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Background: Facilitating the healing process of injured anterior cruciate ligament (ACL) tissue is crucial for patients to safely return to sports. Stem cell derived exosomes have shown positive effects on enhancing the regeneration of injured tendons/ligaments. However, clinical application of exosomes in terms of storage and pre-assembly is challenging. We hypothesized that lyophilized exosomes derived from human umbilical cord stem cells (hUSC-EX) could enhance the cell activity of chronically injured ACL cells. Materials and methods: We harvested the 8 weeks injured ACL cells from rabbit under IACUC (No. 110232) approval. The studied exosomes were purified from the culture medium of human umbilical cord stem cells (IRB approval No. A202205014), lyophilized to store, and hydrated for use. We compared exosome treated cells with non-exosome treated cells (control group) from the same rabbits. We examined the cell viability, proliferation, migration capability and gene expression of type I and III collagen, TGFβ, VEGF, and tenogenesis in the 8 weeks injured ACL cells after hUSC-EX treatment. Results: After hydration, the average size of hUSC-EX was 84.5 ± 70.6 nm, and the cells tested positive for the Alix, TSG101, CD9, CD63, and CD81 proteins but negative for the α-Tubulin protein. After 24 h of treatment, hUSC-EX significantly improved the cell viability, proliferation and migration capability of 8 weeks injured ACL cells compared to that of no exosome treatment group. In addition, the expression of collagen synthesis, TGFβ, VEGF, and tenogenesis gene were all significantly increased in the 8 weeks injured ACL cells after 24 h hUSC-EX delivery. Discussion: Lyophilized exosomes are easily stored and readily usable after hydration, thereby preserving their characteristic properties. Treatment with lyophilized hUSC-EX improved the activity and gene expression of 8 weeks injured ACL cells. Conclusion: Lyophilized hUSC-EX preserve the characteristics of exosomes and can improve chronically injured (8 weeks) ACL cells. Lyophilized hUSC-EX could serve as effective and safe biomaterials that are ready to use at room temperature to enhance cell activity in patients with partial ACL tears and after remnant preservation ACL reconstruction. [ABSTRACT FROM AUTHOR]

To elucidate the possible biological roles of fatty acid-binding protein 5 (FABP5) in the intraocular environment, the cells from which FABP5 originates were determined by using four different intraocular tissue-derived cell types including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cell lines, and the effects of FABP ligand 6, a specific inhibitor for FABP5 and FABP7 were analyzed by RNA sequencing and seahorse cellular metabolic measurements. Among these four different cell types, qPCR analysis showed that FABP5 was most prominently expressed in HNPCE cells, in which no mRNA expression of FABP7 was detected. In RNA sequencing analysis, 166 markedly up-regulated and 198 markedly down-regulated differentially expressed genes (DEGs) were detected between non-treated cells and cells treated with FABP ligand 6. IPA analysis of these DEGs suggested that FABP5 may be involved in essential roles required for cell development, cell survival and cell homeostasis. In support of this possibility, both mitochondrial and glycolytic functions of HNPCE cells, in which mRNA expression of FABP5, but not that of FABP7, was detected, were shown by using a Seahorse XFe96 Bioanalyzer to be dramatically suppressed by FABP ligand 6-induced inhibition of the activity of FABP5. Furthermore, in IPA upstream analysis, various unfolded protein response (UPR)-related factors were identified as upstream and causal network master regulators. Analysis by qPCR analysis showed significant upregulation of the mRNA expression of most of UPR-related factors and aquaporin1 (AQP1). The findings in this study suggest that HNPCE is one of intraocular cells producing FABP5 and may be involved in the maintenance of UPR and AQP1-related functions of HNPCE. [ABSTRACT FROM AUTHOR]

Background: To investigate the effects of Isoorientin on the apoptosis, proliferation, invasion, and migration of human gastric cancer cells (HGC27 cells). Methods: We used network pharmacology to predict the targets of drugs and diseases. The CCK-8 assay was used to determine the effects of Isoorientin on the proliferation of HGC27 cells. Flow cytometry was employed to analyze the effects of Isoorientin on cell apoptosis and cell cycle distribution of HGC27 cells. Scratch test and transwell chamber test were conducted to assess the effects of Isoorientin on invasion and migration, respectively. Additionally, qPCR and western blot were performed to examine the impact of Isoorientin on apoptosis-related genes and protein expression, respectively. Results: The Isoorientin significantly inhibited the proliferation, migration, and invasion of HGC27 cells compared to the control group. Furthermore, Isoorientin induced apoptosis in HGC27 cells by upregulating the relative expression of Bax and caspase-3 while downregulating the relative expression of p-PI3K, p-AKT, and Bcl-2 proteins. Conclusion: The Isoorientin exhibits inhibitory effects on the proliferation, invasion, and migration of HGC27 cells, and induces apoptosis in gastric cancer cells. [ABSTRACT FROM AUTHOR]

