A Pyroglutamate Aminopeptidase 1 Responsive Fluorescence Imaging Probe for Real-Time Rapid Differentiation between Thyroiditis and Thyroid Cancer.

Current diagnostic methods for thyroid diseases, including blood tests, ultrasound, and biopsy, always have difficulty diagnosing thyroiditis accurately, occasionally mistaking it for thyroid cancer. To address this clinical challenge, we developed Ox-PGP1, a novel fluorescent probe realizing rapid, noninvasive, and real-time diagnostic techniques. This is the first imaging tool capable of noninvasively distinguishing between thyroiditis and thyroid cancer. Ox-PGP1 was introduced as a fluorescent probe custom-built for the specific detection and quantification of pyroglutamate aminopeptidase 1 (PGP-1), a known pivotal biomarker of inflammation. Ox-PGP1 overcame the disadvantages of traditional enzyme-responsive fluorescent probes that relied on the intramolecular charge transfer (ICT) mechanism, including the issue of high background fluorescence, while offering exceptional photostability under laser irradiation. The spectral properties of Ox-PGP1 were meticulously optimized to enhance its biocompatibility. Furthermore, the low limit of detection (LOD) of Ox-PGP1 was determined to be 0.09 µg/mL, which demonstrated its remarkable sensitivity and precision. Both cellular and in vivo experiments validated the capacity of Ox-PGP1 for accurate differentiation between normal, inflammatory, and cancerous thyroid cells. Furthermore, Ox-PGP1 showed the potential to rapidly and sensitively differentiate between autoimmune thyroiditis and anaplastic thyroid carcinoma in a mouse model, achieving results in just 5 min. The successful design and application of Ox-PGP1 represent a substantial advancement in technology over traditional diagnostic approaches, potentially enabling earlier interventions for thyroid diseases.

Btbd8 deficiency reduces susceptibility to colitis by enhancing intestinal barrier function and suppressing inflammation.

Introduction: BTBD8 has been identified as a susceptible gene for inflammatory bowel diseases (IBD). However, the function of BTBD8 in normal development and IBD pathogenesis remains unknown. Methods: We administered drinking water with 3% dextran sodium sulfate (DSS) to wild-type (WT) and Btbd8 knockout (KO) mice for seven consecutive days to induce IBD. Subsequently, we further examined whether Btbd8 KO affects intestinal barrier and inflammation. Results: We demonstrated that Btbd8 deficiency partially protects mice from DSS-induced IBD, even though no obvious phenotypes were observed in Btbd8 KO mice. Btbd8 deletion leads to strengthened tight junctions between intestinal epithelial cells, elevated intestinal stem cell activity, and enhanced mucus layer. All these three mechanisms work together to improve the intestinal barrier integrity in Btbd8 KO mice. In addition, Btbd8 deficiency mitigates inflammation by reducing the expression of IL-1ß and IL-6 by macrophages. Discussion: Our studies validate the crucial role of Btbd8 in IBD pathogenesis, and reveal that Btbd8 deficiency may ameliorate DSS-induced IBD through improving the intestinal barrier integrity, as well as suppressing inflammatory response mediated by macrophages. These findings suggest that Btbd8 could be a promising therapeutic target for the treatment of IBD.

Disulfiram Reduces Atherosclerosis and Enhances Efferocytosis, Autophagy, and Atheroprotective Gut Microbiota in Hyperlipidemic Mice.

