Fold Change In Gene Expression Research Articles

149 Development of a leaky gut model: Impact of 24-h feed restriction on ileal expression of tight junction proteins in weaned pigs

Abstract Weanling barrows, 26 to 28 d old [n = 90; Duroc terminal line Fast genetics, initial body weight (BW)= 5.63 ± 0.43 kg] were used to evaluate the effect of 24-h complete feed restriction on growth and intestinal integrity. Upon arrival at the NutriQuest modeling center (Fergus Falls, MN), pigs were weighed, blocked by BW, and randomly assigned to 1 of 30 pens. Five treatments were randomly assigned to 6 pens: 1) Positive Control (PC), and 4 treatments that had 24-hr feed restriction (FR); 2) Negative control (NC), 3) Direct Fed Microbial (DFM) A, 4) DFM B, and 5) DFM C. Feed intake and BW were determined weekly and before and after FR. All pigs were fed a common diet. Fifteen pigs were removed prior to FR due to enteric disease. On d 18, feed was removed from pigs on treatments 2 to 5 for 24 h. After 24 h, 8 pigs per treatment were harvested for tissue collection. Feed access was restored to the remaining pigs who were harvested 3 d later. Ileal samples were collected and immediately frozen. Plasma and fecal samples were collected upon arrival, at FR, and 24 h and 4-d post-FR. Ileal expression of claudin-3, occludin, and tight-junction-1 genes was determined using QuantStudio 3 Real Time PCR system. Fold-change in gene expression compared with NC was calculated for each treatment. Plasma samples were analyzed for LPS binding protein (LBP) concentration, and fecal samples were analyzed for myeloperoxidase (MPO) concentration. Pig (n = 75) was the experimental unit for BW, LBP, MPO, and gene expression data. Pen (n = 30) was the experimental unit for feed intake. Data were analyzed using PROC GLIMMIX of SAS. Preplanned orthogonal contrasts were performed to determine the effect of PC vs. 4 FR treatments. To determine the relationship of tight junction gene expression and LBP and MPO, PROC REG of SAS was used. Data are described as PC vs. FR treatments to focus on model development. Final BW for pigs on PC was greater compared with the FR treatments (P < 0.05; Table 1). From 1 to 4 d post-FR, average daily gain (ADG) for FR treatments was greater than PC (P < 0.01). There was no effect of treatment on feed intake or feed efficiency. Plasma LBP and fecal MPO concentrations were greater 24 h post-FR for pigs on PC compared with FR treatments (P < 0.01). Fold change in occludin and claudin-3 expression was greater for PC compared with FR treatments (P ≤ 0.05). For pigs euthanized 24 h post-FR, there was a positive relationship between LBP concentrations and occludin expression (P < 0.02). Twenty-four hour FR in weaned pigs alters ileal tight junction gene expression and plasma LBP concentrations; therefore, a timed FR event may serve as a model for leaky gut.

Overexpression of WT1 in all molecular subtypes of breast cancer and its impact on survival: exploring oncogenic and tumor suppressor roles of distinct WT1 isoforms.

Breast cancer is a highly heterogeneous solid tumor, posing challenges in developing targeted therapies effective for all mammary carcinoma subtypes. WT1 emerges as a promising target for breast cancer therapy due to its potential oncogenic role in various cancer types. Previous works have yielded inconsistent results. Therefore, further studies are needed to clarify the behavior of this complex gene in breast cancer. In this study, we examined WT1 expression in both Formalin Fixed Paraffin Embedded breast tumors (n = 41) and healthy adjacent tissues (n = 41) samples from newly diagnosed cases of ductal invasive breast cancer. The fold change in gene expression between the tumor and healthy tissue was determined by calculating 2-∆∆Ct. Disease-free survival analysis was computed using the Kaplan-Meier method. To identify the expression levels of different WT1 isoforms, we explored the ISOexpresso database. Relative quantification of the WT1 gene revealed an overexpression of WT1 in most cases. The percentage of patients surviving free of disease at 8years of follow-up was lower in the group overexpressing WT1 compared to the group with down-regulated WT1. Interestingly, this overexpression was observed in all molecular subtypes of invasive breast cancer, underscoring the significance of WT1 as a potential target in all these subtypes. The observed WT1 down-expression in a few cases of invasive breast cancer, associated with better survival outcomes, may correspond to the down-regulation of a particular WT1-KTS (-) isoform: the WT1 A isoform (EX5-/KTS-). The co-expression of this WT1 oncogenic isoform with a regulated WT1- tumor suppressor isoform, such as the major WT1 F isoform (EX5-/KTS +), could also explain such survival outcomes. Due to its capacity to adopt dual roles, it becomes imperative to conduct individual molecular expression profiling of the WT1 gene. Such an approach holds great promise in the development of personalized treatment strategies for breast cancer.

Immunostimulation of Asian elephant (Elephas maximus) blood cells by parapoxvirus ovis and CpG motif-containing bacterial plasmid DNA upregulates innate immune gene expression.

