apoptosis pathway Fold enrichment [FC]: 4.29, P =0.005), plays a crucial role in the anti-tumorigenic effects of these two natural products. Moreover, the combination of ABE and OPCs significantly altered the expression of key proteins involved in cell apoptosis and suppressed the expression level of LMNB1 in HCT116 (Combination vs. ABE, FC= 0.28; Combination vs. OPCs, FC= 0.24) and SW480 (Combination vs. ABE, FC= 0.65; Combination vs. OPCs, FC= 0.47), thereby inhibiting downstream Akt phosphorylation. Finally, we successfully demonstrated that the combined treatment displayed a superior anti-cancer activity in 3D organoids, significantly reducing both their number and size (P < 0.05). Conclusion: We present novel evidence for the synergistic anticancer effects of ABE and OPCs in CRC cells, which are in part achieved by regulating cellular apoptosis and the oncogene LMNB1 within the Akt signaling pathway. Citation Format: Yuan Li, Caiming Xu, Ajay Goel. Aronia berry and oligomeric proanthocyanidins synergistically exhibit anticancer effects in colorectal cancer by inhibiting LMNB1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 523.

Performing Quantitative PCR after Chromatin Immunoprecipitation (ChIP) of Drosophila Antennal and Brain Samples.

Chromatin immunoprecipitation (ChIP) is a technique used to study specific protein-DNA interaction. Briefly, in this technique, antibodies to proteins of interest are used to isolate regions of DNA where these proteins bind. ChIP samples can be processed and analyzed in different ways. One of the approaches for assessing the results of ChIP experiments is quantitative PCR (qPCR). qPCR is used to quantitatively measure the amount of DNA fragments that have been isolated, reflecting the signal of specific proteins interacting with these fragments. This protocol describes both the "percent input" method and the "fold enrichment" method for ChIP-qPCR data analysis using Drosophila tissues as an example. The "percent input" method measures signals of DNA fragments against the input measurement. In contrast, the "fold enrichment" method quantifies the amplified signal strength relative to a background control. Because the quality of primers is critical for the reliability of ChIP-qPCR results, this protocol also describes how to measure primer amplification efficiency using Drosophila genomic DNA.

An unbiased approach of individual tissue-specific autoantibody identification using Immunoprecipitation coupled to Mass-Spectrometry (IP-MS)

Abstract Background and aim Autoreactive antibodies have gained increasing recognition in autoimmune diseases of the central nervous system (CNS) serving as disease drivers and / or biomarkers. Identification of novel autoantibodies therefore is crucial to optimize diagnostic work and therapy of these diseases as well as understanding their pathogenesis. Hence, we aimed to develop an unbiased strategy to identify patients’ individual main autoantibodies directed against brain epitopes. Methods We used murine tissue-based assays to screen patients’ sera and cerebrospinal fluids (CSFs) for autoreactive IgGs with known and unknown antibody targets. IgGs from autoreactive patients’ samples were coupled to magnetic beads and used as a bait to pull down potential targets from homogenized brain tissue of wildtype mice. After precipitation and several washes to reduce unspecific binding, beads were subjected to on-bead tryptic digestion followed by mass spectrometry based proteomic analyses using liquid chromatography tandem mass-spectrometry (LC-MS/MS). For antibody targets identification the fold change enrichment compared to a negative control or to all other pulldown samples, as well as the protein abundance was used. Antibody target validation was done via recombinant expression of the target protein in HEK-293 cells and neutralization with the respective antigen in tissue-based immunofluorescence assays. Results As proof of principle, we were able to identify Grin1 Caspr2, Trim46 and Nf-h [MOU1] as main targets of patients’ sera or CSF with autoimmune encephalopathy. All four antigens showed highest enrichment and/or highest protein abundance in the respective samples and have been confirmed in cell-based assays. In addition, we identified one novel anti-neuronal antibody target in serum and CSF of a patient showing a speckled staining of the granular cerebellar layer. Preabsorbance of the serum and CSF with the recombinant protein fully neutralized the signal of the tissue-based immunofluorescence. Conclusions Our IP-MS approach is suitable to identify the main autoantibody targets in patients’ serum and CSF within murine CNS tissue thereby providing an unbiased and scalable technique allowing for tissue-specific autoantibody determination.

