Transfected HEK293T Cells Research Articles

Luciferase-Based Reporter System for Investigating GPx4-Mediated Ferroptosis and Its Therapeutic Implications in Diabetes.

Ferroptosis, a distinct form of regulated cell death, is characterized by iron-dependent lipid peroxide accumulation in cell membranes from dysregulated cellular iron homeostasis and compromised antioxidant defense mechanisms. Glutathione peroxidase 4 (GPx4) is crucial in the regulation of ferroptosis by controlling lipid peroxide accumulation. Recent research established the association of ferroptosis with several diseases, prompting investigation toward ferroptosis-targeted therapeutic approaches. However, there is a lack of sensor systems designed to evaluate ferroptosis modulation in intact cells. In this study, we developed a highly sensitive luciferase-based reporter system to study GPx4-mediated ferroptosis in cells. We constructed a novel vector flanking the GPx4 promoter driving luciferase gene expression, demonstrating ferroptosis-specific luciferase activity in transfected HEK293T cells. We established stable cells expressing the construct and optimized its suitability for high-throughput screening using well-established ferroptosis modulators. We identified eugenol, a phenolic compound, as a potent ferroptosis inhibitor using the developed reporter system. Eugenol demonstrated dose-dependent protection against ferroptosis-induced damage in pancreatic beta cells, as assessed by the expression of the key markers such as GPx4, SLC7A11, NRF2, and HO1. Further, we showed the regulation of iron levels and total iron-binding capacity of beta cells by eugenol in streptozotocin (STZ) -induced diabetic mice. Additionally, the diabetes-induced downregulation of GPx4 and antioxidant Nrf2 in pancreatic tissue was significantly mitigated by eugenol, as evidenced by both immunohistochemistry and gene expression analysis. This research validates the functionality of the ferroptosis sensor and offers an approach to develop antidiabetic therapy by targeting ferroptosis to protect beta-cell viability and function.

Novel apocynin regulates TRPV1 activity in the trigeminal system and controls pain in a temporomandibular joint neurogenic model

ObjectiveHerein, we investigate the potential analgesic effect of a newly synthesized chalcone-derived apocynin in a neurogenic pain model. MethodsMolecular docking was used to foretell the apocynin binding features and dynamics with the TRPV1 channel, and the activity was tested in vitro, using transfected HEK 293T cells with the rat TRPV1 receptor. The analgesic effect of apocynin was investigated using a capsaicin-induced pain model. The expression of TRPV1, TRPA1, TRPM8, and MAPKs was assessed by electrophoresis, and immunosorbent assays were performed to quantify the neurotransmitters Substance P, Glutamate, and CGRP. A survival assay using Galleria mellonella was carried out to determine the toxicity. ResultsWe observed that apocynin exhibits greater thermodynamic stability. Upon apocynin ligand binding, it changes the electrostatic potential for a predominantly electronegative state in the interior and neutral in its external vanilloid pocket. Treatment of apocynin induces antinociceptive effects against the noxious challenge of capsaicin. Histologically, apocynin decreased the number of TRPV1+ immunopositive cells. Electrophoresis showed reduced phosphorylation of p44/42 (ERK1/2) and decreased protein levels of substance P, and CGRP. In the survival assay, apocynin showed low toxicity. ConclusionsIn conclusion, we provide proof-of-principles that the newly synthesized apocynin compound effectively prevented nociception in a neurogenic model of orofacial pain.

Evaluating SARS-CoV-2 Spike Protein Transfection in HEK-293T Cells for VLP Applications

Evaluating SARS-CoV-2 Spike Protein Transfection in HEK-293T Cells for VLP Applications

Human IL-22 receptor-targeted small protein antagonist suppress murine DSS-induced colitis