Simple Summary: Aquaporins (AQPs) have emerged as potential predictors of response to cancer therapy and as targets for increasing sensitivity to treatment. However, systemic therapeutic inhibition using pan-AQP or AQP-specific inhibitors has not been characterized for efficacy in treating breast cancer or as a component of combination treatment. We evaluated AQP inhibition using established AQP inhibitors in cytotoxicity and pharmacologic assays. This study identified AQPs as a targetable vulnerability in breast cancer. AQP inhibitors can increase therapeutic efficacy, both as single agents and in a combination therapy strategy, in treating breast cancer progression and metastasis. These results provide significant insights that support the future development of improved AQP inhibitors. AQPs contribute to breast cancer progression and metastasis. We previously found that genetic inhibition of Aqp7 reduces primary tumor burden and metastasis in breast cancer. In this study, we utilized two AQP inhibitors, Auphen and Z433927330, to evaluate the efficacy of therapeutic inhibition of AQPs in breast cancer treatment. The inhibitors were evaluated in breast cancer for both cytotoxicity and metabolic stability assays across both murine and human breast cancer cell lines. Both AQP inhibitors also affected the expression of other AQP transcripts and proteins, which demonstrates compensatory regulation between AQP family members. As a single agent, Auphen treatment in vivo extended overall survival but did not impact primary or metastatic tumor burden. However, Auphen treatment made cells more responsive to chemotherapy (doxorubicin) or endocrine treatment (tamoxifen, fulvestrant). In fact, treatment with Tamoxifen reduced overall AQP7 protein expression. RNA-seq of breast cancer cells treated with Auphen identified mitochondrial metabolism genes as impacted by Auphen and may contribute to reducing mammary tumor progression, lung metastasis, and increased therapeutic efficacy of endocrine therapy in breast cancer. Interestingly, we found that Auphen and tamoxifen cooperate to reduce breast cancer cell viability, which suggests that Auphen treatment makes the cells more susceptible to Tamoxifen. Together, this study highlights AQPs as therapeutic vulnerabilities of breast cancer metastasis that are promising and should be exploited. However, the pharmacologic results suggest additional chemical refinements and optimization of AQP inhibition are needed to make these AQP inhibitors appropriate to use for therapeutic benefit in overcoming endocrine therapy resistance. [ABSTRACT FROM AUTHOR]

Background: Neuroblastoma, a prevalent solid tumor in children, often manifests with hidden onset sites, rapid growth, and high metastatic potential. The prognosis for children with high-risk neuroblastoma remains poor, highlighting the urgent need for novel prognostic models and therapeutic avenues. In recent years, puerarin, as a kind of small molecule drug extracted from Chinese medicine Pueraria lobata, has demonstrated significant anticancer effects on various cancer cell types. In this study, through bioinformatics analysis and in vitro experiments, the potential and mechanism of puerarin in the treatment of neuroblastoma were investigated, and a prognostic model was established. Methods: A total of 9 drug-disease related targets were observed by constructing a database of drug targets and disease genes. Besides, GO and KEGG enrichment analysis was performed to explore the potential mechanism of its therapeutic effect. To construct the prognostic model, risk regression analysis and LASSO analysis were carried out for validation. Finally, the prognostic genes were identified. Parachute test and immunofluorescence staining were performed to verify the potential mechanism of puerarin in neuroblastoma treatment. Results: Three prognostic genes, i.e., BIRC5, TIMP2 and CASP9, were identified. In vitro studies verified puerarin's impact on BIRC5, TIMP2, and CASP9 expression, inhibiting proliferation in neuroblastoma SH-SY5Y cells. Puerarin disrupts the cytoskeleton, boosts gap junctional communication, curtailing invasion and migration, and induces mitochondrial damage in SH-SY5Y cells. Conclusions: Based on network pharmacology and bioinformatics analysis, combined with in vitro experimental verification, puerarin was hereby observed to enhance GJIC in neuroblastoma, destroy cytoskeleton and thus inhibit cell invasion and migration, cause mitochondrial damage of tumor cells, and inhibit cell proliferation. Overall, puerarin, as a natural medicinal compound, does hold potential as a novel therapy for neuroblastoma. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa, a nosocomial pathogen, has strong biofilm capabilities, representing the main source of infection in the human body. Repurposing existing drugs has been explored as an alternative strategy to combat emerging antibiotic-resistant pathogens. Fingolimod hydrochloride (FTY720), an immunomodulatory drug for multiple sclerosis, has shown promising antimicrobial effects against some ESKAPE pathogens. Therefore, the effects of FTY720 on the biofilm capabilities of Pseudomonas aeruginosa were investigated in this study. It was determined that FTY720 inhibited the growth of P. aeruginosa PAO1 at 100 µM. The significant reduction in PAO1 cell viability was observed to be dose-dependent. Additional cytotoxicity analysis on human cell lines showed that FTY720 significantly reduced viabilities at sub-inhibitory concentrations of 25–50 µM. Microtiter assays and confocal analysis confirmed reductions in biofilm mass and thickness and the cell survivability ratio in the presence of FTY720. Similarly, virulence production and biofilm-related gene expression (rhlA, rhlB, pilA, pilI, fliC, fliD and algR) were determined. The results demonstrate that pigment production was affected and quantitative real-time PCR analysis showed a variable degree of reduced gene expression in response to FTY720 at 12.5–50 µM. These findings suggest that FTY720 could be repurposed as an alternative antibiofilm agent against Pseudomonas aeruginosa. [ABSTRACT FROM AUTHOR]

Cancer cells modulate their metabolism, creating an acidic microenvironment that, in turn, can favor tumor progression and chemotherapy resistance. Tumor cells adopt strategies to survive a drop in extracellular pH (pHe). In the present manuscript, we investigated the contribution of mitochondrial sirtuin 3 (SIRT3) to the adaptation and survival of cancer cells to a low pHe. SIRT3-overexpressing and silenced breast cancer cells MDA-MB-231 and human embryonic kidney HEK293 cells were grown in buffered and unbuffered media at pH 7.4 and 6.8 for different times. mRNA expression of SIRT3 and CAVB, was measured by RT-PCR. Protein expression of SIRT3, CAVB and autophagy proteins was estimated by western blot. SIRT3-CAVB interaction was determined by immunoprecipitation and proximity ligation assays (PLA). Induction of autophagy was studied by western blot and TEM. SIRT3 overexpression increases the survival of both cell lines. Moreover, we demonstrated that SIRT3 controls intracellular pH (pHi) through the regulation of mitochondrial carbonic anhydrase VB (CAVB). Interestingly, we obtained similar results by using MC2791, a new SIRT3 activator. Our results point to the possibility of modulating SIRT3 to decrease the response and resistance of tumor cells to the acidic microenvironment and ameliorate the effectiveness of anticancer therapy. [ABSTRACT FROM AUTHOR]