The immune system of Asian elephants (Elephas maximus) is poorly studied, compared to that of livestock, rodents or humans. The innate immune response has become a focus of interest in relation to Elephant endotheliotropic herpesviruses (EEHVs). EEHVs cause a fatal hemorrhagic disease (EEHV-HD) and are a significant threat to captive Asian elephant populations worldwide. Similar to other herpesvirus infections, nearly all animals become infected, but only some develop disease. As progression to EEHV-HD is often acute, a robust innate immune response is crucial to control EEHV infections. This is invariably true of the host in the first instance, but it can also potentially be modulated by intervention strategies. Here, two immunostimulant veterinary medicinal products, authorized for use in domestic species, were tested for their ability to induce innate anti-viral immune responses in Asian elephant blood cells. Sequence data were obtained for a range of previously unidentified Asian elephant immune genes, including C-X-C motif chemokine ligand 10 (CXCL10), interferon stimulated gene 15 (ISG15) and myxovirus GTPase 1 (Mx1), and were employed in the design of species-specific qPCR assays. These assays were subsequently used in analyses to determine fold changes in gene expression over a period of 24 hours. This study demonstrates that both immunostimulant medications are capable of inducing significant innate anti-viral immune responses which suggests that both could be beneficial in controlling EEHV infections in Asian elephants.

Plasma Circulating mRNA Profile for the Non-Invasive Diagnosis of Colorectal Cancer Using NanoString Technologies.

Colorectal cancer (CRC) is one of the most prevalent cancers and the second leading cause of cancer deaths in developed countries. Early CRC may have no symptoms and symptoms usually appear with more advanced diseases. Regular screening can identify people who are at increased risk of CRC in order to offer earlier treatment. A cost-effective non-invasive platform for the screening and monitoring of CRC patients allows early detection and appropriate treatment of the disease, and the timely application of adjuvant therapy after surgical operation is needed. In this study, a cohort of 71 plasma samples that include 48 colonoscopy- and histopathology-confirmed CRC patients with TNM stages I to IV were recruited between 2017 and 2019. Plasma mRNA profiling was performed in CRC patients using NanoString nCounter. Normalized data were analyzed using a Mann-Whitney U test to determine statistically significant differences between samples from CRC patients and healthy subjects. A multiple-group comparison of clinical phenotypes was performed using the Kruskal-Wallis H test for statistically significant differences between multiple groups. Among the 27 selected circulating mRNA markers, all of them were found to be overexpressed (gene expression fold change > 2) in the plasma of patients from two or more CRC stages. In conclusion, NanoString-based targeted plasma CRC-associated mRNAs circulating the marker panel that can significantly distinguish CRC patients from a healthy population were developed for the non-invasive diagnosis of CRC using peripheral blood samples.

Comparative transcriptome profiling reveals differential defense responses among Alternaria brassicicola resistant Sinapis alba and susceptible Brassica rapa.

Alternaria blight is a devastating disease that causes significant crop losses in oilseed Brassicas every year. Adoption of conventional breeding to generate disease-resistant varieties has so far been unsuccessful due to the lack of suitable resistant source germplasms of cultivated Brassica spp. A thorough understanding of the molecular basis of resistance, as well as the identification of defense-related genes involved in resistance responses in closely related wild germplasms, would substantially aid in disease management. In the current study, a comparative transcriptome profiling was performed using Illumina based RNA-seq to detect differentially expressed genes (DEGs) specifically modulated in response to Alternaria brassicicola infection in resistant Sinapis alba, a close relative of Brassicas, and the highly susceptible Brassica rapa. The analysis revealed that, at 48 hpi (hours post inoculation), 3396 genes were upregulated and 23239 were downregulated, whereas at 72 hpi, 4023 genes were upregulated and 21116 were downregulated. Furthermore, a large number of defense response genes were detected to be specifically regulated as a result of Alternaria infection. The transcriptome data was validated using qPCR-based expression profiling for selected defense-related DEGs, that revealed significantly higher fold change in gene expression in S. alba when compared to B. rapa. Expression of most of the selected genes was elevated across all the time points under study with significantly higher expression towards the later time point of 72 hpi in the resistant germplasm. S. alba activates a stronger defense response reaction against the disease by deploying an array of genes and transcription factors involved in a wide range of biological processes such as pathogen recognition, signal transduction, cell wall modification, antioxidation, transcription regulation, etc. Overall, the study provides new insights on resistance of S. alba against A. brassicicola, which will aid in devising strategies for breeding resistant varieties of oilseed Brassica.

Astragaloside IV Alleviates Acute Liver Failure Induced by D-GalN/LPS by Upregulating Autophagy and Reducing Inflammation

Abstract Acute liver failure (ALF) is a life-threatening condition that manifests in an extremely serious manner and progresses rapidly. The following study investigated the protective effect of astragaloside IV (AS-IV), a traditional Chinese drug, on ALF, and its underlying mechanisms, focusing on autophagy and inflammation regulation. Mice were randomly divided into a saline group, a D-galactosamine and lipopolysaccharide (D-GalN/LPS) group and an AS-IV group. Biochemical analysis, immunohistochemistry, cytometric bead array, high-throughput quantitative PCR, flow cytometry and Western analysis were used to assess inflammation and liver damage 5 hours after D-GalN/LPS exposure. Astragaloside IV treatment reduced mortality by alleviating D-GalN/LPS–induced hepatic damage and decreasing inflammation (decreasing Ly6c+ monocyte levels, reducing inflammatory cytokines and increasing anti-inflammatory factors) as well as upregulating autophagy. Furthermore, PCR array was used to detect expression of autophagy-related genes, which demonstrated a Log2 fold change in gene expression between the AS-IV and D-GalN/LPS groups ranging from 1.19 to −3.53, with Tnfsf10 showing the largest alteration between the two groups. These data suggest that AS-IV may alleviate ALF by upregulating autophagy and reducing inflammation, and it may therefore be an interesting drug for alleviating ALF.

