3T3 Cells Research Articles

8368 Functional Evaluation Of Novel CYP21A2 Variants: Expanding The Genetic Basis Of Non-classic CAH

Abstract Disclosure: A. Matveeva: None. Y. Xu: None. V. Tardy-Guidollet: None. A.V. Pandey: None. Introduction: Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive genetic disorders affecting the production of glucocorticoids, mineralocorticoids, and gonadocorticoids. In approximately 95% of cases, CAH results from a deficiency in CYP21A2. A Non-Classic form of CAH (NC-CAH) arises when CYP21A2 activity decreases to 20-50% of the normal level, occurring at an estimated frequency of 1 in 200 - 1000 individuals. The clinical picture is characterized by the accumulation of androgen precursors, causing hirsutism, acne, metabolic syndrome, menstrual irregularities, infertility in females, and androgenic alopecia, oligospermia in males. Methods: We selected mutations from CYP21A2 sequence data of patients with NC-CAH symptoms admitted to CHU Lyon, France (L49V, A81T, R125G, I161T, L199F, L289H, L289F, V306D, L462P). Additional variants were selected from the SNP databases (dbSNP, gnomAD, ClinVar) based on a high frequency in humans (H393Q, P387L, T201A, S203G S274Y, S494N, R76K, Leu10del). All variants underwent in silico assessment considering evolutionary, structural, and functional aspects (ConSurf, Predict SNP2, CADD, DANN, FATHMM, FunSeq2, GWAVA, PANTHER, SNPs&GO, PhD-SNP, SIFT, SNAP, Meta-SNP, Polyphen2, HDIV, DUET). The CYP21A2 proteins carrying the mutations were overexpressed in HEK 293T cells. We evaluated CYP21A2 enzyme activities by measuring the conversion of 14C-Progesterone into 11-deoxycortisol by thin layer chromatography and autoradiography. Samples were tested in duplicates, using the activity of WT CYP21A2 as a control. Results: Computational analysis revealed that A81T, R125G, I161T, and L289H substitutions significantly impair protein structure, while other variants showed moderate effects. Except for L199F, all patient-derived variants exhibited reduced 21-hydroxylase (CYP21A2) activity associated with NC-CAH phenotypes. Variants from databases did not demonstrate a significant loss of function related to CAH. Conclusion: In this study we investigate the pathogenicity of uncharacterized CYP21A2 variants in relation to non-classic congenital adrenal hyperplasia (NC-CAH), contributing to a better understanding of associated phenotypes. The findings, particularly the identification of novel mutations and the assessment of their impact on CYP21A2 activity, hold promise for improving diagnosis and treatment strategies aimed at mitigating severe outcomes of NC-CAH, such as miscarriages or irreversible alopecia. It also raises awareness among heterozygous carriers, assisting in family planning and medical decisions. Presentation: 6/1/2024

Multi-omics analysis unveils the predictive value of IGF2BP3/SPHK1 signaling in cancer stem cells for prognosis and immunotherapeutic response in muscle-invasive bladder cancer

BackgroundMuscle invasive bladder cancer (MIBC) is a life-threatening malignant tumor characterized by high metastasis rates, poor prognosis, and limited treatment options. Immune checkpoint inhibitors (ICIs) targeting PD-1 and PD-L1 represent an emerging treatment for MIBC immunotherapy. However, the characteristics of patients likely to benefit from immunotherapy remain unclear.MethodsWe performed single-cell mass cytometry (CyTOF) analysis of 179,483 single cells to characterize potential immunotherapy-related cancer stem cells (CSCs)-like populations in the tumor microenvironment of 38 MIBC tissues. The upregulated expression of IGF2BP3 in CD274 + ALDH + CSC-like cells, which was associated with poor clinical prognosis, was analyzed by bulk RNA-sequencing data from an in-house cohort. The functional role of IGF2BP3 was determined through cell proliferation, colony formation, cell apoptosis and sphere formation assays. The regulation of SPHK1 expression by IGF2BP3 was investigated using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and bulk RNA-sequencing (bulk RNA-seq). We further utilized single-nucleus RNA sequencing (snRNA-seq) data from 67,988 cells of 25 MIBC tissues and single-cell RNA sequencing (scRNA-seq) data from MIBC patient-derived organoids to characterize the molecular features of bladder cancer cells co-expressing IGF2BP3 and SPHK1. Spatial transcriptomics (ST) and co-detection by indexing (CODEX) analysis were used to describe the spatial distribution and interactions of IGF2BP3 + SPHK1 + bladder cancer cells and immune cells.ResultsA subset of CD274 + ALDH + CSC-like cells was identified, associating with immunosuppression and low survival rates in MIBC patients. IGF2BP3, an m6A reader gene, was found to be upregulated in the CD274 + ALDH + CSC-like cell population and linked to poor clinical prognosis in MIBC. Knockout of IGF2BP3 dramatically promoted cell apoptosis and reduced cell proliferation in T24 cells. By integrating MeRIP-seq and bulk RNA-seq analyses, we identified SPHK1 served as a substrate for IGF2BP3 in an m6A-dependent manner. Further snRNA-seq, scRNA-seq, ST, and CODEX analysis revealed a closer topographical distance between IGF2BP3 + SPHK1 + bladder cancer cells and exhausted CD8 + T cells, providing one explanation for the superior response to immunotherapy in IGF2BP3 + SPHK1 + bladder cancer cells-enriched patients. Finally, an ICI-associated signature was developed based on the enriched genes of IGF2BP3 + SPHK1 + bladder cancer cells, and its potential ability to predict the response to immunotherapy was validated in two independent immunotherapy cohort.ConclusionsOur study highlighted the critical involvement of the IGF2BP3/SPHK1 signaling in maintaining the stemness of CSCs and promoting MIBC progression. Additionally, these findings suggested that the IGF2BP3/SPHK1 signaling might serve as a biomarker for prognosis and immunotherapy response in MIBC.

