Baseline Expression Research Articles

DOP067 Selective JAK1 blockade with Upadacitinib is effective in immune checkpoint inhibitor-induced colitis

Abstract Background Immune checkpoint inhibitors (CPI) have transformed cancer outcomes, but frequently trigger autoimmune- like events, including life-threatening colitis. Current immunosuppressive treatments for CPI- colitis (CPI-c) incur significant side effects and may compromise CPI efficacy, highlighting the need for new therapies. Methods RNA sequencing was performed on colonic biopsies from patients with CPI-c (n=12), CPI (no colitis, n=6), healthy controls (n=13), and UC (n=12). Gene expression, pathway level (GSEA) and cellular composition were analysed. Cell-cell communication were analysed using a single-cell RNA sequencing reposited dataset. Colonic organoids from CPI-c and controls were cultured and treated with IFNg ± JAK1-STAT inhibition with Upadacitinib . The impact of JAK1 blockade was evaluated in vivo using pre-clinical models of CPI-c1. Colonic leukocytes were analysed by flow cytometry. Clinical and endoscopic outcomes were defined in five patients with treatment-refractory CPI-c, following treatment with Upadacitinib 45mg for 8 weeks. Results Gene expression profiling of colonic biopsies highlighted overactivity of the JAK1-dependent cytokine IFNg in CPI-c. IFNg responsive transcripts, including CXCL9, CXCL10, IDO1, GZMB and STAT1, were among the most upregulated transcripts, with IFNg signalling identified as the top enriched pathway. Cellular deconvolution revealed enrichment of TH1 and CD8+ memory cells. Cell-cell communication analysis indicated distinct dialogue mediated by IFNg between T-cell subsets in CPI-c, including TH17+PD1+ and epithelial cells, and inflammatory enterocytes. Colonic organoids from CPI-c patients retained high baseline expression of IFNg-responsive transcripts, and significantly over-expressed these following ex vivo stimulation with IFNg, which was corroborated at protein level. Upadacitinib treatment of stimulated organoids reversed these changes. In vivo administration of Upadacitinib significantly reduced pathological features and severity of CPI-c in preclinical models of disease. Encouraged by these findings, we treated 5 patients with severe, treatment refractory CPI-c with Upadacitinib. All achieved rapidly resolution of disease with complete mucosal healing, no adverse events, and favourable 9-month cancer outcomes were sustained. Conclusion IFNg signalling was highly upregulated in CPI-colitis patients, which was recapitulated in a novel CPI-c derived colonic organoid model and in vivo models of disease. Upadacitinib improved organoid morphology, reduced IFNg targets, and ameliorated CPI-c in mice. Moreover, Upadacitinib rapidly rescued treatment refractory patients highlighting its potential as a promising therapeutic approach in patients with severe disease.

Histone Methyltransferase G9a in Primary Sensory Neurons Promotes Inflammatory Pain and Transcription of Trpa1 and Trpv1 via Bivalent Histone Modifications.

Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) channels are crucial for detecting and transmitting nociceptive stimuli. Inflammatory pain is associated with sustained increases in TRPA1 and TRPV1 expression in primary sensory neurons. However, the epigenetic mechanisms driving this upregulation remain unknown. G9a (encoded by Ehmt2) catalyzes H3K9me2 and generally represses gene transcription. In this study, we found that intrathecal administration of UNC0638, a specific G9a inhibitor, or G9a-specific siRNA, substantially reduced complete Freund's adjuvant (CFA)-induced pain hypersensitivity. Remarkably, CFA treatment did not induce persistent pain hypersensitivity in male and female mice with conditional Ehmt2 knockout in dorsal root ganglion (DRG) neurons. RNA sequencing and quantitative PCR analyses showed that CFA treatment caused a sustained increase in mRNA levels of Trpa1 and Trpv1 in the DRG. Ehmt2 knockout in DRG neurons elevated baseline Trpa1 and Trpv1 mRNA levels but notably reversed CFA-induced increases in their expression. Chromatin immunoprecipitation revealed that CFA treatment reduced G9a and H3K9me2 levels while increasing H3K9ac and H3K4me3-activating histone marks-at Trpa1 and Trpv1 promoters in the DRG. Strikingly, conditional Ehmt2 knockout in DRG neurons not only diminished H3K9me2 but also reversed CFA-induced increases in H3K9ac and H3K4me3 at Trpa1 and Trpv1 promoters. Our findings suggest that G9a in primary sensory neurons constitutively represses Trpa1 and Trpv1 transcription under normal conditions but paradoxically enhances their transcription during tissue inflammation. This latter action accounts for inflammation-induced TRPA1 and TRPV1 upregulation in the DRG. Thus, G9a could be targeted for alleviating persistent inflammatory pain.Significance statement This study demonstrates for the first time that G9a, a histone methyltransferase, in sensory neurons is crucial for the persistent pain development following inflammation. Inhibiting or knockdown of G9a at the spinal cord level reduced inflammation-induced pain hypersensitivity. Remarkably, mice lacking G9a in sensory neurons failed to develop persistent pain hypersensitivity after inflammation. Ablating G9a in sensory neurons increased baseline expression of the TRPA1 and TRPV1 ion channels but reversed their upregulation induced by inflammation. Additionally, G9a deletion blocked the inflammation-driven enrichment of activating histone marks at Trpa1 and Trpv1 promoters. These findings highlight a dual role of G9a in sensory neurons: suppressing Trpa1 and Trpv1 transcription under normal conditions while promoting their transcription during inflammation through bivalent histone modifications.