BACKGROUND: Pyroptosis executor GsdmD (gasdermin D) promotes atherosclerosis in mice and humans. Disulfiram was recently shown to potently inhibit GsdmD, but the in vivo efficacy and mechanism of disulfiram's antiatherosclerotic activity is yet to be explored. METHODS AND RESULTS: We used human/mouse macrophages, endothelial cells, and smooth muscle cells and a hyperlipidemic mouse model of atherosclerosis to determine disulfiram antiatherosclerotic efficacy and mechanism. The effects of disulfiram on several atheroprotective pathways such as autophagy, efferocytosis, phagocytosis, and gut microbiota were determined. Atomic force microscopy was used to determine the effects of disulfiram on the biophysical properties of the plasma membrane of macrophages. Disulfiram-fed hyperlipidemic apolipoprotein E-/- mice showed significantly reduced interleukin-1ß release upon in vivo Nlrp3 (NLR family pyrin domain containing 3) inflammasome activation. Disulfiram-fed mice showed smaller atherosclerotic lesions (~27% and 29% reduction in males and females, respectively) and necrotic core areas (~50% and 46% reduction in males and females, respectively). Disulfiram induced autophagy in macrophages, smooth muscle cells, endothelial cells, hepatocytes/liver, and atherosclerotic plaques. Disulfiram modulated other atheroprotective pathways (eg, efferocytosis, phagocytosis) and gut microbiota. Disulfiram-treated macrophages showed enhanced phagocytosis/efferocytosis, with the mechanism being a marked increase in cell-surface expression of efferocytic receptor MerTK. Atomic force microscopy analysis revealed altered biophysical properties of disulfiram-treated macrophages, showing increased order-state of plasma membrane and increased adhesion strength. Furthermore, 16sRNA sequencing of disulfiram-fed hyperlipidemic mice showed highly significant enrichment in atheroprotective gut microbiota Akkermansia and a reduction in atherogenic Romboutsia species. CONCLUSIONS: Taken together, our data show that disulfiram can simultaneously modulate several atheroprotective pathways in a GsdmD-dependent as well as GsdmD-independent manner.

Extracranial Vascular Anomalies Driven by RAS/MAPK Variants: Spectrum and Genotype-Phenotype Correlations.

BACKGROUND: We aimed to correlate alterations in the rat sarcoma virus (RAS)/mitogen-activated protein kinase pathway in vascular anomalies to the clinical phenotype for improved patient and treatment stratification. METHODS AND RESULTS: This retrospective multicenter cohort study included 29 patients with extracranial vascular anomalies containing mosaic pathogenic variants (PVs) in genes of the RAS/mitogen-activated protein kinase pathway. Tissue samples were collected during invasive treatment or clinically indicated biopsies. PVs were detected by the targeted sequencing of panels of genes known to be associated with vascular anomalies, performed using DNA from affected tissue. Subgroup analyses were performed according to the affected genes with regard to phenotypic characteristics in a descriptive manner. Twenty-five vascular malformations, 3 vascular tumors, and 1 patient with both a vascular malformation and vascular tumor presented the following distribution of PVs in genes: Kirsten rat sarcoma viral oncogene (n=10), neuroblastoma ras viral oncogene homolog (n=1), Harvey rat sarcoma viral oncogene homolog (n=5), V-Raf murine sarcoma viral oncogene homolog B (n=8), and mitogen-activated protein kinase kinase 1 (n=5). Patients with RAS PVs had advanced disease stages according to the Schobinger classification (stage 3-4: RAS, 9/13 versus non-RAS, 3/11) and more frequent progression after treatment (RAS, 10/13 versus non-RAS, 2/11). Lesions with Kirsten rat sarcoma viral oncogene PVs infiltrated more tissue layers compared with the other PVs including other RAS PVs (multiple tissue layers: Kirsten rat sarcoma viral oncogene, 8/10 versus other PVs, 6/19). CONCLUSIONS: This comparison of patients with various PVs in genes of the RAS/MAPK pathway provides potential associations with certain morphological and clinical phenotypes. RAS variants were associated with more aggressive phenotypes, generating preliminary data and hypothesis for future larger studies.

Electrochemical Impedimetric Platform Based on Con A@MIL-101 for Glycoprotein Detection.