BackgroundHuman interleukin-22 (IL-22) is known as a “dual function” cytokine that acts as a master regulator to maintain homeostasis, structural integrity of the intestinal epithelial barrier, and shielding against bacterial pathogens. On the other hand, the overexpression of IL-22 is associated with hyper-proliferation and recruitment of pathologic effector cells, leading to tissue damage and chronic inflammation in specific diseases including inflammatory bowel disease (IBD). To study a role of IL-22-mediated signaling axis during intestinal inflammation, we generated a set of small protein blockers of IL-22R1 and verified their inhibitory potential on murine model of colitis.MethodsWe used directed evolution of proteins to identify binders of human IL-22 receptor alpha (IL-22R1), designated as ABR ligands. This approach combines the assembly of a highly complex combinatorial protein library derived from small albumin-binding domain scaffold and selection of promising protein variants using ribosome display followed by large-scale ELISA screening. The binding affinity and specificity of ABR variants were analyzed on transfected HEK293T cells by flow cytometry and LigandTracer. Inhibitory function was further verified by competition ELISA, HEK-Blue IL-22 reporter cells, and murine dextran sulfate sodium (DSS)-induced colitis.ResultsWe demonstrate that ABR specifically recognizes transgenic IL-22R1 expressed on HEK293T cells and IL-22R1 on TNFα/IFNγ-activated HaCaT cells. Moreover, some ABR binders compete with the IL-22 cytokine and function as IL-22R1 antagonists in HEK-Blue IL22 reporter cells. In a murine model of DSS-induced acute intestinal inflammation, daily intraperitoneal administration of the best IL-22R1 antagonist, ABR167, suppressed the development of clinical and histological markers of colitis including prevention of mucosal inflammation and architecture deterioration. In addition, ABR167 reduces the DSS-induced increase in mRNA transcript levels of inflammatory cytokines such as IL-1β, IL-6, IL-10, and IL-17A.ConclusionsWe developed small anti-human IL-22R1 blockers with antagonistic properties that ascertain a substantial role of IL-22-mediated signaling in the development of intestinal inflammation. The developed ABR blockers can be useful as a molecular clue for further IBD drug development.

A homozygous nonsense variant in HENMT1 causes male infertility in humans and mice.

HENMT1 encodes a small RNA methyltransferase that plays a crucial role in mouse spermatogenesis through the methylation of the 3' end of PIWI-interacting RNAs. Our study aims to elucidate the relationship between HENMT1 and male infertility in humans. A consanguineous family, having a single non-obstructive azoospermia patient was recruited for pathogenic variants screening. The research includes genetic analysis and experimental validation using mouse models. The patient was diagnosed with non-obstructive azoospermia. Whole-exome sequencing and subsequent bioinformatic analyses were performed to screen for candidate pathogenic variants. The pathogenicity of the identified variant was assessed and studied in vivo using a mouse model that mimicked the patient's mutation. Through whole-exome sequencing, we identified a homozygous nonsense variant (c.555G>A, p.Trp185*) in HENMT1 in the patient. The presence of the mutant HENMT1 mRNA was detected in the patient's blood, and the truncated HENMT1 protein was observed in transfected HEK293T cells. The mutant mice modeling this HENMT1 variant displayed an infertile phenotype similar to that of the patient, characterized by spermiogenesis arrest. Further analysis revealed a significant derepression of retrotransposon LINE1 in the testes of the Henmt1 mutant mice, and increased apoptosis of spermatids. Our findings provide the evidence of pathogenicity of the identified HENMT1 variant, thus shedding light on the indispensable role of HENMT1 in human spermatogenesis.

Development of in vitro model of exosome transport in microfluidic gut-brain axis-on-a-chip.

The gut communicates with the brain in a variety of ways known as the gut-brain axis (GBA), which is known to affect neurophysiological functions as well as neuronal disorders. Exosomes capable of passing through the blood-brain-barrier (BBB) have received attention as a mediator of gut-brain signaling and drug delivery vehicles. In conventional well plate-based experiments, it is difficult to observe the exosome movement in real time. Here, we developed a microfluidic-based GBA chip for co-culturing gut epithelial cells and neuronal cells and simultaneously observing exosome transport. The GBA-chip is aimed to mimic the in vivo situation of convective flow in blood vessels and convective and diffusive transport in the tissue interstitium. Here, fluorescence-labeled exosome was produced by transfection of HEK-293T cells with CD63-GFP plasmid. We observed in real time the secretion of CD63-GFP-exosomes by the transfected HEK-293T cells in the chip, and transport of the exosomes to neuronal cells and analyzed the dynamics of GFP-exosome movement. Our model is expected to enhance understanding of the roles of exosome in GBA.

