Expression Of Target Proteins Research Articles

Development and application of an uncapped mRNA platform

Background A novel uncapped mRNA platform was developed. Methods Five lipid nanoparticle (LNP)-encapsulated mRNA constructs were made to evaluate several aspects of our platform, including transfection efficiency and durability in vitro and in vivo and the activation of humoral and cellular immunity in several animal models. The constructs were eGFP-mRNA-LNP (for enhanced green fluorescence mRNA), Fluc-mRNA-LNP (for firefly luciferase mRNA), SδT-mRNA-LNP (for Delta strain SARS-CoV-2 spike protein trimer mRNA), gDED-mRNA-LNP (for truncated glycoprotein D mRNA coding ectodomain from herpes simplex virus type 2 (HSV2)) and gDFR-mRNA-LNP (for truncated HSV2 glycoprotein D mRNA coding amino acids 1–400). Results Quantifiable target protein expression was achieved in vitro and in vivo with eGFP- and Fluc-mRNA-LNP. SδT-mRNA-LNP, gDED-mRNA-LNP and gDFR-mRNA-LNP induced both humoral and cellular immune responses comparable to those obtained by previously reported capped mRNA-LNP constructs. Notably, SδT-mRNA-LNP elicited neutralizing antibodies in hamsters against the Omicron and Delta strains. Additionally, gDED-mRNA-LNP and gDFR-mRNA-LNP induced potent neutralizing antibodies in rabbits and mice. The mRNA constructs with uridine triphosphate (UTP) outperformed those with N1-methylpseudouridine triphosphate (N1mψTP) in the induction of antibodies via SδT-mRNA-LNP. Conclusions Our uncapped, process-simplified and economical mRNA platform may have broad utility in vaccines and protein replacement drugs. KEY MESSAGES The mRNA platform described in our paper uses internal ribosome entry site (IRES) (Rapid, Amplified, Capless and Economical, RACE; Register as BH-RACE platform) instead of caps and uridine triphosphate (UTP) instead of N1-methylpseudouridine triphosphate (N1mψTP) to synthesize mRNA. Through the self-developed packaging instrument and lipid nanoparticle (LNP) delivery system, mRNA can be expressed in cells more efficiently, quickly and economically. Particularly exciting is that potent neutralizing antibodies against Delta and Omicron real viruses were induced with the new coronavirus S protein mRNA vaccine from the BH-RACE platform.

DJ-1-mediated repression of the RNA-binding protein FMRP is predicted to impact known Alzheimer's disease-related protein networks.

RNA-binding proteins (RBPs) modulate the synaptic proteome and are instrumental in maintaining synaptic homeostasis. Moreover, aberrant expression of an RBP in a disease state would have deleterious downstream effects on synaptic function. While many underlying mechanisms of synaptic dysfunction in Alzheimer's disease (AD) have been proposed, the contribution of RBPs has been relatively unexplored. To investigate alterations in RBP-messenger RNA (mRNA) interactions in AD, and its overall impact on the disease-related proteome. We first utilized RNA-immunoprecipitation to investigate interactions between RBP, DJ-1 (Parkinson's Disease protein 7) and target mRNAs in controls and AD. Surface Sensing of Translation - Proximity Ligation Assay (SUnSET-PLA) and western blotting additionally quantified alterations in mRNA translation and protein expression of DJ-1 targets. Finally, we utilized an unbiased bioinformatic approach that connects AD-related pathways to two RBPs, DJ-1 and FMRP (Fragile X messenger ribonucleoprotein 1). We find that oligomeric DJ-1 in AD donor synapses were less dynamic in their ability to bind and unbind mRNA compared to synapses from cognitively unimpaired, neuropathologically-verified controls. Furthermore, we find that DJ-1 associates with the mRNA coding for FMRP, Fmr1, leading to its reduced synaptic expression in AD. Through the construction of protein-protein interaction networks, aberrant expression of DJ-1 and FMRP are predicted to lead to the upregulation of key AD-related pathways, such as thyroid hormone stimulating pathway, autophagy, and ubiquitin mediated proteolysis. DJ-1 and FMRP are novel targets that may restore established neurobiological mechanisms underlying AD.