Identifying Inflammatory Pathways That Promote RBC Alloimmunization in Patients with Sickle Cell Disease

Introduction: During red blood cell (RBC) transfusion, exposure to unmatched non-ABO antigens can lead to production of alloantibodies that promote potentially fatal hemolytic events. Patients with Sickle Cell Disease (SCD) have the highest incidence of RBC alloimmunization. Prior studies indicate that an altered immuno-regulatory and/or pro-inflammatory state may determine the risk of RBC alloimmunization. For example, one study reported that patients with SCD have inflammatory cytokine gene polymorphisms that could be associated with diverse disease presentations and alloimmunization. We previously reported that inflammatory type 1 interferons (IFNα/β) produced by innate immune cells are required for RBC alloimmunization in mice, while others have suggested that NFkB-induced cytokines play a role in murine transfusion models. However, the inflammatory factors that regulate RBC alloimmunization in patients with SCD are poorly understood. Alloimmunized patients with SCD also have elevated levels of anti-RBC autoantibodies, which facilitate erythrophagocytosis. Given the role of erythrophagocytosis by spleen macrophages in processing RBC antigens, we hypothesized that erythrophagocytosis of SCD RBCs may promote inflammatory pathways that regulate alloimmune responses. Methods: RBCs from control subjects (AA), and reticulocytes and mature RBCs from patients with SCD, were opsonized or not with anti-RBC antibodies (anti-RhD) and co-cultured for 3 hrs with human macrophages (hMac) differentiated from monocytes of healthy subjects. Phagocytosis of labeled RBCs was analyzed by flow cytometry. Bulk mRNA sequencing of co-cultured macrophages was performed to identify differential gene expression. Ingenuity Pathway Analysis (IPA) software was used to identify relevant pathways and upstream regulators of differentially expressed genes. To validate sequencing results, cytokine levels from co-culture supernatants were measured by multiplex cytokine assays. Results: Flow cytometric analysis revealed that opsonization was required for erythrophagocytosis in an FcR dependent manner. RNA-seq Differential Expression analysis of macrophages co-cultured with AA opsonized RBCs vs. non-opsonized RBCs identified 166 significantly upregulated and 24 downregulated genes (p<0.0001). IPA analysis revealed that the top 12 canonical pathways were related to inflammation, including TNF, IL-17, NF-kappa-B and NOD-like receptor signaling pathways. TNF signaling was the top predicted upstream regulator pathway with a fold enrichment score of 10.3 (16 genes) (p<0.0001). Co-cultures of mature RBCs from patients with SCD showed comparable results. When comparing macrophages from co-cultures of opsonized reticulocytes vs. non-opsonized reticulocytes from patients with SCD, 174 genes were upregulated and 31 were downregulated (p<0.0001). Pathway analysis also revealed unique signatures for opsonized reticulocytes from patients with SCD. The top 12 canonical pathways included TNF, NF-kappa-B and NOD-like receptor signaling pathways (Figure 1). The top predicted upstream regulators were TNF and the NF-kappa B signaling pathway with a fold enrichment score of 12.3 (22 genes) (p<0.0001) and 10.9 (18 genes), respectively (Figure 1). Finally, to validate the sequencing results, cytokines levels from supernatants of hMac co-cultures with the RBCs from the patients with SCD and control subjects were measured. Erythrophagocytosis of opsonized reticulocytes and AA RBCs, compared to non-opsonized co-cultures, led to production of NFκB-mediated cytokines, including TNF. Our results show that erythrophagocytosis of SCD RBCs promote NFκB-mediated TNF production. These results warrant further investigation of the role of NFκB-mediated inflammation in RBC alloimmunization in patients with SCD.