APOBEC3B expression in 293T viral producer cells drives mutations in chimeric antigen receptors and reduces CAR T cell efficacy

Chimeric antigen receptor (CAR) Tcells are a clinically approved therapy for blood cancers. To produce clinical-grade CAR Tcells, a retroviral or lentiviral vector is used to deliver the CAR and associated genes to patient Tcells. Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) enzymes are known to be upregulated after transfection and retroviral infection and to deaminate cytidine to uracil in nucleic acids, resulting in cytidine-to-thymine mutations in DNA. Here, we hypothesized that APOBEC3 enzymes, induced during the production of CAR Tcells, impact the efficacy of the resulting CAR Tcells. We demonstrated that APOBEC3 family member APOBEC3B was upregulated at the RNA and protein levels after transfection of HEK293T cells with plasmids to make lentivirus, and that APOBEC3 signature mutations were present in the CAR construct. APOBEC3B overexpression in HEK293T cells led to further mutations in the resulting CAR Tcells, and significantly decreased CAR Tcell killing. APOBEC3B knockout in HEK293T cells led to reduced mutations in the CAR construct and significantly increased in CAR Tcell killing. These results suggest that generation of CAR-expressing viruses from producer cell lines deficient in genome-modifying proteins such as APOBEC3B could enhance the quality of CAR Tcell production.

Discovery and structure − activity relationships of 2,4,5-trimethoxyphenyl pyrimidine derivatives as selective D5 receptor partial agonists

Dopamine receptors are therapeutic targets for the treatment of various neurological and psychiatric disorders, including Parkinson’s and Alzheimer’s. Previously, PF-06649751 (tavapadon), PF-2562 and PW0464 have been discovered as potent and selective G protein-biased D1/D5 receptor agonists with optimal pharmacokinetic properties. However, no selective D5R agonist has been reported yet. In this context, we designed and synthesized forty non-catecholamines-based pyrimidine derivatives and identified four pyrimidine derivatives as selective D5R partial agonists. Using cAMP-based GloSensor assay in transiently transfected HEK293T cells with human D1 or D5 receptors, we discovered that compound 5c (4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine) exhibited modest D5R agonist activity. This leads us to explore various modifications of this scaffold to improve the D5 agonist potency and efficacy. Using molecular docking, and rational design followed by their evaluation at D1 and D5 receptors for agonist activity, we identified three new derivatives, 5j, 5h, and 5e. The most potent compound of this series 5j (4-(4-iodophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine), exhibited EC50 of 269.7 ± 6.6 nM. Mice microsomal stability studies revealed that 5j is quite stable (>70 % at 1 hr). Furthermore, pharmacokinetic analysis of 5j (20 mg/kg, p.o) in C57BL/6j mice showed that 5j is readily absorbed via oral route of dosing and also enters into the brain (plasma Tmax: 1 h, Cmax: 51.10 ± 13.51 ng/ml; Brain Tmax: 0.5 h, Cmax: 22.54 ± 4.08 ng/ml). We further determined the in-vivo effect of 5j on cognition in scopolamine-induced amnesia in C57BL/6j mice. We observed that 5j (10 mg/kg, p.o) alleviated scopolamine-induced impairment in short-term memory and social recognition, which were blocked by D1/D5 antagonist SCH23390 (0.1 mg/kg, i.p.). Furthermore, 5j did not exhibit any cytotoxicity (up to 10 µM) or in vivo acute toxicity up to 200 mg/kg (p.o). These results strongly suggest that 5j could be further developed for treating neurological disorders wherein the D5 receptors play pivotal roles.

Anti-DEFA5 Monoclonal Antibody Clones 1A8 and 4F5 Immunoreactive Bioassay for Diagnosing Inflammatory Bowel Disease.

