3T3 Cells Research Articles

OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids

Abstract Disclosure: M. Laulhé: None. E. Kuhn: None. J. Bouligand: None. L. Amazit: None. J. Perrot: None. P. Kamenicky: None. M. Lombes: None. J. Fagart: None. S. Viengchareun: None. L. Martinerie: None. Introduction: Constitutive glucocorticoid resistance is associated with mutations in the NR3C1 gene, encoding the Glucocorticoid Receptor (GR). We identified a novel heterozygous point mutation causing fluctuating resistance to glucocorticoid depending on ligand concentrations. Initially, the propositus, a 45-year-old man, was diagnosed with a right adrenal incidentaloma and an ACTH-independent biological hypercortisolism. Shortly after adrenal surgery, plasma cortisol increased again (612 nmol/L) associated with inadequate ACTH levels (122 pg/mL) without any evidence of pituitary adenoma or ectopic ACTH-secreting tumor, suggesting a glucocorticoid resistance syndrome. Methods: We sequenced NR3C1 gene and performed in vitro analysis to assess contribution of this variant to the patient’s phenotype and how it affects biological activity of GR. Results: Exon sequencing of the NR3C1 gene identified a novel missense variant located on exon 5 (NM_000176.3, c.1706 G>A) resulting in substitution of arginine to glutamine at amino acid 569 (GR R569Q) in the Ligand-Binding Domain (LBD) of GR. We characterized transcriptional activity of GR R569Q in HEK 293T cells transiently co-transfected with either GR WT or GR R569Q, together with a luciferase reporter gene construct placed under control of a promoter containing two glucocorticoid responsive elements. We observed a decreased transcriptional activity of GR R569Q (EC50 4.55 nM [3.8;5.3]) compared to GR WT (EC50 0.19 nM [0.15; 0.24]), in response to dexamethasone. Interestingly, GR R569Q exerted a dominant negative effect on GR WT, with a decrease in transcriptional activity of 80% when stimulated with 1 nM dexamethasone (2-way ANOVA test, P<0.0001), which was blunted when stimulated with a 100-fold higher concentration of dexamethasone. As the variant is located within the second nuclear localization signal, we studied nuclear translocation in response to 1 nM cortisol for 1 h. As expected, nuclear-cytoplasmic ratio was lower in patients’ fibroblasts (2.3 ± 0.14 vs 3.0 ± 0.98, ANOVA 2-Way, P < 0.0001) compared with control fibroblasts, as quantified by automated high-throughput microscopy. Three-dimensional modeling revealed that arginine substitution leads to loss of a hydrogen bond stabilizing the LBD structure. This may result in a lower ligand affinity that can be bypassed by high concentrations of agonist. We are currently testing receptor affinity and reversibility of phenotype after hydrogen bond restoration by directed mutagenesis (c.1705 C>A, p.Q569K). Conclusion: Overall, we identified a novel variant of the NR3C1 gene associated with fluctuating GR sensitivity depending on ligand concentration. This finding may provide new insights into mechanisms involved in GR sensitivity. Presentation: Friday, June 16, 2023

The combined plasma membrane coating and cluster bombing strategy for improved tumor-targeting gene delivery of silicon nanoclusters

With the promising biosafety and favorable cell imaging efficiency, silicon quantum dots (SiQDs) was broadly exploited as non-viral gene carriers in recent years. However, the low transfection efficiency and weak targeting ability hindered its further clinical applications. In this study, the combined plasma membrane coating and cluster bombing strategy was adopted to enhance the gene delivery potential of silicon quantum dots nanoclusters (SiNC). Initially, SiNC was generated via 3, 3′-Dithiodipropionic acid (DipA) crosslinking of SiQDs, then the obtained nanoclusters were coated by distinct plasma membrane. Interestingly, cell membrane coated SiNC (CM-SiNC) underwent particle size change, the typical character of “cluster bombing”, when exposed to high GSH concentration, which was observed in the tumor microenvironment. Meanwhile, CM-SiNC can be efficiently uptaken by HEK 293T and HeLa cells, therefore transferring DNA into those cells. More importantly, among the particles coated by HeLa (HeLa-M), Red Blood (RBC-M) or RAW267.4 (RAW-M) cell membrane, HeLa cell membrane coating exhibited better cellular uptake and transfection efficiency in HeLa cells, which suggested the encouraging tumor targeting ability. In sum, these data suggested that cluster bombing of SiNC could be beneficial for physical stability and biodistribution, the additional plasma membrane coating further endowed SiNC the efficient gene delivery and tumor targeting ability. Therefore, CM-SiNC had the potential as a gene delivery vector and its application should be further addressed in vivo.