Epigenetic regulation of PCSK9 expression by statins

Abstract Background Statins are lipid-lowering drugs widely used to reduce the risk of cardiovascular diseases. Although they inhibit HMG-CoA, statins are known to induce the expression of PCSK9, a key regulator of plasma LDL-cholesterol. However, the underlying epigenetic mechanisms remain unknown. Objective This study aimed at investigating the histone modification-mediate epigenetic regulation of PCSK9 and the underlying mechanisms during statin treatment. Methods Using immunoblotting, qPCR, and immunofluorescence techniques we assessed various histone modifications and expression levels of PCSK9 and other cholesterol-regulating candidates upon statin treatment. Further, 4-6 weeks old C57BL/6 mice were subjected to high-fat diet in combination with atorvastatin for 10 weeks. We then performed ChIP-qPCR to evaluate the fold enrichment of targeted histone marks at the gene promoter regions of PCSK9 and SREBP. Results Atorvastatin and cholesterol-treated HepG2 cells were reported to have an increased transcript and protein expression of PCSK9. Further, screening of different histone methylation and acetylation marks under atorvastatin treatment revealed significant enrichment in the levels of the activating H3K4me3 and H3K9ac marks. ChIP-qPCR analysis suggested significantly higher enrichment of H3K4me3 and H3K9ac at the gene promoter region of PCSK9 and SREBP2 (Figure 1&2). In vitro pharmacological intervention against these histone modifications not only significantly lowered their activation but also attenuated statin-induced PCSK9 expression. Conclusion Together, these findings suggest that statins modulate the expression of PCSK9 by regulating H3K4me3 and H3K9ac levels at the PCSK9 gene promoter. Inhibition of these activating histone modifications repressed statin-dependent PCSK9 expression. Thus, targeting chromatin remodeling may help negate the adverse effects of statin treatment, primarily of increasing PCSK9 during hypercholesterolemia.

ALKBH5 promotes the development of lung adenocarcinoma by regulating the polarization of M2 macrophages through CDCA4

ObjectiveLung adenocarcinoma (LUAD) is the most common subtype of lung cancer, with high morbidity and mortality. N6-methyladenosine (m6A) is an important regulator of LUAD progression. Here, we investigated the potential biological functions of ALKBH5 (a m6A demethylated enzyme) and cell division cycle associated protein 4 (CDCA4) in the progression of LUAD. MethodsThe expressions of CDCA4, METTL3, ALKBH5, FTO, YTHDC2 and YTHDC1 mRNA and proteins in LUAD and adjacent tissues, as well as NCI-H1299 and NCI-H157 cells were detected by RT-qPCR and western blot. Meanwhile, the role of ALKBH5 and CDCA4 in macrophage polarization was explored through tumor formation in Lewis lung carcinoma (LLC) mice and the co-culture system of NCI-H1299 and NCI-H157/THP-1 cells. Cell characterization was further analyzed. The expression of Ki-67 in tumor tissue was tested by immunohistochemistry. The scale of M1 and M2 macrophages was determined by flow cytometry. ResultsCDCA4 was significantly overexpressed in NCI-H1299 and NCI-H157 cell lines compared with BEAS-2B cells. The fold enrichment of CDCA4 m6A level in the overexpression (oe)-METTL3 or short hairpin (sh)-ALKBH5 cells was enhanced. Overexpression of CDCA4 promoted the cell viability, proliferation and migration, and inhibited apoptosis, which was reversed by sh-ALKBH5 intervention. Overexpression of YTHDC2 (not YTHDC1) inhibited the effect of CDCA4 on sh-ALKBH5 cells. sh-CDCA4 inhibited tumor growth and weight of LLC cells in mice, and promoted M1/M2 ratio in LLC mice and NCI-H1299/THP-1 and NCI-H157/THP-1 co-culture systems. Oe-CDCA4 promoted the volume and weight of tumor and inhibited the M1/M2 ratio of tumor tissue in LLC mice, but was reversed by sh-ALKBH5 intervention. Conclusionm6A demethylase ALKBH5 promotes the development of LUAD through CDCA4 regulation of malignant characterization and M1/M2 macrophage polarization.

Abstract 13116: Profiling the Role of Gut Microbiota-Derived Trimethylamine- N -Oxide in Cardiac Fibrosis — Evidence From a Primary Human in vitro Model