Small Molecules Inducing Autophagic Degradation of Expanded Polyglutamine Protein through Interaction with Both Mutant ATXN3 and LC3.

Polyglutamine (polyQ)-mediated spinocerebellar ataxia (SCA), including SCA1, 2, 3, 6, 7, and 17, are caused by mutant genes with expanded CAG repeats, leading to the intracellular accumulation of aggregated proteins, the production of reactive oxygen species, and cell death. Among SCA, SCA3 is caused by a mutation in the ATXN3 (ataxin-3) gene. In a circumstance of polyQ aggregation, the autophagic pathway is induced to degrade the aggregated proteins, thereby suppressing downstream deleterious effects and promoting neuronal survival. In this study, we tested the effects of synthetic indole (NC009-1, -2, -3, -6) and coumarin (LM-022, -031) derivatives as chemical chaperones to assist mutant ATXN3-Q75 folding, as well as autophagy inducers to clear aggregated protein. Among the tested compounds, NC009-1, -2, and -6 and LM-031 interfered with Escherichia coli-derived ATXN3-Q75 aggregation in thioflavin T binding and filter trap assays. In SH-SY5Y cells expressing GFP-fused ATXN3-Q75, these compounds displayed aggregation-inhibitory and neurite growth-promoting potentials compared to untreated cells. Furthermore, these compounds activated autophagy by increasing the phosphatidylethanolamine-conjugated LC3 (microtubule associated protein 1 light chain 3)-II:cytosolic LC3-I ratio in these cells. A biochemical co-immunoprecipitation assay by using a mixture of HEK 293T cell lysates containing recombinant ATXN3-Q75-Venus-C-terminus (VC) or Venus-N-terminus (VN)-LC3 protein indicated that NC009-1 and -2 and LM-031 served as an autophagosome-tethering compound (ATTEC) to interact with ATXN3-Q75 and LC3, and the interaction was further confirmed by bimolecular fluorescence complementation analysis in cells co-expressing both ATXN3-Q75-VC and VN-LC3 proteins. The study results suggest the potential of NC009-1 and -2 and LM-031 as an ATTEC in treating SCA3 and, probably, other polyQ diseases.

Macrophage erythropoietin signaling promotes macrophage-myofibroblast transformation and fibroblast-myofibroblast differentiation

While myofibroblasts are the key cause of abnormal extracellular matrix accumulation, the origin of which has not yet been fully elucidated. Recently, it has been found that macrophage-myofibroblast transformation (MMT) defined by the expression of both macrophage markers (F4/80 or CD68) and myofibroblast markers (α-SMA) is one of its important sources. In the process of MMT, it is unclear whether epor is involved. In this study, when BMDM was induced by tgf-β1, the number of F4/80+α-SMA+ cells increased, the cells polarized toward M2, and the expression of tgf-β1 increased. After the activation of epor, the number of F4/80 +α-SMA + cells and the polarization level of M2 were further increased. At the same time, we found that the conditioned medium from MMT cells could induce the activation of 3T3 cells with increased the expression of α-SMA and col-1. In contrast, the number of F4/80+α-SMA + cells, the polarization of M2, and the expression of Tgf-β1 decreased after epor was inhibited by siRNA. Our results demonstrate that the activation of epor in BMDMs could promote the transformation of macrophage-myofibroblast induced by TGF-β1.