Transglutaminase 2 regulates endothelial cell calcification via IL-6-mediated autophagy.

Endothelial cell (EC) calcification is an important marker of atherosclerotic calcification. ECs play a critical role not only in atherogenesis but also in intimal calcification, as they have been postulated to serve as a source of osteoprogenitor cells that initiate this process. While the role of transglutaminase 2 (TG2) in cellular differentiation, survival, apoptosis, autophagy, and cell adhesion is well established, the mechanism underlying the TG2-mediated regulation of EC calcification is yet to be fully elucidated. The TG2 gene was overexpressed or silenced by using siRNA and recombinant adenovirus. RT-PCR and WB were used to analyze the relative expression of target genes and proteins. 5-BP method analyzed TG2 activity. mCherry-eGFP-LC3 adenovirus and transmission electron microscopy analyzed EC autophagy level. Calcium concentrations were measured by using a calcium colorimetric assay kit. Alizarin red S staining assay analyzed EC calcification level. Elisa analyzed IL-6 level. Establishing EC calcification model by using a calcification medium (CM). Our findings demonstrated that CM increased TG2 activity and expression, which activated the NF-κB signaling pathway, and induced IL-6 autocrine signaling in ECs. Furthermore, IL-6 activated the JAK2/STAT3 signaling pathway to suppress cell autophagy and promoted ECs calcification. ECs are not only critical for atherogenesis but also believed to be a source of osteoprogenitor cells that initiate intimal calcification. Previous research has shown that TG2 plays an important role in the development of VC, but the mechanism by which it exerts this effect is not yet fully understood. Our results demonstrated that TG2 forms complexes with NF-κB components inhibition of autophagy promoted endothelial cell calcification through EndMT. Therefore, our research investigated the molecular mechanism of EC calcification, which can provide new insights into the pathogenesis of atherosclerosis.

M6A-modified circCacna1c regulates necroptosis and ischemic myocardial injury by inhibiting Hnrnpf entry into the nucleus

BackgroundCircular RNAs (circRNAs) are differentially expressed in various cardiovascular diseases, including myocardial infarction (MI) injury. However, their functional role in necroptosis-induced loss of cardiomyocytes remains unclear. We identified a cardiac necroptosis-associated circRNA transcribed from the Cacna1c gene (circCacna1c) to investigate the involvement of circRNAs in cardiomyocyte necroptosis.MethodsTo investigate the role of circCacna1c during oxidative stress, H9c2 cells and neonatal rat cardiomyocytes were treated with hydrogen peroxide (H2O2) to induce reactive oxygen species (ROS)-induced cardiomyocyte death. The N6-methyladenosine (m6A) modification level of circCacna1c was determined by methylated RNA immunoprecipitation quantitative polymerase chain reaction (MeRIP–qPCR) analysis. Additionally, an RNA pull-down assay was performed to identify interacting proteins of circCacna1c in cardiomyocytes, and the regulatory role of circCacna1c in target protein expression was tested using a western blotting assay. Furthermore, the MI mouse model was constructed to analyze the effect of circCacna1c on heart function and cardiomyocyte necroptosis.ResultsThe expression of circCacna1c was found to be reduced in cardiomyocytes exposed to oxidative stress and in mouse hearts injured by MI. Overexpression of circCacna1c inhibited necroptosis of cardiomyocytes induced by hydrogen peroxide and MI injury, resulting in a significant reduction in myocardial infarction size and improved cardiac function. Mechanistically, circCacna1c directly interacts with heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in the cytoplasm, preventing its nuclear translocation and leading to reduced Hnrnpf levels within the nucleus. This subsequently suppresses Hnrnpf-dependent receptor-interacting protein kinase 1 (RIPK1) expression. Furthermore, fat mass and obesity-associated protein (FTO) mediates demethylation of m6A modification on circCacna1c during necrosis and facilitates degradation of circCacna1c.ConclusionOur study demonstrates that circCacna1c can improve cardiac function following MI-induced heart injury by inhibiting the Hnrnpf/RIPK1-mediated cardiomyocyte necroptosis. Therefore, the FTO/circCacna1c/Hnrnpf/RIPK1 axis holds great potential as an effective target for attenuating cardiac injury caused by necroptosis in ischemic heart disease.Graphical