The aim of the study was to explore the therapeutic effect of ultrasound targeted destruction of schisandrin A contrast microbubbles on liver cancer and its related mechanism. The Span-PEG microbubbles loaded with schisandrin A were prepared using Span60, NaCl, PEG-1500, and schisandrin A. The loading rate of schisandrin A in Span-PEG composite microbubbles was determined by ultraviolet spectrophotometry method. The Walker-256 cell survival rate of schisandrin A was determined by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay. The content of schisandrin A in the cells was determined by high performance liquid chromatography. Ultrasound imaging was used to evaluate the therapeutic effect in situ. Enzyme linked immunosorbent assay (ELISA) was used to measure the content of inflammatory factors in serum. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of experimental animals in each group. Immunohistochemistry was used to detect the expression of hypoxia inducible factor-1α (HIF-1α), vascular endothlial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR-2) in tumor tissues, and western blot was used to detect the protein expression of phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway in tumor tissues. The composite microbubbles were uniform in size, and the particle size distribution was unimodal and stable, which met the requirements of ultrasound contrast agents. The loading rate of schisandrin A in Span-PEG microbubbles was 8.84 ± 0.14%, the encapsulation efficiency was 82.24±1.21%. The IC50 value of schisandrin A was 2.87 μg/mL. The drug + microbubbles + ultrasound (D+M+U) group had the most obvious inhibitory effect on Walker-256 cancer cells, the highest intracellular drug concentration, the largest reduction in tumor volume, the most obvious reduction in serum inflammatory factors, and the most obvious improvement in pathological results. The results of immunohistochemistry showed that HIF-1α, VEGF and VEGFR-2 protein decreased most significantly in D+M+U group (P < 0.01). WB results showed that D+M+U group inhibited the PI3K/AKT/mTOR signaling pathway most significantly (P < 0.01). Schisandrin A had an anti-tumor effect, and its mechanism might be related to the inhibition of the PI3K/AKT/mTOR signaling pathway. The schisandrin A microbubbles could promote the intake of schisandrin A in tumor cells after being destroyed at the site of tumor under ultrasound irradiation, thus playing the best anti-tumor effect. [ABSTRACT FROM AUTHOR]

Objective: Melanoma, with its high degree of malignancy, stands as one of the most dangerous skin cancers and remains the primary cause of death from skin cancer. With studies demonstrating the potential of traditional Chinese medicine to intervene and treat melanoma, we turned our attention to celastrol. Celastrol is a triterpene compound extracted from the traditional Chinese medicine derived from Tripterygium wilfordii. Previous studies have shown that celastrol exerts inhibitory effects on various malignant tumors, including melanoma. Hence, our goal was to clarify the impact of celastrol on cell viability, apoptosis, and cell cycle progression by elucidating its effects on the PI3K/AKT/mTOR pathway. Methods: CCK-8 and wound healing assays were used to determine the effect of celastrol on the viability and migration of B16-F10 cells. Changes in cell apoptosis, cell cycle, reactive oxygen species (ROS), and mitochondrial membrane potential were detected by flow cytometry. PI3K/AKT/mTOR pathway proteins and HIF-α mRNA expression in B16-F10 cells were detected by western blotting and qPCR. Moreover, the addition of a PI3K activator demonstrated that celastrol could inhibit the function of B16-F10 cells via the PI3K/AKT/mTOR pathway. Results: Celastrol inhibited the viability and migration of B16-F10 cells. Through the inhibition of the PI3K/AKT/mTOR pathway down-regulates the expression of HIF-α mRNA, thereby causing an increase of ROS in cells and a decrease in the mitochondrial membrane potential to promote cell apoptosis and cell cycle arrest. The inhibitory effect of celastrol on B16-F10 cells was further demonstrated by co-culturing with a PI3K activator. Conclusion: Celastrol inhibits the function of B16-F10 cells by inhibiting the PI3K/AKT/mTOR cellular pathway and regulating the expression of downstream HIF-α mRNA. [ABSTRACT FROM AUTHOR]

Simple Summary: The use of antiretroviral therapy has shown promising antineoplastic effects in multiple cancers; however, its efficacy in glioblastoma is unknown. We conducted an unbiased screen of 16 antiretroviral medications in 40 glioma cell lines and validated their efficacy in patient-derived glioma neurospheres and established cell lines. Our study provides the first mechanistic and functional insight into the utility of drug repurposing for malignant gliomas, which supports the current literature. Given their safety profile, preclinical efficacy, and neuropenetrance, antiretroviral therapy may be a promising adjuvant treatment for glioblastoma. Outcomes for glioblastoma (GBM) remain poor despite standard-of-care treatments including surgical resection, radiation, and chemotherapy. Intratumoral heterogeneity contributes to treatment resistance and poor prognosis, thus demanding novel therapeutic approaches. Drug repositioning studies on antiretroviral therapy (ART) have shown promising potent antineoplastic effects in multiple cancers; however, its efficacy in GBM remains unclear. To better understand the pleiotropic anticancer effects of ART on GBM, we conducted a comprehensive drug repurposing analysis of ART in GBM to highlight its utility in translational neuro-oncology. To uncover the anticancer role of ART in GBM, we conducted a comprehensive bioinformatic and in vitro screen of antiretrovirals against glioblastoma. Using the DepMap repository and reversal of gene expression score, we conducted an unbiased screen of 16 antiretrovirals in 40 glioma cell lines to identify promising candidates for GBM drug repositioning. We utilized patient-derived neurospheres and glioma cell lines to assess neurosphere viability, proliferation, and stemness. Our in silico screen revealed that several ART drugs including reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs) demonstrated marked anti-glioma activity with the capability of reversing the GBM disease signature. RTIs effectively decreased cell viability, GBM stem cell markers, and proliferation. Our study provides mechanistic and functional insight into the utility of ART repurposing for malignant gliomas, which supports the current literature. Given their safety profile, preclinical efficacy, and neuropenetrance, ARTs may be a promising adjuvant treatment for GBM. [ABSTRACT FROM AUTHOR]