An electrochemical impedimetric biosensing platform with lectin as a molecular recognition element has been established for the sensitive detection of glycoproteins, a class of important biomarkers in clinical diagnosis. One of the representative metal-organic framework materials, MIL-101(Cr)-NH2, was utilized as the supporting matrix, and its amino groups served as the anchors to immobilize the lectins of concanavalin A (Con A), constituting Con A@MIL-101(Cr)-NH2 for the determination of invertase (INV) as a model glycoprotein. The Con A concentration, immobilization time, and incubation time with INV were optimized. Under the optimal conditions, the degree of impedance increase was linearly proportional to the logarithm of INV concentration between 1.0 × 10-16 and 1.0 × 10-11 M, affording a limit of detection as low as 3.98 × 10-18 M. Good specificity, stability, reproducibility, and repeatability were demonstrated for the fabricated biosensing platform. Moreover, real mouse serum samples were spiked with different concentrations of INV. Excellent recoveries were obtained, which demonstrated the biosensing platform's capability of analyzing glycoproteins within a complex matrix.

Iron Oxide Nanoparticles Inhibit Tumor Progression and Suppress Lung Metastases in Mouse Models of Breast Cancer.

Systemic exposure to starch-coated iron oxide nanoparticles (IONPs) can stimulate antitumor T cell responses, even when little IONP is retained within the tumor. Here, we demonstrate in mouse models of metastatic breast cancer that IONPs can alter the host immune landscape, leading to systemic immune-mediated disease suppression. We report that a single intravenous injection of IONPs can inhibit primary tumor growth, suppress metastases, and extend survival. Gene expression analysis revealed the activation of Toll-like receptor (TLR) pathways involving signaling via Toll/Interleukin-1 receptor domain-containing adaptor-inducing IFN-ß (TRIF), a TLR pathway adaptor protein. Requisite participation of TRIF in suppressing tumor progression was demonstrated with histopathologic evidence of upregulated IFN-regulatory factor 3 (IRF3), a downstream protein, and confirmed in a TRIF knockout syngeneic mouse model of metastatic breast cancer. Neither starch-coated polystyrene nanoparticles lacking iron, nor iron-containing dextran-coated parenteral iron replacement agent, induced significant antitumor effects, suggesting a dependence on the type of IONP formulation. Analysis of multiple independent clinical databases supports a hypothesis that upregulation of TLR3 and IRF3 correlates with increased overall survival among breast cancer patients. Taken together, these data support a compelling rationale to re-examine IONP formulations as harboring anticancer immune (nano)adjuvant properties to generate a therapeutic benefit without requiring uptake by cancer cells.

Positive allosteric modulators of the GABAB receptor: a new class of ligands with therapeutic potential for alcohol use disorder.

BACKGROUND: Positive allosteric modulators (PAMs) of the GABAB receptor constitute a new class of GABAB-receptor ligands. GABAB PAMs reproduce several pharmacological effects of the orthosteric GABAB receptor agonist, baclofen, although displaying a better safety profile. AIMS: This paper reviews the reducing or, frequently, even suppressing effects of all GABAB PAMs tested to date on multiple alcohol-related behaviours in laboratory rodents exposed to validated experimental models of human alcohol use disorder. RESULTS: Acute or repeated treatment with CGP7930, GS39783, BHF177, rac-BHFF, ADX71441, CMPPE, COR659, ASP8062, KK-92A, and ORM-27669 reduced excessive alcohol drinking, relapse- and binge-like drinking, operant alcohol self-administration, reinstatement of alcohol seeking, and alcohol-induced conditioned place preference in rats and mice. CONCLUSIONS: These effects closely mirrored those of baclofen; notably, they were associated to remarkably lower levels of tolerance and toxicity. The recent transition of ASP8062 to clinical testing will soon prove whether these highly consistent preclinical data translate to AUD patients.

Self-Assembled STING-Activating Coordination Nanoparticles for Cancer Immunotherapy and Vaccine Applications.