Robust evidence suggests that the aberrant expression of α defensin 5 protein (DEFA5) in colon inflammatory bowel diseases (IBDs) underlies the distinct pathogenesis of Crohn's colitis, can be exploited as a reliable diagnostic biomarker to differential diagnosis of Crohn's colitis (CC) from Ulcerative colitis (UC) in otherwise indeterminate colitis (IC). We evaluated the specificity of the commercially available anti-DEFA5 antibodies and showed further validation of their appropriateness for a given application is required. We established two mouse monoclonal DEFA5 antibody clones 1A8 and 4F5 by immunizing the mice with purified recombinant protein and validated the specificity, selectivity and cross reactivity in recognizing the endogenous and recombinant DEFA5 protein, especially for Immunohistochemistry, Western blot, Immunoprecipitation, or enzyme-linked immunosorbent assay. Clones 1A8 and 4F5 recognized effectively the endogenous DEFA5 in active human diverticulitis (DV), UC, CC or IC disease samples, including transiently transfected HEK293T cells expressing DEFA5 with high degree of specificity and minimal non-confounding cross reactivity. 1A8 and 4F5 clones are worth studying in larger IBD cohorts to fully address whether DEFA5 expression may be used as a diagnostic biomarker to discrimination of the diagnosis of UC from CC or IC into authentic CC or UC or a colitis with different pathological characteristics.

PBAE-PEG based lipid nanoparticles for lung cell-specific gene delivery.

Delivery of modified mRNA encapsulated in lipid nanoparticles, exemplified by their successful use in COVID-19 vaccination, provides a framework for treating various genetic and acquired disorders. Herein, we developed PEGylated(PBAE-PEG) and non-PEGylated(PBAE) PBAE with lipids 4A3-SC8/DOPE/cholesterol/DOTAP to form lipid nanoparticles (LNPs) for mRNA delivery into different types of pulmonary cells in vivo. PBAE-PEG/LNP were highly active in transfecting HEK293T cells and air-liquid interfaced H441 cells in vitro. PBAE-PEG/LNP were used to express Cre-recombinase after administration to mice by intravenous injection, resulting in high transfection levels in pulmonary vascular endothelial cells. Intratracheal injection of both PBAE-PEG/LNP and PBAE/LNPs resulted in efficient and selective transfection of lung epithelial cells, identified by the expression of stabilized Cre-recombinase mRNA in club cells and alveolar type 2 cells. PBAE-PEG/LNP were most effective in transfecting alveolar type 2 cells after intratracheal injection, while PBAE/LNPs administered intratracheally were more effective in transfecting secretory airway cells. Cre-mediated recombination was specific to lung epithelial cells after intratracheal administration. Likewise, intravenous administration resulted in selective transfection of endothelial cells but not other pulmonary cell types, indicating their failure to cross the pulmonary endothelial-to-epithelial barrier. Moreover, 5-methoxyuridine modified mRNA was more efficient than unmodified mRNA in vivo but not in vitro.

Alamandine, a protective component of the renin-angiotensin system, reduces cellular proliferation and interleukin-6 secretion in human macrophages through MasR–MrgDR heteromerization

Alamandine (ALA) exerts protective effects similar to angiotensin (Ang) (1–7) through Mas-related G protein-coupled receptor type D receptor (MrgDR) activation, distinct from Mas receptor (MasR). ALA induces anti-inflammatory effects in mice but its impact in human macrophages remains unclear. We aimed to investigate the anti-inflammatory effects of ALA in human macrophages. Interleukin (IL)-6 and IL-1β were measured by ELISA in human THP-1 macrophages and human monocyte-derived macrophages exposed to lipopolysaccharide (LPS). Consequences of MasR–MrgDR heteromerization were investigated in transfected HEK293T cells. ALA decreased IL-6 and IL-1β secretion in LPS-activated THP-1 macrophages. The ALA-induced decrease in IL-6 but not in IL-1β was prevented by MasR blockade and MasR downregulation, suggesting MasR–MrgDR interaction. In human monocyte-derived M1 macrophages, ALA decreased IL-1β secretion independently of MasR. MasR–MrgDR interaction was confirmed in THP-1 macrophages, human monocyte-derived macrophages, and transfected HEK293T cells. MasR and MrgDR formed a constitutive heteromer that was not influenced by ALA. ALA promoted Akt and ERK1/2 activation only in cells expressing MasR–MrgDR heteromers, and this effect was prevented by MasR blockade. While Ang-(1–7) reduced cellular proliferation in MasR −but not MrgDR- expressing cells, ALA antiproliferative effect was elicited in cells expressing MasR–MrgDR heteromers. ALA also induced an antiproliferative response in THP-1 cells and this effect was abolished by MasR blockade, reinforcing MasR–MrgDR interaction. MasR–MrgDR heteromerization is crucial for ALA-induced anti-inflammatory and antiproliferative responses in human macrophages. This study broaden our knowledge of the protective axis of the RAS, thus enabling novel therapeutic approaches in inflammatory-associated diseases.