Synthesis, Characterization, Bioavailability and Antimicrobial Studies of Cefuroxime-Based Organic Salts and Ionic Liquids

Low oral bioavailability is a common feature in most drugs, including antibiotics, due to low solubility in physiological media and inadequate cell permeability, which may limit their efficacy or restrict their administration in a clinical setting. Cefuroxime is usually administered in its prodrug form, cefuroxime axetil. However, its preparation requires further reaction steps and additional metabolic pathways to be converted into its active form. The combination of Active Pharmaceutical Ingredients (APIs) with biocompatible organic molecules as salts is a viable and documented method to improve the solubility and permeability of a drug. Herein, the preparations of five organic salts of cefuroxime as an anion with enhanced physicochemical characteristics have been reported. These were prepared via buffer-assisted neutralization methodology with pyridinium and imidazolium cations in quantitative yields and presented as solids at room temperature. Cell viability studies on 3T3 cells showed that only the cefuroxime salts combined with longer alkyl chain cations possess higher cytotoxicity than the original drug, and while most salts lost in vitro antibacterial activity against E. coli, P. aeruginosa and B. subtilis, one compound, [PyC10Py][CFX]2, retained the activity. Cefuroxime organic salts have a water solubility 8-to-200-times greater than the original drug at 37 °C. The most soluble compounds have a very low octanol-water partition, similar to cefuroxime, while more lipophilic salts partition predominantly to the organic phase.

Activation of SIRT3 by Tanshinone IIA ameliorates renal fibrosis by suppressing the TGF-β/TSP-1 pathway and attenuating oxidative stress.

Although doxorubicin (DOX) is a common chemotherapeutic drug, the serious nephrotoxicity caused by DOX-induced renal fibrosis remains a considerable clinical problem. Tanshinone IIA (Tan IIA), a compound extracted from Salvia miltiorrhiza, has been reported to have an anti-fibrotic effect. Therefore, this study investigated the molecular pathway whereby Tan IIA protects the kidneys from DOX administration. DOX (3mg/kg body weight) was intraperitoneally administered every 3 d for a total of 7 injections (cumulative dose of 21mg/kg) to induce nephrotoxicity. Then, Tan IIA (5 or 10mg/kg/d) was administered by intraperitoneal injection for 28 d. In an in vitro study, 293T cells were cultured and treated with DOX and Tan IIA for 24h. Tan IIA reduced the blood urea nitrogen levels elevated by DOX while increasing superoxide dismutase activity, down-regulating reactive oxygen species, ameliorating renal-tubule thickening, and rescuing mitochondrial morphology. Additionally, Tan IIA reduced the renal collagen deposition, increased ATP production and complex-I activity, down-regulated transforming growth factor-β1 (TGF-β1) and thrombospondin-1 (TSP-1), and up-regulated sirtuin 3 (SIRT3). Tan IIA significantly increased cell viability. Additionally, RNA interference was employed to silence the expression of SIRT3, which eliminated the effect of Tan IIA in suppressing the expression of TGF-β1 and TSP-1. In conclusion, Tan IIA ameliorated DOX-induced nephrotoxicity by attenuating oxidative injury and fibrosis. The Tan IIA-induced rescue of mitochondrial morphology and function while alleviating renal fibrosis may be associated with the activation of SIRT3 to suppress the TGF-β/TSP-1 pathway.

MMP7, Regulated by c-Jun, is Involved in Oral Squamous Cell Carcinoma and Associated with Cancer-Related Fibroblasts Infiltration.

This study aimed to analyze the expression of Matrix Metalloproteinase 7 (MMP7) and molecular mechanism at the Transcription Factor (TF) level in Oral Squamous Cell Carcinoma (OSCC). MMP7 expression was preliminarily explored in Head and Neck Squamous Cell Car-cinoma (HNSCC) in the online database, followed by functional analysis and prediction of TF of MMP7. IHC was employed to detect MMP7 levels in OSCC samples. SCC9 and 293T cells were used to explore the transcriptional and regulatory effects of predicted TF on MMP7 by reporter double luciferase assay, RT-qPCR, western blotting, and cellular immunofluorescence. Transwell and TUNEL were employed to detect the migration and apoptosis. MMP7 was significantly up-regulated in HNSCC and OSCC tissues. Moreover, MMP7 was positively correlated with CAFs and significantly enriched in the signaling pathway of RNA degradation. The c-Jun pathway was also up-regulated in OSCC tissues, and predicted to be optimal TF of MMP7 with positive regulatory relationship. In OSCC, silencing and over-expression of c-Jun significantly decreased and increased the level of MMP7. Meanwhile, c-Jun affected the behavior of SCC9 cells, which showed that after c-Jun gene silencing, the ability of cell migration was weakened, while apoptosis was enhanced. When c-Jun gene was overexpressed, the migration ability was enhanced, but apoptosis was not significantly affected. MMP7 has been proven to be a key protein in the development of OSCC, and has the potential to become a biological marker and therapeutic target. It has been found that c-Jun could bind to the MMP7 promoter region, and the silencing or overexpression of c-Jun can positively regulate the expression of MMP7.

