Reporter Gene Research Articles

Extracellular Vesicles From Dental Pulp Cells Promote Osteogenic Differentiation in Periodontal Ligament Cells.

Periodontal osseous defects are mainly caused by periodontitis, which seriously affects the quality of patient life. Dental pulp cells (DpCs)-derived extracellular vesicles (EVs) can effectively promote tissue regeneration. Homeobox A9 (HOXA9) mRNA is abundant in EVs derived from DSCs, which may be related to promoting alveolar bone regeneration, but the specific mechanism is unclear. We aimed to elucidate the mechanism through which HOXA9 from DPCs-derived EVs can impact the osteogenic differentiation of periodontal ligament cells (PDLCs). DPCs-derived EVs were isolated and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot. Lipopolysaccharide (LPS) was employed to induce the inflammatory environment. Cell viability was assessed by CCK8 assay. Calcium deposition was determined by Alizarin red staining. H3K27ac enrichment in the FLI1 enhancer region and the interaction between C/EBPα, HOXA9, and FLI1 were analyzed by ChIP assay. The interaction between HOXA9 and FLI1 enhancer in 293T cells was analyzed by dual luciferase reporter gene assay. DPCs-derived EVs promoted PDLC osteogenesis under LPS treatment and increased HOXA9 expression in PDLCs. HOXA9 knockdown in DPCs reversed the promoting effect of DPCs-derived EVs on PDLC osteogenic differentiation. HOXA9 from DPCs-derived EVs promoted H3K27ac enrichment in the FLI1 enhancer region by facilitating HOXA9 competitively binding FLI1 enhancer region with C/EBPα. Moreover, HOXA9 from DPCs-derived EVs promoted PDLC osteogenesis by activating the PI3K/AKT pathway through upregulating FLI1. HOXA9 from DPCs-derived EVs promoted PDLC osteogenic differentiation by activating the PI3K/AKT pathway through promoting H3K27ac enrichment in the FLI1 enhancer region and upregulating FLI1. Our study identified a previously unknown mechanism that HOXA9/FLI1 signaling axis participates in the processes of EVs derived from DPCs to treat bone tissue injury. Our research presents a theoretical basis for using EVs derived from DPCs to treat bone tissue injury.

Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3to Inhibit Pulmonary Vascular Remodeling.

The function of super-enhancers (SEs) in pulmonary hypertension (PH), especially in the proliferation of pulmonary artery smooth muscle cells (PASMCs), is currently unknown. We identified SEs-targeted genes in PASMCs with chromatin immunoprecipitation (ChIP)-sequence by H3K27ac antibody and proved that CBP/p300-interacting transactivator with Glu/Asp-rich C-terminal domain, 2 (CITED2) is an SEs-targeted gene through bioinformatics prediction, ChIP-PCR, dual-luciferase reporter gene assays and other experimental methods. We also found that the expression of CITED2 and the transcription factor Forkhead Box J3 (FOXJ3) was reduced in hypoxic mouse PASMCs. In addition, the expression of CITED2 and FOXJ3 also decreased in both the patients with idiopathic pulmonary arterial hypertension (iPAH) and the human PASMCs exposed to hypoxia. The decreased expression of CITED2 was reversed by co-transfection of FOXJ3 and SEs plasmids. Overexpressing of CITED2 attenuated the PASMCs proliferation induced by hypoxia. Lentiviral overexpression of CITED2 also reversed hypoxia-induced pulmonary hypertension mice model. Mechanically, the expression of CITED2 by affecting by FOXJ3, which binding with three SEs located in the about 2000 bp of TSS. In conclusion, we first identified that CITED2 is a kind of SEs-targeted gene, modulated by FOXJ3. The FOXJ3/SEs/CITED2 axis may become a new therapeutic target of PH.