In cis “benign” SOCS1 variants linked to enhanced interferon signaling and autoimmunity

We aimed to characterize the genetic basis of disease in a family with multiple autoimmune manifestations, including systemic lupus erythematosus (SLE), immune thrombocytopenia, and autoimmune thyroiditis. Whole exome sequencing (WES) was conducted to identify candidate variants, which were analyzed by flow cytometry, immunoblotting, immunoprecipitation, and luciferase reporter assay in transfected 293T cells. Gene expression in peripheral blood mononuclear cells (PBMC) was profiled by bulk RNA sequencing and plasma cytokines were measured by proximity extension assay.In two siblings with early-onset SLE and immune thrombocytopenia, WES identified two maternally inherited in cis variants (p. Pro50Leu and p.Ala76Gly) in Suppressor of cytokine signaling 1 (SOCS1), flanking the kinase inhibitory domain that interacts with Janus kinases (JAK). Both variants were predicted to be benign by most in silico algorithms and neither alone affected the ability of SOCS1 to inhibit JAK-STAT1 signaling by functional studies. When both variants were expressed in cis, the mutant SOCS1 protein displayed decreased binding to JAK1 and reduced capacity to inhibit type I interferon (IFN–I) signaling by ∼20–30% compared to the wildtype protein. PBMC from the probands and their mother showed increased expression of interferon-inducible genes compared to healthy controls, supporting defective regulation of IFN-I signaling. Cells from all three subjects displayed heightened sensitivity to IFN-I stimulation, while response to IFN-γ, IL-4, and IL-6 was comparable to healthy controls.Our work illustrates the critical fine-tuning of IFN-I signaling by SOCS1 to prevent autoimmunity. We show that a combination of genetic variants that are individually benign may have deleterious consequences.

Characterization of gelatin-based wound dressing biomaterials containing increasing coconut oil concentrations

This study determined the influence and ideal ratios of various coconut oil (CO) amounts in gelatin (G) based-films as wound dressings since there are limited comparative studies to evaluate the sole effect of increasing CO on protein-based biomaterials. Homogenous films at G:CO ratio of 4:0,4:2,4:3,4:4 (w:w) corresponding to CO-0, CO-2, CO-3, CO-4, respectively, were obtained using solution casting. SEM showed CO caused rougher surfaces decreasing mechanical strength. However, no pores were observed in CO-4 due to bigger clusters of oil improving stretchability compared to CO-3; and durability since aging of CO-4 was >10% lower than CO-0 in aqueous media. FTIR showed triglycerides’ band only in CO films with increasing amplitude. Moreover, amide-I of CO-2 was involved in more hydrogen bonding, therefore, CO-2 had the highest melt-like transition temperatures (Tmax) at ∼163 °C while others’ were at ∼133 °C; and had more ideal mechanical properties among CO films. XTT showed that increased CO improved 3T3 cell viability as CO-0 significantly decreased viability at 10,50,75,100 μg/mL (p < 0.05), whereas CO-2 and CO-3 within 5-75 μg/mL and CO-4 within 5-100 μg/mL range increased viability ≥100% suggesting proliferation. All CO samples at 25 μg/mL stimulated 3T3 cell migration in Scratch Assay indicating wound healing. CO amounts mainly improved thermal and healing properties of gelatin-based biomaterial. CO-2 was more thermally stable and CO-4 had better influence on cell viability and wound healing than CO-0. Therefore, increased CO ratios, specifically 4:2 and 4:4, G:CO (w:w), in gelatin-based films can be ideal candidates for wound dressing materials.