Adiponectin receptor 1 regulates endometrial receptivity via the adenosine monophosphate‑activated protein kinase/E‑cadherin pathway.

Endometrial receptivity is essential for successful embryo implantation and pregnancy initiation and is regulated via various signaling pathways. Adiponectin, an important adipokine, may be a potential regulator of reproductive system functions. The aim of the present study was to elucidate the regulatory role of adiponectin receptor 1 (ADIPOR1) in endometrial receptivity. The endometrial receptivity between RL95‑2 and AN3CA cell lines was confirmed using an in vitro JAr spheroid attachment model. 293T cells were transfected with control or short hairpin (sh)ADIPOR1 vectors and RL95‑2 cells were transduced with lentiviral particles targeting ADIPOR1. Reverse transcription‑quantitative PCR and immunoblot assays were also performed. ADIPOR1 was consistently upregulated in the endometrium during the mid‑secretory phase compared with that in the proliferative phase and in receptive RL95‑2 cells compared with that in non‑receptive AN3CA cells. Stable cell lines with diminished ADIPOR1 expression caused by shRNA showed reduced E‑cadherin expression and attenuated in vitro endometrial receptivity. ADIPOR1 regulated AMP‑activated protein kinase (AMPK) activity in endometrial epithelial cells. Regulation of AMPK activity via dorsomorphin and 5‑aminoimidazole‑4‑carboxamide ribonucleotide affected E‑cadherin expression and in vitro endometrial receptivity. The ADIPOR1/AMPK/E‑cadherin axis is vital to endometrial receptivity. These findings can help improve fertility treatments and outcomes.

Fabrication of highly flexible biopolymeric chitosan/agarose based bioscaffold with Matricaria recutita herbal extract for antimicrobial wound dressing applications

A flexible biopolymer-based antimicrobial wound dressing has the potential to alleviate the burden of bacterial infections in wounds by enhancing antimicrobial effectiveness and promoting faster wound healing. This study focuses on the development of a highly flexible chitosan-agarose (CS-AG) bioscaffold, incorporating Matricaria recutita chamomile flower extract (CH) through a conventional casting method. The flexible CS-AG bioscaffold's physiochemical properties were confirmed by FTIR, indicating secondary interactions, and XRD, showing its crystalline structure. The addition of CH to the optimized CS-AG bioscaffold resulted in significant tensile strength (17.28 ± 0.33 MPa), distinctive structural morphology (SEM), surface roughness (AFM), contact angle, improved thermal properties (DSC), and enhanced thermal stability (TGA). Furthermore, the CH-infused bioscaffold significantly increased swelling capacity (~81.09 ± 1.74 % over 48 h), and degradation profile (~52 % over 180 h). The release studies of CS-AG-CH bioscaffold demonstrate controlled release of CH with in the bioscaffold at different pH conditions. The bioscaffold demonstrated effective antibacterial activity against S. aureus and E. coli strains. Additionally, cytotoxicity assays indicated that the bioscaffold supports better cell viability and proliferation in fibroblast (NIH 3T3) cell lines. Consequently, this antimicrobial bioscaffold shows promise as a drug release system and biocompatible wound dressing suitable for tissue engineering applications.

Isolation and Characterization of Cytotoxic Compounds from Detarium microcarpum Guill. and Perr. Stem Bark.