The cGAS-STING pathway plays a crucial role in innate immune activation against cancer and infections, and STING agonists based on cyclic dinucleotides (CDN) have garnered attention for their potential use in cancer immunotherapy and vaccines. However, the limited drug-like properties of CDN necessitate an efficient delivery system to the immune system. To address these challenges, we developed an immunostimulatory delivery system for STING agonists. Here, we have examined aqueous coordination interactions between CDN and metal ions and report that CDN mixed with Zn2+ and Mn2+ formed distinctive crystal structures. Further pharmaceutical engineering led to the development of a functional coordination nanoparticle, termed the Zinc-Mn-CDN Particle (ZMCP), produced by a simple aqueous one-pot synthesis. Local or systemic administration of ZMCP exerted robust antitumor efficacy in mice. Importantly, recombinant protein antigens from SARS-CoV-2 can be simply loaded during the aqueous one-pot synthesis. The resulting ZMCP antigens elicited strong cellular and humoral immune responses that neutralized SARS-CoV-2, highlighting ZMCP as a self-adjuvant vaccine platform against COVID-19 and other infectious pathogens. Overall, this work establishes a paradigm for developing translational coordination nanomedicine based on drug-metal ion coordination and broadens the applicability of coordination medicine for the delivery of proteins and other biologics.

Charting the cellular biogeography in colitis reveals fibroblast trajectories and coordinated spatial remodeling.

Gut inflammation involves contributions from immune and non-immune cells, whose interactions are shaped by the spatial organization of the healthy gut and its remodeling during inflammation. The crosstalk between fibroblasts and immune cells is an important axis in this process, but our understanding has been challenged by incomplete cell-type definition and biogeography. To address this challenge, we used multiplexed error-robust fluorescence in situ hybridization (MERFISH) to profile the expression of 940 genes in 1.35 million cells imaged across the onset and recovery from a mouse colitis model. We identified diverse cell populations, charted their spatial organization, and revealed their polarization or recruitment in inflammation. We found a staged progression of inflammation-associated tissue neighborhoods defined, in part, by multiple inflammation-associated fibroblasts, with unique expression profiles, spatial localization, cell-cell interactions, and healthy fibroblast origins. Similar signatures in ulcerative colitis suggest conserved human processes. Broadly, we provide a framework for understanding inflammation-induced remodeling in the gut and other tissues.

Suppression of the growth and metastasis of mouse melanoma by Taenia crassiceps and Mesocestoides corti tapeworms.

Cancer is still one of the leading causes of death, with an estimated 19.3 million new cases every year. Our paper presents the tumor-suppressing effect of Taenia crassiceps and Mesocestoides corti on B16F10 melanoma, the intraperitoneal application of which followed the experimental infection with these tapeworms, resulting in varying degrees of effectiveness in two strains of mice. In the case of M. corti-infected ICR mice, a strong tumor growth suppression occurred, which was accompanied by a significant reduction in the formation of distant metastases in the liver and lung. Tapeworm-infected C57BL/6J mice also showed a suppression of tumor growth and, in addition, the overall survival of infected C57BL/6J mice was significantly improved. Experiments with potential cross-reaction of melanoma and tapeworm antigens with respective specific antibodies, restimulation of spleen T cells, or the direct effect of tapeworm excretory-secretory products on melanoma cells in vitro could not explain the phenomenon. However, infections with T. crassiceps and M. corti increased the number of leukocytes possibly involved in anti-tumor immunity in the peritoneal cavity of both ICR and C57BL/6J mice. This study unveils the complex interplay between tapeworm infections, immune responses, and melanoma progression, emphasizing the need for further exploration of the mechanisms driving observed tumor-suppressive effects.

Preparation of PLCL/ECM nerve conduits by electrostatic spinning technique and evaluationin vitroandin vivo.