Methionine restriction inhibits pancreatic cancer proliferation while suppressing JAK2/STAT3 pathway

BackgroundMethionine restriction (MR) has been demonstrated to exhibit anti-tumor effects in various types of cancer, including pancreatic cancer (PC). However, the detailed mechanism induced by MR remains still unclear. This study aims to reveal the underlying mechanism of MR on PC by proteomic analysis. Material & MethodsHuman PC cell lines were cultured in both standard and MR media to evaluate the effect of MR. The differences in protein expression were evaluated through proteomic analysis. Ingenuity Pathway Analysis (IPA) was performed to identify proteins potentially associated with tumor growth in vitro. The proteins associated with the anti-tumor effect were validated using western blotting, real-time PCR, and ELISA. An experimental model involving subcutaneous PC mice was established for the assessment of the effectiveness of the MR diet and the expression of target proteins through immunohistochemical staining. ResultsCell proliferation was suppressed in the MR media compared to the standard media. IPA analysis showed that STAT3 was decreased in the Apoptotic Pathway of Pancreatic Cancer Cell lines in the MR group. Western blotting showed MR decreased STAT3 expression. Real-time PCR showed that MR decreased JAK2 and STAT3 mRNA expression in Panc-1 and Mia-PaCa 2, but not in Capan-1. ELISA revealed that NF-kB expression was decreased in the MR group. In the in vivo study, the final estimated tumor volume in the MR group was significantly lower than the control group (p < 0.01). Immunostaining of resected specimens showed that STAT3 expression was suppressed in the MR group. ConclusionMR suppressed the JAK2/STAT3 pathway and decreased NF-kB in some PC cell lines.

Isoxanthohumol improves hepatic lipid metabolism via regulating the AMPK/PPARα and PI3K/AKT signaling pathways in hyperlipidemic mice.

Hyperlipidemia presents a significant global healthcare challenge, necessitating innovative therapeutic strategies for more effective outcomes. Recent studies have highlighted the beneficial impact of moderate beer intake on metabolic diseases. The purpose of this research is to explore the possible molecular mechanisms of isoxanthohumol (IXN), the major hop flavonoid in beer, in the treatment of hyperlipidemia. The mice model of acute hyperlipidemia was constructed by intraperitoneal injection of Triton WR-1339. The therapeutic effect of IXN was assessed by biochemical and histological analyses. Furthermore, comprehensive data mining across various public databases was conducted to identify underlying therapeutic targets of IXN on hyperlipidemia. A protein-protein interaction network was constructed to pinpoint hub targets, and subsequent GO and KEGG enrichment analyses were used to elucidate underlying biological functions. Molecular docking was utilized to validate the binding affinity between hub targets and IXN. Western blotting analysis further verified the protein expression of potential IXN targets. IXN administration significantly improved blood lipid and hepatic lipid levels, alongside increased SOD activity and decreased MDA content in hyperlipidemia mice. Histological analyses, including H&E and Oil Red O staining, showed the improvement of hepatic steatosis with IXN treatment. At the molecular level, IXN significantly increased protein levels of p-AMPK, PPARα, p-PI3K, and p-AKT. IXN activates AMPK/PPARα and PI3K/AKT signaling pathways, leading to reduction in lipid accumulation and oxidative stress, and ultimately ameliorating hyperlipidemia.

Nodularin-R Synergistically Enhances Abiraterone Against Castrate- Resistant Prostate Cancer via PPP1CA Inhibition.