Near-Infrared-Induced NO-Releasing Photothermal Adhesive Hydrogel with Enhanced Antibacterial Properties.

Bacterial infection and the development of antibiotic-resistant bacteria have decreased the effectiveness of traditional antibiotic treatments for wound healing. The design of a multifunctional adhesive hydrogel with antibacterial activity, self-healing properties, and on-demand removability to promote wound healing is highly desirable. In this work, a photothermal cyclodextrin with a NO-releasing moiety has been incorporated within an oxidized sodium alginate conjugated polyacrylamide (OS@PA) hydrogel to get a photothermal NO-releasing GSNOCD-OS@PA hydrogel. Such a multifunctional hydrogel has the unique feature of combined antibacterial activity as a result of a controlled photothermal effect and NO gas release under an 808 near-infrared laser. Because of oxidized sodium alginate (OSA), the hydrogel matrix easily adheres to the skin under twisted and bent states. In vitro cytotoxicity analysis against 3T3 cells showed that the hydrogels OS@PA and GSNOCD-OS@PA are noncytotoxic under laser exposure. The temperature-induced NO release by GSNOCD-OS@PA reached 31.7 mg/L when irradiated with an 808 nm laser for 10 min. The combined photothermal therapy and NO release from GSNOCD-OS@PA effectively reduced viability of both Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) to 3 and 5%, respectively. Importantly, the phototherapeutic NO-releasing platform displayed effective fibroblast proliferation in a cell scratch assay.

Development of a biomimetic DNA delivery system by encapsulating polyethyleneimine functionalized silicon quantum dots with cell membranes.

Quantum dots (QDs) are renowned for their remarkable optoelectronic properties, making them suitable for applications such as bioimaging and optoelectronics. However, their use in gene delivery has been restricted due to the low DNA loading capacity. This study aimed to develop a biomimetic DNA delivery system by encapsulating polyethyleneimine (PEI) functionalized silicon QDs (SiQDs) with cell membranes and evaluate its potential as a gene vector in vitro. To achieve this, hydrophilic dispersed silicon QDs (PQDs) were prepared through a one-pot hydrothermal reaction of PEI and 3-Aminopropyltrimethoxysilane (APTMS). Subsequently, red blood cell membrane (RBCM) encapsulated biomimetic QDs (CM-PQDs) was obtained through the extrusion method. The CM-PQDs exhibited higher DNA loading capacity and better stability than naked SiQDs. The CM-PQDs/DNA complex was effectively taken up by cells, as observed through the fluorescence characteristics of QDs themselves. Both CM-P10QDs (prepared with PEI10k) and CM-P25QDs (prepared with PEI25k) could deliver DNA into cells and express the reporter protein successfully. CM-P25QDs showed a higher transfection efficiency of 77.32% in 293T cells and 47.11% in HeLa cells than SiQDs and CM-P10QDs. The results also indicated that cell membrane encapsulation could effectively reduce the cytotoxicity of SiQDs further. Therefore, the study concludes that CM-PQDs have the potential to serve as a safe and traceable biomimetic gene delivery system.

Hydroxamic Acids as PARP‐1 Inhibitors: Molecular Design and Anticancer Activity of Novel Phenanthridinones

AbstractPoly(ADP‐ribose)polymerase‐1 (PARP‐1) is a promising target for antitumor agents. This study presents the first evidence of hydroxamic acids as efficient PARP inhibitors. Molecular docking and molecular dynamics simulations revealed that N−O substituted phenanthridinones form a complex interplay with PARP‐1. A series of cyclic aryl hydroxamic acids, N‐(benzyloxy)‐ and N‐(hydroxy)phenanthridinones, were prepared through a ligand‐free methodology from N‐(benzyloxy)benzamides using dual C−H/N−H bond activation. Three of the computed hit compounds exhibited significant activity in cell‐based and enzymatic assays, inhibiting PARP‐1 in the low‐nanomolar range. The antiproliferative activity of all prepared compounds and the reference compounds PJ34 and Olaparib was evaluated in cancer cells (HepG2, BxPC3, MDA‐MD‐231, and HeLa) and in noncancer cell lines (NIH 3T3 and HEK 293). An N‐(benzyloxy)‐ and an N‐(hydroxy)phenanthridinone showed the most promising properties as leads for developing therapeutics with a submicromolar activity window. The study highlights the potential utility of this scaffold for PARP inhibitors and the importance of target‐specific design to minimize toxicity and side effects.