Globally, about 8.2 million cancer-related deaths are recorded annually. Sadly, most of the deaths result from the toxicity of most chemotherapeutic agents. Hence, there are growing demands for chemotherapeutic agents with high specificity and selectivity. This study was designed to assess the cytotoxic potential of Detarium microcarpum and isolate cytotoxic compounds with better selectivity profiles. Detarium microcarpum Stem bark (DMS) was collected and authenticated at the Forest Herbarium Ibadan (FHI), and a voucher (FHI-111954) was issued. Dried DMS was pulverized and extracted into 70% methanol. The extract was partitioned into hexane, dichloromethane, and ethyl acetate fractions. The cytotoxicities of the extract, fractions, and isolated compounds were determined. The cytotoxicity of the isolated compounds was tested against different cell lines, including human breast (AU565 and MDA MB231), oral adenosquamous (CAL27), and cervical (HeLa) cancer cells, as well as healthy (3T3) non-cancer cells. Methyl gallate, eriodictyol, quercetin, quebrachitol, catechin, catechin gallate, and gallic acid, isolated from dichloromethane and ethyl acetate fractions, displayed weak cytotoxicity against breast (AU565 and MDAMD-231) and cervical (HeLa) cancer cell lines. Interestingly, all the compounds, except gallic acid (48.91±4.51% inhibition), displayed potent cytotoxicity on oral cancer cells. Methyl gallate and quercetin displayed the highest activity, with IC50 values of 89.57±1.98µM and 78.19±1.49µM, respectively. Interestingly, all the compounds were not toxic to healthy non-cancer (3T3) cells. The compounds displayed anticancer activity specific to oral cancer cells and were highly selective for cancer cells without causing significant toxicity to healthy non-cancer cells.

Fish Oil Alginate Microspheres Produced via Wave-Based Drop-on-Demand Jetting Electrospray: Improving Stability and Palatability

Despite the proven health benefits of fish oil to the human body, the unpleasant aftertaste and short shelf-life limit the public acceptability as supplementation. Microencapsulation of the fish oil into microspheres is an approach to overcome these limitations. This study aims to prepare controllable-size fish oil alginate microspheres using pulsed electrospray technique for enhancing fish oil stability and palatability. Fish oil alginate microspheres were prepared and optimized by bipolar wave-based drop on demand jetting electrospray. The microspheres were characterized in terms of particle size, aspect ratio, encapsulation efficiency (EE), loading capacity (LC), Fourier transform infrared spectroscopy (FTIR) and drug release. The stability of the fish oil microspheres was assessed based on specific characteristics and antioxidant activity over a period of 6 months. The anticancer effectiveness of encapsulated and unencapsulated fish oil was evaluated using HCT116 and 3T3 cell lines. Additionally, a human panel palatability test was conducted to evaluate the taste masking ability of fish oil microspheres. The optimal formulation exhibited an average particle size of 344.75 ± 10.84 μm, aspect ratio of 1.03 ± 0.02, EE of 98.98 ± 0.72%, and LC of 22.60 ± 0.62%. The FTIR analysis revealed that the chemical properties of the fish oil were preserved after microencapsulation and remained stable after 6 months of storage. The fish oil microspheres showed a superior anticancer effect compared to the free ones. In addition, the panellists of the palatability test of the fish oil microspheres showed an excellent taste masking ability. Hence, this study formulated fish oil microspheres with enhanced stability, efficacy, and palatability, suggesting their potential utility as dietary supplements to fulfil the daily recommended intake of fish oil.

A heterozygous SPRY4 variant identified in female infertility characterized by reduced oocyte potential and early embryonic arrest.