PSV-26 Use of Garlic Drench in the Modulation of Galectin Gene Expression in St Croix Sheep

Abstract St. Croix sheep are parasite resistant and can live in a wide variety of climates. Dietary supplements can influence innate immunity. Garlic (Allium sativum) is used as an immune modulator and an alternative antibiotic. Lectins in garlic could interfere with animal lectins such as Galectins (Gals). Galectins bind β-galactosides and modulate pathological processes and immunity. The effect of garlic drench on Gal (1,3,9) gene expression in sheep blood was evaluated. All protocols were approved by the IACUC. Clinically healthy, adult St. Croix sheep were divided into two groups. The treatment group (n = 6) was drenched with 5 mL garlic barrier at a rate of 10 mL diluted in sterile distilled water 1:1. The control group (n = 6) was drenched with 10 mL sterile distilled water for four weeks. Cells and plasma were separated by centrifugation of whole blood. The concentration of total plasma protein was determined using the BCA assay kit (Pierce). Secretion of Galectins 1,3,9 was evaluated using Sheep-specific ELISA(My-BioSource). Data were analyzed using SAS Statistical analysis software 9.4 (SAS Institute). Nucleic acid purity and concentrations were assessed using the Nanodrop spectrophotometer. Galectin gene transcription was evaluated using sheep Gal 1,3 and 9 specific primers commercially sequenced (Eurofins Genomics). The house keeping genes, Ribosomal Protein Lateral Stalk Subunit P0 and Ubiquitin C-Terminal Hydrolase L5, were used as internal controls. Samples were amplified in duplicate using the CFX Connect real-time PCR system. The Livak Method was used to calculate the fold change in gene expression. Galectin 1, 3, and 9 were transcribed and translated. Transcription and translation were differentially affected by garlic. Galectin 1 transcription increased; secretion decreased. Galectin 3 transcription and secretion increased at P < 0.05. There was no change in the transcription of galectin 9, however, secretion was increased. Thus, garlic drench deferentially impacted transcription and secretion of structurally distinct Gal in sheep blood. Further studies are needed to determine the effect of Garlic on Gal-mediated functions important to sheep health and production.

Protective Effect of Crocin on Cadmium Induced-Overexpression of Matrix Metalloproteinases (MMPs) Gene Family in Rat Liver

Background: Metal pollutants such as Cadmium are known as carcinogenic agents and correlated with metastatic potential and tumor aggressiveness. Matrix metalloproteinases (MMPs) are taken into account as the focal points of metastasis. Crocin, a carotenoid chemical compound in Saffron, has been proved to have anti-cancerous properties. However, there is no reliable evidence yet to prove its anti-metastatic activity. Objective: The evaluation of the protective effect of Crocin against Cadmium through monitoring the expression of MMP genes. Materials and Methods: The in-silico analysis based on the GEO database was done for purposefully choosing MMPs. At the wet lab step, after 8 weeks of treatment of the animal (40 female Wistar rats divided into four groups of 10) with Cadmium, Crocin, complex (Cadmium+Crocin) and placebo (control), RNA extraction followed by cDNA synthesizing was done from rats’ liver tissues. Using primers for MMP genes (MMP-1, 3, 7, 12 and 13), the relative fold change of gene expression was studied. Ultimately, the statistical analysis determined the difference in gene expression between groups. Results: Cadmium significantly upregulated the selected MMP genes in comparison with controls. In contrast, Crocin significantly downregulated the expression of all studied MMP genes. Complex administration of Cadmium and Crocin displayed a controlled increase of expression in all MMPs except MMP-1, which was lower than the Cadmium group. Conclusion: These results indicated that the elevation of MMPs expression level by Cadmium was significantly suppressed by Crocin. This could promisingly demonstrate that Crocin possesses a modulating role in inhibiting overexpression of MMPs that might be considered as the anti-metastatic agent.