Clinically, most prostate cancer (PCa) patients inevitably progress to castration-resistant prostate cancer (CRPC) with poor prognosis after androgen deprivation therapy (ADT), including abiraterone, the drug of choice for ADT. Therefore, it is necessary to explore the resistance mechanism of abiraterone in depth. Genome-wide CRISPR/Cas9 knockout technology was used to screen CRPC cell line 22Rv1 for abiraterone-resistant genes. Combined with bioinformatics, a key gene with high expression and poor prognosis in CRPC patients was screened. Then, the effects of target gene on abiraterone-resistant 22Rv1 cell function were explored by silencing and overexpression. Further, a natural product with potential targeting effect was identified and validated by molecular docking and protein expression. Molecular dynamics simulations revealed potential mechanism for the natural product affecting target protein expression. Finally, the combined anti-CRPC effects of the natural product and abiraterone were validated by cellular and invivo experiments. Five common resistance genes (KCNJ3, COL2A1, PPP1CA, MDH2 and EXOSC5) were identified successfully, among which high PPP1CA expression had the worst prognosis for disease-free survival. Moreover, PPP1CA was highly expressed in abiraterone-resistant 22Rv1 cells. Silencing PPP1CA increased cell sensitivity to abiraterone while promoting apoptosis and inhibiting clone formation. Overexpressing PPP1CA exerted the opposite effects. Molecular docking revealed the binding mode of the natural product nodularin-R to PPP1CA with a dose-dependent manner for inhibition. Mechanistically, nodularin-R attenuates the interaction between PPP1CA and USP11 (deubiquitinating enzyme), potentially promoting PPP1CA degradation. Additionally, combination of 2.72 μM nodularin-R and 54.5 μM abiraterone synergistically inhibited the resistant 22Rv1 cell function. Invivo experiments also revealed that combination therapy significantly inhibited tumour growth and reduced inducible expression of PPP1CA. PPP1CA is a key driver for abiraterone resistance, and nodularin-R enhances the anti-CRPC effects of abiraterone by inhibiting PPP1CA.

Cordycepin Alleviates Lipopolysaccharides-Induced Preeclampsia-Like Impairments in Rats

ABSTRACT Introduction Preeclampsia is a serious pregnancy complication that can lead to life-threatening conditions such as seizures, strokes, and even death. A dysregulated inflammatory response in the placenta plays a crucial role in the development of preeclampsia. Cordycepin, known for its anti-inflammatory and antioxidant properties, was the focus of this study, which aimed to investigate its effects on preeclampsia. Methods A preeclampsia-like rat model was established via tail vein injection of lipopolysaccharides (LPS) at a dose of 1 μg/kg in pregnant rats. These rats were then treated with cordycepin at doses of 5, 25, or 50 mg/kg from embryonic day 6 (E6) today 18 (E18). Systolic blood pressures and urinary protein levels were monitored, and pregnancy outcomes, such as fetal body length and weight, were measured. The expression of target genes or proteins was assessed by qPCR, ELISA, and Western blot. Results Our findings revealed that cordycepin significantly reduced systolic blood pressure and proteinuria in preeclampsia-like rats. Additionally, cordycepin improved pregnancy outcomes, as shown by increased fetal body length and weight. The treatment also lowered serum sFlt-1 levels, elevated PIGF levels, decreased placental pro-inflammatory cytokine levels (IL-1β, TNF-α, IL-6, MCP-1, and MIP-2), and raised levels of anti-inflammatory cytokine IL-10 level in preeclampsia-like rats. Furthermore, cordycepin helped restore macrophage population imbalances, increasing M1-type macrophage markers (iNOS, TNF-α, and IL-1β) and reducing M2-type macrophage markers (Arg 1, IL-10, and TGF-β). Conclusion This study suggests that cordycepin alleviates LPS-induced preeclampsia by reducing placental inflammation and correcting the M1/M2 macrophage imbalance, offering potential therapeutic benefits for managing preeclampsia.

High-efficiency expression of alginate lyase in Pichia pastoris facilitated by Vitreoscilla hemoglobin

Vitreoscilla hemoglobin (VHb) can enhance the ability of recombinant strains to express heterologous proteins under low-oxygen conditions. However, its mechanism of action in the Pichia pastoris expression system remains unclear. In this study, three VHb construction strategies were designed to elucidate the mechanisms by which VHb promotes heterologous protein expression in P. pastoris. Notably, the co-expression pattern involving the sequential expression of the 102C300C gene followed by the Vgb gene significantly improved enzyme activity in the recombinant strain X33-102C300C-Vgb. The enzyme activity was 203.4 ± 0.57 U/mL at 180 h of fermentation in the 5-L system, which was 20.7 % higher than that of the starting strain X33-102C300C. Fluorescent labeling experiments revealed for the first time that a dual-transcription unit approach achieved superior VHb expression, indicating its potential for further development. Furthermore, transcriptomic and metabolomic analyses demonstrated that VHb enhanced the growth of recombinant yeast colonies by improving respiration-related metabolism under low-oxygen conditions. This, in turn, alleviated the repression of the expression alcohol oxidase (AOX) at high methanol concentrations, resulting in increased alginate lyase activity. This study provides a theoretical foundation for improving the target protein expression in recombinant P. pastoris during high-density fermentation.