Introduction: The gut derived trimethylamine- N -oxide (TMAO) is correlated with increased atrial inflammation and fibrosis and associated with atrial fibrillation, heart failure, and stroke. However, the mechanisms mediating these links remain unresolved. Hypothesis: TMAO and its precursor metabolite L-carnitine (LCART) induce the transformation of atrial fibroblasts into pro-fibrotic phenotypes. Aim: To characterise the association between TMAO and cardiac fibrosis. Methods: Primary human cardiac (atrial) fibroblasts (hCFs) were sourced from healthy donors. hCFs were starved for 24 h and treated with PBS (control), 20 ng/mL transforming growth factor beta 1 (TGFβ1), 10 mM TMAO, or 1.0 mM LCART for 72 h (n=6 in each), then analysed by flow cytometry for α-smooth muscle actin (αSMA) expression. Unbiased proteomics was performed using LC-MS/MS to determine protein expression profile. Results: Analysis of αSMA expression revealed 3 hCF states: quiescent (Fbs), quiescent-to-myofibroblast (intermediate [Fbs-myoFbs]), and fully activated myofibroblast (myoFbs), (Fig-A). Compared to controls, there was no difference in the intermediate state after TGFβ1, TMAO, or LCART treatment (p=0.30). TMAO and LCART resulted in a significant induction of myofibroblast states compared to controls and TGFβ1 group. Unbiased proteomics identified 92, 65, and 43 proteins to be overexpressed and 84, 46, and 78 proteins downregulated following TGFβ1, TMAO, and LCART treatments (Fig-B). Both TGFβ1 and TMAO demonstrated shared enrichment for oxidative stress-regulated ferroptosis pathway (fold enrichment [FE] 31.8 and 22.2, p <.01 and 0.03); LCART showed enrichment for cell motility (FE 3.8, p=0.03). Conclusions: TMAO and its precursor L-carnitine are associated with activation of pro-fibrotic states, which may be due to oxidative stress and abnormal cell migration related mechanisms.

Genetic Liability to Posttraumatic Stress Disorder Symptoms and Its Association With Cardiometabolic and Respiratory Outcomes

Posttraumatic stress disorder (PTSD) has been reported to be a risk factor for several physical and somatic symptoms. However, the genetics of PTSD and its potential association with medical outcomes remain unclear. To examine disease categories and laboratory tests from electronic health records (EHRs) that are associated with PTSD polygenic scores. This genetic association study was conducted from July 15, 2021, to January 24, 2023, using EHR data from participants across 4 biobanks. The polygenic scores of PTSD symptom severity (PGS-PTSD) were tested with all available phecodes in Vanderbilt University Medical Center's biobank (BioVU), Mass General Brigham (MGB), Michigan Genomics Initiative (MGI), and UK Biobank (UKBB). The significant medical outcomes were tested for overrepresented disease categories and subsequently tested for genetic correlation and 2-sample mendelian randomization (MR) to determine genetically informed associations. Multivariable MR was conducted to assess whether PTSD associations with health outcomes were independent of the genetic effect of body mass index and tobacco smoking. Polygenic score of PTSD symptom severity. A total of 1680 phecodes (ie, International Classification of Diseases, Ninth Revision- and Tenth Revision-based phenotypic definitions of health outcomes) across 4 biobanks and 490 laboratory tests across 2 biobanks (BioVU and MGB). In this study including a total of 496 317 individuals (mean [SD] age, 56.8 [8.0] years; 263 048 female [53%]) across the 4 EHR sites, meta-analyzing associations of PGS-PTSD with 1680 phecodes from 496 317 individuals showed significant associations to be overrepresented from mental health disorders (fold enrichment = 3.15; P = 5.81 × 10-6), circulatory system (fold enrichment = 3.32; P = 6.39 × 10-12), digestive (fold enrichment = 2.42; P = 2.16 × 10-7), and respiratory outcomes (fold enrichment = 2.51; P = 8.28 × 10-5). The laboratory measures scan with PGS-PTSD in BioVU and MGB biobanks revealed top associations in metabolic and immune domains. MR identified genetic liability to PTSD symptom severity as an associated risk factor for 12 health outcomes, including alcoholism (β = 0.023; P = 1.49 × 10-4), tachycardia (β = 0.045; P = 8.30 × 10-5), cardiac dysrhythmias (β = 0.016, P = 3.09 × 10-5), and acute pancreatitis (β = 0.049, P = 4.48 × 10-4). Several of these associations were robust to genetic effects of body mass index and smoking. We observed a bidirectional association between PTSD symptoms and nonspecific chest pain and C-reactive protein. Results of this study suggest the broad health repercussions associated with the genetic liability to PTSD across 4 biobanks. The circulatory and respiratory systems association was observed to be overrepresented in all 4 biobanks.