Osteoinductive effect β-TCP and vitamin D3 on RUNX2 mRNA expression

Although the autograft is regarded as the gold standard bone graft, β-TCP artificial alloplastic is widely used in bone defect healing and has been accepted in human and animal studies to be resorbed and interchanged by vital bone. The active form of vitamin D3 is calcitriol, on mesenchymal stem cells (MSCs) it has a direct influence on encouraging their osteogenic differentiation which is identified as osteoinductive. The need for osteoinductive ability is a demanding issue regarding medical and dental care. RUNX2 regards an essential transcriptional factor for osteoblast differentiation. Adult male New Zealand rabbits (Twenty-four) with an average weight of (1.5-2 kg) were elected in this study. Four holes were made intra-bone in both tibias of each animal. The experimental groups divide as follows: Group C: Bone defect will be left to heal naturally as control; Group TCP: Bone defect will be occupied with β-TCP. Group vitamin D3: Bone defect will be filled with vitamin D3; Group TCPD: Bone defect will be filled with a combination of β-TCP and vitamin D3. Animals sacrificed at (1 and 3 weeks). Purification of total RNA from formalin fixed paraffin embedded (FFPE), The cDNA of RUNX2 and VDR amplify in RT-PCR and detected with sybr green. One-way Analysis of Variance (ANOVA) and least significant difference (LSD) was used as a tool for statistical analysis. Results at 7 days highest mean value of mRNA RUNX2 gene expression Fold Change estimated in TCP followed by TCPD experimental group while at 21 days, the highest mean value in TCPD. All measured parameters showed highly significant differences in the course of healing periods from 7 days to 21 days. In mRNA VDR gene expression parameters showed a highly significant difference in 7days and 21 days durations among experimental groups and the highest mean value of mRNA VDR gene expression Fold Change is recorded in TCPD followed by vitamin D3 groups in both durations. These findings suggest positive inductive effects of local application of vitamin D3 combined with β-TCP as well as vitamin D3 on osteoblast cells and induced bone defect healing.

Dynamic Translational Landscape Revealed by Genome-Wide Ribosome Profiling under Drought and Heat Stress in Potato.

The yield and quality of potatoes, an important staple crop, are seriously threatened by high temperature and drought stress. In order to deal with this adverse environment, plants have evolved a series of response mechanisms. However, the molecular mechanism of potato's response to environmental changes at the translational level is still unclear. In this study, we performed transcriptome- and ribosome-profiling assays with potato seedlings growing under normal, drought, and high-temperature conditions to reveal the dynamic translational landscapes for the first time. The translational efficiency was significantly affected by drought and heat stress in potato. A relatively high correlation (0.88 and 0.82 for drought and heat stress, respectively) of the fold changes of gene expression was observed between the transcriptional level and translational level globally based on the ribosome-profiling and RNA-seq data. However, only 41.58% and 27.69% of the different expressed genes were shared by transcription and translation in drought and heat stress, respectively, suggesting that the transcription or translation process can be changed independently. In total, the translational efficiency of 151 (83 and 68 for drought and heat, respectively) genes was significantly changed. In addition, sequence features, including GC content, sequence length, and normalized minimal free energy, significantly affected the translational efficiencies of genes. In addition, 28,490 upstream open reading frames (uORFs) were detected on 6463 genes, with an average of 4.4 uORFs per gene and a median length of 100 bp. These uORFs significantly affected the translational efficiency of downstream major open reading frames (mORFs). These results provide new information and directions for analyzing the molecular regulatory network of potato seedlings in response to drought and heat stress.

Examining the effect of Helicobacter pylori cagPAI variety on gene expression pattern related to gastric cancer.

We aimed to determine possible association between heterogeneity of Helicobacter pylori cytotoxin-associated gene pathogenicity island and gene expression profiles in patients with distinct histopathological changes. Gastric biopsies were obtained from seventy five patients. Microbiological and pathological examinations were done and intactness of Helicobacter pylori cagPAI was determined by PCR using 11 pairs of primers flanking cagζ-cagA regions and cagPAI empty site. Alterations at mRNA levels of eight genes were investigated by real-time PCR and their association with cagPAI intactness and histopathological changes examined statistically. A larger proportion of cagPAI positive strains colonized patients with SAG (52.4%), followed by CG (33.3%), and IM (14.3%). Intact cagPAI was found in 87.5% of the strains obtained from patients with SAG, while significantly lower frequency was detected among those with CG (12.5%) and IM (0%). No significant difference was found among thestudied histological groups and fold changes in gene expression of gastric biopsies of Helicobacter pylori infected patients with distinct cagPAI status. However, in each histological group, the strains with more complete gene cluster induced (ErbB2, CCNE1, CTNNB1, and MMP7 in SAG and IM groups) or reduced (TP53, in CG group) expression of the GC associated genes in relatively higher levels. APC, TP53 and E-cadherin were down-regulated in patients with SAG and IM compared with CG patients, irrespective to the status of cagPAI integrity. Helicobacter pylori strains that carry more complete cagPAI segment could induce remarkably higher levels of mRNA changes of GC associated genes in all histopathological groups.

Hepatic genomic assessment of dietary ingestion of 2-aminoanthracene in Sprague Dawley rats

This research aims to investigate the effects of 2-aminoanthracene (2-AA), a polycyclic aromatic hydrocarbon (PAH), on the liver. PAH is a by-product of the incomplete combustion of fossil fuels. Specifically, the impact of 2-AA on different body tissues in animals has been reported. The liver is an organ central to the metabolism of PAHs, including 2-AA. Sprague Dawley rats ingested a well-defined dose of 2-AA in their diet (0, 50, and 100 mg/kg 2-AA) for 12 weeks. Hepatic global gene expression using Affymetrix Rat Genome 230 2.0 microarray was performed. Overall, more than 17,000 genes were expressed. Approximately 70 genes were upregulated, while 65 were downregulated when control rats were compared with low-dose animals. Similarly, 103 genes were upregulated and 49 downregulated when the high-concentration 2-AA group was compared with the control group rats. This result suggests that the magnitude of gene expression fold change depends on the dose of 2-AA ingested. Several differentially expressed genes are involved in biological processes such as gene transcription, cell cycle, and immune system function, indicating that the ingestion of 2-AA could impact these processes. The over-expression of genes related to liver inflammation, nonalcoholic liver disease, hepatic glucose processing, and PAH metabolism were noted.