Can novel genetic factors contributing to early embryonic arrest in infertile patients be identified, along with the underlying mechanisms of the pathogenic variant? We identified a heterozygous variant in the SPRY4 (sprouty RTK signaling antagonist 4) in infertile patients and conducted in vitro and in vivo studies to investigate the effects of the variant/deletion, highlighting its critical role in female reproductive health. SPRY4 acts as a negative regulator of receptor tyrosine kinases (RTKs) and functions as a tumor suppressor. Its abnormal expression can lead to recurrent miscarriage by affecting trophoblast function. In mice, Spry4 knockout (KO) leads to craniofacial anomalies and growth defects. A human study links the SPRY4 variant to a male patient with isolated hypogonadotropic hypogonadism (IHH), hypothetically impacting gonadotropin-releasing hormone (GnRH) neurons, and causing reproductive dysfunctions. SPRY4 is thus potentially integral in regulating endocrine homeostasis and reproductive function. To date, no study has reported SPRY4 variants associated with female fertility, and a causal relationship has not been established with functional evidence. Whole-exome sequencing (WES) was performed in 392 infertile women who suffered from primary infertility of unknown reason, and the heterozygous SPRY4 variant were identified in one independent family. The infertile patients presenting were recruited from July 2017 to November 2023. Women diagnosed with primary infertility were recruited from the Reproduction Center of Zhongshan Hospital, Fudan University. Genomic DNA was extracted from peripheral blood for WES analysis. The SPRY4 variant were identified through WES, in silico analysis, and variant screening. All variants were confirmed by Sanger sequencing. The effects of the variants were investigated in human embryonic kidney (HEK) 293T (HEK293T) cells via western blotting, and in mouse oocytes and embryos through complementary RNA (cRNA) injection, RNA sequencing, fluorescence, absorbance, and RT-qPCR assays. Gene function was further examined in Spry4 KO mice via histology, western blotting, ELISA, and RT-qPCR assays. We identified a missense heterozygous pathogenic variant in SPRY4 (GRCh38, GenBank: NM_030964.5, c.157C>T p.(Arg53Trp), rs200531302) that reduces SPRY4 protein levels in HEK293T cells and disrupts the redox system and mitochondrial function in mouse oocyte, and perturbs developmental potential in mouse embryos. These phenotypes could be partially reversed by the exogenous addition of Nrf1 cRNA. Additionally, Spry4-/- mice exhibit ovarian oxidative stress and decreased ovarian function. Due to the limited WES data and population, we identified only one family with a SPRY4 mutation. The deeper mechanism and therapeutic strategy should be further investigated through mutant mice and recovery experiment. Our study has identified a pathogenic variant in SPRY4 associated with early embryonic arrest in humans. These findings enhance our understanding of the role of SPRY4 in early embryonic development and present a new genetic marker for female infertility. This work was supported by the National Natural Science Foundation of China (82071643 and 82171655) and Natural Science Foundation of Shanghai (22ZR1456200). None of the authors have any competing interests. N/A.

Extract from Falcaria vulgaris loaded with exosomes for the treatment of hypertension in pregnant mice: In vitro and In vivo investigations.

Hypertensive disorders during pregnancy pose significant risks to both maternal and fetal health, necessitating safe and effective therapeutic interventions. This study aimed to investigate the potential of an extract derived from Falcaria vulgaris (FV), loaded with exosomes to form the Exo/FV complex, as a novel therapeutic agent for the management of hypertension in pregnant mice: antioxidants, antimicrobials, and phenolic compounds present in FV lower blood pressure. The isolation of exosomes was done by ultracentrifugation methods and the FV was loaded into the exosomes by electroporation method. The Exo/FV was found to be spherical with diameter ranges from 20 to 30nm and they were tested for biocompatibility in NHI 3T3 cell lines and found to be effective. This research investigated invivo hypertension in mice induced by L-NAME and treated with FV and Exo/FV and found that AChE and MAO determine mice's redox state tends to reduce blood pressure. Increased non-protein thiol (NP-SH) and decreased lipid peroxidation were also found, and PDE-5, ACE, Arginase, and MDA activity has also been tested. This analysis showed that Exo/FV effectively treated hypertension during pregnancy.

USP5 promotes tumor progression by stabilizing SLUG in bladder cancer.

Bladder cancer ranks as the second most prevalent urology malignancy globally. Invasive metastasis is a significant contributor to mortality among patients with bladder cancer, yet the underlying mechanisms remain elusive. Deubiquitinases are pivotal in carcinogenesis, with USP5 implicated in the malignant progression of hepatocellular carcinoma, colorectal cancer and non-small cell lung cancer. The present study assessed the role and mechanism of ubiquitin-specific proteinase 5 (USP5) in the malignant progression of bladder cancer. The association between USP5 expression and bladder cancer prognosis and stage was analyzed using The Cancer Genome Atlas database. Moreover, to elucidate the role of USP5 in bladder cancer, USP5 overexpression and knockdown cell lines were established using T24 cells. Cell viability, proliferation and migration were assessed using Cell Counting Kit-8, Transwell and scratch assays, respectively. Cyclohexanamide was used to evaluate the effect of USP5 expression on Snail family zinc finger 2 (SLUG) stability. Immunoprecipitation and immunofluorescence co-localization were utilized to probe the interaction between USP5 and SLUG. Changes in mRNA and protein levels were assessed using reverse transcription-quantitative PCR and western blotting, respectively. The results revealed that patients with bladder cancer with high USP5 expression had significantly shorter survival (P<0.05) and a higher clinicopathologic stage (P<0.05) than those with low USP5 expression. T24 cells overexpressing USP5 demonstrated significantly increased proliferation (P<0.05), invasion (P<0.05) and expression of epithelial-mesenchymal transition markers (P<0.05); whereas T24 cells with knocked-down USP5 expression revealed significantly reduced proliferation (P<0.05), invasion (P<0.05) and epithelial-mesenchymal transition markers (P<0.05). Immunoprecipitation experiments demonstrated the binding of USP5 to SLUG in bladder cancer cells, with further analysis revealing that USP5 upregulated protein levels of SLUG by inhibiting its ubiquitination. Furthermore, the treatment of bladder cancer cells with Degrasyn, a USP5 inhibitor, was associated with a significant inhibition of the proliferation (P<0.05) and invasion (P<0.05) of T24 cells. In conclusion, the findings of the present study underscore the role of USP5 in promoting the malignant progression of bladder cancer through the stabilization of SLUG. Targeting USP5 holds promise as a strategy for inhibiting bladder cancer progression.