Identification of CYCLE targets that contribute diverse features of circadian rhythms in the mosquito Culex pipiens

Culex pipiens demonstrates robust circadian rhythms in adult eclosion, flight activity, mating, and development. These rhythmic patterns are believed to be controlled by the endogenous light-entrainable circadian clock that consists of positive and negative regulators working in a transcription-translation feedback loop. Moreover, these mosquitoes undergo seasonal diapause in exposure to the short photoperiod of late summer or early fall. However, the exact genetic and cellular mechanism behind the clock gene-mediated activity pattern, seasonal time measurement, and subsequent diapause initiation still need to be unraveled. To determine the possible linkage between clock genes and downstream processes, here we employed ChIP-sequencing to identify the direct targets of one of the core clock proteins, Cycle (CYC). The nearest genes with peaks mapping to their 1Kb upstream region of the transcription start site were extracted and scanned for consensus E box sequences, resulting in a dataset comprising the target genes possibly regulated by CYC. Based on the highest fold enrichment and functional relevance, we identified genes relating to five gene categories of potential interest, including peptide/receptors, neurotransmission, olfaction, immunity, and reproductive growth. Of these, we validated fourteen genes with ChIP-qPCR and qRT-PCR. These genes showed a significantly high expression in dusk compared to dawn in concert with the activity level of the CYC transcription factor and are thus strong candidates for mediating circadian rhythmicity and possibly regulating seasonal shifts in mosquito reproductive activity.

Development of a Plasmodium vivax biobank for functional ex vivo assays

BackgroundPlasmodium vivax is the second most prevalent cause of malaria yet remains challenging to study due to the lack of a continuous in vitro culture system, highlighting the need to establish a biobank of clinical isolates with multiple freezes per sample for use in functional assays. Different methods for cryopreserving parasite isolates were compared and subsequently the most promising one was validated. Enrichment of early- and late-stage parasites and parasite maturation were quantified to facilitate assay planning.MethodsIn order to compare cryopreservation protocols, nine clinical P. vivax isolates were frozen with four glycerolyte-based mixtures. Parasite recovery post thaw, post KCl-Percoll enrichment and in short-term in vitro culture was measured via slide microscopy. Enrichment of late-stage parasites by magnetic activated cell sorting (MACS) was measured. Short and long-term storage of parasites at either − 80 °C or liquid nitrogen were also compared.ResultsOf the four cryopreservation mixtures, one mixture (glycerolyte:serum:RBC at a 2.5:1.5:1 ratio) resulted in improved parasite recovery and statistically significant (P < 0.05) enhancement in parasite survival in short-term in vitro culture. A parasite biobank was subsequently generated using this protocol resulting in a collection of 106 clinical isolates, each with 8 vials. The quality of the biobank was validated by measuring several factors from 47 thaws: the average reduction in parasitaemia post-thaw (25.3%); the average fold enrichment post KCl-Percoll (6.65-fold); and the average percent recovery of parasites (22.0%, measured from 30 isolates). During short-term in vitro culture, robust maturation of ring stage parasites to later stages (> 20% trophozoites, schizonts and gametocytes) was observed in 60.0% of isolates by 48 h. Enrichment of mature parasite stages via MACS showed good reproducibility, with an average of 30.0% post-MACS parasitaemia and an average of 5.30 × 105 parasites/vial. Finally, the effect of storage temperature was tested, and no large impacts from short-term (7 days) or long-term (7–10 years) storage at − 80 °C on parasite recovery, enrichment or viability was observed.ConclusionsHere, an optimized freezing method for P. vivax clinical isolates is demonstrated as a template for the generation and validation of a parasite biobank for use in functional assays.

DeepGenePrior: A deep learning model for prioritizing genes affected by copy number variants.

The genetic etiology of brain disorders is highly heterogeneous, characterized by abnormalities in the development of the central nervous system that lead to diminished physical or intellectual capabilities. The process of determining which gene drives disease, known as "gene prioritization," is not entirely understood. Genome-wide searches for gene-disease associations are still underdeveloped due to reliance on previous discoveries and evidence sources with false positive or negative relations. This paper introduces DeepGenePrior, a model based on deep neural networks that prioritizes candidate genes in genetic diseases. Using the well-studied Variational AutoEncoder (VAE), we developed a score to measure the impact of genes on target diseases. Unlike other methods that use prior data to select candidate genes, based on the "guilt by association" principle and auxiliary data sources like protein networks, our study exclusively employs copy number variants (CNVs) for gene prioritization. By analyzing CNVs from 74,811 individuals with autism, schizophrenia, and developmental delay, we identified genes that best distinguish cases from controls. Our findings indicate a 12% increase in fold enrichment in brain-expressed genes compared to previous studies and a 15% increase in genes associated with mouse nervous system phenotypes. Furthermore, we identified common deletions in ZDHHC8, DGCR5, and CATG00000022283 among the top genes related to all three disorders, suggesting a common etiology among these clinically distinct conditions. DeepGenePrior is publicly available online at http://git.dml.ir/z_rahaie/DGP to address obstacles in existing gene prioritization studies identifying candidate genes.