Liver macrophage-derived exosomal miRNA-342-3p promotes liver fibrosis by inhibiting HPCAL1 in stellate cells

BackgroundThe progression of liver fibrosis involves complex interactions between hepatic stellate cells (HSCs) and multiple immune cells in the liver, including macrophages. However, the mechanism of exosomes in the crosstalk between liver macrophages and HSCs remains unclear.MethodExosomes were extracted from primary mouse macrophages and cultured with HSCs, and the differential expression of microRNAs was evaluated using high-throughput sequencing technology. The functions of miR-342-3p in exosomes were verified by qPCR and luciferase reporter gene experiments with HSCs. The function of the target gene Hippocalcin-like protein 1 (HPCAL1) in HSCs was verified by Western blotting, qPCR, cellular immunofluorescence and co-IP in vivo and in vitro.ResultsWe demonstrated that exosomal microRNA-342-3p derived from primary liver macrophages could activate HSCs by inhibiting the expression of HPCAL1 in HSCs. HPCAL1, which is a fibrogenesis suppressor, could inhibit TGF-β signaling in HSCs by regulating the ubiquitination of Smad2 through direct interactions with its EF-hand 4 domain.ConclusionThis study reveals a previously unidentified profibrotic mechanism of crosstalk between macrophages and HSCs in the liver and suggests an attractive novel therapeutic strategy for treating fibroproliferative liver diseases.Graphical

A Reporter Gene Assay for Measuring the Biological Activity of PEGylated Recombinant Human Growth Hormone

PEGylated recombinant human growth hormone (PEG-rhGH) has garnered significant interest in growth hormone research due to its prolonged half-life and improved patient compliance. An accurate evaluation of its biological activity is critical for ensuring the quality of PEG-rhGH-based therapeutics. In this study, we established an in vitro bioactivity assay using a reporter gene method based on the HepG2/IGF-1 cell line. Key assay parameters, including the initial concentration of PEG-rhGH, serial dilution ratios, cell density, and incubation time, were systematically optimized to generate robust dose–response curves. The assay demonstrated high sensitivity, precision, and reproducibility across multiple batches of PEG-rhGH. The validation results showed an excellent correlation with traditional in vivo animal studies and the Nb2-11 cell proliferation assay, highlighting its suitability for quality control. Furthermore, we developed an ion exchange chromatography (IEC) method to separate five positional isomers of PEG-rhGH, revealing significant differences in bioactivity depending on the PEG modification site. This study demonstrates that the optimized reporter gene assay is not only effective for quality control of PEG-rhGH but also serves as a valuable tool for evaluating and optimizing PEGylated long-acting growth hormone therapeutics.

TAZ (WWTR1) Interactome Analysis using nanotrap based affinity purification-mass spectrometry.

Characterization of protein interactions is a fundamental goal in the post-genomic era. Here, we document a generally applicable approach to identify cellular protein interactomes using a combination of nanobody based affinity purification (A-P) coupled with tandem mass spectrometry (MS/MS). The Hippo signalling regulator TAZ functions as a transcriptional co-repressor/activator depending on its protein:protein interaction (PPI) network, we therefore undertook an unbiased proteomic screen to identify TAZ PPI's in striated muscle cells. A GFP-Nanotrap based A-P approach coupled with LC-MS/MS protein identification was used to document a comprehensive list of known and novel TAZ interactome components. Informatic analysis of the interactome documented known components of Hippo signalling and multiple epigenetic regulators such as the NuRD, FACT, SWI/SNF complexes and the pro-myogenic CARM1 methyltransferase. Hippo pathway reporter gene (HOP/HIP) analysis indicated that CARM1 represses TAZ transcriptional co-activator function, promoting TAZ Ser89 phosporylation and TAZ cytoplasmic sequestration. M-S analysis revealed that CARM1 dimethylates TAZ at Arg77 in a PGPR*LAGG consensus peptide resulting in enhanced TAZ Ser89 phoshorylation. These studies underline the utility of a nanobody based A-P approach for interactome analysis.

Identification of compounds contributing to glucocorticoid activity in indoor dust supported by orthogonal fractionation.