Genistein inhibited endocytosis and fibrogenesis in keloid via CTGF signaling pathways

BackgroundThis study aimed to evaluate soy isoflavones' effect and potential use—specifically genistein—in treating human keloid fibroblast cells (KFs) and in a keloid tissue culture model.MethodsTo investigate the effects of genistein on keloid, a wound-healing assay was performed to detect cell migration. Flow cytometry was used to measure apoptosis. Western blotting and immunofluorescence staining were performed to detect the expression of target proteins. KF tissues were isolated, cultured, and divided into the control, silenced connective tissue growth factor (CTGF) proteins, and shNC (negative control) groups.ResultsGenistein suppressed cell proliferation and migration, triggering the cell cycle at the G2/M phase and increasing the expression of p53 dose-dependent in keloids. Genistein inhibited the expression of COL1A1, FN, and CTGF mRNA and protein. Knockdown CTGF reduced the migrated ability in KFs. Genistein also abated TGF-β1-induced keloid fibrosis through the endocytosis model. Separated and cultured the keloid patient’s tissues decreased the cell migration ability by genistein treatment and was time-dose dependent.ConclusionsThis study indicated that genistein-induced p53 undergoes cell cycle arrest via the CTGF pathway-inhibited keloid cultured cells, and genistein suppressed the primary keloid cell migration, suggesting that our research provides a new strategy for developing drugs for treating keloids.

Establishing the green algae Chlamydomonas incerta as a platform for recombinant protein production.

Chlamydomonas incerta, a genetically close relative of the model green alga Chlamydomonas reinhardtii, shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii, this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta's capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii, C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii. For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii, as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta's potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.

Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines

The technology for obtaining the high-efficiency expression of target proteins through site-specific recombination has made progress. However, using the CRISPR/Cas9 system for site-specific integration of long fragments and the expression of active proteins remains a challenge. This study optimized the linear DNA circularization system, eliminated the prokaryotic plasmid backbone on the traditional foreign gene vector, and generated a homologous arm-free circular donor template with a single guide RNA target site (sgRNA TS). This strategy significantly increased the co-transfection efficiency of the 1.6 kb template and Cas9 plasmid by 1.15-fold, and the average knock-in (KI) efficiency of the 4.7 kb long-fragment template for the two target gene sites increased by 1.3-fold. Subsequently, we used rhBCHE as a reporter gene to efficiently integrate the 5.4 kb fragment containing the gene of interest (GOI) into specific sites in the HEK293T cell line to detect the expression of the circular template at different target sites. Overall, this study further verifies that the length of the circular donor is more conducive to non-homologous integration, and more importantly, we provide a simple and optimized strategy for the construction of long-fragment site integration cell lines.

In Vitro Experiment Present ROCK2 Inhibition Promotes the Therapeutic Effect of Bevacizumab in the Treatment of Glioblastoma Multiforme

Objective Gliomas are a general designation for neuroepithelial tumors derived from the glial cells of the central nervous system. According to the histopathological and immunohistochemical features, the World Health Organization classifies gliomas into four grades. Bevacizumab is a monoclonal antibody targeting vascular endothelial growth factor that has been approved for the treatment of glioblastoma multiforme (GBM) as a second-line therapy. However, its efficacy remains limited. Rho/Rho-associated kinase (ROCK) is a downstream molecule of small guanosine triphosphatases (GTPases) that regulates multiple cellular processes, including motility, migration, and proliferation. Thus, ROCK has been regarded as a therapeutic target for cardiovascular diseases, neurological diseases, immune diseases, and cancer, and ROCK inhibitors have high potential clinical value. Methods Viability rate of cells was detected using MTT assay, and apoptosis of cells was detected using FACS. Expression of target genes and proteins was detected using qPCR and western blotting analysis. Concentration of cytokines was detected using ELISA methods. Results Viability and migration of GBM cells were reduced after bevacizumab treatment and that these effects were enhanced by ROCK2 inhibition. We further found that ROCK2 inhibition promoting the effect of bevacizumab was mainly mediated by the RhoA/ROCK2 pathway, further inducing apoptosis in GBM cells. In addition, we found that angiogenesis and degradation of cellular matrix-related cytokines were reduced by ROCK2 inhibition. Conclusions ROCK2 inhibition contributes to the therapeutic effects of bevacizumab.