HomeRNA self-blood collection enables high-frequency temporal profiling of presymptomatic host immune kinetics to respiratory viral infection: a prospective cohort study.

Early host immunity to acute respiratory infections (ARIs) is heterogenous, dynamic, and critical to an individual's infection outcome. Due to limitations in sampling frequency/timepoints, kinetics of early immune dynamics in natural human infections remain poorly understood. In this nationwide prospective cohort study, we leveraged a Tasso-SST based self-blood collection and stabilization tool (homeRNA) to profile detailed kinetics of the presymptomatic to convalescence host immunity to contemporaneous respiratory pathogens. We enrolled non-symptomatic adults with recent exposure to ARIs who subsequently tested negative (exposed-uninfected) or positive for respiratory pathogens. Participants self-collected blood and nasal swabs daily for seven consecutive days followed by weekly blood collection for up to seven additional weeks. Symptom burden was assessed during each collection. Nasal swabs were tested for SARS-CoV-2 and common respiratory pathogens. 92 longitudinal blood samples spanning the presymptomatic to convalescence phase of eight participants with SARS-CoV-2 infection and 40 interval-matched samples from four exposed-uninfected participants were subjected to high-frequency longitudinal profiling of 785 immune genes. Generalized additive mixed models (GAMM) were used to identify temporally dynamic genes from the longitudinal samples and linear mixed models (LMM) were used to identify baseline differences between exposed-infected (n=8), exposed-uninfected (n=4), and uninfected (n=13) participant groups. Between June 2021 and April 2022, 68 participants across 26 U.S. states completed the study and self-collected a total of 691 and 466 longitudinal blood and nasal swab samples along with 688 symptom surveys. SARS-CoV-2 was detected in 17 out of 22 individuals with study-confirmed respiratory infection, of which five were still presymptomatic or pre-shedding, enabling us to profile detailed expression kinetics of the earliest blood transcriptional response to contemporaneous variants of concern. 51% of the genes assessed were found to be temporally dynamic during COVID-19 infection. During the pre-shedding phase, a robust but transient response consisting of genes involved in cell migration, stress response, and T cell activation were observed. This is followed by a rapid induction of many interferon-stimulated genes (ISGs), concurrent to onset of viral shedding and increase in nasal viral load and symptom burden. Finally, elevated baseline expression of antimicrobial peptides was observed in exposed-uninfected individuals. We demonstrated that unsupervised self-collection and stabilization of capillary blood can be applied to natural infection studies to characterize detailed early host immune kinetics at a temporal resolution comparable to that of human challenge studies. The remote (decentralized) study framework enables conduct of large-scale population-wide longitudinal mechanistic studies. This study was funded by R35GM128648 to ABT for in-lab developments of homeRNA and data analysis, a Packard Fellowship for Science and Engineering from the David and Lucile Packard Foundation to ABT for the study execution, sample collection, and analysis, and R01AI153087 to AW for data analysis.

Transformer-based modeling of Clonal Selection and Expression Dynamics reveals resistance mechanisms in breast cancer

Understanding transcriptional heterogeneity in cancer cells and its implication for treatment response is critical to identify how resistance occurs and may be targeted. Such heterogeneity can be captured by in vitro studies through clonal barcoding methods. We present TraCSED (Transformer-based modeling of Clonal Selection and Expression Dynamics), a dynamic deep learning approach for modeling clonal selection. Using single-cell gene expression and the fitness of barcoded clones, TraCSED identifies interpretable gene programs and the time points at which they are associated with clonal selection. When applied to cells treated with either giredestrant, a selective estrogen receptor (ER) antagonist and degrader, or palbociclib, a CDK4/6 inhibitor, pathways dynamically associated with resistance are revealed. For example, ER activity is associated with positive selection around day four under palbociclib treatment and this adaptive response can be suppressed by combining the drugs. Yet, in the combination treatment, one clone still emerged. Clustering based on partial least squares regression found that high baseline expression of both SNHG25 and SNCG genes was the primary marker of positive selection to co-treatment and thus potentially associated with innate resistance — an aspect that traditional differential analysis methods missed. In conclusion, TraCSED enables associating features with phenotypes in a time-dependent manner from scRNA-seq data.

Identification of aberrant plasma vesicles containing AAK1 and CCDC18-AS1 in adolescents with major depressive disorder and preliminary exploration of treatment efficacy.