Indoor dust contains various endocrine-disrupting contaminants, yet the effect drivers of observed glucocorticoid activity are completely unknown. This study conducted an effect-directed analysis using orthogonal fractionation to identify effect drivers of glucocorticoid activity in indoor dust. After the detection of bioactivity using a human cell line stably transfected with a reporter gene, the sample underwent parallel HPLC fractionations with octadecyl, pentafluorophenyl, and aminopropyl columns to obtain orthogonal fractions. The bioassays were utilized to screen the fractions and guide efforts towards prioritization of the bioactive chemicals using targeted and non-targeted analysis with LC-HRMS. The glucocorticoid activity of the identified potential candidates was confirmed by their testing in the same bioassay. To assess their contribution to the detected mixture effects, we calculated their relative potencies. This approach led to the identification of two pharmaceuticals, clobetasol propionate and mometasone furoate, at concentrations ranging from ng to μg per gram of dust, which together accounted for up to 77% of the observed glucocorticoid activity. This is the first report documenting the effect drivers of glucocorticoid receptor agonism in indoor dust; however, together with previous studies of various environmental samples, it documents that in cases when glucocorticoid receptor-agonistic activity is detected, drugs should be considered as likely relevant contaminants. The discovery of potent drugs in household dust highlights concerns for individuals exposed within domestic environments and emphasizes the need to consider pharmaceuticals as relevant contributors to indoor contamination.

Establishment of an Agrobacterium-mediated transformation system for the genetic engineering of Linum grandiflorum Desf.

Genetic transformation is a powerful tool in plant biotechnology. However, its application is limited to species that are well-studied and easy to transform. There is a critical need to establish transformation protocols for non-model species. A stable transformation method using Agrobacterium rhizogenes for hairy root transformation and regeneration of transgenic Linum grandiflorum was established. This protocol shows the successful co-transformation of different T-DNA fragments from both the native Ri plasmid and the binary vector with the reporter gene. Hairy roots were produced after inoculation with Agrobacterium rhizogenes from which later shoots were formed from the callus, and subsequently, whole plants were regenerated. This protocol significantly facilitates genomic studies in Linum grandiflorum, particularly in investigating genes at the S-locus supergene, which are crucial for understanding self-incompatibility. Moreover, the established transformation method enables the production of hairy root lines, which can be utilized for the biosynthesis of medically useful and commercially valuable plant metabolites.

MoCloro: an extension of the Chlamydomonas reinhardtii modular cloning toolkit for microalgal chloroplast engineering.

Photosynthetic microalgae are promising green cell factories for the sustainable production of high-value chemicals and biopharmaceuticals. The chloroplast organelle is being developed as a chassis for synthetic biology as it contains its own genome (the plastome) and some interesting advantages, such as high recombinant protein titers and a diverse and dynamic metabolism. However, chloroplast engineering is currently hampered by the lack of standardized cloning tools and Design-Build-Test-Learn workflows to ease genomic and metabolic engineering. The MoClo (Modular Cloning) toolkit based on Golden Gate assembly was recently developed in the model eukaryotic green microalgae Chlamydomonas reinhardtii to facilitate nuclear transformation and engineering. Here, we present MoCloro as an extension of the MoClo that allows chloroplast genome engineering. Briefly, a Golden Gate-compatible chloroplast transformation vector (pWF.K.4) was constructed, which contains homologous arms for integration at the petA site in the plastome. A collection of standardized parts (promoters, terminators, reporter and selection marker genes) was created following the MoClo syntax to enable easy combinatorial assembly of multi-cassettes in the destination pWF.K.4 vector. The functionality of the biobricks was assayed by constructing and assessing the expression of several multigenic constructs. Finally, a generic vector pK4 was constructed for easy Golden Gate cloning of 5' and 3' homologous arms, allowing targeting at alternative plastome integration sites. This work represents a significant advancement in technology aimed at facilitating more efficient and rapid chloroplast transformation and engineering of green microalgae.

Intravital imaging reveals glucose-dependent cilia movement in pancreatic islets in vivo.