Level of expression of MHCI-presented neoepitopes influences tumor rejection by neoantigen-specific CD8+ T cells.

Neoantigen-targeted therapy holds an array of benefits for cancer immunotherapy, but the identification of peptide targets with tumor rejection capacity remains a limitation. To better define the criteria dictating tumor rejection potential, we examined the capacity of high-magnitude T cell responses induced towards several distinct neoantigen targets to regress MC38 tumors. Surprisingly, despite their demonstrated immunogenicity, vaccine-induced T-cell responses were unable to regress established MC38 tumors or prevent tumor engraftment in a prophylactic setting. However, T cells were functional with robust killing capacity towards neoantigen peptide-loaded cells. Furthermore, tumor cell killing was rescued in proportion to the expression level or saturation of target peptide-loaded MHCs on the cell surface. Overall, this study demonstrates a pivotal role for target protein expression levels in modulating the tumor rejection capacity of neoantigens. Thus, inclusion of this metric, in addition to immunogenicity analysis, may benefit antigen prediction techniques to ensure the full anti-tumor effect of cancer vaccines.

Long noncoding RNA MEG3: an active player in fibrosis.

Fibrosis, characterized by excess accumulation of extracellular matrix components, disrupts normal tissue structure and causes organ dysfunction. Long noncoding RNAs (lncRNAs) are a subset of RNAs longer than 200 nucleotides that are not converted into proteins. The increasing research indicated that lncRNA maternally expressed gene 3 (MEG3) was dysregulated in the pathologic process of fibrosis in several tissues. LncRNA MEG3 was revealed to regulate the expression of target proteins or serve as a miRNAs sponge to control the development of fibrosis, which was involved in NF-ҡB, PI3K/AKT, JAK2/STAT3, Wnt/β-catenin, ERK/p38, and Hh pathway. Importantly, the interference of MEG3 level ameliorated fibrosis. The present review summarized available studies of lncRNA MEG3 in fibrosis, which is helpful for a deeper understanding of the roles of MEG3 in fibrosis.

Effects of BRCA1 overexpression via the NRF2 / HO1 / NQO1 pathway on oral cancer cells proliferation, migration, and apoptosis

ObjectiveHerein, we explored the influences of breast cancer susceptibility gene 1 (BRCA1) overexpression on oral cancer cells proliferation, migration, and apoptosis via evaluation of its interactions with nuclear factor erythroid 2-like 2 (NRF2). DesignCAL-27 and DOK cells were transfected with a BRCA1 overexpressing lentivirus. Next, we utilized Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses to evaluate BRCA1, NRF2, and their target gene expressions. Using cell counting kit-8 (CCK-8) assessment, we assessed cell proliferation and a scratch test detected CAL-27 cell migration. Additionally, flow cytometry was employed used to examine cell apoptosis, while an enzyme-linked immunosorbent assa (ELISA) was employed for evaluation of 8-hydroxy-2′- deoxyguanosine (8-OHdG) expression. An immunohistochemical analysis was employed to determine the NRF2 target genes and Ki-67 expressions. ResultsBRCA1 overexpression increased the NRF2 and its target gene transcript and protein expressions. CCK-8 and scratch test results showed that BRCA1 overexpression decreased cell proliferation and weakened CAL-27 cell migratory ability. Flow cytometry results showed that BRCA1 overexpression promoted cell apoptosis in a time-dependent manner, while enzyme-linked immunosorbent assay results showed that BRCA1 overexpression decreased 8-OHdG expression levels in CAL-27 and DOK cells. Immunohistochemical analysis results showed higher expression of NRF2 target genes and Ki-67 in oral squamous cell carcinoma cells. ConclusionsExperiments involving oral cancer cells confirmed that BRCA1 overexpression could up-regulate the NRF2 signalling pathway, reduce oxidative damage, and inhibit cell proliferation and other biological behaviours. The BRCA1 and NRF2 pathways might be associated with oral cancer occurrence and development.