Characterizing ER Retention Defects of PDZ Binding Deficient Cx36 Mutants Using Confocal Microscopy.

Overexpression of proteins in transiently transfected cells is a simple way to study basic transport mechanisms and the underlying protein-protein interactions. While expression systems have obvious drawbacks compared to in vivo experiments, they allow a quick assessment of more conserved functions, for instance, ER export or sorting of proteins in the Golgi. In a previous study, our group described the formation of ER-derived removal vesicles for the gap junction protein Cx36 in transfected HEK293T cells. These removal vesicles, termed "whorls" because of their concentric structure, were formed by Cx36 channels that failed to escape the ER. In this article, we describe an imaging protocol that can be used to determine these ER retention defects for Cx36 expressed in cultured cells. The protocol we provide here employs regular confocal microscopy, which allows for sufficient resolution to reveal the characteristic shape of ER whorls.

Anti-SARS-CoV-2 Activity of Andrographis paniculata (Burm.f.) Nees Extract via Inhibition of Spike-mediated Syncytia Formation in HEK293T Cell Model

The resurgence of COVID-19 endemic cases at the end of 2023 has underscored the need for effective treatments. Some severe cases of COVID-19 are often characterized by the formation of multinucleated syncytial pneumocytes in the lungs. Therefore, our study aimed to explore the potential of Andrographis paniculata (Burm. f) Nees as an antivirus against SARS-CoV-2, which involves syncitia formation. We utilized the non-toxic concentrations of A. paniculata extract on HEK293T cells determined by MTT assay, which were 1 μg/ml (cell viability 97.96%) and 10 μg/ml (cell viability 95.24%) for further assays. First, we conducted a pseudovirus cellular entry assay as a model of SARS-CoV-2 infection in HEK293T cells expressing hACE2/TMPRSS2. The HEK293T cells were co-transfected with plasmids expressing hACE2 and TMPRSS2, then infected with pseudotyped spike*∆G-GFP rVSV with or without A. paniculata extract. The internalized pseudovirus would trigger GFP expression as a reporter of the infected cells. Next, we performed a syncytia assay by transfecting HEK293T cells with hACE2, TMPRSS2, and SARS-CoV-2 spike expression vectors to induce syncytia formation as a model of intercellular viral transmission. As the results, 10 μg/mL of the extract significantly lowered the number of SARS-CoV-2 pseudovirus-infected cells by 54.69% (P = 0.02) and spike-mediated syncytia formation by 42.39% (P<0.001). In conclusion, our results suggested that A. paniculata has a potential antiviral activity against SARS-CoV-2 by hindering virus infection and cell-to-cell transmission.

Sparsely PEGylated poly(beta-amino ester) polyplexes enhance antigen specific T-cell response of a bivalent SARS-CoV-2 DNA vaccine

DNA technology has emerged as a promising route to accelerated manufacture of sequence agnostic vaccines. For activity, DNA vaccines must be protected and delivered to the correct antigen presenting cells. However, the physicochemical properties of the vector must be carefully tuned to enhance interaction with immune cells and generate sufficient immune response for disease protection. In this study, we have engineered a range of polymer-based nanocarriers based on the poly(beta-amino ester) (PBAE) polycation platform to investigate the role that surface poly(ethylene glycol) (PEG) density has on pDNA encapsulation, formulation properties and gene transfectability both in vitro and in vivo. We achieved this by synthesising a non-PEGylated and PEGylated PBAE and produced formulations containing these PBAEs, and mixed polyplexes to tune surface PEG density. All polymers and co-formulations produced small polyplex nanoparticles with almost complete encapsulation of the cargo in all cases. Despite high gene transfection in HEK293T cells, only the fully PEGylated and mixed formulations displayed significantly higher expression of the reporter gene than the negative control in dendritic cells. Further in vivo studies with a bivalent SARS-CoV-2 pDNA vaccine revealed that only the mixed formulation led to strong antigen specific T-cell responses, however this did not translate into the presence of serum antibodies indicating the need for further studies into improving immunisation with polymer delivery systems.