Major depressive disorder (MDD) during adolescence significantly jeopardizes both mental and physical health. However, the etiology underlying MDD in adolescents remains unclear. A total of 74 adolescents with MDD and 40 health controls (HCs) who underwent comprehensive clinical and cognitive assessments were enrolled. Differential expression analysis was conducted on plasma extracellular vesicles (EVs) carrying long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) by microarray analysis. Two possible lncRNA-miR-mRNA networks were established and candidate regulatory axes were generated using the StarBase, miRDB, and TargetScan bioinformatics databases. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the candidate molecules and signaling axes in a clinical cohort. A total of 3752 dysregulated lncRNAs and 1789 dysfunctional mRNAs were identified. Two candidate regulatory axes (AC156455.1/miR-126-5p/AAK1 and CCDC18-AS1/miR-6835-5p/CCND2) with potential connections with MDD were selected. The candidate molecules exhibit differential expression patterns among adolescents with MDD and HCs, as well as before and after treatment with sertraline in adolescents with MDD. Furthermore, AAK1, CCDC18-AS1, and miR-6835-5p expressions exhibited significant differences between the response and non-response groups. Baseline expression of CCDC18-AS1, miR-6835-5p, and CCND2 could predict the therapeutic effect of sertraline, which may be associated with reducing suicidal ideation and improving cognitive function. Our study may provide insights into the understanding of the underlying pathological mechanisms in adolescents with MDD.

Targeting histone acetylation to overcome drug resistance in the parasite Trichomonas vaginalis.

Trichomoniasis, caused by the parasite Trichomonas vaginalis, is the most common non-viral sexually transmitted infection. Current treatment relies exclusively on 5-nitroimidazole drugs, with metronidazole (MTZ) as the primary option. However, the increasing prevalence of MTZ-resistant strains poses a significant challenge, particularly in the current absence of alternative therapies. Several studies have revealed that the development of metronidazole resistance in T. vaginalis is linked to genomic and transcriptional alterations. Given the role of epigenetic regulation in controlling gene expression, we investigated whether targeting histone deacetylase (HDAC) enzymes could influence drug resistance. Treatment of an MTZ-resistant strain (B7268) with the HDAC inhibitor, trichostatin A (TSA), in combination with MTZ enhanced drug sensitivity and induced significant genome-wide transcriptional changes, as revealed by RNA-seq analysis. To identify drug-related genes epigenetically silenced in the resistant strain but highly active in a sensitive strain, we compared the expression levels of the genes affected by TSA and MTZ treatment with their baseline expression profiles in both resistant and sensitive strains. This analysis identified 130 candidate genes differentially expressed in the sensitive strain NYH209, less expressed in the resistant B7268 strain, that exhibited significant expression changes upon TSA and MTZ treatment. Functional validation involved transfecting the B7268 strain with plasmids encoding four individual candidate genes: a thioredoxin reductase (TrxR), a cysteine synthase (CS), and two genes containing Myb domains (Myb5 and Myb6). Overexpression of three of these genes resulted in a marked reduction in MTZ resistance, demonstrating their role in modulating drug sensitivity. Our findings identified three novel genes that modulate drug resistance in T. vaginalis. This study reveals a previously unknown epigenetic mechanism underlying drug resistance and highlights the therapeutic potential of targeting epigenetic factors, such as HDACs, to overcome resistance and improve treatment efficacy.

Using flow-cytometry in measuring platelet activity in type 2 diabetes and predicting macrovascular complications

Platelets are hyperactive in patients with type2 diabetes (T2DM), they adhere to vascular endothelium and play a key role in macrovascular complications. Platelets activity can be measured by flow-cytometry (cluster of differentiation (CD) 41, CD 42, CD 62, CD 63), which allows detection of surface antigens in a sensitive and specific manner. This study aimed to describe platelets activity in T2DM in association with cardiovascular and cerebrovascular complications in relation to duration of diabetes (DM). This was a case-control study with 130 participants (65 diabetic cases and 65 normal controls). All cases were subjected to history and clinical examination, base-line laboratory investigations and surface expression of platelets receptors e.g. CD 41% and mean fluorescent intensity (MFI), CD 42% and MFI, CD 62% and MFI, CD 63% and MFI were determined by flow-cytometry. There was a statistically significant higher expression of CD 62%, CD 62 MFI, CD 63% and CD 63 MFI (p<0.001 for all) in diabetic cases compared to controls. There were significantly higher CD 62 %, CD 62 MFI, CD 63% and CD 63 MFI in cases with cardiovascular complications (p=0.001, p<0.001, p=0.05 and p=0.007, respectively) and in cases with cerebrovascular complications compared to cases without complications (p=0.05, p=0.008, p=0.035, p=0.017, respectively). A significant positive correlation was found between glycated hemoglobin, body mass index and CD 62 %, CD 62 MFI and CD 63%. Using the receiver operating characteristic curve showed that CD 62 %, CD 62 MFI, CD 63 % and CD 63 MFI have a diagnostic ability to early predict DM (area under the curve (AUC)=0.998) as well as cardiovascular (AUC=0.855) and cerebrovascular (AUC=0.765) complications. In conclusion CD 62%, CD 62 MFI, CD 63% and CD 63 MFI markers have a diagnostic ability for early prediction of cardiovascular and cerebrovascular complications among diabetic patients.