Simple Summary: Retinoblastoma (RB) is a malignant childhood eye cancer. In search for new or adjuvant treatment options, the gastric inhibitory polypeptide receptor (GIPR), upregulated upon the overexpression of trefoil factor family peptide 1 (TFF1), a diagnostic and prognostic biomarker for advanced RBs, came into our focus of interest. The overexpression of GIPR, found to be co-expressed with TFF1 in RB tumors, significantly reduced RB cell viability and growth and increased apoptosis levels. Moreover, GIPR-overexpressing RB cells developed significantly smaller tumors in vivo, indicating a tumor suppressor role of GIPR in RB. Although our data revealed that GIPR is not a direct TFF1 receptor, TFF1 and GIPR seem to be involved in the same signaling cascades. GIPR expression in RB cells seems to be regulated by miR-542-5p, and p53 is involved in GIPR downstream signaling, together providing potential targets for novel retinoblastoma treatment approaches. Retinoblastoma (RB) is the most common malignant intraocular tumor in early childhood. Gene expression profiling revealed that the gastric inhibitory polypeptide receptor (GIPR) is upregulated following trefoil factor family peptide 1 (TFF1) overexpression in RB cells. In the study presented, we found this G protein-coupled transmembrane receptor to be co-expressed with TFF1, a new diagnostic and prognostic RB biomarker for advanced subtype 2 RBs. Functional analyses in two RB cell lines revealed a significant reduction in cell viability and growth and a concomitant increase in apoptosis following stable, lentiviral GIPR overexpression, matching the effects seen after TFF1 overexpression. In chicken chorioallantoic membrane (CAM) assays, GIPR-overexpressing RB cells developed significantly smaller CAM tumors. The effect of GIPR overexpression in RB cells was reversed by the GIPR inhibitor MK0893. The administration of recombinant TFF1 did not augment GIPR overexpression effects, suggesting that GIPR does not serve as a TFF1 receptor. Investigations of potential GIPR up- and downstream mediators suggest the involvement of miR-542-5p and p53 in GIPR signaling. Our results indicate a tumor suppressor role of GIPR in RB, suggesting its pathway as a new potential target for future retinoblastoma therapy. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a heterogenous pathology with high incidence and mortality rates globally, but it is also preventable so finding the most promising candidates (natural compounds or repurposed drugs) to be chemopreventive alternatives has become a topic of interest in recent years. The present work aims to elucidate the potential effects of a combination between genistein (GEN), an isoflavone of natural origin, and aspirin (ASA) in CRC prevention/treatment by performing an in vitro evaluation in human colorectal cancer cells (HCT-116) and an in ovo analysis using the chick embryo chorioallantoic membrane (CAM) model. Cell viability was verified by an MTT (migratory potential by scratch) assay, and the expressions of MMP-2 and MMP-9 were analyzed using RT-qPCR. Our results indicated a dose-dependent cytotoxic effect of ASA (2.5 mM) + GEN (10–75 µM) combination characterized by reduced cell viability and morphological changes (actin skeleton reorganization and nuclei deterioration), an inhibition of HCT-116 cells' migratory potential by down-regulating MMP-2 and MMP-9 mRNA expressions, and an antiangiogenic effect by modifying the vascular network. These promising results raise the possibility of future in-depth investigations regarding the chemopreventive/therapeutical potential of ASA+GEN combination. [ABSTRACT FROM AUTHOR]

Connarus semidecandrus Jack (Family: connaraceae) is a medicinal plant known for its wide distribution throughout Southeast Asia. Renowned for its diverse therapeutic properties, it has been traditionally used for treating fever, skin irritation, and colic. Numerous individuals suffer from skin issues, including wrinkles, hyperpigmentation, and inflammation, due to environmental factors. Although many drugs are available to treat skin problems, chemical drugs have many shortcomings and side effects. Therefore, natural products are attractive potential medicines for alleviating skin troubles. We recently showed that Connarus semidecandrus Jack ethanol extract (Cs-EE) has anti-alopecia potential. This paper aims to explore the potential skin-protective effects and underlying molecular mechanisms of Connarus semidecandrus Jack in UVB-induced human keratinocytes (HaCaT). Before utilization, Cs-EE was dissolved in dimethyl sulfoxide (DMSO) and was preserved at a temperature of −20 °C. The phytochemical constituents of Cs-EE were detected by gas chromatography–mass spectrometry analysis (GC-MS). Sequentially, HaCaT cells were exposed to varying concentrations of Cs-EE prior to ultraviolet B (UVB) irradiation. Evaluations of cellular responses in HaCaT cells, including assessments of cell viability, deoxyribonucleic acid (DNA) damage, and gene and protein expressions, were carried out. To explore the specific signaling pathway involved, we conducted a luciferase assay in addition to validating these pathways using Western blot analysis. Nitric oxide (NO) and intracellular reactive oxygen species were decreased. Melanin production through the activation of melanocytes by α-melanocyte-stimulating hormone (MSH) was also inhibited by Cs-EE. Furthermore, the mRNA expression levels of key factors such as cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), MMP-1, MMP-3, and MMP-9 exhibited a remarkable decrease. In addition, the phosphorylation of TAK1 within the signaling cascade exhibited a decline, and the activities of the transcription factor AP-1 were decreased according to a luciferase reporter assay. Taken together, these findings suggest that the anti-inflammatory, anti-aging, and anti-apoptotic effects of Cs-EE indicate the compound's potential usefulness as a natural component in pharmaceutical and cosmetic products. [Display omitted] • Connarus semidecandrus Jack ethanol extract (Cs-EE) shows radical scavenging activity. • Cs-EE reduces UVB-induced cell death and apoptosis. • Cs-EE reduces the expression of genes involved in UVB-induced inflammatory response and wrinkle formation. • Cs-EE downregulates α-MSH-induced melanogenic responses of melanocyte. • Cs-EE inhibits AP-1 activity via suppression of TAK1/MKK3/6/p38 pathway. [ABSTRACT FROM AUTHOR]