Rare genetic variants of NLRP12 in Admixed Latino-American Children with SARS-CoV-2-related Multisystem Inflammatory Syndrome.

Multisystem Inflammatory Syndrome in Children (MIS-C) is a rare, potentially fatal complication of SARS-CoV-2 infection. Genetic defects in inflammation-related pathways have been linked to MIS-C, but additional research is needed, especially in diverse ethnic groups. The present study aimed to identify genetic variants underlying MIS-C in Brazilian patients. Whole-exome sequencing was performed, focusing on genes involved in the host immune response to SARS-CoV-2. Functional assays assessed the impact of selected variants on NF-κB signaling. Nine rare, potentially deleterious variants were found in eight of 21 patients, located in IL17RC, IFNA10, or NLRP12 genes. Unlike the wild-type NLRP12 protein, which inhibits NF-κB activation in HEK 293T cells, the mutant NLRP12 proteins have significantly reduced inhibitory properties. In conclusion, our results indicate that rare autosomal variants in immune-related genes may underlie MIS-C, highlighting the potential role of NLRP12 in its predisposition. These findings provide new insights for the appropriate management of MIS-C.

In Situ Synthesis and Visualization of Membrane SNAP25 Nano-Organization.

Cryo-electron tomography (cryo-ET) can provide insights into the structure and states of natural membrane environments to explore the role of SNARE proteins at membrane fusion and understand the relationship between their subcellular localization/formation and action mechanism. Nevertheless, the identification of individual molecules in crowded and low signal-to-noise ratio membrane environments remains a significant challenge. In this study, cryo-ET is employed to image near-physiological state 293T cell membranes, specifically utilizing in situ synthesized gold nanoparticles (AuNPs) bound with cysteine-rich protein tags to single-molecularly labeled synaptosomal-associated protein 25 (SNAP25) on the membrane surface. The high-resolution images reveal that SNAP25 is predominantly located in regions of high molecular density within the cell membrane and aggregates into smaller clusters, which may increase the fusion efficiency. Remarkably, a zigzag arrangement of SNAP25 is observed on the cell membrane. These findings provide valuable insights into the functional mechanisms of SNARE proteins.

Anti-inflammatory and Anti-angiogenic Effects of Bioconverted Picrasma quassioides Extract: A Comparative Study on NF-κB Signaling Pathway Inhibition and Angiogenesis Suppression

Purpose: This study aimed to compare the anti-inflammatory and anti-angiogenic properties of bioconverted Picrasma quassioides extract (B-P.Q) with those of the non-bioconverted extract (P.Q) and peony root bark extract (BOT).Methods: The research utilized several experimental techniques, including cell viability assays on NIH 3T3 fibroblasts, measurement of NF-κB-related signaling protein activation, chorioallantoic membrane (CAM) assays, tube formation assays, and cell migration inhibition assays using human umbilical vein endothelial cells (HUVECs). These methods were employed to evaluate cytotoxicity, anti-inflammatory effects, and anti-angiogenic properties of the extracts.Results: B-P.Q demonstrated no cytotoxicity at the tested concentrations and significantly inhibited the activation of NF-κB-related signaling proteins (p-p38, p-Akt-1, p-SAPK/JNK, p-MEK1/2, and p-IκB) compared to P.Q and BOT. In angiogenesis experiments, B-P.Q showed superior inhibition of new blood vessel formation in CAM assays, effectively suppressed tube formation, and significantly inhibited HUVEC migration compared to controls and P.Q.Conclusion: The study found that bioconverted Picrasma quassioides extract (B-P.Q) has strong anti-inflammatory and anti-angiogenic properties, consistently outperforming non-bioconverted P.Q and BOT in various experimental models. These results suggest that B-P.Q holds significant promise as a therapeutic agent for chronic inflammatory conditions, particularly those involving excessive angiogenesis. The research underscores the effectiveness of bioconversion in enhancing the bioactive properties of natural extracts and opens up new possibilities for developing treatments for inflammatory skin problems.