Intermittent Hypoxia Promotes TAM-Induced Glycolysis in Laryngeal Cancer Cells via Regulation of HK1 Expression through Activation of ZBTB10.

Obstructive sleep apnea (OSA), characterized by intermittent hypoxia (IH), may increase the risk of cancer development and a poor cancer prognosis. TAMs of the M2 phenotype, together with the intermittent hypoxic environment within the tumor, drive tumor aggressiveness. However, the mechanism of TAMs in IH remains unclear. In our study, IH induced the recruitment of macrophages, and IH-induced M2-like TAMs promoted glycolysis in laryngeal cancer cells through hexokinase 1. The hexokinase inhibitor 2-deoxy-D-glucose and HK1 shRNA were applied to verify this finding, confirming that M2-like TAMs enhanced glycolysis in laryngeal cancer cells through HK1 under intermittent hypoxic conditions. Comprehensive RNA-seq analysis disclosed a marked elevation in the expression levels of the transcription factor ZBTB10, while evaluation of a laryngeal cancer patient tissue microarray demonstrated a positive correlation between ZBTB10 and HK1 expression in laryngeal carcinoma. Knockdown of ZBTB10 decreased HK1 expression, and overexpression of ZBTB10 increased HK1 expression in both laryngeal cancer cells and 293T cells. The luciferase reporter assay and Chromatin immunoprecipitation assay confirmed that ZBTB10 directly bound to the promoter region of HK1 and regulated the transcriptional activity of HK1. Finally, the CLEC3B level of the M2 supernatant is significantly higher in the IH group and showed a protumor effect on Hep2 cells. As ZBTB10-mediated regulation of HK1 affects glycolysis in laryngeal cancer, our findings may provide new potential therapeutic targets for laryngeal cancer.

Gamma Secretase Inhibition Sensitizes Pancreatic Adenocarcinoma Tumors to RT In Vivo

Pancreatic adenocarcinoma (PDAC) is an extremely aggressive cancer that lacks curative treatment options. Almost half of patients present with unresectable disease limiting treatment to non-curative options. Patients treated with neoadjuvant radiation therapy (RT) exhibit increases in fibrosis and epithelial-to-mesenchymal transition (EMT). ADAM10, an extracellular sheddase, can stimulate stromal fibrosis, EMT, and radioresistance. ADAM10 also mediates EMT through Notch signaling by cleaving its extracellular domain. Further cleavage by gamma secretase produces the Notch intracellular domain (NICD), which translocates to the nucleus and activates downstream transcriptional targets. Here, we explore whether inhibition of Notch cleavage by gamma secretase radiosensitizes PDAC tumors. Bilateral flank subcutaneous PDAC isografts were produced in 40 mice using PK5L1940 KPC cells. Intraperitoneal injections of the gamma secretase inhibitor, DAPT (5 mg/kg), were delivered daily for 7 days, starting 3 days prior to RT. A single dose of 20 Gy was administered to each flank tumor, and volumes were measured twice weekly. Colony formation assays of KPC cells were performed after RT, in the presence or absence of DAPT. Since stromal fibrosis can mediate radio-resistance in the tumor microenvironment (TME), the effect of tumor cells on Notch pathway activation in mouse fibroblasts (3T3 cells) was investigated using a luciferase reporter assay. Thus, 3T3 cells transfected with a Notch pathway luciferase reporter were incubated with PDAC cells for 48 h, followed by measurement of luciferase activity. In vivo, the combination of DAPT and RT significantly delayed tumor growth, and some tumors were completely eradicated. Mean tumor size for the combination at 21 days was 21 mm3 (range = 0-53, p = 0.005), while tumor size was 577 mm3 (range = 217-955, p = 0.69) for DAPT alone, 435 mm3 for RT alone (range = 51-932, p = 0.79), and 367 mm3 for untreated vehicle (range = 97-1144). Surprisingly, DAPT did not reduce clonogenic survival in vitro. Both ADAM10 knockout and DAPT decreased NICD cleavage and transcription of the downstream target Hes1 in vivo and in vitro. Co-culture with PDAC cells increased Notch luciferase reporter activity in fibroblasts. This effect was not mimicked by PDAC-conditioned media, suggesting a requirement for intercellular contact. Notch pathway inhibition sensitizes PDAC tumors to RT in vivo, but not in vitro, suggesting involvement of the TME. Indeed, co-culture with PDAC cells stimulates notch signaling in fibroblasts, suggesting non-cell autonomous mechanisms mediating fibrosis in the TME driving radioresistance. Future studies will determine if ADAM10 inhibition targeting PDAC cells and/or gamma secretase inhibition targeting the TME enhances radiation sensitivity in vivo by blocking fibroblast Notch signaling.