Objective. Artificial nerve scaffolds composed of polymers have attracted great attention as an alternative for autologous nerve grafts recently. Due to their poor bioactivity, satisfactory nerve repair could not be achieved. To solve this problem, we introduced extracellular matrix (ECM) to optimize the materials.Approach.In this study, the ECM extracted from porcine nerves was mixed with Poly(L-Lactide-co-ϵ-caprolactone) (PLCL), and the innovative PLCL/ECM nerve repair conduits were prepared by electrostatic spinning technology. The novel conduits were characterized by scanning electron microscopy (SEM), tensile properties, and suture retention strength test for micromorphology and mechanical strength. The biosafety and biocompatibility of PLCL/ECM nerve conduits were evaluated by cytotoxicity assay with Mouse fibroblast cells and cell adhesion assay with RSC 96 cells, and the effects of PLCL/ECM nerve conduits on the gene expression in Schwann cells was analyzed by real-time polymerase chain reaction (RT-PCR). Moreover, a 10 mm rat (Male Wistar rat) sciatic defect was bridged with a PLCL/ECM nerve conduit, and nerve regeneration was evaluated by walking track, mid-shank circumference, electrophysiology, and histomorphology analyses.Main results.The results showed that PLCL/ECM conduits have similar microstructure and mechanical strength compared with PLCL conduits. The cytotoxicity assay demonstrates better biosafety and biocompatibility of PLCL/ECM nerve conduits. And the cell adhesion assay further verifies that the addition of ECM is more beneficial to cell adhesion and proliferation. RT-PCR showed that the PLCL/ECM nerve conduit was more favorable to the gene expression of functional proteins of Schwann cells. Thein vivoresults indicated that PLCL/ECM nerve conduits possess excellent biocompatibility and exhibit a superior capacity to promote peripheral nerve repair.Significance.The addition of ECM significantly improved the biocompatibility and bioactivity of PLCL, while the PLCL/ECM nerve conduit gained the appropriate mechanical strength from PLCL, which has great potential for clinical repair of peripheral nerve injuries.

PSMA2 promotes glioma proliferation and migration via EMT.

BACKGROUND: Gliomas advance rapidly and are associated with a poor prognosis. Epithelial-mesenchymal transition (EMT) accelerates the progression of gliomas, exerting a pivotal role in glioma development. Proteasome subunit alpha type-2 (PSMA2) exhibits high expression levels in gliomas. however, its specific involvement in glioma progression and its correlation with EMT remain elusive. This study aims to elucidate the role of PSMA2 in glioma progression and its potential association with EMT. METHODS: Online tools were employed to analyze the expression patterns and survival curves of PSMA2 in gliomas. The relationship between PSMA2 and various characteristics of glioma patients was investigated using data from the TCGA and CGGA databases. In vitro, cell proliferation and migration were assessed through CCK-8, colony formation, and transwell assays. Furthermore, a tumor xenograft model in nude mice was established to evaluate in vivo tumorigenesis. Protein binding to PSMA2 was scrutinized using co-immunoprecipitation MS (co-IP MS). The potential biological functions and molecular pathways associated with PSMA2 were explored through GO analysis and KEGG analysis, and the correlation between PSMA2 and EMT was validated through correlation analysis and Western blot experiments. RESULTS: Bioinformatics analysis revealed a significant upregulation of PSMA2 across various cancers, with particularly heightened expression in gliomas. Moreover, elevated PSMA2 levels were correlated with advanced tumor stages and diminished survival rates among glioma patients. Inhibition of PSMA2 demonstrated a pronounced suppressive effect on glioma cell proliferation, both in vitro and in vivo. Knockdown of PSMA2 also impeded the migratory capacity of glioma cells. GO and KEGG enrichment analyses indicated that PSMA2-binding proteins (identified through Co-IP-MS) were associated with cell adhesion molecule binding and cadherin binding. Western blot results further confirmed the role of PSMA2 in promoting epithelial-mesenchymal transition (EMT) in glioma cells. CONCLUSION: Our study provides evidence supporting the role of PSMA2 as a regulatory factor in EMT and suggests its potential as a prognostic biomarker for glioma progression.

Mycobacterium bovis BCG as immunostimulating agent prevents the severe form of chronic experimental Chagas disease.

Introduction: There is currently no vaccine against Chagas disease (ChD), and the medications available confer multiple side effects. Mycobacterium bovis Bacillus Calmette-Guérin (BCG) produces balanced Th1, Th2, and Th17 modulatory immune responses and has improved efficacy in controlling chronic infections through nonspecific immunity. We aimed to improve the response to infection by inducing a stronger immune response and greater protection against the parasite by trained immunity. Methods: BALB/c mice were immunized with BCG subcutaneously, and 60 days later, they were infected with Trypanosoma cruzi intraperitoneally. An evaluation of the progression of the disease from the acute to the chronic stage, analyzing various aspects such as parasitemia, survival, clinical status, and humoral and cellular immune response, as well as the appearance of visceral megas and the histopathological description of target organs, was performed. Results: Vaccination reduced parasitemia by 70%, and 100% survival was achieved in the acute stage; although the presentation of clinical signs was reduced, there was no increase in the antibody titer or in the differential production of the isotypes. Conclusion: Serum cytokine production indicated a proinflammatory response in infected animals, while in those who received BCG, the response was balanced by inducing Th1/Th2-type cytokines, with a better prognosis of the disease in the chronic stage.