Screening and identification of gene expression in large cohorts of clinical tissue samples unveils the major involvement of EZH2 and SOX2 in lung cancer

Lung adenocarcinoma (LUAD), the primary subtype of Non-Small Cell Lung Cancer (NSCLC), accounts for 80% to 85% of cases. Due to suboptimal screening method, LUAD is often detected in late stage, leading to aggressive progression and poor outcomes. Therefore, early disease prognosis for the LUAD is high priority. In order to identify early detection biomarkers, we conducted a meta-analysis of mRNA expression TCGA and GTEx datasets from LUAD patients. A total of 795 differentially expressed genes (DEGs) were identified by exploring the Network-Analyst tool and utilizing combined effect size methods. DEGs refer to genes whose expression levels are significantly different (either higher or lower) compared to their normal baseline expression levels. KEGG pathway enrichment analysis highlighted the TNF signaling pathway as being prominently associated with these DEGs. Subsequently, using the MCODE and CytoHubba plugins in Cytoscape software, we filtered out the top 10 genes. Among these, SOX2 was the only gene exhibiting higher expression, while the others were downregulated. Consequently, our subsequent research focused on SOX2. Further transcription factor-gene network analysis revealed that enhancer of zeste homolog 2 (EZH2) is a significant partner of SOX2, potentially playing a crucial role in euchromatin-heterochromatin dynamics. Structure of SOX2 protein suggest that it is a non-druggable transcription factor, literature survey suggests the same. SOX2 is considered challenging to target directly, or "non-druggable," because of several intrinsic properties that make it difficult to design effective therapeutic agents against it. The primary function of SOX2 is to bind DNA and regulates gene expression. Unlike enzymes or receptors with defined active sites or binding pockets, transcription factors typically have relatively flat or diffuse surfaces that do not offer obvious "pockets" for small molecules to bind effectively. Hence, we drove our focus to investigate on potential drug(s) targeting EZH2. Molecular docking analyses predicted most probable inhibitors of EZH2. We employed several predictive analysis tools and identified GSK343, as a promising inhibitor of EZH2.

Synaptic protein expression in bipolar disorder patient-derived neurons implicates PSD-95 as a marker of lithium response.

Bipolar disorder (BD) is a severe mental illness characterized by recurrent episodes of depression and mania. Lithium is the gold standard pharmacotherapy for BD, but outcomes are variable, and the relevant therapeutic mechanisms underlying successful treatment response remain uncertain. To identify synaptic markers of BD and lithium response, we measured the effects of lithium on induced pluripotent stem cell-derived neurons from BD patients and controls. We determined that baseline expression of synapsin I (SYN1) and PSD-95 is reduced in BD neurons compared to controls. In control neurons, lithium treatment had modest, transient effects increasing SYN1 and PSD-95 expression. In BD neurons, lithium increased SYN1 expression regardless of lithium response history. However, lithium only increased PSD-95 expression selectively in neurons from lithium-responders and not in neurons from lithium non-responders, leading to group differences in the colocalization of SYN1 and PSD-95. In conclusion, this preliminary work indicates synaptic protein markers are associated with BD pathology and correction of post-synaptic protein expression may be an important mechanism underlying lithium response.

Chromatin enables precise and scalable gene regulation with factors of limited specificity

Biophysical constraints limit the specificity with which transcription factors (TFs) can target regulatory DNA. While individual nontarget binding events may be low affinity, the sheer number of such interactions could present a challenge for gene regulation by degrading its precision or possibly leading to an erroneous induction state. Chromatin can prevent nontarget binding by rendering DNA physically inaccessible to TFs, at the cost of energy-consuming remodeling orchestrated by pioneer factors (PFs). Under what conditions and by how much can chromatin reduce regulatory errors on a global scale? We use a theoretical approach to compare two scenarios for gene regulation: one that relies on TF binding to free DNA alone and one that uses a combination of TFs and chromatin-regulating PFs to achieve desired gene expression patterns. We find, first, that chromatin effectively silences groups of genes that should be simultaneously OFF, thereby allowing more accurate graded control of expression for the remaining ON genes. Second, chromatin buffers the deleterious consequences of nontarget binding as the number of OFF genes grows, permitting a substantial expansion in regulatory complexity. Third, chromatin-based regulation productively co-opts nontarget TF binding for ON genes in order to establish a "leaky" baseline expression level, which targeted activator or repressor binding subsequently up- or down-modulates. Thus, on a global scale, using chromatin simultaneously alleviates pressure for high specificity of regulatory interactions and enables an increase in genome size with minimal impact on global expression error.