Pancreatic islet cells harbor primary cilia, small sensory organelles that detect environmental changes to regulate hormone secretion and intercellular communication. While the sensory and signaling capacity of primary cilia are well-appreciated, it is less recognized that these organelles also possess active motility, including in dense multicellular tissues such as the pancreatic islet. In this manuscript, we use transgenic cilia reporter mice and an intravital imaging approach to quantitate primary cilia dynamics as it occurs in live mouse pancreatic islets. We validate this imaging workflow as suitable for studying islet cilia motion in real time in vivo and demonstrate that glucose stimulation corresponds to a change in cilia motility, which may be a physiologic measure of nutrient-dependent fluxes in islet cell function. Complementary ex vivo analysis of isolated islets further demonstrates that metabolic stress in the form of lipotoxicity impairs cilia motility and these effects can be reversed by glucose elevation. These findings suggest that cilia motility is sensitive to metabolic stress and highlight its potential functional role in beta cell adaptation.

Fluoxetine-induced downregulation of circMap2k1 signaling cascade to improve neurological function after ischemic stroke.

Ischemic stroke (IS) is known for its high incidence, disability, and mortality, and there is an urgent need to investigate the pathophysiological mechanisms and develop novel treatment strategies. We aimed to investigate the mechanisms of the novel circMap2k1/miR-135b-5p/Pidd1 axis in the treatment of IS progression with fluoxetine. The middle cerebral artery occlusion (MCAO) model was done in adult male Sprague-Dawley (SD) rats and followed by fluoxetine treatment and the injection of adeno-associated virus (AAV)-sh-ctr and AAV-sh-circMap2k1 into the bilateral hippocampal tissues of rats. Dual-luciferase reporter gene assay was employed to confirm the binding between miR-135b-5p and Pidd1. Enzyme-linked immunosorbent assay was performed to measure the concentrations of the inflammatory factors TNF-α, IL-6, and IL-1β in the plasma. The role of circMap2k1 in cells was tested by overexpression of circMap2k1. Cell viability was assessed using Cell Counting Kit-8 assay, while apoptosis was measured by flow cytometry. Knockdown of circMap2k1 enhanced the therapeutic and protective effect of fluoxetine on IS injury in rats. Dual-luciferase reporter gene assay confirmed the targeting of miR-135b-5p to Pidd1. Additionally, fluoxetine deactivated the adsorption of miR-135b-5p by downregulating circMap2k1, and miR-135b-5p further exerted its inhibitory effect on Pidd1 and finally attenuated the inflammatory response caused by microglial polarization after IS. Cell experiments revealed that overexpression of circMap2k1 repressed cell viability and promoted cell apoptosis. Fluoxetine downregulated of circMap2k1 was associated ameliorate neurological injury and inflammatory responses induced by microglial polarization after IS. The manuscript is available as a preprint at this link: doi.org/10.21203/rs.3.rs-3209057/v1.

CARM1-induced lncRNA NEAT1 synchronously activates MYCN and GalNAcT-I to accelerate the progression of neuroblastoma.

Long non-coding RNAs (lncRNAs) play important roles in progression of neuroblastoma (NB). LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been shown to affect the development of multiple tumors. However, the effect of NEAT1 on NB remain unclear. In this study, the new mechanisms whereby how NEAT1 influences tumor progression in NB was investigated. RT-qPCR, western blot, bioinformatics, cell growth, Transwell, and flow cytometric analyses were performed to determine how NEAT1 synchronously regulates the miR-873-5p/MYCN proto-oncogene(MYCN) and miR-873-5p/polypeptide N-acetylgalactosaminyltransferase 1(GalNAcT-I) axes to accelerate the progression of NB. NB-bearing animal models were established to evaluate the function of NEAT1 in NB. The relationships between transcription factor coactivatorassociated arginine methyltransferase 1 (CARM1) and NEAT1, NEAT1 and miR-873-5p, miR-873-5p and GalNacT-I or MYCN, were verified using luciferase reporter gene assay, respectively. Our study revealed elevated levels of NEAT1 expression in NB cells and tissues which was associated with an advanced pathological stage and poor prognostic outcomes. According to in vitro gain- and loss- of function experiments, NEAT1 enhances progression of NB. NEAT1 silencing was found to inhibit NB proliferation in vivo. Mechanistically, to achieve upstream regulation, epigenetic downregulation of NEAT1 was achieved via the inhibition of CARM1. NEAT1 was found to positively regulate MYCN and GalNAcT-I levels as a competitive sponge of miR-873-5p. Activity of the lncRNA NEAT1 can be triggered via CARM1, which synchronously promotes NB development via the miR-873-5p/MYCN and miR-873-5p/GalNAcT-I axes. These findings shed light on the novel molecular mechanisms underlying NB progression.