An in vivo tumour organoid model based on the chick embryonic chorioallantoic membrane mimics key characteristics of the patient tissue: a proof-of-concept study

BackgroundPatient-derived tumour organoids (PDOs) are highly advanced in vitro models for disease modelling, yet they lack vascularisation. To overcome this shortcoming, organoids can be inoculated onto the chorioallantoic membrane (CAM); the highly vascularised, not innervated extraembryonic membrane of fertilised chicken eggs. Therefore, we aimed to (1) establish a CAM patient-derived xenograft (PDX) model based on PDOs generated from the liver metastasis of a colorectal cancer (CRC) patient and (2) to evaluate the translational pipeline (patient – in vitro PDOs – in vivo CAM-PDX) regarding morphology, histopathology, expression of C-X-C chemokine receptor type 4 (CXCR4), and radiotracer uptake patterns.ResultsThe main liver metastasis of the CRC patient exhibited high 2-[18F]FDG uptake and moderate and focal [68Ga]Ga-Pentixafor accumulation in the peripheral part of the metastasis. Inoculation of PDOs derived from this region onto the CAM resulted in large, highly viable, and extensively vascularised xenografts, as demonstrated immunohistochemically and confirmed by high 2-[18F]FDG uptake. The xenografts showed striking histomorphological similarity to the patient’s liver metastasis. The moderate expression of CXCR4 was maintained in ovo and was concordant with the expression levels of the patient’s sample and in vitro PDOs. Following in vitro re-culturing of CAM-PDXs, growth, and [68Ga]Ga-Pentixafor uptake were unaltered compared to PDOs before transplantation onto the CAM. Although [68Ga]Ga-Pentixafor was taken up into CAM-PDXs, the uptake in the baseline and blocking group were comparable and there was only a trend towards blocking.ConclusionsWe successfully established an in vivo CAM-PDX model based on CRC PDOs. The histomorphological features and target protein expression of the original patient’s tissue were mirrored in the in vitro PDOs, and particularly in the in vivo CAM-PDXs. The [68Ga]Ga-Pentixafor uptake patterns were comparable between in vitro, in ovo and clinical data and 2-[18F]FDG was avidly taken up in the patient’s liver metastasis and CAM-PDXs. We thus propose the CAM-PDX model as an alternative in vivo model with promising translational value for CRC patients.Graphical

The identification of key molecules and pathways in the crosstalk of calcium oxalate-treated TCMK-1 cells and macrophage via exosomes

The interplay between crystals and epithelial cells forms the cornerstone of kidney stone development, communication between epithelial cells and macrophages emerging as a pivotal role in this process. We conducted next-generation sequencing on the secreted exosomes of TCMK-1 cells treated with calcium oxalate monohydrate (OX_EXO) or controls (NC_EXO), and on the macrophage cell line RAW264.7 stimulated with OX_EXO or NC_EXO, followed by validation of differentially expressed target proteins and miRNAs through Western blot and PCR. UPSET plots were employed to identify genes co-targeted by exosomal miRNAs. Various bioinformatic analyses were employed to predict potential mechanisms of the dysregulated genes. We integrated sequencing data from the GEO database, and validated findings using clinical patient urine and kidney tissues. We identified 665 differentially expressed exosomal miRNAs between OX_EXO and NC_EXO. Among the top 10 down-regulated miRNAs, the most targeted genes were AAK1 and NUFIP2, whereas PLCB1 was significantly targeted among the top 10 up-regulated miRNAs. In clinical specimens, we confirmed the differential expressions of five homologous miRNAs, as well as CNOT3, CNCNA1C, APEX1, and TMEM199. In conclusion, treatment of TCMK-1 cells with calcium oxalate significantly alerted the expression profile of exosomal miRNAs, subsequently influencing gene expression in macrophages, thereby modulating the processes of kidney stone formation.