Differences in constitutive gene expression of cytochrome P450 enzymes and ATP-binding cassette transporter gene expression between a susceptible and a highly macrocyclic lactone-resistant Haemonchus contortus isolate in the absence of drug-inducible expression

BackgroundAnthelmintic resistance in ruminants is a widespread problem that has a severe impact on productivity and animal welfare. The helminth Haemonchus contortus is generally considered the most important parasite in small ruminants due to its high pathogenicity and the widespread occurrence of anthelmintic resistance in it. Although the molecular mechanisms associated with resistance against the anthelmintics benzimidazoles (BZs) and levamisole are relatively well understood, the resistance mechanisms against the widely used anthelmintic macrocyclic lactones (MLs) ivermectin (IVM) and moxidectin (MOX) remain poorly understood. Detoxifying enzymes and xenobiotic transporters have been frequently proposed to play a role in ML resistance in multiple organisms, including nematodes.MethodsThe reference genome of H. contortus was screened for cytochrome P450 genes (cyp genes) by using the Basic Local Alignment Search Tool, and maximum-likelihood phylogenetic analysis was used to assign the sequences to gene families. Fourth-stage larvae of the susceptible (McMaster) and the ML-resistant (Berlin-selected) H. contortus isolates were generated in vitro and compared regarding basal expression levels of cyp genes and ATP-binding cassette (ABC) transporters by using RNA sequencing. The resistant isolate was further incubated with 100 nM IVM or MOX for 3, 6 and 12 h, and the effects of incubation time and drugs were evaluated.ResultsTwenty-five cyp genes were identified in the H. contortus genome and assigned to 13 different families. The ML-resistant isolate showed significantly higher and lower constitutive expression of 13 and four cyp genes, respectively. Out of the 50 ABC transporter genes, only six showed significantly higher expression in the ML-resistant isolate, while 12 showed lower expression. The fold changes were in general low (range 0.44–5.16). Only pgp-13 showed significant downregulation in response to IVM (0.77 fold change at 6 h, 0.96 fold change at 12 h) and MOX (0.84 fold change at 12 h). In contrast, mrp-5 was significantly, albeit minimally, upregulated in the presence of IVM, but not MOX, after 12 h (1.02 fold change).ConclusionsDespite little observable ML-inducible gene expression in the isolate examined here, some of the changes in the baseline expression levels might well contribute to ML resistance in the context of additional changes in a multigenic resistance model. However, neither cyp genes nor the ABC transporters appear to be the main drivers that can explain the high levels of resistance observed in the resistant isolate examined here.Graphical

Loss of Pigment Epithelium Derived Factor Sensitizes C57BL/6J Mice to Light-Induced Retinal Damage.

Pigment epithelium-derived factor (PEDF) is a neurotrophic glycoprotein secreted by the retinal pigment epithelium (RPE) that supports retinal photoreceptor health. Deficits in PEDF are associated with increased inflammation and retinal degeneration in aging and diabetic retinopathy. We hypothesized that light-induced stress in C57BL/6J mice deficient in PEDF would lead to increased retinal neuronal and RPE defects, impaired expression of neurotrophic factor Insulin-like growth factor 1 (IGF-1), and overactivation of Galectin-3-mediated inflammatory signaling. C57BL/6J mice expressing the RPE65 M450/M450 allele were crossed to PEDF KO/KO and wildtype (PEDF +/+) littermates. Mice were exposed to 50,000 lux light for 5 hours to initiate acute damage. Changes in visual function outcomes were tracked via electroretinogram (ERG), confocal scanning laser ophthalmoscopy(cSLO), and spectral domain optical coherence tomography (SD-OCT) on days 3, 5, and 7 post-light exposure. Gene and protein expression of Galectin-3 were measured by digital drop PCR (ddPCR) and western blot. To further investigate the role of galectin-3 on visual outcomes and PEDF expression after damage, we also used a small-molecule inhibitor to reduce its activity. Following light damage, PEDF KO/KO mice showed more severe retinal thinning, impaired visual function (reduced a-, b-, and c-wave amplitudes), and increased Galectin-3 expressing immune cell infiltration compared to PEDF +/+. PEDF KO/KO mice had suppressed damage-associated increases in IGF-1 expression. Additionally, baseline Galectin-3 mRNA and protein expression were reduced in PEDF KO/KO mice compared to PEDF +/+. However, after light damage, Galectin-3 expression decreases in PEDF +/+ mice but increases in PEDF KO/KO mice without reaching PEDF +/+ levels. Galectin-3 inhibition worsens retinal degeneration, reduces PEDF expression in PEDF +/+ mice, and mimics the effects seen in PEDF knockouts. Loss of PEDF alone does not elicit functional defects in C57BL/6J mice. However, under light stress, PEDF deficiency significantly increases severe retinal degeneration, visual deficits, Galectin-3 expression, and suppression of IGF-1 than PEDF +/+. PEDF deficiency reduced baseline expression of Galectin-3, and pharmacological inhibition of Galectin-3 worsens outcomes and suppresses PEDF expression in PEDF +/+, suggesting a novel co-regulatory relationship between the two proteins in mitigating light-induced retinal damage.