Claudin-6 enhances the malignant progression of gestational trophoblastic neoplasm by promoting proliferation and metastasis.

Choriocarcinoma (CC) is a rare and highly malignant epithelial tumour. However, the mechanism underlying its occurrence and development remains unknown. We aimed to reveal the biological significance and prognostic value of Claudin-6 (CLDN6) in gestational trophoblastic disease (GTD). We collected clinical GTD specimens from 2011 to 2019 and measured CLDN6 gene expression by immunohistochemistry (IHC). High-throughput mRNA sequencing (RNA-seq) revealed a GTD progression-associated gene. CCK-8, wound healing, and flow cytometry assays were used to assess the biological effects of CLDN6 overexpression and knockdown. The medical records of 118 GTD patients from 2011 to 2019 were retrospectively analysed to identify correlations between CLDN6 expression and GTD patient clinical-pathological parameters; these correlations were analysed using the chi-square test and one-way ANOVA. Univariate logistic regression was used to analyse various prognostic parameters of patients with post-molar GTN. CLDN6 had the second highest fold change in gene expression between GTN and normal samples. CLDN6 was highly expressed in GTN tissues and CC cell lines, and silencing CLDN6 inhibited the proliferation and migration and promoted the apoptosis of CC cells. CLDN6 overexpression was significantly correlated with uterine size (p = 0.01) and ovarian cysts > 6cm (p = 0.027), CLDN6 expression was significantly higher in HR-GTNs than in low-risk GTNs (LR-GTNs) (p = 0.008), and logistic regression analysis showed that CLDN6 expression in hydatidiform moles (HMs) was related to a high risk of developing post-molar GTN (OR = 2.393, p = 0.03). We propose that CLDN6 participates in the development of GTD and may become a new therapeutic target for CC.

Sterilization Procedures for Titanium Alloy Surfaces Leads to Higher Expression of Biofilm-Related Staphylococcus aureus Genes.

Background: Around 1-2% of all implantation surgeries lead to implant-related infections, incurring costs of $40,000-$160,000 per total hip PJI. The 5-year mortality rate of prosthetic joint infections is up to 21%. To prevent infections during surgery, sterile surgery rooms and procedures have been developed and certified standards have been established. To guarantee the sterility, implants can be acquired already sterile from manufacturers. Some titanium implants can be delivered unsterilized with a manual for sterilization procedure in compliance with ISO 17664. The aim of this study is to evaluate if the most used sterilization methods (steam sterilization in an autoclave and UV light sterilization) of titanium alloys, can influence the biofilm forming capacity of Staphylococcus aureus. In this study, we examined the influence of sterilization methods on the gene expression of biofilm-associated genes and regulators. Methods: We compared gene expression of icaADBC, SarA, SigB, and SodA on titanium CP4 and Ti6Al4V alloys sterilized by UV-light and pressurized saturated steam sterilization. We performed RT-qPCR after RNA extraction of Staphylococcus aureus ATCC 29213. In addition, bacterial cell growth on the sterilized titanium surfaces was examined by colony forming unit counting on agar plates after 24 h of incubation. Results: Colony forming units of S. aureus on titanium CP4 samples showed a higher tendency in colony counts when sterilized with UV light than with pressurized saturated steam (autoclaved). Similarly, colony forming unit counts on Ti6Al4V samples showed tendencies of higher numbers on UV light sterilized samples than on autoclaved samples. Gene expression of icaADBC, SarA and SodA between steamed samples and UV light sterilized samples showed no difference on titanium CP4 samples, whereas SigB showed higher gene expression on titanium CP4 samples when sterilized with UV light than in an autoclave. On autoclaved Ti6Al4V samples, all examined genes showed 4 to 9 times higher fold changes in gene expression than on UV light sterilized samples. Conclusions: This study indicates that steam sterilization of Ti6Al4V can increase biofilm formation of S. aureus on its surface. The significantly increased gene expression of biofilm responsible genes may indicate a modification of titanium surfaces on alloy components. This may promote biofilm formation that can lead to implant-infections in vivo.