New insights into the regulation of cyp3a65 expression in transgenic tg(cyp3a65:GFP) zebrafish embryos.

Facing the need for alternative models allowing assessment of metabolic-endocrine disrupting chemicals (MDCs), especially in poorly investigated tissues such as the intestine, we recently developed a transgenic zebrafish embryo in vivo model, tg(cyp3a65:GFP), expressing the Green Fluorescent Protein (GFP) under the control of the zebrafish cyp3a65 promoter, ortholog of human cyp3a4, a gene coding for a key enzyme of intestinal xenobiotic and endobiotic metabolism. In this study, we aimed to better understand the regulation of cyp3a65 expression by zfPXR, zfAhR2, and zfGR zebrafish orthologs of well-known human xenosensors PXR and AhR, and steroid nuclear receptor GR. For this purpose, we performed zebrafish embryo tg(cyp3a65:GFP) (co)exposures to a variety of agonists (clotrimazole, TCDD, fluticasone propionate) and antagonists (econazole nitrate, CH223181, RU486), which were characterized using in vitro zebrafish reporter gene assays. We show that zfPXR and zfAhR2 cooperate to positively regulate cyp3a65 expression, involving different transcription factors and their interaction. Moreover, for the first time, we show that zfGR agonist strongly inhibits the constitutive expression of cyp3a65, and we hypothesized the possible involvement of the transcriptional factor zfHNF4α. These results provide a better understanding of the regulation of zebrafish cyp3a65 expression, highlighting the complex interaction between different transcription factors, which is consistent with the multiple regulatory pathways of cyp3a4 in humans. Our data support the idea that this gene is a target of multiple contaminants capable of interacting with zfPXR, zfAhR2 and zfGR and highlights the relevance of the tg(cyp3a65:GFP) model to screen chemicals potentially acting as MDCs based on their modes of action at the intestinal level, which could be relevant for hazard assessment of chemicals for human and environmental health.

The characterization of a novel IRF8-like homolog and its role in the immune modulation of the sea urchin Strongylocentrotus intermedius.

Interferon regulatory factor (IRF) proteins, functioning as transcription factors, are essential for various animal species' innate immune defense and stress responses. However, further research is required to elucidate the roles of IRF in echinoderms. In this study, a new IRF gene (SiIRF8-like) was obtained from the sea urchin (Strongylocentrotus intermedius). The open reading frame for SiIRF8-like spanned 2004 bp and encoded a protein composed of 667 amino acids. Domain prediction analysis revealed a typical IRF domain at the N-terminus and an IRF3 domain at the C terminus of the SiIRF8-like protein, exhibiting similar amino acid sequences across different species. Phylogenetic analyses indicated that SiIRF8-like proteins were closely related to mollusk IRF8 proteins. Quantitative real-time PCR revealed detectable levels of SiIRF8-like mRNA in all sea urchin tissues examined, with the highest expression observed in coelomocytes. Furthermore, lipopolysaccharide and polyinosinic-polycytidylic acid treatments significantly increased transcript expression levels of SiIRF8-like. Subcellular localization experiments revealed that SiIRF8-like is mainly localized in the nucleus. Additionally, dual-luciferase reporter assays indicated that overexpression of SiIRF8-like in HEK293T cells could specifically activate reporter genes such as interleukin 6, interferon α/β/γ, activating protein 1, and interferon-stimulated response element. Finally, the overexpressed SiIRF8-like could promote the phosphorylation of protein kinases (JNK and Erk1/2). These preliminary findings regarding the immune functions linked to the SiIRF8-like protein offer valuable insights into the innate immunity mechanisms of invertebrate IRFs and provide theoretical support for developing disease-resistant strains of sea urchins.