Distinct Hemostasis and Blood Composition in Spiny Mouse Acomys cahirinus.

The spiny mouse (Acomys species) is capable of scarless wound regeneration through largely yet unknown mechanisms. To investigate whether this capacity is related to peculiarities of the hemostatic system, we studied the blood of Acomys cahirinus in comparison to Mus musculus (Balb/c) to reveal differences in blood composition and clotting in both males and females. In response to surgical manipulations, blood clots formed in wounds of Acomys comprised a stronger hemostatic seal with reduced surgical bleeding in comparison with Balb/c. Acomys demonstrated notably shorter tail bleeding times and elevated clottable fibrinogen levels. Histological analysis revealed that clots from Acomys blood had densely packed fibrin-rich clots with pronounced fibrin segregation from erythrocytes. Acomys exhibited superior plasma clot stiffness as revealed with thromboelastography. The latter two characteristics are likely due to hyperfibrinogenemia. Light transmission platelet aggregometry demonstrated that ADP-induced platelet aggregates in Acomys males are stable, unlike the aggregates formed in the plasma of Balb/c undergoing progressive disaggregation over time. There were no apparent distinctions in platelet contractility and baseline expression of phosphatidylserine. Hematological profiling revealed a reduced erythrocytes count but increased mean corpuscular volume and hemoglobin content in Acomys. These results demonstrate the distinctive hemostatic potential of Acomys cahirinus, which may contribute to their remarkable regenerative capacity.

Assessment of a Novel Stress and Immune Gene Panel on the Development of Australasian Snapper (Chrysophrys auratus) Larvae.

Larvae development is a critical step in aquaculture, yet the development of immune and stress responses during this early phase of life is not well understood. Snapper is a species that has been selected as a candidate for aquaculture in New Zealand. In this study we explore a set of 18 genes identified as potentially being involved in the stress and immune responses of snapper larvae during the first 30 days of development. Larvae were collected between 11:45 a.m. and 16:10 p.m. each day. Most genes did not deviate from baseline expression throughout the 30 days, with some exceptions between Days 0 and 6 with glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase, mitochondrial uncoupling protein 2-like, peroxiredoxin-5 mitochondrial, and hepcidin, which predominantly increased and then stabilized by Day 6 until Day 30. Some genes were affected by the time of day, such as actin cytoplasmic 1 and catalase isoform X2. This exploratory study is the first to look at a panel of stress- and immune-related marker gene expression during early snapper development. It sets methods in place to explore the expression of these markers and determine the impact of different potential stressors, such as alternative food sources and other environmental changes. It also highlights the importance of same time of day collections for gene expression studies.

Targeted Variant Assessments of Human Endogenous Retroviral Regions in Whole Genome Sequencing Data Reveal Retroviral Variants Associated with Papillary Thyroid Cancer.

Papillary thyroid cancer (PTC) is one of the fastest-growing cancers worldwide, lacking established causal factors or validated early diagnostics. Human endogenous retroviruses (HERVs), comprising 8% of human genomes, have potential as PTC biomarkers due to their comparably high baseline expression in healthy thyroid tissues, indicating homeostatic roles. However, HERV regions are often overlooked in genome-wide association studies because of their highly repetitive nature, low sequence coverage, and decreased sequencing quality. Using targeted whole-genome sequence analysis in conjunction with high sequencing depth to overcome methodological limitations, we identified associations of specific HERV variants with PTC. Analyzing WGS data from 138 patients with PTC generated through The Cancer Genome Atlas project and 2015 control samples from the 1000 Genomes Project, we examined the mutational variation in HERVs within a 20 kb radius of known cancer predisposition genes (CPGs) differentially expressed in PTC. We discovered 15 common and 13 rare germline HERV variants near or within 20 CPGs that distinguish patients with PTC from healthy controls. We identified intragenic-intronic HERV variants within RYR2, LRP1B, FN1, MET, TCRVB, UNC5D, TRPM3, CNTN5, CD70, RYR1, RUNX1, CRLF2, and PCDH1X, and three variants downstream of SERPINA1 and RUNX1T1. Sanger sequencing analyses of 20 thyroid and 5 non-thyroid cancer cell lines confirmed associations with PTC, particularly for MSTA HERV-L variant rs200077102 within the FN1 gene and HERV-L MLT1A LTR variant rs78588384 within the CNTN5 gene. Variant rs78588384, in particular, was shown in our analyses to be located within a POL2 binding site regulating an alternative transcript of CNTN5. In addition, we identified 16 variants that modified the poly(A) region in Alu elements, potentially altering the potential to retrotranspose. In conclusion, this study serves as a proof-of-concept for targeted variant analysis of HERV regions and establishes a basis for further exploration of HERVs in thyroid cancer development.