EFS24 Failure, BRD4 Expression, and TP53 Are Linked to Reduced CDKN1A Expression in DLBCL and May be Targeted By Single-Agent Protac ARV-825 or in Synergistic Combination with the CDK4/6 Inhibitor Abemaciclib

Background: Genomically-targeted Diffuse Large B-cell (DLBCL) treatments for refractory or relapsing patients remain a clinical need alongside the growing success of Chimeric Antigen T-cells (CAR-T), which have ushered in durable responses in 40-70% of patients. Recent studies have identified factors associated with progression, including alterations to the TP53 tumor suppressor and a cold immune environment, among others. The BRD4 bromodomain protein has emerged as an actionable target in recent years given its role orchestrating numerous oncogenic processes that drive aggressive DLBCL capable of rejecting R-CHOP regimens. Specifically, Proteolysis targeting chimera technology - PROTACs - have arisen as a novel mechanism to degrade target oncogenes in hematological malignancies. The BRD4-degrading PROTAC ARV-825 represents the next generation of this technology. Herein, we report integrative analyses supporting that CDKN1A restoration via BRD4 degradation represents a treatment avenue for DLBCL patients with a cold immune microenvironment predictive of inferior CAR-T response, the first observations of single-agent ARV-825 efficacy vs. DLBCL cell lines, and in-vitro synergy between ARV-825 and the CDK4/6 inhibitor Abemaciclib. Methods: We analyzed 2 de-novo DLBCL patient cohorts (Xu-Monette et. al. 2020 and the TCGA) for differential expression based on Event-free 24-month survival (EFS24), TP53 alteration status, and BRD4 overexpression. Profiles expressing 1+ Standard deviations above the BRD4 mean were classified as BRD4-High. We integrated post-BRD4-degradation gene expression fold change values from Jain et. al. 2019 alongside these data. We next assayed ARV-825 vs. 7 DLBCL cell lines, 5 of which bear TP53 alterations, across a series of 9 concentrations (78nM-20µM) across 3-9 replicates. We followed these results by combining ARV-825 with Abemaciclib in the TP53-impaired DHL6 cell lines, analyzing results using the BLISS synergy model. Results: Integrative analyses revealed that EFS24 failure was most associated with alterations to TP53 (P = 0.0069) and greater BRD4 expression (P = 0.0217), with BRD4 as the greatest differentially expressed gene in these patients. Overall survival matched this trend, with TP53 alteration (0.0005) and High BRD4-expressing (P = 0.0352) patients facing inferior outlooks. Interestingly, these factors were associated with each other, as BRD4 expression was greater in TP53-mutant profiles (FDR = 0.0663). Tumors harboring either factor were associated with significantly reduced expression of Regulation of Programmed Cell Death (FDR = 9.080E-19) and Cytokine Signaling in Immune System (FDR = 1.029E-12) genes. Genes with significantly greater expression were unsurprisingly most associated with the cell cycle (FDR = 2.550E-12). Notably, the CDK1NA tumor suppressor responsible for inhibiting the CDK4/6 oncogenes was significantly reduced in patients with TP53 alterations and/or High BRD4 expression (FDR = 9.840E-09), collectively serving as the greatest loss within this population. Significant renewal of CDKN1A expression may be accomplished via BRD4 inhibition (q-value = 0.0132, Fold Change = 2.024) based on post-ARV-771 DLBCL cell line treatment results from Jain et. al. 2019. Following these results, we assayed ARV-825 vs. DLBCL cell lines, demonstrating sub-micromolar efficacy in all 7 models. ED50 values ranged between 41nM and 440nM. ARV-825 was next combined with the CDK4/6 inhibitor Abemaciclib in the TP53-imparied DHL6 cell line, resulting in synergistic reduction of cell viability across a range of doses and a BLISS synergy score of 12.946. Conclusions: Our results suggest that increased BRD4 expression alongside TP53 alterations are associated with EFS24 failure, a cold immune microenvironment, and the loss of critical tumor suppressors, namely CDKN1A. We assayed the BRD4-degrading PROTAC ARV-825 vs. cell line models and are the first to report the substantial preclinical efficacy of this molecule in DLBCL. We are also the first to report synergistic reductions in cell viability in a TP53-imparied DLBCL cell line when ARV-825 was combined with the CDK4/6 inhibitor Abemaciclib. These results highlight the potential efficacy of PROTACs within DLBCL and how targeted treatment of a population facing inferior CAR-T prognosis may emerge via the restoration of CDKN1A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

OR04-3 Genetic Alterations of ARMC5 and KDM1A Are Associated With Different Expression Profiles of Illegitimate Receptors in Primary Bilateral Macronodular Adrenal Hyperplasia