Prognostic Significance and Immune Landscape of an Efferocytosis-Related Gene Signature in Bladder Cancer.

Bladder cancer poses a significant global health challenge, underscoring the imperative for precise prognostic instruments to advance patient care. Against the backdrop of efferocytosis's increasingly recognized role in cancer, this research endeavors to develop and authenticate a prognostic signature intricately linked to efferocytosis in bladder cancer. LASSO-COX regression analysis crafted an efferocytosis-related genes risk prognostic model, followed by the construction of a column chart. External validation sets confirmed the predictive accuracy of both the model and chart. Clinical, tumor microenvironment, drug sensitivity, and immunotherapy analyses were employed to comprehensively assess efferocytosis-related scores. The expression of TGFB3 key genes was validated via RT-PCR and western blotting. Further validation included Transwell, Wound healing, Colony formation, and EDU assays. We formulated and validated an efferocytosis-related genes risk model in bladder cancer, comprising 13 core genes. The risk model demonstrated autonomous prognostic significance in both univariate and multivariate Cox analyses. Following the multivariate analysis, we devised a nomogram. Moreover, by utilizing individual risk scores derived from this risk model, we successfully stratified patients into two discernible risk cohorts, unveiling noteworthy variances in immune infiltration profiles and responsiveness to immunotherapy. Notably, the model's key gene TGFB3 was validated through comprehensive experimental investigations, including Transwell assays for migration and invasion and Wound healing assays for motility on the T24 and BIU cell lines. This study has furnished innovative perspectives on an efferocytosis-related prognostic signature, elucidating the prognosis and immune milieu intricacies in patients with bladder cancer.

Automated Microfluidics-Assisted Hydrogel-Based Wet-Spinning for the Biofabrication of Biomimetic Engineered Myotendinous Junction.

The muscle-tendon junction (MTJ) plays a pivotal role in efficiently converting the muscular contraction into a controlled skeletal movement through the tendon. Given its complex biomechanical intricacy, the biofabrication of such tissue interface represents a significant challenge in the field of musculoskeletal tissue engineering. Herein, a novel method to produce MTJ-like hydrogel yarns using a microfluidics-assisted 3D rotary wet-spinning strategy is developed. Optimization of flow rates, rotational speed, and delivery time of bioinks enables the production of highly compartmentalized scaffolds that recapitulate the muscle, tendon, and the transient MTJ-like region. Additionally, such biofabrication parameters are validated in terms of cellular response by promoting an optimal uniaxial alignment for both muscle and tendon precursor cells. By sequentially wet-spinning C2C12 myoblasts and NIH 3T3 fibroblasts, a gradient-patterned cellular arrangement mirroring the intrinsic biological heterogeneity of the MTJ is successfully obtained. The immunofluorescence assessment further reveals the localized expression of tissue-specific markers, including myosin heavy chain and collagen type I/III, which demonstrate muscle and tenogenic tissue maturation, respectively. Remarkably, the muscle-tendon transition zone exhibits finger-like projection of the multinucleated myotubes in the tenogenic compartment, epitomizing the MTJ signature architecture.

Low-energy electric shock ameliorates cell proliferation, morphallaxis, and regeneration via driving key regenerative proteins in earthworm and 3T3 cells

Electric stimulation regulates many cellular processes like cell proliferation, differentiation, apoptosis and cellular migration. Despite its crucial role in regulating stem cells and regeneration, it remains underexplored in both in-vivo and in-vitro settings. In this study, Eudrilus eugeniae are subjected to electric stimulation (1.5 V) prior and after amputation and which augments regeneration up to double-time. Blocking epimorphosis using 2 M thymidine retracts regeneration kinetics to one-third but such inhibition was rescued by applying electric stimulation which propels an overactive morphallaxis pattern of regeneration. Excreting electric stimulation on control worms shows minimal impact, whereas it enhances the key regenerative proteins like VEGF, COX2, YAP, c-Myc, and Wnt3a on amputated worms. Upon blocking epimorphosis, all these key regenerative proteins are down-regulated but through electric stimulation, the cells are reprogrammed to express a triple fold of the mentioned regenerative proteins, that further promotes morphallaxis. In 3T3 cells, electric stimulation accelerates cell proliferation and migrations in 5 secs exposure and it exerts its function by overexpressing VEGF mediated by MEK1. Wnt3a expression was gradually upregulated in increasing exposure (5 and 25 secs) which aids in maintaining the stemness property. The molecular mechanism underlying regeneration capability can assist in designing novel therapeutic applications.