Compound 225# inhibits the proliferation of human colorectal cancer cells by promoting cell cycle arrest and apoptosis induction.

Colorectal cancer (CRC) ranks as the second leading cause of cancer­related death worldwide due to its aggressive nature. After surgical resection, >50% of patients with CRC require adjuvant therapy. As a result, eradicating cancer cells with medications is a promising method to treat patients with CRC. In the present study, a novel compound was synthesized, which was termed compound 225#. The inhibitory activity of compound 225# against CRC was determined by MTT assay, EdU fluorescence labeling and colony formation assay; the effects of compound 225# on the cell cycle progression and apoptosis of CRC cells were detected by flow cytometry and western blotting; and the changes in autophagic flux after the administration of compound 225# were detected using the double fluorescence fusion protein mCherry­GFP­LC3B and western blotting. The results demonstrated that compound 225# exhibited antiproliferative properties, inhibiting the proliferation and expansion of CRC cell lines in a time­ and dose­dependent manner. Furthermore, compound 225# triggered G2/M cell cycle arrest by influencing the expression of cell cycle regulators, such as CDK1, cyclin A1 and cyclin B1, which is also closely related to the activation of DNA damage pathways. The cleavage of PARP and increased protein expression levels of PUMA suggested that apoptosis was triggered after treatment with compound 225#. Moreover, the increase in LC3­II expression and stimulation of autophagic flux indicated the activation of an autophagy pathway. Notably, compound 225# induced autophagy, which was associated with endoplasmic reticulum (ER) stress. In accordance with the in vitro findings, the in vivo results demonstrated that compound 225# effectively inhibited the growth of HCT116 tumors in mice without causing any changes in their body weight. Collectively, the present results demonstrated that compound 225# not only inhibited proliferation and promoted G2/M­phase cell cycle arrest and apoptosis, but also initiated cytoprotective autophagy in CRC cells by activating ER stress pathways. Taken together, these findings provide an experimental basis for the evaluation of compound 225# as a novel potential medication for CRC treatment.

Pellino1 orchestrates gut-kidney axis to perpetuate septic acute kidney injury through activation of STING pathway and NLRP3 inflammasome.

AIMS: Intestinal barrier dysfunction is the initial and propagable factor of sepsis in which acute kidney injury (AKI) has been considered as a common life-threatening complication. Our recent study identifies the regulatory role of Pellino1 in tubular death under inflammatory conditions in vitro. The objective of our current study is to explore the impact of Pellino1 on gut-kidney axis during septic AKI and uncover the molecular mechanism (s) underlying this process. MATERIALS AND METHODS: Immunohistochemistry (IHC) was conducted to evaluate Pellino1 and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) levels in renal biopsies from critically ill patients with a clinical diagnosis of sepsis. Functional and mechanistic studies were characterized in septic models of the Peli-knockout (Peli1-/-) mice by histopathological staining, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunofluorescence, biochemical detection, CRISPR/Cas9-mediated gene editing and intestinal organoid. KEY FINDINGS: Pellino1, together with NLRP3, are highly expressed in renal biopsies from critically ill patients diagnosed with sepsis and kidney tissues of septic mice. The Peli1-/- mice with sepsis become less prone to develop AKI and have markedly compromised NLRP3 activation in kidney. Loss of Peli1 endows septic mice refractory to intestinal inflammation, barrier permeability and enterocyte apoptosis that requires stimulator of interferons genes (STING) pathway. Administration of STING agonist DMXAA deteriorates AKI and mortality of septic Peli1-/- mice in the presence of kidney-specific NLRP3 reconstitution. SIGNIFICANCE: Our studies suggest that Pellino1 has a principal role in orchestrating gut homeostasis towards renal pathophysiology, thus providing a potential therapeutic target for septic AKI.