MiR-6760-5p suppresses neoangiogenesis by targeting Yes-associated protein 1 in patients with moyamoya disease undergoing indirect revascularization.

The aim of this research was to investigate the specific regulatory role of miR-6760-5p in angiogenesis in moyamoya disease. HUVECs were transfected with miR-6760-5p inhibitor and mimics fragments, then subjected to assays for cell proliferation, migration, and tube formation. Subsequently, downstream target genes of miR-6760-5p were predicted and the protein expression levels of these genes were evaluated. The presence of miR-6760-5p and YAP1 was verified by a dual luciferase reporter gene test, followed by an assessment of the effects of YAP1 and miR-6760-5p on the HUVECs. Comparatively to the control group, increased expression of miR-6760-5p decreased cell growth, movement, and tube formation. YAP1 gene was discovered as a target controlled by miR-6760-5p, with subsequent investigation confirming YAP1 as a gene regulated by miR-6760-5p. Additionally, miR-6760-5p was found to counteract the angiogenesis-promoting effect of YAP1. The results of this research suggest a possible link between the miR-6760-5p gene found in the cerebrospinal fluid of individuals with moyamoya disease and the process of vascularization in this particular condition. The findings indicate that miR-6760-5p may be a new molecular indicator and potential target for the diagnosis of moyamoya disease.

Transcriptomic landscapes of STING-mediated DNA-sensing reveal cellular response heterogeneity.

Transfection of plasmid DNA (pDNA) encoding target genes is a routine tool in gene function studies and therapeutic applications. However, nucleic acid-sensing-mediated innate immune responses influence multiple intracellular signaling pathways. The stimulator of interferon genes (STING) is a crucial adapter protein for DNA sensors in mammalian cells. In this study, we explored the molecular mechanisms underlying DNA sensing by investigating the relationship between mRNA and protein expression levels and the STING pathway using single-cell analysis. We observed that reporter gene expression was dose-nonlinear after transfection of pDNA in cells with intact DNA-sensing pathways. Moreover, blocking the STING pathway in THP-1 cells significantly downregulated innate immune responses, upregulated exogenous gene expression, and mitigated the effects of innate immune responses on cell and gene function, but did not affect the proportion of reporter protein-positive cells. We elucidated the mechanisms of DNA sensing-induced innate immune response and cell death by analyzing heterozygous cellular responses to DNA transfection and transcriptome changes in positive cells. These findings suggest that the regulation of STING-mediated nucleic acid-sensing pathways is crucial for the accuracy of gene function studies and could enhance the efficacy of gene therapy.

Effect of tRF-28-ZJ47D112Z4DZ on the pathogenesis of gastric cancer through targeting cyclin D1/CDK4 and its early diagnostic value.