Abstract Introduction Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) is a heterogeneous disease characterized by bilateral adrenal macronodules responsible for adrenal Cushing. To date, two genetic causes of PBMAH are known: germline inactivating variants of the tumor suppressor genes ARMC5 identified in 2013 (Assié, N Eng J Med 2013), responsible for 20 to 25% of index cases, and KDM1A, identified recently (Vaczlavik, GIM 2021; Chasseloup, Lancet D&E 2021), responsible for the rare presentation associated with food-dependent Cushing's syndrome (FDCS) due to aberrant expression of the GIP receptor (GIPR) in adrenocortical cells. Multiple other illegitimate receptors are known to be responsible for abnormal cortisol response to various physiological stimuli in PBMAH. A recent multiomic analysis, identified three distinct molecular PBMAH groups: G1 with ARMC5-mutated tumors, G2 with KDM1A-mutated tumors from FDCS patients, and G3 with no identified genetic cause at present. We aimed to identify specific expression profiles of illegitimate receptors in the three groups. Methods Based on the transcriptome data obtained by RNA sequencing (Illumina) of the tumors from 31 patients (G1/ARMC5, 16 patients; G2/KDM1A, 6 patients; G3, 9 patients), expression of the following genes, encoding potential illegitimate receptors, were compared: ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3, AVPR1A, AVPR1B, AVPR2, GCGR, GIPR, HTR4, HTR7, LHCGR. Calculations were performed using R statistical software. The Bioconductor limma package was used to analyze mRNA differential expression. Results G1/ARMC5 tumors showed a relative overexpression of the vasopressin receptors AVPR1A and AVPR1B compared to the two other groups (fold-change [FC] =7.39, p<0.001 and 3.98, p<0.001, respectively) but a lower expression of AVPR2 (FC=0.43, p=0.015). G2/KDM1A tumors showed a dramatic overexpression of GIPR compared to the two other groups (FC=105.02, p<0.001) but also of the adrenergic receptors ADRA1D and ADRA2A (FC=2.93, p=0.027 and 9.99, p<0.001, respectively) and of the LH/hCG receptor (LHCGR) (FC=12.20, p<0.001). G3 tumors showed a slight overexpression of the adrenergic receptor ADRA1B (FC=3.49, p=0.001) and in few tumors ADRA1D, AVPR2 and LHCGR were highly expressed suggesting molecular heterogeneity in G3. Conclusion This study reveals specific expression profiles of illegitimate receptors related to the three molecular groups. ARMC5 tumors are associated with the overexpression of two vasopressin receptors, while, besides GIPR, KDM1A inactivation seems to drive the overexpression of the LH/hCG receptor, as previously suggested in patients with FDCS (Bertherat, JCE&M 2005), potentially responsible for Cushing's syndrome associated with pregnancy and menopause. These molecular patterns need to be corroborated by clinical data with a systematic testing of the aberrant cortisol responses. Additionally, further studies would be needed to investigate the clinical relevance and significance of moderate fold-changes in gene expression (e.g. <4). Presentation: Saturday, June 11, 2022 12:00 p.m. - 12:15 p.m.

Expression of IL1β Gene in the Placentas from Humans and Cows during Brucella Infection.

Brucellosis is an important contagious disease affecting most domestic and mature animals. Since the impact of IL-1β in B. abortus invasion and survival remains elusive, the current study sought to elucidate the actual roles of these potent cytokines in the modulation of the initial immune response to Brucella infection. Therefore, this study aimed to detect Brucella abortus in the placenta of aborted women and cows and estimate the expression of the interleukin 1β (IL1β) gene associated with immune response mechanisms to Brucella abortus infection. The detection of Brucella abortus was performed by Rose Bengal Test (RBT) and Polymerase Chain Reaction based AlkB gene (AlkB-PCR) in the sera and placenta samples of aborted women and cows, respectively. The overall percentage of Brucella abortus infection was 13.1% and 5% as determined by RBT and AlkB-PCR in aborted women's sera and placentas, respectively. On the other hand, the overall percentage rates of Brucella abortus infection in the sera and placentas from aborted cows were 30% and 11% as estimated by RBT and AlkB-PCR, respectively. The results of RBT demonstrated that the association between Brucella abortus and abortion in cows was statistically significant. On the other hand, it was found that the association between Brucella abortus and abortion in women was not significant. Moreover, according to the results of AlkB-based PCR, the association between Brucella abortus and abortion was statistically significant in aborted cows, while it was not significant in aborted women. The sensitivity, specificity, and accuracy of RBT were calculated as 60.00, 53.85, and 54.55%, respectively. Moreover, positive and negative predictive values were reported as 14.33% and 91.28%, respectively. Regarding RBT for aborted cows, the sensitivity, specificity, and accuracy of the test were 81.82%, 57.78%, and 62.49%, respectively. The positive predictive value was reported as 32.08%, while the negative predictive value was reported as 92.88%. Quantitative PCR (qPCR) was carried out for the evaluation of Interleukin 1 Beta (IL1β) gene expression. The qPCR result was presented as a fold change in gene expression. A significant increment of IL1β gene expression was observed in aborted women (114.905±99.661) and cows (22.454 ±18.528), compared to non-aborted women (4.953±5.564) and cows (2.033±1.845). Statistical comparison of IL1β gene expression between aborted women and cows illustrated a non-significant increment in IL1β gene expression in aborted women (114.905±99.661), compared to aborted cows (22.454 ±18.528).