Antibody-Free Fluorine-Assisted Metabolic Sequencing of RNA N4-Acetylcytidine.

N4-Acetylcytidine (ac4C) has been found to affect a variety of cellular and biological processes. For a mechanistic understanding of the roles of ac4C in biology and disease, we present an antibody-free, fluorine-assisted metabolic sequencing method to detect RNA ac4C, called "FAM-seq". We successfully applied FAM-seq to profile ac4C landscapes in human 293T, HeLa, and MDA cell lines in parallel with the reported acRIP-seq method. By comparison with the classic ac4C antibody sequencing method, we found that FAM-seq is a convenient and reliable method for transcriptome-wide mapping of ac4C. Because this method holds promise for detecting nascent RNA ac4C modifications, we further investigated the role of ac4C in regulating chemotherapy drug resistance in chronic myeloid leukemia. The results indicated that drug development or combination therapy could be enhanced by appreciating the key role of ac4C modification in cancer therapy.

Circadian ribosome profiling reveals a role for the Period2 upstream open reading frame in sleep

Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered posttranscriptionally. We used ribosome profiling to examine posttranscriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An increase in the number of uORFs in the 5'UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced ribosome binding on Per2, and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA posttranscriptionally, which can impact physiological rhythms and sleep.

Single neurons on microelectrode array chip: manipulation and analyses.

Chips-based platforms intended for single-cell manipulation are considered powerful tools to analyze intercellular interactions and cellular functions. Although the conventional cell co-culture models could investigate cell communication to some extent, the role of a single cell requires further analysis. In this study, a precise intercellular interaction model was built using a microelectrode array [microelectrode array (MEA)]-based and dielectrophoresis-driven single-cell manipulation chip. The integrated platform enabled precise manipulation of single cells, which were either trapped on or transferred between electrodes. Each electrode was controlled independently to record the corresponding cellular electrophysiology. Multiple parameters were explored to investigate their effects on cell manipulation including the diameter and depth of microwells, the geometry of cells, and the voltage amplitude of the control signal. Under the optimized microenvironment, the chip was further evaluated using 293T and neural cells to investigate the influence of electric field on cells. An examination of the inappropriate use of electric fields on cells revealed the occurrence of oncosis. In the end of the study, electrophysiology of single neurons and network of neurons, both differentiated from human induced pluripotent stem cells (iPSC), was recorded and compared to demonstrate the functionality of the chip. The obtained preliminary results extended the nature growing model to the controllable level, satisfying the expectation of introducing more elaborated intercellular interaction models.