Andrographolide suppresses SARS-CoV-2 infection by downregulating ACE2 expression: A mechanistic study.

Angiotensin-converting enzyme 2 (ACE2) is the receptor that enables SARS-CoV-2 to invade host cells. Previous studies have reported that reducing ACE2 expression may have an anti-SARS-CoV-2 effect. In this study, we constructed a pGL4.10-F2-ACE2 vector with double luciferase genes (firefly and Renilla luciferase) under the control of the ACE2 promoter and used it to screen compounds from Chinese traditional medicinal herbs (CTMHs) that can inhibit ACE2 transcription in human cells. We transfected HEK293T cells with pGL4.10-F2-ACE2 and treated them with CTMH compounds and then measured fluorescence to evaluate the indirect inhibition of ACE2 transcription. Out of 37 compounds tested, andrographolide demonstrated a dose-dependent inhibition of ACE2 transcription. We further confirmed by RT-qPCR and Western blot assays that andrographolide also reduced ACE2 expression in BEAS-2B cells in a dose-dependent manner. Moreover, pseudovirus infection assays in BEAS-2B cells demonstrated that andrographolide can inhibit SARS-CoV-2 infection in a dose-dependent manner. These results suggest that andrographolide has potential anti-SARS-CoV-2 activity and could be a candidate drug for COVID-19 prevention and treatment.

Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging

BackgroundProgrammed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging.MethodsWe designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1.ResultsBinding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7–99.3% and in vitro stability in human serum after 120 min was in the range 94.6–98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors.ConclusionsUsing directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.Graphical

Dysregulation of TNF-induced protein 3 and CCAAT/enhancer-binding protein β in alveolar macrophages: Implications for systemic sclerosis-associated interstitial lung disease.

This study investigates the role of TNF-induced protein 3 (TNFAIP3) and CCAAT/enhancer-binding protein β (C/EBPβ) in alveolar macrophages (AMs) of patients with systemic sclerosis-associated interstitial lung disease (SSc-ILD) and their influence on pulmonary fibrosis. Transfection of HEK293T cells and AMs with plasmids carrying TNFAIP3 and C/EBPβ was performed, followed by co-culturing AMs with pulmonary fibroblasts. Immunoblotting analysis was then utilized to assess the expression of TNFAIP3, C/EBPβ, and collagen type 1 (Col1). Quantitative PCR analysis was conducted to quantify the mRNA levels of C/EBPβ, IL-10, and TGF-β1. STRING database analysis, and immunoprecipitation assays were employed to investigate the interactions between TNFAIP3 and C/EBPβ. TNFAIP3 expression was significantly reduced in SSc-ILD AMs, correlating with increased Col1 production in fibroblasts. Overexpression of TNFAIP3 inhibited this pro-fibrotic activity. Conversely, C/EBPβ expression was elevated in SSc-ILD AMs, and its reduction through TNFAIP3 restoration decreased pro-fibrotic cytokines IL-10 and TGFβ1 levels. Protein-protein interaction studies confirmed the regulatory relationship between TNFAIP3 and C/EBPβ. This study highlights the important role of TNFAIP3 in regulating pulmonary fibrosis in SSc-ILD by modulating C/EBPβ expression in AMs. These findings suggest that targeting TNFAIP3 could be a potential therapeutic strategy for managing SSc-ILD patients.

439 Extracellular-in-frame deletions and kinase domain duplications are novel, gain-of-function mutations in fibroblast growth factor receptor genes in cancer