Sex, age and menopause differentially impact gut barrier function and proteomic profile

Cardiovascular disease (CVD) and metabolic syndrome (MetS) depends on sex, age and hormonal status and is worsened by comprised gut barrier function. Previous work establishes that loss of estrogen and age comprises intestinal barrier function permeability exacerbating systemic inflammation, increasing gut oxidative stress, and elevating pro-inflammatory cytokines. We hypothesize that loss of estrogen during menopause underlies sex- and age-dependent pathological remodeling in gut barrier function leading to increased risk for CVD and MetS. C57BL-6 male and female mice were fed a high-fat-diet (HFD, 45% fat); menopause was induced via 21 days of 4-vinylcycohexane diepoxide (VCD) intraperitoneal injections (controls were injected with sesame oil) in adult and aged female mice. Diestrus was reached 14+days after the end of VCD injections and menopause was determined by cytology. Upon sacrifice, plasma was collected, and intestines were flash frozen. LPS assays were performed on plasma samples of premenopausal (n=8), menopausal (n=10), aged menopausal (n=6), and male mice (n=11). Male mice, and aged menopausal mice had significantly higher circulating endotoxin levels than premenopausal mice, while menopausal mice did not significantly differ from pre-menopause. These results indicate that age and sex impact barrier leakiness. Further, these results highlight that barrier integrity in menopause is distinctly different from aged menopause mice.Quantitative proteomics of ileum samples in premenopausal (n=5), menopausal (n=5), aged menopausal (n=5), and male mice (n=5) identified 7664 proteins with &gt;1 unique peptide of which 134 proteins were significant (p&lt;0.05) with a max fold change ˂1.27 across all groups. Protein expression of all groups was assessed using principal component analysis. Aged menopausal mice cluster closer to premenopausal mice than menopausal mice. These results indicate that changes in the ileum during menopause are different than those observed in aging. Compared to premenopausal mice, menopausal mice have significantly increased in proteins involved in the Go-biological processes including an enrichment in proteins involved in the innate immune system (6.3 fold enrichment, FE, -log10p=0.00006), phagocytosis recognition (19.82 FE, -log10p=1.14), defense response to bacterium (10.066 FE, -log10p=9.56), and B cell receptor signaling pathways (20.311 FE, -log10p=9.33). In contrast, male mice compared to premenopausal mice show a significant decrease in proteins involved in phagocytosis recognition (22.57 FE, -log10p=4.28), defense response to bacterium (12.898 FE, -log10p=2.76), and B cell receptor signaling pathway (20.56 FE, -log10p=8.12). These results indicate an increase in immune activity in menopausal female mice and a decrease in male mice compared to premenopausal female mice. In summary, these data demonstrate the change within the gut during menopause is dissimilar to those seen in male and aging mice. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lymphoid Follicle Transcriptome Reveals Maladaptive Immune Responses In Severe Chronic Obstructive Pulmonary Disease (COPD).