Super-enhancer-associated SNHG15 cooperating with FOSL1 contributes to bladder cancer progression through the WNT pathway

Small nucleolar RNA host gene 15 (SNHG15) plays an oncogenic role in many cancers. However, the role of SNHG15 in bladder cancer (BLCA) remains unclear. In this study, the regulation of SNHG15 on the activities of BLCA cells (T24 and RT112) was investigated. In detail, super-enhancers (SEs), differentially expressed genes, and functional enrichment were detected by bioinformatic analyses. Mutant cell lines lacking SNHG15-SEs were established using CRISPR-Cas9. Relative gene expression was detected by quantitative polymerase chain reaction (qPCR), western blot, in situ hybridization, and immunohistochemistry assays. Cell senescence, apoptosis, viability, and proliferation were measured. Chromatin immunoprecipitation (ChIP)-qPCR and luciferase reporter gene assays were conducted to analyze the interactions between genes. A novel super-enhancer of SNHG15 (SNHG15-SEs) was discovered in several BLCA datasets. The deletion of SNHG15-SEs resulted in a significant downregulation of SNHG15. Mechanistically, the core active region of SNHG15-SEs recruited the transcription factor FOSL1 to facilitate the SNHG15 transcription, thereby inducing the proliferation and metastasis of BLCA cells. Deletion of SNHG15-SEs inhibited the growth and metastasis of T24 and RT112 cells by inactivating the WNT/CTNNB1 pathway activation. Overexpression of FOSL1 in SNHG15-SEs restored the cell proliferation and metastasis. Next, a xenograft mouse model showed that SNHG15-SEs deletion inhibited the proliferation and metastasis of BLCA cells in vivo. Collectively, our data indicate that SNHG15-SEs recruit FOSL1 to promote the expression of SNHG15 which interacts with CTNNB1 in the nucleus to activate the transcription of ADAM12, leading to the malignance of BLCA cells.

The Impact of Adding Chitosan Nanoparticles on Biofilm Formation, Cytotoxicity, and Certain Physical and Mechanical Aspects of Directly Printed Orthodontic Clear Aligners.

Aligner treatment is associated with bacterial colonization, leading to enamel demineralization. Chitosan nanoparticles have been demonstrated to have antibacterial properties. This in vitro study aims to determine the effect of adding chitosan nanoparticles to directly 3D-printed clear aligner resin with regard to antibiofilm activity, cytotoxicity, degree of conversion, accuracy, deflection force, and tensile strength. Different concentrations (2%, 3%, and 5% w/w) of chitosan nanoparticles were mixed with the clear resin, and the samples were then 3D printed. Additionally, the thermoforming technique for aligner manufacturing was utilized. The obtained specimens were evaluated for antibiofilm activity against Streptococcus mutans bacteria and cytotoxicity against L929 and 3T3 cell lines. Additionally, Fourier transform infrared spectroscopy via attenuated total reflection analysis was used to assess the degree of conversion. Geomagic Control X software was utilized to analyze the accuracy. In addition, the deflection force and tensile strength were evaluated. The results indicated a notable reduction in bacterial colonies when the resin was incorporated with 3 and 5% chitosan nanoparticles. No significant changes in the cytotoxicity or accuracy were detected. In conclusion, integrating biocompatible chitosan nanoparticles into the resin can add an antibiofilm element to an aligner without compromising the material's certain biological, mechanical, and physical qualities at specific concentrations.

Non-enzymatic electrochemical sensor based on Pt/MoSe2 nanomesh for the detection of hydrogen peroxide

The development of diagnostic methods that are sensitive and selective for detection of reactive oxygen species (ROS) is crucial for early diagnosis of bladder cancer. Herein, a new electrochemical sensing platform for hydrogen peroxide (H2O2) was created based on the Pt/MoSe2 nanomesh with more active sites and larger specific surface area. The Pt/MoSe2 nanomesh sensors exhibited highly sensitive and selective for H2O2, wherein the correlation coefficient (R2) and the limit of detection (LOD) were 0.9983 and 2.56 μM obtained at the signal/noise (S/N) ratio of 3, respectively. Additionally, Pt/MoSe2 nanomesh possessed a good anti-interference performance even in the presence of potentially interfering small molecules within cells, such as ascorbic acid (AA), glucose (Glc) and so on. More importantly, the detection of H2O2 in tap, rain Water, urine FBS, human serum and bladder cancer T24 cells could be successfully detected by the Pt/MoSe2 nanomesh electrochemical sensor.