Novel Inhibitory Actions of Neuroactive Steroid [3α,5α]-3-Hydroxypregnan-20-One on Toll-like Receptor 4-Dependent Neuroimmune Signaling.

The endogenous neurosteroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) modulates inflammatory and neuroinflammatory signaling through toll-like receptors (TLRs) in human and mouse macrophages, human blood cells and alcohol-preferring (P) rat brains. Although it is recognized that 3α,5α-THP inhibits TLR4 activation by blocking interactions with MD2 and MyD88, the comprehensive molecular mechanisms remain to be elucidated. This study explores additional TLR4 activation sites, including TIRAP binding to MyD88, which is pivotal for MyD88 myddosome formation, as well as LPS interactions with the TLR4:MD2 complex. Both male and female P rats (n = 8/group) received intraperitoneal administration of 3α,5α-THP (15 mg/kg; 30 min) or a vehicle control, and their hippocampi were analyzed using immunoprecipitation and immunoblotting techniques. 3α,5α-THP significantly reduces the levels of inflammatory mediators IL-1β and HMGB1, confirming its anti-inflammatory actions. We found that MyD88 binds to TLR4, IRAK4, IRAK1, and TIRAP. Notably, 3α,5α-THP significantly reduces MyD88-TIRAP binding (Males: -31 ± 9%, t-test, p < 0.005; Females: -53 ± 15%, t-test, p < 0.005), without altering MyD88 interactions with IRAK4 or IRAK1, or the baseline expression of these proteins. Additionally, molecular docking and molecular dynamic analysis revealed 3α,5α-THP binding sites on the TLR4:MD2 complex, targeting a hydrophobic pocket of MD2 usually occupied by Lipid A of LPS. Surface plasmon resonance (SPR) assays validated that 3α,5α-THP disrupts MD2 binding of Lipid A (Kd = 4.36 ± 5.7 μM) with an inhibition constant (Ki) of 4.5 ± 1.65 nM. These findings indicate that 3α,5α-THP inhibition of inflammatory mediator production involves blocking critical protein-lipid and protein-protein interactions at key sites of TLR4 activation, shedding light on its mechanisms of action and underscoring its therapeutic potential against TLR4-driven inflammation.

IMMU-46. CROSS-TALK OF INNATE IMMUNE CELLS IN GLIOBLASTOMA PATHOPHYSIOLOGY: UNDERSTANDING NLRP3 AND NLRP12

Abstract Glioblastoma (GBM) are malignant gliomas with an average survival of less than 14 months after diagnosis, despite surgery, chemotherapy, and radiotherapy. Tumor survival is aided by the heterogeneous cell populations in the tumor microenvironment. Two crucial innate immune cells of the brain, namely microglia and macrophages, favor a tumor-promoting microenvironment IN GBM. The nucleotide-binding domain leucine-rich repeat-containing receptor (NLR) family in astrocytes and microglia cells recognizes pathogen- and damage-associated molecular patterns that induce inflammation. NLRP3, upon activation, leads to cleavage and activation of Caspase-1, causing the proteolytic cleavage-mediated activation and conversion of pro-IL-1ß and pro-IL-18 to IL-1ß and IL-18, leading to pyroptosis. NLRP12 downregulates inflammatory responses in microglia and macrophages by regulating the NF-kB pathway. NLRP3 and NLRP12 have tumor-promoting and anti-tumour functions in various cancers. NLRP12 is a prognostic marker for glioblastoma, and its increased expression correlates with poor survival in GBM patients. However, the cellular and molecular mechanisms of NLRP3 and NLRP12 in GBM pathophysiology are largely unknown. This study aims to understand the effect of NLRP3 and NLRP12 expression in GBM, to interpret the pathophysiology, and to help develop future targeted therapeutic interventions. To understand the baseline expression of NLRP3 and NLRP12 in cell lines and patient-derived GBM cells, PCR, western blotting, and Immunocytochemistry were performed comparing WT and nlrp3-/- and nlrp12-/- cells. Differential expression of NLRP3 and NLRP12 in patient-derived cells can be compared to GBM, astrocyte, and microglia cell lines. This may be the underlying cause for intra and inter-tumor heterogeneity and resultant differential therapy outcomes. To understand cell-to-cell communication in GBM-like tumor microenvironments, heterocellular 3D organoids, and patient-derived tumor organoids were developed. Cell viability in 3D patient-derived tumor organoids decreased by up to 10% with Doxorubicin and 40% with Carboplatin treatment decreases compared to untreated controls. This suggests the scope of developing drugs targeting inflammation regulatory genes.

ENaC contributes to macrophage dysfunction in cystic fibrosis.