Abstract Rationale: Increases in the number and size of lymphoid follicles (LFs) are present in severe stages of COPD and have not been fully characterized. HLA complex is crucial in enabling the immune system to recognize “self” versus “non-self” antigens and plays a major role in immune responses to cigarette smoke exposure. Methods: Lung follicles from COPD patients with milder (GOLD I–II) and more severe (GOLD III–IV) COPD were sampled using GeoMX® Digital Spatial Transcriptional Profiling. Data were analyzed and normalized using GeoMx® DSP Analysis Suite. Results: Pathway analysis showed that B cell activation pathways had the highest fold enrichment. GOLD I–II compared to GOLD III–IV COPD patients had the highest expression of HLA-Cand its ligands (KIR2DS2, KIR2DS1, KIR3DL1; p&amp;lt;0.0001) which facilitate CD8-mediated cytotoxicity, and HLA-Gand its receptor LILRB4(p=0.01) which are key modulators of immune tolerance. GOLD III–IV COPD patients had the highest HLA enrichment in LFs, with upregulation of transcription factors (HIF1A, IRF1, NFYBand NFYC) and assembly components (CALR, TAPBP, ERP27, MARCHF1, B2M, and CIITA) of HLA (p&amp;lt;0.01). Compared to GOLD I–II, LFs from GOLD III–IV COPD patients had much higher expression of HLAs involved in autoimmune responses to self-antigens and adaptive immune evasion, such HLA-DRA, HLA-DRB1, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-Eand HLA-B(p&amp;lt;0.0001). Conclusions: We show for the first time that LFs from patients with severe COPD are characterized by expression of HLAs associated with immune evasion and autoimmune responses, whereas LFs in milder stages of COPD are characterized by induction of immune responses controlled by tolerance mechanisms R01 HL 149744-04/06

Abstract 3825: Andrographis reverses gemcitabine-associated chemoresistance by regulating calcium signaling in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the lethal malignancies due to the acquisition of intrinsic or acquired resistance to chemotherapeutic drugs, such as Gemcitabine (Gem). Recent evidence revealed that calcium signaling significantly contributes to Gem-associated chemoresistance (Gem resistance) in PDAC cells. Andrographis, a labdane diterpenoid isolated from the traditional herb Andrographis paniculate, is known to have a potent calcium channel-blocking potential. Accordingly, we hypothesized that Andrographis might facilitate the reversal of Gem resistance in PDAC. Methods: The critical regulatory pathways associated with the Gem resistance in PDAC were identified by analyzing publicly available cell line-based transcriptomic profiling. Gem resistant (Gem-R) PDAC cells (Gem-R MIA PaCa-2 and BxPC-3) were established by continuously culturing cells with Gem. A series of cell culture experiments were performed using Gem-R PDAC cells to evaluate the Andrographis-mediated reversal of Gem resistance in PDAC cells. At last, the cell culture findings were validated in patient-derived PDAC organoids. Results: Transcriptomic profiling identified the calcium signaling pathway as one of the key regulators of Gem resistance (Fold enrichment: 2.8, P =0.002). The combination of Andrographis and Gem revealed a superior anti-cancer potential in Gem-R PDAC cells on cell viability, clonogenicity, and migration compared to individual treatments with Andrographis or Gem. The combined treatment potentiated cellular apoptosis in Gem-R MIA PaCa-2 (Andro, Gem vs. Combination, 20.3%, 10.9% vs. 31.4%) and Gem-R BxPC-3 (Andro, Gem vs. Combination, 11.3%, 6.8% vs. 16.1%). The fluo-4 assay demonstrated that Andrographis decreased intracellular calcium concentration in Gem-R PDAC cells, while the combined treatment with Andrographis and Gem decreased its concentration more in Gem-R MIA PaCa-2 (fold change [FC] =0.79 vs. Andrographis; FC =0.54 vs. Gem) and Gem-R BxPC-3 (FC =0.80 vs. Andrographis; FC =0.73 vs. Gem). Finally, the combined treatment with Andrographis and Gem showed anti-cancer activity in patient-derived PDAC organoids. Conclusions: We have firstly investigated the Andrographis-mediated reversal of chemoresistance against Gem in PDAC cells, possibly through the regulation of cellular apoptosis and calcium signaling, using a systematic series of Gem resistant cell cultures and patient-derived tumor organoid models. Our findings could provide evidence of the combined treatment with Andrographis and Gem as a complementary therapeutic modality against PDAC. Citation Format: Keisuke Okuno, Caiming Xu, Masanori Tokunaga, Cristina Fillat, Haiyong Han, Yusuke Kinugasa, Ajay Goel. Andrographis reverses gemcitabine-associated chemoresistance by regulating calcium signaling in pancreatic ductal adenocarcinoma. [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 3825.