Construction and functional analysis of EGFRvIII CAR-T cells co-expressing IL-15 and CCL19

The aim of this study was to investigate the functional characteristics and in vitro specific killing effect of EGFRvIII CAR-T cells co-expressing interleukin-15 and chemokine CCL19, in order to optimize the multiple functions of CAR-T cells and improve the therapeutic effect of CAR-T cells targeting EGFRvIII on glioblastoma (GBM). The recombinant lentivirus plasmid was obtained by genetic engineering, transfected into 293T cells to obtain lentivirus and infected T cells to obtain the fourth generation CAR-T cells targeting EGFRvIII (EGFRvIII-IL-15-CCL19 CAR-T). The expression rate of CAR molecules, proliferation, chemotactic ability, in vitro specific killing ability and anti-apoptotic ability of the fourth and second generation CAR-T cells (EGFRvIII CAR-T) were detected by flow cytometry, cell counter, chemotaxis chamber and apoptosis kit. The results showed that compared with EGFRvIII CAR-T cells, EGFRvIII-IL-15-CCL19 CAR-T cells successfully secreted IL-15 and CCL19, and had stronger proliferation, chemotactic ability and anti-apoptosis ability in vitro (all P < 0.05), while there was no significant difference in killing ability in vitro. Therefore, CAR-T cells targeting EGFRvIII and secreting IL-15 and CCL19 are expected to improve the therapeutic effect of glioblastoma and provide an experimental basis for clinical trials.

Oryza sativa L. Indica Seed Coat Ameliorated Concanavalin A-Induced Acute Hepatitis in Mice via MDM2/p53 and PKCα/MAPK1 Signaling Pathways.

Acute hepatitis (AH) is a common liver disease with an increasing number of patients each year, requiring the development of new treatments. Hence, our work aimed to evaluate the therapeutic effect of Oryza sativa L. indica (purple rice) seed coat on concanavalin A (ConA)-induced AH and further reveal its potential mechanisms. Purple rice seed coat extract (PRE) was extracted with hydrochloric acid ethanol and analyzed through a widely targeted components method. We evaluated the effects of PRE on AH through histopathological examination, liver function, gut microbiota composition, and the intestinal barrier. The potential targets of PRE on AH were predicted by bioinformatics. Western blotting, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining, and corresponding kits were used to investigate PRE effects on predicting targets and associated signaling pathways in AH mice. In AH model mice, PRE treatment increased transformed mouse 3T3 cell double minute 2 (MDM2) expression to inhibit apoptosis; it also markedly downregulated protein kinase C alpha (PKCα), prostaglandin-endoperoxide synthase 1 (PTGS1), and mitogen-activated protein kinase 1 (MAPK1) activity to alleviate inflammation. Thus, PRE treatment also recovered the intestinal barrier, decreased the lipopolysaccharide (LPS) levels of plasma and the liver, enhanced liver function, and improved the composition of intestinal microbiota. In general, PRE targeting MDM2, PKCα, MAPK1, and PTGS1 ameliorated ConA-induced AH by attenuating inflammation and apoptosis, restoring the intestinal barrier, enhancing the liver function, and improving the gut microbiota, which revealed that the purple rice seed coat might hold possibilities as a therapeutic option for AH.

Downregulation of CRTAC1 in Urothelial Carcinoma Promotes Tumor Aggressiveness and Confers Poor Prognosis.