Small extracellular vesicles derived from lipopolysaccharide-preconditioned dental follicle cells inhibit cell apoptosis and alveolar bone loss in periodontitis.

OBJECTIVE: This study aimed to explore the effects of small extracellular vesicles derived from lipopolysaccharide-preconditioned dental follicle cells (L-D-sEV) on periodontal ligament cells from periodontitis affected teeth (p-PDLCs) in vitro and experimental periodontitis in mice. DESIGN: In vitro, the biological function of p-PDLCs and the underlying molecular mechanism were investigated by flow cytometry, Western blot, and quantitative real-time PCR (qRT-PCR) analysis. Eighteen-eight-week-old male C57BL/6 mice were randomly divided into three groups: control (Con), periodontitis (Peri), and L-D-sEV groups. Mice periodontitis model was induced by placing the 5-0 silk thread (around the maxillary second molar) and P.gingivalis (1 ×107 CFUs per mouse). In vivo, the alveolar bone loss, osteoclast activity, and macrophage polarization were measured by micro-computed tomography and histological analysis. RESULTS: In vitro, the RANKL/OPG ratio and phosphorylation of JNK and P38 protein levels of p-PDLCs were significantly decreased after L-D-sEV administration. Besides, flow cytometry and qRT-PCR analysis showed that L-D-sEV reduced apoptosis of p-PDLCs, down-regulated apoptosis-related genes Caspase-3 and BCL-2-Associated X expression, and up-regulated B-cell lymphoma-2 gene levels. In vivo, L-D-sEV administration significantly reduced alveolar bone loss, inhibited osteoclast activity, and induced M2 polarization. The histological analysis showed that iNOS/CD206, RANKL/OPG, p-JNK/JNK, and p-P38/P38 ratios were significantly lower in the L-D-sEV group than in the Peri group. CONCLUSIONS: L-D-sEV administration alleviated alveolar bone loss by mediating RANKL/OPG-related osteoclast activity and M2 macrophage polarization, alleviating p-PDLCs apoptosis and proliferation via the JNK and P38 pathways.

Endothelial DR6 in blood-brain barrier malfunction in Alzheimer's disease.

The impairment of the blood-brain barrier (BBB) has been increasingly recognised as a critical element in the early pathogenesis of Alzheimer's disease (AD), prompting a focus on brain endothelial cells (BECs), which serve as the primary constituents of the BBB. Death receptor 6 (DR6) is highly expressed in brain vasculature and acts downstream of the Wnt/ß-catenin pathway to promote BBB formation during development. Here, we found that brain endothelial DR6 levels were significantly reduced in a murine model of AD (APPswe/PS1dE9 mice) at the onset of amyloid-ß (Aß) accumulation. Toxic Aß25-35 oligomer treatment recapitulated the reduced DR6 in cultured BECs. We further showed that suppressing DR6 resulted in BBB malfunction in the presence of Aß25-35 oligomers. In contrast, overexpressing DR6 increased the level of BBB functional proteins through the activation of the Wnt/ß-catenin and JNK pathways. More importantly, DR6 overexpression in BECs was sufficient to rescue BBB dysfunction in vitro. In conclusion, our findings provide new insight into the role of endothelial DR6 in AD pathogenesis, highlighting its potential as a therapeutic target to tackle BBB dysfunction in early-stage AD progression.

Nr1h4 and Thrb ameliorate ER stress and provide protection in the MPTP mouse model of Parkinson's.