Identification of novel pQTL-SNPs associated with lung adenocarcinoma risk: A multi-stage study.

To explore the association between protein quantitative trait loci (pQTL-SNPs) and the risk of LUAD. "Blood +" high depth blood proteomics analysis was performed on plasma from female LUAD patients and female healthy controls, and combined with proteomics data from tumors and adjacent non-tumor tissues of female LUAD patients to screen proteins uniformly expressed in plasma and tissues. pQTL-SNPs were then screened through multiple databases and subjected to multilevel screening. The associations between selected pQTL-SNPs and LUAD risk were evaluated by Female Lung Cancer Consortium in Asia GWAS (FLCCA GWAS). Enzyme linked immunosorbent assay (ELISA) is used to determine the levels of candidate protein. A total of 7 pQTL-SNPs were significantly associated with altered LUAD risk (p < 0.05). Meanwhile, the expression of their corresponding target proteins were all decreased in both plasma and tumor tissues of LUAD cases, which may play a role of tumor suppressor proteins. After mutation of 3 pQTL-SNPs (rs7683000, rs73224660, and rs2776937), the expression of corresponding target proteins BST1 and NRP1 decreased, and as potential tumor suppressor proteins, which may promote tumorigenesis and further increasing the risk of developing LUAD (OR >1, p < 0.05); while after mutation the other pQTL-SNP rs62069916, the corresponding target protein APOH expression was increased, while as a potential tumor suppressor protein, which may inhibit tumorigenesis and further reduced the risk of developing LUAD (OR <1, p < 0.05). In addition, the expression of NRP1 and APOH were significant decreased in LUAD cell lines and validated in plasma of LUAD patients. A total of 4 pQTL-SNPs (rs7683000, rs73224660, rs2776937, and rs62069916) may associate with altered LUAD risk by regulating the expression of target proteins (BST1, NRP1, and APOH) after mutation.

Neuroinflammation and Neurometabolomic Profiling in Fentanyl Overdose Mouse Model Treated with Novel β-Lactam, MC-100093, and Ceftriaxone.

Opioid-related deaths are attributed to overdoses, and fentanyl overdose has been on the rise in many parts of the world, including the USA. Glutamate transporter 1 (GLT-1) has been identified as a therapeutic target in several preclinical models of substance use disorders, and β-lactams effectively enhance its expression and function. In the current study, we characterized the metabolomic profile of the nucleus accumbens (NAc) in fentanyl-overdose mouse models, and we evaluated the protective effects of the functional enhancement of GLT-1 using β-lactams, ceftriaxone, and MC-100093. BALB/c mice were divided into four groups: control, fentanyl, fentanyl/ceftriaxone, and fentanyl/MC-100093. While the control group was intraperitoneally (i.p.) injected with normal saline simultaneously with other groups, all fentanyl groups were i.p. injected with 1 mg/kg of fentanyl as an overdose after habituation with four repetitive non-consecutive moderate doses (0.05 mg/kg) of fentanyl for a period of seven days. MC-100093 (50 mg/kg) and ceftriaxone (200 mg/kg) were i.p. injected from days 5 to 9. Gas chromatography-mass spectrometry (GC-MS) was used for metabolomics, and Western blotting was performed to determine the expression of target proteins. Y-maze spontaneous alternation performance and the open field activity monitoring system were used to measure behavioral manifestations. Fentanyl overdose altered the abundance of about 30 metabolites, reduced the expression of GLT-1, and induced the expression of inflammatory mediators IL-6 and TLR-4 in the NAc. MC-100093 and ceftriaxone attenuated the effects of fentanyl-induced downregulation of GLT-1 and upregulation of IL-6; however, only ceftriaxone attenuated fentanyl-induced upregulation of TRL4 expression. Both of the β-lactams attenuated the effects of fentanyl overdose on locomotor activities but did not induce significant changes in the overall metabolomic profile. Our findings revealed that the exposure to a high dose of fentanyl causes alterations in key metabolic pathways in the NAc. Pretreatment with ceftriaxone and MC-100093 normalized fentanyl-induced downregulation of GLT-1 expression with subsequent attenuation of neuroinflammation as well as the hyperactivity, indicating that β-lactams may be promising drugs for treating fentanyl use disorder.