475 Background: Gastric cancer exhibits high incidence and mortality rates worldwide. Current research on gastric cancer aims to discover stable and effective early diagnostic markers, as well as uncover the disease's pathogenesis. tRNA-derived fragment (tRF) is a newly discovered regulatory non-coding RNA, which plays an important role in tumor diagnosis and treatment. Methods: This study, through transcriptome sequencing and MINTBase v2.0 database, screened out a new molecule tRF-28-ZJ47D112Z4DZ (tRF-28) related to gastric cancer. An absolute quantitative method was established to quantify the level of tRF-28 in plasma. ROC curve, survival curve, Cox regression model were constructed, and clinicopathological factors were collected to analyze the diagnostic value of tRF-28. "Overexpression" and "knockdown" tRF-28 gastric cancer cell models and xenograft tumor models were established to study the effects of tRF-28 on cell proliferation. Using bioinformatics, qRT-PCR, Western blot, dual luciferase reporter gene, fluorescence in situ hybridization, RNA binding protein immunoprecipitation assay, rescue experiment and immunohistochemistry to study the regulatory role of tRF-28 by binding to the 3'UTR of CCND1. Results: This study clarified the tRF-28 levels in the plasma of healthy individuals, and gastric benign and malignant lesions, providing a new candidate biomarker for the early diagnosis of gastric cancer. Through upregulating and downregulating the level of tRF-28 in gastric cancer cells, it was revealed that tRF-28 inhibits the proliferation of gastric cancer cells in vivo and in vitro by regulating key proteins such as Cyclin D1, CDK4, Rb, and p-Rb. In studying specific molecular mechanisms, tRF-28 and CCND1 were co-located in the cytoplasm. tRF-28 combines with Ago2 protein to form a complex and plays a role in silencing the target gene CCND1 in the cytoplasm. The rescue experiments revealed that silencing the target gene CCND1 can reverse the carcinogenesis effect of tRF-28 inhibitors, clarifying the biological functions of tRF-28 by regulating CCND1. Conclusions: Based on a series of research findings, we elucidated that tRF-28 form a complex with Ago2 protein, binding to the 3' UTR of CCND1 to downregulate Cyclin D1, CDK4, and other critical proteins, thereby inhibiting the proliferation of gastric cancer cells. Our study aims to offer new insights into identifying potential early diagnostic markers and therapeutic targets for gastric cancer.

Novel Reporter Constructs to Accelerate Antiviral and Therapeutic Discovery for Enterovirus-A71.

Enterovirus A71 (EV-A71) is an important human pathogen and 'prototype pathogen' for studies of other Enteroviruses of pandemic potential. Understanding the biology of EV-A71 would inform generalizable strategies for antiviral drug, vaccine, and monoclonal antibody development. Such studies are accelerated by robust reagents to evaluate efficacy. Here, we describe and evaluate a suite of synthetic reporter constructs to accelerate EV-A71 research and therapeutic discovery. These constructs include replicons and infectious clones carrying luminescent and fluorescent reporter proteins. Among the reporters we tested were shorter luminescent and de novo-designed synthetic fluorescent proteins, which enhance genetic stability, reduce reporter gene loss and improve the utility of these reporters. This toolbox provides free access to robust and flexible assays for EV-A71 infection and replication through public repositories, promoting and accelerating open scientific discovery for this understudied emerging pathogen.

Quantitative amplification ratio for established and emerging biomarkers in gastroesophageal (GE) cancer.

470 Background: Gene copy number (CN) amplifications (amps) and/or protein overexpression in ERBB2 , EGFR , FGFR2 , and MET represent established and emerging clinical targets in GE. Various methodologies are used to report gene amp or overexpression with some data suggesting FISH ratio may predict therapy response. Quantitative methods for NGS CN and relationship to ISH/IHC remains largely unresolved. We studied the landscape of targetable CN amps in GE to understand correlations of quantitative CN amp ratio and outcomes. Methods: GE tissue (N = 20,468) and liquid (N = 1,760) samples underwent NGS-based comprehensive genomic profiling (CGP). A genome-wide CN model for each sample was generated, segmented, and combined with variant allele frequencies of heterozygous SNPs to estimate the sample purity, ploidy, and CN for each segment. Genes were considered amplified at CN ≥ ploidy + 3 for ERBB2 and + 4 for other genes. Sub-analysis using amp ratio (ratio of the gene CN to the sample ploidy) ≥ 3 was performed. ctDNA tumor fraction (TF) was quantified using a composite algorithm (PMID: 38990098). The Flatiron Health-Foundation Medicine clinico genomic database, a nationwide (US-based) de-identified EHR-derived database linked to CGP data, was queried to assess external HER2 FISH or IHC and describe real-world (rw) progression-free survival (rwPFS) and overall survival (rwOS) of GE patients (pts) with ERBB2 amp on trastuzumab-based regimens. Results: ≥1 gene amps were detected in 15,341 (75%) GE tissue cases, and in 67% of cases using an amp ratio ≥3 threshold. The rates of amps in current clinical targets using each threshold were ERBB2 : 17%/12%, KRAS : 13%/12%, EGFR: 8.7%/7.2%, MET: 5.2%/4.0% , FGFR2: 3.1%/2.7% of tissue samples. The median amp ratio and percent of amps with ratio ≥ 3 were ERBB2 : 7.1/73%, KRAS : 14/92%, EGFR : 13/82%, MET : 6.3/76%, and FGFR2 : 29/88%. Amp ratio ≥ 3 was associated with focal amplicons vs amp ratio < 3 (median amplicon size 0.95 vs. 3.8 Mbp, p < 0.001). Of 1,382 pts with HER2 NGS and IHC results, concordance with IHC3+ was 68% and increasing ERBB2 amp ratio was associated with IHC 3+ status. Of 604 pts with HER2 NGS and FISH, concordance was 80% (118 positive, 366 negative). Higher ERBB2 amp ratio was also associated with longer rwOS and rwPFS as a continuous variable on first-line trastuzumab-based regimens. ≥1 gene amps were detected in 597 (34%) liquid samples (median TF 18%) and 32% with the amp ratio ≥3 threshold, increasing to 79% and 73% with TF ≥ 20% (n=355). Conclusions: Gene amps are common established or emerging GE targets and ERBB2 amp ratio is associated with outcomes for pts on trastuzumab-based regimens. Amp detection in liquid correlates with higher TF and amp ratio. As the number of therapies targeting gene amps and overexpression grows, quantification of the degree of amplification and harmonization across methodologies may be an important tool to select pts for optimal outcomes.