Liver cancer is a common malignant tumor worldwide, traditional Chinese medicine is one of the treatment measures for liver cancer because of its good anti-tumor effects and fewer toxic side effects. Ginsenoside CK (CK) is an active component of ginseng. This study explored the mechanism by which CK induced ferroptosis in liver cancer cells. We found that CK inhibited the proliferation of HepG2 and SK-Hep-1 cells, induced ferroptosis of cells. Ferrostatin-1, an ferroptosis inhibitor, was used to verify the role of CK in inducing ferroptosis of liver cancer cells. Network pharmacological analysis identified the FOXO pathway as a potential mechanism of CK, and western blot showed that CK inhibited p-FOXO1. In cells treated with the FOXO1 inhibitor AS1842856, further verify the involvement of the FOXO pathway in regulating CK-induced ferroptosis in HepG2 and SK-Hep-1 cells. A HepG2 cell–transplanted tumor model was established in nude mice, and CK inhibited the growth of transplanted tumors in nude mice, p-FOXO1 was decreased in tumor tissues, and SLC7A11 and GPX4 expressions were also down-regulated after CK treatment. These findings suggested that CK induces ferroptosis in liver cancer cells by inhibiting FOXO1 phosphorylation and activating the FOXO signaling pathway, thus playing an antitumor role. [ABSTRACT FROM AUTHOR]

Background: Polygonum multiflorum (PM), a widely used traditional Chinese medicine herb, is divided into two forms, namely raw polygonum multiflorum (RPM) and polygonum multiflorum praeparata (PMP), according to the processing procedure. Emerging data has revealed the differential hepatotoxicity of RPM and PMP, however, its potential mechanism is still unclear. Methods: In our study, we investigated the differential hepatotoxicity of RPM and PMP exerted in C57BL/6 mice. First, sera were collected for biochemical analysis and HE staining was applied to examine the morphological alternation of the liver. Then we treated L02 cells with 5 mg / mL of RPM or PMP. The CCK8 and EdU assays were utilized to observe the viability and proliferation of L02 cells. RNA sequencing was performed to explore the expression profile of L02 cells. Western blotting was performed to detect the expression level of ferroptosis-related protein. Flow cytometry was used to evaluate ROS accumulation. Results: In our study, a significant elevation in serum ALT, AST and TBIL levels was investigated in the RMP group, while no significant differences were observed in the PMP group, compared to that of the CON group. HE staining showed punctate necrosis, inflammatory cell infiltration and structural destruction can be observed in the RPM group, which can be significantly attenuated after processing. In addition, we also found RPM could decrease the viability and proliferation capacity of L02 cells, which can be reversed by ferroptosis inhibitor. RNA sequencing data revealed the adverse effect of PM exerted on the liver is closely associated with ferroptosis. Western blotting assay uncovered the protein level of GPX4, HO-1 and FTL was sharply decreased, while the ROS content was dramatically elevated in L02 cells treated with RPM, which can be partially restored after processing. Conclusions: The hepatotoxicity induced by RPM was significantly lower than the PMP, and its potential mechanism is associated with ferroptosis. [ABSTRACT FROM AUTHOR]

While previous reports have established the anti-inflammatory effects of hangeshashinto, the intracellular signal transduction pathways involved have yet to be elucidated. We aim to employ an experimental system using oral cancer cells to assess the impact of hangeshashinto on intracellular signal transduction pathways in response to stimulation by Porphyromonas gingivalis pathogen-associated molecular patterns (PAMP). Hangeshashinto demonstrated the ability to inhibit the production of interleukin (IL)-6 and IL-8 induced by P. gingivalis PAMP. Furthermore, hangeshashinto suppressed the activation of the IL-6 promoter stimulated by PAMP. Hangeshashinto, like Toll-like receptor (TLR) signaling inhibitors (resatorvid and C29) and an immunosuppressant (dexamethasone), exhibited the ability to suppress TLR-mediated activation of the transcription factor nuclear factor-κB (NF-κB) in response to PAMP stimulation. This study suggests that the anti-inflammatory effects of hangeshashinto may be attributed to the inhibition of TLR signal transduction pathways including NF-κB activation, thereby suppressing NF-κB-dependent gene expression. [ABSTRACT FROM AUTHOR]

Background: The intricate and complex molecular mechanisms that underlie the progression of Alzheimer's disease (AD) have prompted a concerted and vigorous research endeavor aimed at uncovering potential avenues for therapeutic intervention. Objective: This study aims to elucidate the role of miRNA PC-5P-12969 in the pathogenesis of AD. Methods: We assessed the differential expression of miRNA PC-5P-12969 in postmortem AD brains, AD animal and cell models using real-time reverse-transcriptase RT-PCR, we also checked the gene and protein expression of GSK3α and APP. Results: Our investigation revealed a notable upregulation of miRNA PC-5P-12969 in postmortem brains of AD patients, in transgenic mouse models of AD, and in mutant APP overexpressing-HT22 cells. Additionally, our findings indicate that overexpression of miRNA PC-5P-12969 exerts a protective effect on cell survival, while concurrently mitigating apoptotic cell death. Further-more, we established a robust and specific interaction between miRNA PC-5P-12969 and GSK3α. Our luciferase reporter assays provided confirmation of the binding between miRNA PC-5P-12969 and the 3′-UTR of the GSK3α gene. Manipulation of miRNA PC-5P-12969 levels in cellular models of AD yielded noteworthy alterations in the gene and protein expression levels of both GSK3α and APP. Remarkably, the manipulation of miRNA PC-5P-12969 levels yielded significant enhancements in mitochondrial respiration and ATP production, concurrently with a reduction in mitochondrial fragmentation, thus unveiling a potential regulatory role of miRNA PC-5P-12969 in these vital cellular processes. Conclusions: In summary, this study sheds light on the crucial role of miRNA PC-5P-12969 and its direct interaction with GSK3α in the context of AD. [ABSTRACT FROM AUTHOR]