Abstract P3077: The Role Of Neutrophils In Initiating Type I Interferon Response In Myocardial Ischemia/reperfusion

Background: Inflammation succeeding myocardial ischemia/reperfusion (MI/R) plays a significant role in damaging cardiomyocytes and influencing infarct size, with neutrophils being the most rapid and most numerous cells in the post-MI/R heart. While neutrophils play a role in wound healing, they can cause further damage through the generation of reactive oxygen species, proteolytic enzymes, and neutrophil extracellular traps (NETs). We thus hypothesize that double stranded DNA stemming from these NETs activates cGAS-STING signaling in a murine model of MI/R, leading to the production of type I interferons by neutrophils, which worsens reperfusion injury. Methods: Single-cell RNA sequencing was performed on neutrophils isolated from the hearts of mice 24 hours post-MI/R. Additionally, mice underwent MI/R and heart tissues were harvested 6- and 24- hours after reperfusion for ELISA and qPCR analysis. Results: Utilizing single-cell RNA sequencing to better characterize the role of neutrophils and NETs in MI/R, we identified a significant population of neutrophils with a strong type I interferon (IFN) signature recruited to the area of infarct 24 hours post MI/R in a murine model. To further investigate the IFN response seen in this population of neutrophils, we preformed protein and gene expression analysis on heart tissue samples isolated from mice at timepoints in which neutrophils would be present in the area of infarct. Analysis by qPCR shows an increased fold change in gene expression of IFNα (7.22 ± 6.05, n = 3; 3.83 ± 2.22, n = 4) and IFNβ (6.74 ± 5.39, n = 3; 3.64 ± 2.25, n = 4) at 6- and 24- hours post MI/R respectively, compared to sham mice. Furthermore, analysis by ELISA shows a significant increase in IFNβ protein levels in the heart after 6- hours (0.41pg/mg ± 0.04, n =8) compared to sham mice (0.16 pg/mg ± 0.06, n = 7; p ≤ 0.01, Student’s t-test). Conclusions: These data show a rapid increase in type I IFN in the hearts of mice post-MI/R, which implicates neutrophils may be participating in the IFN signaling pathway. Identifying neutrophils as an early source of IFN can provide a potentially new therapeutic target in treating MI/R.

Heat-induced increases in body temperature in lactating dairy cows: impact on the cumulus and granulosa cell transcriptome of the periovulatory follicle.

Cows acutely heat stressed after a pharmacologically induced luteinizing hormone (LH) surge had periovulatory changes in the follicular fluid proteome that may potentiate ovulation and impact oocyte developmental competence. Because the cellular origins of differentially abundant proteins were not known, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows exhibiting varying levels of hyperthermia when occurring after the LH surge. After pharmacological induction of a dominant follicle, lactating dairy cows were administered gonadotropin releasing hormone (GnRH) and maintained in thermoneutral conditions (~67 temperature-humidity index [THI]) or heat stress conditions where THI was steadily increased for ~12 h (71 to 86 THI) and was sufficient to steadily elevate rectal temperatures. Cumulus-oocyte complexes and mural granulosa cells were recovered by transvaginal aspiration of dominant follicle content ~16 h after GnRH. Rectal temperature was used as a continuous, independent variable to identify differentially expressed genes (DEGs) increased or decreased per each 1 °C change in temperature. Cumulus (n = 9 samples) and granulosa (n = 8 samples) cells differentially expressed (false discovery rate [FDR] < 0.05) 25 and 87 genes, respectively. The majority of DEGs were upregulated by hyperthermia. Steady increases in THI are more like the "turning of a dial" than the "flipping of a switch." The moderate but impactful increases in rectal temperature induced modest fold changes in gene expression (<2-fold per 1 °C change in rectal temperature). Identification of cumulus DEGs involved in cell junctions, plasma membrane rafts, and cell-cycle regulation are consistent with marked changes in the interconnectedness and function of cumulus after the LH surge. Depending on the extent to which impacts may be occurring at the junctional level, cumulus changes may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. Two granulosa cell DEGs have been reported by others to promote ovulation. Based on what is known, several other DEGs are suggestive of impacts on collagen formation or angiogenesis. Collectively these and other findings provide important insight regarding the extent to which the transcriptomes of the components of the periovulatory follicle (cumulus and mural granulosa cells) are affected by varying degrees of hyperthermia.

The optimal electrical stimulation for neural differentiation of conjunctiva mesenchymal stem cells.

The combination of biomaterial conductive scaffolds and electrical stimulation (ES) dramatically promotes stem cell differentiation into electro-responsive cells like neural cells. In this study, we aimed to fabricate PCL/PPY nanofiber scaffolds through the electrospinning method and investigate the effect of ES duration on neural differentiation of Conjunctiva Mesenchymal Stem Cells (CJMSCs). The topography of the fabricated scaffold was characterized using SEM and TEM microscopy, and its mechanical and other properties were determined by tensile, TGA, FTIR, and Contact angle tests. CJMSCs were seeded on the scaffolds and then subjected to electrical current (115 V m-1 at 100 Hz) with durations of 1, 3, and 7 min for 3 days. Then the effect of nanofiber scaffold and electrical currents on cell viability and expression of neural marker genes (Nestin, β-tubulin, MAP-2) was investigated by MTT assay and qPCR analysis. Our results revealed the good biocompatibility of the PCL-PPy nanofiber scaffold, and according to q-PCR results, the electrical stimulation of 1 min day-1 for 3 days can induce neural differentiation of CJMSCs as indicated by the fold change of gene expression of Nestin (~127), B-tubulin (~30), and MAP-2 (~52). This study emphasizes that the utilization of an electrically conductive nanofibrous scaffold in conjunction with electrical current has potential applications in the field of neural tissue engineering.