Cystic fibrosis (CF) is a chronic systemic disease caused by dysfunctional or absent cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed in human immune cells and plays a role in regulating innate immunity both directly and indirectly. Besides CFTR, research indicates that the epithelial sodium channel (ENaC) also contributes to dysfunction in CF airway epithelial cells. However, the impact of non-CFTR ion channel dysfunction on CF immune responses is not yet fully understood. A precise understanding of how CF immune function is regulated by ion channels may allow antibiotic-and mutation-agnostic treatment approaches to chronic bacterial infection and inflammation. Therefore, we hypothesized that ENaC is aberrantly expressed in CF macrophages and directly contributes to impaired phagocytic and inflammatory functions. ENaC expression was characterized in human immune cells isolated from CF and non-CF blood donors. Monocyte-derived macrophage (MDM) function and bacterial killing was tested in the setting of ENaC modulation. Baseline expression of ENaC in human CF MDMs, lymphocytes, and granulocytes was increased at both the transcript and protein level relative to non-CF controls and persisted after exposure to bacteria. Inhibition of CFTR in non-CF MDMs resulted in ENaC overexpression.CFTR modulator treatment reduced but did not eliminate ENaC overexpression in CF MDMs. Interestingly, ENaC inhibition with Amiloride increased CFTR expression. Amiloride-treated CF MDMs also showed normalized ROS production, improved autophagy, and decreased pro-inflammatory cytokine production. Finally, results from an ion channel microarray indicated that sodium channel expression in CF MDMs normalized after Amiloride treatment with minimal effect on other ion channels. ENaC is overexpressed in CF immune cells and is associated with abnormal macrophage function. ENaC modulation in immune cells is a novel potential therapeutic target for infection control in CF, either in combination with CFTR modulators, or as a sole agent for patients not currently eligible for CFTR modulators.

Global loss of promoter–enhancer connectivity and rebalancing of gene expression during early colorectal cancer carcinogenesis

Although three-dimensional (3D) genome architecture is crucial for gene regulation, its role in disease remains elusive. We traced the evolution and malignant transformation of colorectal cancer (CRC) by generating high-resolution chromatin conformation maps of 33 colon samples spanning different stages of early neoplastic growth in persons with familial adenomatous polyposis (FAP). Our analysis revealed a substantial progressive loss of genome-wide cis-regulatory connectivity at early malignancy stages, correlating with nonlinear gene regulation effects. Genes with high promoter–enhancer (P–E) connectivity in unaffected mucosa were not linked to elevated baseline expression but tended to be upregulated in advanced stages. Inhibiting highly connected promoters preferentially represses gene expression in CRC cells compared to normal colonic epithelial cells. Our results suggest a two-phase model whereby neoplastic transformation reduces P–E connectivity from a redundant state to a rate-limiting one for transcriptional levels, highlighting the intricate interplay between 3D genome architecture and gene regulation during early CRC progression.

Transmembrane Serine Protease 2 and Proteolytic Activation of the Epithelial Sodium Channel in Mouse Kidney.

The renal epithelial sodium channel (ENaC) is essential for sodium balance and blood pressure control. ENaC undergoes complex proteolytic activation by not yet clearly identified tubular proteases. Here, we examined a potential role of transmembrane serine protease 2 (TMPRSS2). Murine ENaC and TMPRSS2 were (co-)expressed in Xenopus laevis oocytes. ENaC cleavage and function were studied in TMPRSS2-deficient murine cortical collecting duct (mCCDcl1) cells and TMPRSS2-knockout (Tmprss2-/-) mice. Short-circuit currents (ISC) were measured to assess ENaC-mediated transepithelial sodium transport of mCCDcl1 cells. The mCCDcl1 cell transcriptome was studied using RNA sequencing. The effect of low-sodium diet with or without high potassium were compared in Tmprss2-/- and wildtype mice using metabolic cages. ENaC-mediated whole-cell currents were recorded from microdissected tubules of Tmprss2-/- and wildtype mice. In oocytes, co-expression of murine TMPRSS2 and ENaC resulted in fully cleaved γ-ENaC and ∼2-fold stimulation of ENaC currents. High baseline expression of TMPRSS2 was detected in mCCDcl1 cells without a stimulatory effect of aldosterone on its function or transcription. TMPRSS2 knockout in mCCDcl1 cells compromised γ-ENaC cleavage and reduced baseline and aldosterone-stimulated ISC which could be rescued by chymotrypsin. A compensatory transcriptional upregulation of other proteases was not observed. Tmprss2-/- mice kept on standard diet exhibited no apparent phenotype, but renal γ-ENaC cleavage was altered. In response to a low-salt diet, particularly with high potassium intake, Tmprss2-/- mice increased plasma aldosterone significantly more than wildtype mice to achieve a similar reduction of renal sodium excretion. Importantly, the stimulatory effect of trypsin on renal tubular ENaC currents was much more pronounced in Tmprss2-/- mice than that in wildtype mice. This indicated the presence of incompletely cleaved and less active channels at the cell surface of TMPRSS2-deficient tubular epithelial cells. TMPRSS2 contributes to proteolytic ENaC activation in mouse kidney in vivo.