TFAP4 regulates the progression of liver fibrosis through the STING signaling pathway.

To investigate the mechanism by which the transcription factor TFAP4 promotes the progression of liver fibrosis through the STING signaling pathway. The expression of STING and TFAP4 in liver fibrosis mouse tissue was upregulated, AAV8-TFAP4 promoted the activation of the STING signaling pathway, and promoted the progression of liver fibrosis and tissue inflammation. In STING-KO mice, AAV8-TFAP4 could not further increase the level of liver fibrosis and tissue inflammation. Luciferase reporter gene experiments showed that there is an interactive relationship between TFAP4 and STING.TFAP4 can act as a transcription factor for STING, promote the activation of the STING signaling pathway, thereby exacerbating the progression of liver fibrosis and tissue inflammation in mice.

Innovative transgenic zebrafish biosensor for heavy metal detection.

Heavy metal contamination is an urgent environmental issue that poses a significant threat to human health and the ecosystem. To mitigate the adverse impacts of heavy metal pollution, the aim of this research was to develop genetically engineered zebrafish as biosensors, which offer a promising alternative for detecting heavy metal exposure, specifically Cd2⁺ and Zn2⁺. A novel heavy metal-sensitive gene construct metallothionine 2 promoter with DsRed reporter gene (mt2-DsRed2) was synthesized and integrated into zebrafish embryos using a Tol2 transposon transposase system with the transgenic zebrafish line subjected to biosensing applications for Cd2+ and Zn2+. The biosensor showed specific responses with linear correlation heavy metal concentration and DsRed fluorescence signal for Cd2+ and Zn2+ with (p<0.01) a minimum detection limit of 4ppb for each metal ion but not for the non-specific metal ion Ni2+, which makes it suitable for laboratory-based heavy metal assessment assays in the low heavy metal concentration ranges of 0-10ppb. Additionally, the study investigated the toxicity of heavy metals on zebrafish early developmental stages applying a modified version of the OECD Fish Embryo Toxicity (FET-236) test. Accordingly, Cd2+, Zn2+, and Ni2+ exhibited no significant toxicity effects on zebrafish embryos within the low dose range of 2-10ppb confirming the biocompatibility of the transgenic zebrafish biosensor for heavy metal sensing applications. Thus, the developed transgenic zebrafish line can accurately sense heavy metals Cd2+ and Zn2+ within the low dose range, making it a promising alternative laboratory assay for environmental monitoring and risk assessment.