Simple Summary: Anthracycline-induced cardiomyopathies and sarcopenia are often seen in cancer patients, affecting their quality of life and overall survival. Translational research aimed to find new cardioprotective strategies is strictly needed in cardioncology. Soluble guanylate cyclase activator vericiguat reduces heart failure rates in patients with reduced ejection fraction. In this study, we highlighted the cardioprotective and anti-inflammatory properties of vericiguat during exposure to anthracyclines and demonstrated its preventive properties of sarcopenia induced by doxorubicin (DOXO), which can be exploited for potential cardioprotective strategies in cancer patients. Furthermore, vericiguat reduces chemokines and cytokines involved in cardiomyopathies through NLR family pyrin domain containing 3 (NLRP-3) pathways in human cardiomyocytes and skeletal muscle cells. The findings that emerged from this study could provide the rationale for further preclinical and clinical investigations aimed at reducing anthracycline cardiotoxicity and sarcopenia in cancer patients. Anthracycline-induced cardiomyopathies and sarcopenia are frequently seen in cancer patients, affecting their overall survival and quality of life; therefore, new cardioprotective and anti-sarcopenic strategies are needed. Vericiguat is a new oral guanylate cyclase activator that reduces heart failure hospitalizations or cardiovascular death. This study highlighted the potential cardioprotective and anti-sarcopenic properties of vericiguat during anthracycline therapy. Human cardiomyocytes and primary skeletal muscle cells were exposed to doxorubicin (DOXO) with or without a pre-treatment with vericiguat. Mitochondrial cell viability, LDH, and Cytochrome C release were performed to study cytoprotective properties. Intracellular Ca++ content, TUNEL assay, cGMP, NLRP-3, Myd-88, and cytokine intracellular levels were quantified through colorimetric and selective ELISA methods. Vericiguat exerts significant cytoprotective and anti-apoptotic effects during exposure to doxorubicin. A drastic increase in cGMP expression and reduction in NLRP-3, MyD-88 levels were also seen in Vericiguat-DOXO groups vs. DOXO groups (p < 0.001) in both cardiomyocytes and human muscle cells. GCa vericiguat reduces cytokines and chemokines involved in heart failure and sarcopenia. The findings that emerged from this study could provide the rationale for further preclinical and clinical investigations aimed at reducing anthracycline cardiotoxicity and sarcopenia in cancer patients. [ABSTRACT FROM AUTHOR]

Introduction. Diabetic cataract (DC) is a common ocular complication of diabetes. Mitofusin 2 (MFN2), a mitochondrial fusion protein, is involved in the pathogenesis of cataract and diabetic complications. However, its role and molecular mechanisms in DC remain unclear. Materials and methods. DC models in rats were induced by intraperitoneal injection of streptozocin (STZ) for 12 weeks. We measured the body weight of rats, blood glucose concentrations, sorbitol dehydrogenase (SDH) activity and advanced glycation end products (AGE) content in the lenses of rats. MFN2 mRNA and protein expression levels in the lenses were detected by RT-qPCR and western blot assays. In vitro, human lens epithelial (HLE) B3 cells were treated for 48 h with 25 mM glucose (high glucose, HG) to induce cell damage. To determine the role of MFN2 in HG-induced cell damage, HLE-B3 cells were transfected with lentivirus loaded with MFN2 overexpression plasmid or short hairpin RNA (shRNA) to overexpress or knock down MFN2 expression, followed by HG exposure. Cell viability was assessed by CCK-8 assay. Flow cytometry was used to detect cell apoptosis and reactive oxygen species (ROS) level. JC-1 staining showed the changes in mitochondrial membrane potential (Δψm). The mediators related to apoptosis, mitochondrial damage, and autophagy were determined. Results. STZ-administrated rats showed reduced body weight, increased blood glucose levels, elevated SDH activity and AGE content, suggesting successful establishment of the DC rat model. Interestingly, MFN2 expression was significantly downregulated in DC rat lens and HG-induced HLE-B3 cells. Further analysis showed that under HG conditions, MFN2 overexpression enhanced cell viability and inhibited apoptosis accompanied by decreased Bax, cleaved caspase-9 and increased Bcl-2 expression in HLE-B3 cells. MFN2 overexpression also suppressed the mitochondrial damage elicited by HG as manifested by reduced ROS production, recovered Δψm and increased mitochondrial cytochrome c (Cyto c) level. Moreover, MFN2 overexpression increased LC3BⅡ/LC3BⅠ ratio and Beclin-1 expression, but decreased p62 level, and blocked the phosphorylation of mTOR in HG-treated HLE-B3 cells. In contrast, MFN2 silencing exerted opposite effects. Conclusions. Presented findings indicate that MFN2 expression may be essential for preventing lens epithelial cell apoptosis during development of diabetic cataract. [ABSTRACT FROM AUTHOR]