OBJECTIVES/GOALS: There are gain-of-function genomic alterations in FGFR genes that guide personalized treatment in some patients with cholangiocarcinoma (10%) and bladder cancer (30%) who can benefit from targeted therapies. We sought to evaluate other genomic alterations in cancer involving FGFRs and assess whether they are gain-of-function. METHODS/STUDY POPULATION: We collaborated with Foundation Medicine Inc (FMI), for the assessment of 300,000 sequenced tumors and a retrospective analysis of recent publications, to identify novel candidate FGFR alterations. We propose to transiently transfect HEK293T cells with an empty vector (EV), FGFR1-4 wild-type (WT), and these variants and use a luminescent-proximity based high-throughput assay, AlphaLISA, and Western blot to assess FGFR and phosphorylated downstream signaling proteins, FRS2, AKT and ERK, and their sensitivity to FGFR inhibitors: pemigatinib, erdafitinib, futibatinib, RLY-4008, and TYRA-200. RESULTS/ANTICIPATED RESULTS: Through our collaboration we identified >100 novel candidate FGFR1-4 variants of unknown significance (VUS) including extracellular-in-frame deletions (EIDs), kinase domain duplications (KDDs), insertions/deletions (INDELs), short number variants (SNVs), and truncations. Immunoblot analysis confirmed the presence of desired EV, FGFR WT, and VUS’ in HEK293T cells. We anticipate the FGFR EIDs and KDDs to display an increased presence of in the respective pFGFR, pFRS2, pERK, and pAKT as compared to the EV and FGFR WT by both immunoblot and AlpahLISA analysis. Additionally, we anticipate the VUS’ to be sensitive to FGFR inhibitors: pemigatinib, erdafitinib, futibatinib, RLY-4008, and TYRA-200 using the AlphaLISA assay. DISCUSSION/SIGNIFICANCE: These findings suggest that the novel FGFR VUS’ are capable of constitutive activation of FGFR kinase activity, and they preliminary demonstrate that these newly identified FGFR alterations are therapeutically targetable. Thus, providing rationale for further clinical evaluation to identify new cohorts of FGFR inhibitor responders.

Understanding and Rescuing the Splicing Defect Caused by the Frequent ABCA4 Variant c.4253+43G>A Underlying Stargardt Disease.

Pathogenic variants in ABCA4 are the underlying molecular cause of Stargardt disease (STGD1), an autosomal recessive macular dystrophy characterized by a progressive loss of central vision. Among intronic ABCA4 variants, c.4253+43G>A is frequently detected in STGD1 cases and is classified as a hypomorphic allele, generally associated with late-onset cases. This variant was previously reported to alter splicing regulatory sequences, but the splicing outcome is not fully understood yet. In this study, we attempted to better understand its effect on splicing and to rescue the aberrant splicing via antisense oligonucleotides (AONs). Wild-type and c.4253+43G>A variant-harboring maxigene vectors revealed additional skipping events, which were not previously detected upon transfection in HEK293T cells. To restore exon inclusion, we designed a set of 27 AONs targeting either splicing silencer motifs or the variant region and screened these in maxigene-transfected HEK293T cells. Candidate AONs able to promote exon inclusion were selected for further testing in patient-derived photoreceptor precursor cells. Surprisingly, no robust splicing modulation was observed in this model system. Overall, this research helped to adequately characterize the splicing alteration caused by the c.4253+43G>A variant, although future development of AON-mediated exon inclusion therapy for ABCA4 is needed.

The Expression and Functionality of CB1R-NMDAR Complexes Are Decreased in A Parkinson's Disease Model.

One of the hallmarks of Parkinson's disease (PD) is the alteration in the expression and function of NMDA receptor (NMDAR) and cannabinoid receptor 1 (CB1R). The presence of CB1R-NMDAR complexes has been described in neuronal primary cultures. The activation of CB1R in CB1R-NMDAR complexes was suggested to counteract the detrimental NMDAR overactivation in an AD mice model. Thus, we aimed to explore the role of this receptor complex in PD. By using Bioluminescence Resonance Energy Transfer (BRET) assay, it was demonstrated that α-synuclein induces a reorganization of the CB1R-NMDAR complex in transfected HEK-293T cells. Moreover, α-synuclein treatment induced a decrease in the cAMP and MAP kinase (MAPK) signaling of both CB1R and NMDAR not only in transfected cells but also in neuronal primary cultures. Finally, the interaction between CB1R and NMDAR was studied by Proximity Ligation Assay (PLA) in neuronal primary cultures, where it was observed that the expression of CB1R-NMDAR complexes was decreased upon α-synuclein treatment. These results point to a role of CB1R-NMDAR complexes as a new therapeutic target in Parkinson's disease.