Cartilage acidic protein 1 (CRTAC1) is a glycosylated calcium-binding extracellular matrix protein. The oncological functions of CRTAC1 in urothelial carcinoma (UC) of the urinary bladder (UB) and upper urinary tract (UT) have not yet been elucidated. Based on the published UBUC transcriptome data, we re-evaluated the differential expression profile of calcium ion binding-related genes (GO:0005509), and we found that CRTAC1 was the most significantly downregulated gene in UBUC progression. Therefore, we analyzed the prognostic value and biological significance of CRTAC1 expression in UC. We used immunohistochemistry to determine the CRTAC1 expression levels in 340 patients with UTUC and 295 patients with UBUC. The CRTAC1 expression was compared with the clinicopathological characteristics, and the prognostic impact of CRTAC1 on metastasis-free survival (MFS) and disease-specific survival (DSS) was evaluated. To study the biological functions of CRTAC1, the proliferation, migration, invasion, and tube formation abilities of UC-derived cells were evaluated. A low CRTAC1 expression significantly correlated with high tumor stage, high histological grade, perineural invasion, vascular invasion, nodal metastasis, and high mitotic rate (all p < 0.01). Moreover, the CRTAC1 immunoexpression status was an independent prognostic factor for MFS and DSS in UBUC and UTUC patients (all p < 0.001) in the multivariate analysis. The exogenous expression of CRTAC1 suppressed the cell proliferation, invasion, and angiogenesis, and downregulated the matrix metallopeptidase 2 (MMP2) level in BFTC909 and T24 cells. CRTAC1 may participate in progression of UC and serve as a prognostic marker for metastasis. Low CRTAC1 expression was significantly associated with aggressive UC characteristics and worse clinical outcomes. The inclusion of CRTAC1 immunoexpression in the standard pathological variables may optimize the risk stratification of patients.

Metabolites analysis and new bioactive compounds from the medicine food homology product of Cordyceps chanhua on artificial media

Cordyceps chanhua on artificial media has been approved as a medicine food homology product. However, the metabolites have not been extensively studied. HPLC-HRMS analysis showed that there were 11 main metabolites in the EtOAc extract including 4 probable unknown compounds. Fumosoroseain A with anti-aging effects is the most abundant known compound and was identified from C. chanhua for the first time. The second abundant compound is N6-(2-Hydroxyethyl) adenosine, a typical metabolite of C. chanhua. All the known compounds have consistent health function with that of the fungus. HRMS, 1D and 2D NMR analyses revealed that compounds 2, 3, and 4 are new nucleosides named as chanhuanosides A, B, and C. Compound 1 is the known compound cordyrrole B isolated from C. chanhua for the first time whose structure is firstly confirmed by single crystal X-ray analysis. Bioactivity analysis revealed that 1-4 significantly inhibited pancreatic lipase activity, and strongly promoted the proliferation of RAW264.7 and 293T cells, suggesting that they might have ant-obesity, immunoregulation, and renal protection functions. Structure-bioactivity analysis revealed that the esterification on ribose can increase their bioactivity. Present metabolites study suggests that C. chanhua cultured on the artificial medium is a promising health food.

Development of a prognostic signature for immune-associated genes in bladder cancer and exploring potential drug findings.

Bladder cancer, predominantly affecting men, is a prevalent malignancy of the urinary system. Although platinum-based chemotherapy has demonstrated certain enhancements in overall survival when compared to surgery alone, the efficacy of treatments is impeded by the unfavorable side effects of conventional chemotherapy medications. Nonetheless, immunotherapy exhibits potential in the treatment of bladder cancer. To create an immune-associated prognostic signature for bladder cancer, bioinformatics analyses were performed utilizing The Cancer Genome Atlas (TCGA) database in this study. By identifying differential gene expressions between the high-risk and low-risk groups, a potential therapeutic drug was predicted using the Connectivity Map database. Subsequently, the impact of this drug on the growth of T24 cells was validated through MTT assay and 3D cell culture techniques. The signature included 1 immune-associated LncRNA (NR2F1-AS1) and 16 immune-associated mRNAs (DEFB133, RBP7, PDGFRA, CGB3, PDGFD, SCG2, ADCYAP1R1, OPRL1, PGR, PSMD1, TANK, PRDX1, ADIPOR2, S100A8, AHNAK, EGFR). Based on the assessment of risk scores, the patients were classified into cohorts of low-risk and high-risk individuals. The cohort with low risk demonstrated a considerably higher likelihood of survival in comparison to the group with high risk. Furthermore, variations in immune infiltration were noted among the two categories. Cephaeline, a possible medication, was discovered by analyzing variations in gene expression. It exhibited promise in suppressing the viability and growth of T24 bladder cancer cells. The novel predictive pattern allows for efficient categorization of patients with bladder cancer, enabling focused and rigorous treatment for those expected to have a worse prognosis. The discovery of a possible curative medication establishes a basis for forthcoming immunotherapy trials in bladder cancer.