Background: Ferroptosis is known to play a crucial role in diabetic osteopathy. However, key genes and molecular mechanisms remain largely unclear. This study aimed to identify a crucial ferroptosis-related differentially expressed gene (FR-DEG) in diabetic osteopathy and investigate its potential mechanism. Methods: We identified fibronectin type III domain-containing protein 5 (FNDC5)/irisin as an essential FR-DEG in diabetic osteopathy using the Ferroptosis Database (FerrDb) and GSE189112 dataset. Initially, a diabetic mouse model was induced by intraperitoneal injection of streptozotocin (STZ), followed by intraperitoneal injection of irisin. MC3T3-E1 cells treated with high glucose (HG) were used as an in vitro model. FNDC5 overexpression plasmid was used to explore underlying mechanisms in vitro experiments. Femurs were collected for micro-CT scan, histomorphometry, and immunohistochemical analysis. Peripheral serum was collected for ELISA analysis. Cell viability was assessed using a CCK-8 kit. The levels of glutathione (GSH), malondialdehyde (MDA), iron, reactive oxygen species (ROS), and lipid ROS were detected by the corresponding kits. Mitochondria ultrastructure was observed through transmission electron microscopy (TEM). Finally, mRNA and protein expressions were examined by quantitative real‐time PCR (qRT‐PCR) and western blot analysis. Results: The expression of FNDC5 was found to be significantly decreased in both in vivo and in vitro models. Treatment with irisin significantly suppressed ferroptosis and improved bone loss. This was demonstrated by reduced lipid peroxidation and iron overload, increased antioxidant capability, as well as the inhibition of the ferroptosis pathway in bone tissues. Furthermore, in vitro studies demonstrated that FNDC5 overexpression significantly improved HG-induced ferroptosis and promoted osteogenesis. Mechanistic investigations revealed that FNDC5 overexpression mitigated ferroptosis in osteoblasts by inhibiting the eukaryotic initiation factor 2 alpha (eIF2α)/activated transcription factor 4 (ATF4)/C/EBP-homologous protein (CHOP) pathway. Conclusions: Collectively, our study uncovered the important role of FNDC5/irisin in regulating ferroptosis of diabetic osteopathy, which might be a potential therapeutic target. [ABSTRACT FROM AUTHOR]

The immune infiltration profiles of the tumor microenvironment have effects on the prognosis of head and neck squamous cell carcinoma (HNSCC). Whereas, HNSCC is a heterogeneous group of tumors, but past work has not taken this into consideration. Herein, we investigate the associations between survival and the function of immune cells in different tumorigenic sites of HNSCC. 1149 samples of HNSCC were collected from publicly accessible databases. Based on gene expression data, CIBERSORTx was applied to determine the proportion of 22 immune cell subpopulations. In the Cox regression model, the associations between overall survival, disease-free survival, and immune cells were examined, modeling gene expression and immune cell proportion as quartiles. Consensus cluster analysis was utilized to uncover immune infiltration profiles. Regardless of tumor sites, CD8+ T cells and activated CD4 memory T cells were associated with favorable survival, while eosinophils were the opposite. The survival of the hypopharynx, oral cavity, and larynx subsites was somewhat affected by immune cells, while the survival of the oropharynx subsite potentially was the most impacted. High expression of TIGIT, CIITA, and CXCR6 was linked to better survival, mainly in the oropharynx subsite. Immune cell clusters with four distinct survival profiles were discovered, of which the cluster with a high CD8+ T cell content had a better prognosis. The immune-infiltration pattern is related to the survival of HNSCC to varying degrees depending on the tumor sites; forthcoming studies into immune-mediated infiltration profiles will lay the groundwork for treating HNSCC with precision therapy. [ABSTRACT FROM AUTHOR]

Phragmenthera capitata (Spreng.) Balle and Globimetula braunii (Engler.) Van Tiegh are African mistletoe traditionally used in cancers treatment. Thus, the aim of the study was to assess the anti-melanoma potential of the methanol extract of Phragmenthera capitata (Spreng.) Balle (PCMe-OH) and Globimetula braunii (Engler.) (GBMe-OH) Van Tiegh. Antioxidant potential was evaluated using DPPH, FRAP and hydroxyl assays. Total flavonoid and phenolic contents was also determined. MTT assay was used to estimate the effects on cell viability using SK-MLE28 and B16-F10 cell lines. Colony formation and wound healing were also assessed. Fluorometry methods were used for qualitative analysis of apoptosis and estimate ROS production. Western blot analysis was used for protein expression. Phragmenthera capitata (PCMe-OH) showed the highest antioxidant activity and possess the highest phenolic contents (1,490.80 ± 55 mgGAE/g extract) in comparison with G. braunii (GBMe-OH) and (1,071.40 ± 45 mgGAE/g extract). Flavonoid content was similar in both extracts (11.63 ± 5.51 mg CATE/g of extract and 12.46 ± 2.58 mg CATE/g of extract respectively). PC-MeOH showed the highest cytotoxicity effect (IC50 of 55.35 ± 1.17 μg/mL) and exhibited anti-migrative potential on B16-F10 cells. Furthermore, PC-MeOH at 55.35 and 110.7 μg/mL; promoted apoptosis-induced cell death in B16-F10 cells by increasing intracellular ROS levels and reducing Bcl-2 expression level at 110.7 μg/mL. Significant upregulation of P-PTEN expression was recorded with PC-MeOH at 110.7 μg/mL; inhibiting therefore PI3K/AKT/m-Tor signaling pathway. Moreover, at 55.37 μg/mL significant reduction of c-myc and cyclin D1 was observed; dysregulating the MAPK kinase signaling pathway and cell cycle progression. Phragmenthera capitata may be developed into selective chemotherapy to fight against melanoma. [ABSTRACT FROM AUTHOR]