Abstract 6561: Specific binding mode of RNA binding protetin FAM120A to lncRNA

Abstract Long noncoding RNAs (lncRNAs) are defined as transcripts &amp;gt;200 nucleotides in length with no protein-coding potential. LncRNAs play diverse roles in regulating gene transcription, post-transcription, translation, and epigenetic modification through interaction with RNA binding proteins (RBPs). Here, we analyzed binding motif of a RBP, family with sequence similarity 120A (FAM120A), which is highly expressed in multiple cancer types and associated with poor prognosis. To investigate FAM120A interacting RNAs genome-wide, we conducted cross-linking immunoprecipitation combined with deep sequencing (CLIP-seq) using an anti-FAM120A antibody in osteosarcoma cell line, U2OS. We identified 37,151 FAM120A-binding clusters by a peak detection algorithm. Up to 92% of these clusters were found within about 5,700 mRNAs, with the highest enrichment at 3'-UTRs. Interestingly, other clusters located within 959 lncRNAs, with peaks at 5’ and 3’ end. Fold enrichment analysis of CLIP reads in comparison with input reads revealed that FAM120A binds more preferentially to lncRNA than mRNA (median fold enrichments, 4.81 (lncRNA) versus 4.41 (mRNA), P&amp;lt;0.001, Mann-Whitney U test). Motif analysis using the HOMER algorithm showed that FAM120A enriched sequence motifs in 3’ of mRNAs, 5’ and 3’ of lncRNAs were completely distinct. These results suggest that FAM120A may exhibit a motif-dependent selective binding to lncRNA in cancer cells. Given that RBPs bind to RNA molecules immediately after their transcription on chromatin, FAM120A may also selectively recognize RNA species and classify lncRNA and mRNA at the transcriptional stage. Since lncRNAs play pivotal roles in cancers FAM120A may exert oncogenic effects by the post-transcriptional regulation of a certain set of lncRNAs. Citation Format: Kenichiro Kawai. Specific binding mode of RNA binding protetin FAM120A to lncRNA [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 6561.

Abstract 4607: TRPS1 inhibits angiogenesis in triple negative breast cancer down regulating JAG1 and TYMP genes involved in angiogenesis pathways

Abstract Background. Triple-negative breast cancer (TNBC), the most difficult subtype to treat, is defined as estrogen receptor, HER2, and progesterone receptor negative. TNBC constitutes 10-20% of all breast cancer and has a higher rate of distal recurrence and a poorer prognosis than other breast cancer subtypes. Less than 30% of women with metastatic TNBC survive 5 years and almost all die from their disease despite adjuvant chemotherapy. Angiogenesis is a critical factor in the development of tumors and metastases in numerous cancers. Clinical trials with anti-angiogenic drugs showed to be inadequate, with moderate response rates and insignificant survival gains for patients. To understand the genetic forces involved in TNBC, we performed a transposon mutagenesis screen in Pten mutant mice that identified several candidate trunk drivers and progression genes. A major finding of our screen was the discovery and functional validation of, Trichorhinophalangeal syndrome type 1 (TRPS1), a GATA-like transcription factor, which functions as a transcriptional repressor or activator, depending on cell type, stage of development, or pathological conditions. Based on this data, we explored the role of TRPS1 in angiogenesis. Methods and Results. Tube formation and sprouting assays were performed using overexpression and inactivation of TRPS1 in MDA-MD-231 and HCC70 cells, respectively. Interestingly, inactivation of TRPS1 expression accelerates tube formation structures compared to the vector control as well as cell branching in the sprouting assay. Overexpression of TRPS1 prevents tubing and branching formation in vitro assays. Moreover, immunohistochemistry staining of CD31 detected a reduced number of blood vessels in MDA-MB-231 tumor xenografts overexpressing TRPS1, and an increase of angiogenic vasculature in HCC70 TRPS1-shRNA tumor xenografts. In vitro and in vivo assays demonstrate the role of TRPS1 in tumor angiogenesis. Furthermore, human ChIP qPCR angiogenesis array identified 10 top candidate genes potentially regulated by TRPS1 transcription factor. Of these candidates, JAG1 and TYMP showed to have a higher fold enrichment compared to other angiogenic related genes. Finally, we validated its direct functional binding by using luciferase reporter assay, such demonstrated that TRPS1 is a direct transcriptional regulator of JAG1 and TYMP. Conclusion. TRPS1 is a tumor suppressor involved in the mechanisms regulating tumor angiogenesis by repressing the expression of JAG1 and TYMP genes in TNBC. Citation Format: Liliana Guzman. TRPS1 inhibits angiogenesis in triple negative breast cancer down regulating JAG1 and TYMP genes involved in angiogenesis pathways. [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 4607.