Elevated ER stress has been linked to the pathogenesis of several disease conditions including neurodegeneration. In this study, we have holistically determined the differential expression of all the nuclear receptors (NRs) in the presence of classical ER stress inducers. Activation of Nr1h4 and Thrb by their cognate ligands (GW4064 and T3) ameliorates the tunicamycin (TM)-induced expression of ER stress genes. A combination of both ligands is effective in mitigating cell death induced by TM. Further exploration of their protective effects in the Parkinson's disease (PD) model shows that they reduce MPP+-induced dissipation of mitochondrial membrane potential and ROS generation in an in vitro PD model in neuronal cells. Furthermore, the generation of an experimental murine PD model reveals that simultaneous treatment of GW4064 and T3 protects mice from ER stress, dopaminergic cell death, and functional deficits in the MPTP mouse model of PD. Thus, activation of Nr1h4 and Thrb by their respective ligands plays an indispensable role in ER stress amelioration and mounts protective effects in the MPTP mouse model of PD.

The Janus face of endogenous neuronal tPA: promoting self-protection and worsening the death of neighboring neurons.

Recombinant tissue-type plasminogen activator (r-tPA/Actilyse) stands as the prevailing pharmacological solution for treating ischemic stroke patients, of whom because their endogenous circulating tPA alone is not sufficient to rescue reperfusion and to promote favorable outcome. Beyond the tPA contributed by circulating endothelial cells and hepatocytes, neurons also express tPA, sparking debates regarding its impact on neuronal fate ranging from pro-survival to neurotoxic properties. In order to investigate the role of neuronal tPA during brain injuries, we developed models leading to its conditional deletion in neurons, employing AAV9-pPlat-GFP and AAV9-pPlat-Cre-GFP along with tPA floxed mice. These models were subjected to N-methyl-D-aspartate (NMDA)-induced excitotoxicity or thromboembolic ischemic stroke in mice. Initially, we established that our AAV9 constructs selectively transduce neurons, bypassing other brain cell types. Subsequently, we demonstrated that tPA-expressing neurons exhibit greater resistance against NMDA-induced excitotoxicity compared to tPA negative neurons. The targeted removal of tPA in neurons heightened the susceptibility of these neurons to cell death and prevented a paracrine neurotoxic effect on tPA non-expressing neurons. Under ischemic conditions, the self-neuroprotective influence of tPA encompassed both excitatory (GFP+/Tbr1+) and inhibitory (GFP+/GABA+) neurons. Our data indicate that endogenous neuronal tPA is a protective or deleterious factor against neuronal death in an excitotoxic/ischemic context, depending on whether it acts as an autocrine or a paracrine mediator.

RUVBL1 ubiquitination by DTL promotes RUVBL1/2-ß-catenin-mediated transcriptional regulation of NHEJ pathway and enhances radiation resistance in breast cancer.

Radiotherapy effectiveness in breast cancer is limited by radioresistance. Nevertheless, the mechanisms behind radioresistance are not yet fully understood. RUVBL1 and RUVBL2, referred to as RUVBL1/2, are crucial AAA+ ATPases that act as co-chaperones and are connected to cancer. Our research revealed that RUVBL1, also known as pontin/TIP49, is excessively expressed in MMTV-PyMT mouse models undergoing radiotherapy, which is considered a murine spontaneous breast-tumor model. Our findings suggest that RUVBL1 enhances DNA damage repair and radioresistance in breast cancer cells both in vitro and in vivo. Mechanistically, we discovered that DTL, also known as CDT2 or DCAF2, which is a substrate adapter protein of CRL4, promotes the ubiquitination of RUVBL1 and facilitates its binding to RUVBL2 and transcription cofactor ß-catenin. This interaction, in turn, attenuates its binding to acetyltransferase Tat-interacting protein 60 (TIP60), a comodulator of nuclear receptors. Subsequently, ubiquitinated RUVBL1 promotes the transcriptional regulation of RUVBL1/2-ß-catenin on genes associated with the non-homologous end-joining (NHEJ) repair pathway. This process also attenuates TIP60-mediated H4K16 acetylation and the homologous recombination (HR) repair process. Expanding upon the prior study's discoveries, we exhibited that the ubiquitination of RUVBL1 by DTL advances the interosculation of RUVBL1/2-ß-catenin. And, it then regulates the transcription of NHEJ repair pathway protein. Resulting in an elevated resistance of breast cancer cells to radiation therapy. From the aforementioned, it is evident that targeting DTL-RUVBL1/2-ß-catenin provides a potential radiosensitization approach when treating breast cancer.