Simple Summary: The interaction between the highly aggressive triple-negative breast cancer, and the neutrophils is connected to the poor outcomes of this tumor. Using a wide array of cellular and molecular essays, we found that triple-negative breast cancer cells stressed out the neutrophil-like cells and reduced their viability in cell culture conditions. Furthermore, growing neutrophil-like cells with breast cancer cells enhanced the inflammatory activity of the neutrophil-like cells through the release and expression of chemical or structural inflammatory mediators. Given that cancer cells can exploit the active inflammatory milieu to the advantage of their growth and spread, targeting the crosstalk mechanisms that we illustrated here could help in defining potential therapeutic options in breast cancer management. Breast cancer (BC) is one of the most common types of cancer in women in the United Arab Emirates. Immunogenic tumours, such as triple-negative breast cancer (TNBC), show increased neutrophil infiltration, which is associated with poor prognosis and limited efficacy of immunotherapy. This study aims to investigate in vitro the bidirectional effect of neutrophils on metastatic TNBC (MDA-MB-231) compared to less-metastatic luminal breast cancer (MCF-7) cell lines. We found that BC cells or their conditioned medium (CM) reduced the viability of neutrophil-like cells (HL60). This was supported by increased cellular stress and NETosis in differentiated HL60 cells (dHL60) upon exposure to MDA-MB-231 compared to MCF-7-CM using nucleic acid staining essays. Flow cytometry showed comparable expression of inflammatory markers by polymorphonuclear cells (PMN) when treated with MDA-MB-231-CM and standard polarizing cocktails. Furthermore, MDA-MB-231-CM triggered an inflammatory pattern with evidence of stronger adhesion (CD62L) and degranulation (CD11b and CD66b) phenotypes. The proinflammatory polarization of dHL60 by MDA-MB-231-CM was additionally confirmed by the elevated CD54 expression, myeloperoxidase, and CD11b protein levels, which matched an increased transwell migratory capacity. In conclusion, BC might use neutrophils to their benefit through NETosis and complement system activation, which makes this crosstalk a potential mechanism for understanding tumour progression. [ABSTRACT FROM AUTHOR]

Colorectal carcinoma (CRC) is a common cancer associated with poor prognosis, and cudraflavone C (Cud C) is a natural flavonol with reported anti-CRC capacity. However, the precise mechanisms underlying the anti-CRC effect require further demonstration. The aim of present study was to evaluate the impact of Cud C on the cell viability and apoptosis of CRC cells and to determine the underlying mechanisms. The Human Protein Atlas (THPA) and Gene Expression Profiling Interactive Analysis (GEPIA) databases were used to analyze the expression status of glutathione peroxidase 2 (GPX2) in CRC. Cell viability was examined using cell counting kit-8 (CCK-8) assay. Flow cytometry was utilized to evaluate apoptosis. The levels of gene transcription and protein expression of GPX2, caspase-3, cleaved caspase-3), β-catenin, and c-Myc were determined by RT-qPCR and Western blotting. Our results showed that GPX2 was overexpressed in CRC as compared to normal tissue and the extent of GPX2 overexpression is greatest in CRC when compared with other cancers according to GEPIA and THPA databases. GPX2 knockdown significantly suppressed the cell viability, induced apoptosis of CRC cell lines, and restrained the activity of Wnt/β-catenin pathway. Cud C treatment decreased cell viability, induced apoptosis in CRC cell lines, and diminished the expression level of GPX2-dependent activation of Wnt/β-catenin pathway, while such effects can be abolished by GPX2 overexpression. In conclusion, Cud C suppressed GPX2-dependent Wnt/β-catenin pathway to exert anti-CRC function. [ABSTRACT FROM AUTHOR]

Simple Summary: This research explores how the low-molecular microphthalmia-associated transcription factor (MITF) specific inhibitor ML329 affects malignant melanoma (MM) cells' biology. ML329 significantly reduced cell viability in specific melanoma cell lines and differently influenced their metabolic functions. ML329 also substantially and differently affected the ability to form 3D spheroids and altered the expression of genes related to regulatory and signaling factors among various MM cell lines. These diverse effects across different melanoma types underscore the complexity of MITF-related activities among various types of MM, aiding the development of a more effective understanding of MMs' pathophysiology as well as their targeted therapies. To study the inhibitory effects on microphthalmia-associated transcription factor (MITF)-related biological aspects in malignant melanomas (MMs) in the presence or absence of the low-molecular MITF specific inhibitor ML329, cell viability, cellular metabolic functions, and three-dimensional (3D) spheroid formation efficacy were compared among MM cell lines including SK-mel-24, A375, dabrafenib- and trametinib-resistant A375 (A375DT), and WM266-4. Upon exposure to 2 or 10 μM of ML329, cell viability was significantly decreased in WM266-4, SK-mel-24, and A375DT cells, but not A375 cells, in a dose-dependent manner, and these toxic effects of ML329 were most evident in WM266-4 cells. Extracellular flux assays conducted using a Seahorse bioanalyzer revealed that treatment with ML329 increased basal respiration, ATP-linked respiration, proton leakage, and non-mitochondrial respiration in WM266-4 cells and decreased glycolytic function in SK-mel-24 cells, whereas there were no marked effects of ML329 on A375 and A375DT cells. A glycolytic stress assay under conditions of high glucose concentrations also demonstrated that the inhibitory effect of ML329 on the glycolytic function of WM266-4 cells was dose-dependent. In addition, ML329 significantly decreased 3D-spheroid-forming ability, though the effects of ML329 were variable among the MM cell lines. Furthermore, the mRNA expression levels of selected genes, including STAT3 as a possible regulator of 3D spheroid formation, KRAS and SOX2 as oncogenic-signaling-related factors, PCG1a as the main regulator of mitochondrial biogenesis, and HIF1a as a major hypoxia transcriptional regulator, fluctuated among the MM cell lines, possibly supporting the diverse ML329 effects mentioned above. The findings of diverse ML329 effects on various MM cell lines suggest that MITF-associated biological activities are different among various types of MM. [ABSTRACT FROM AUTHOR]

Background: Clostridioides difficile infection (CDI) is one of the most common health care-acquired infections. The dramatic increase in antimicrobial resistance of C. difficile isolates has led to growing demand to seek new alternative medicines against CDI. Achillea millefolium L. extracts exhibit strong biological activity to be considered as potential therapeutic agents. In this work, the inhibitory effects of A. millefolium, its decoction (DEC) and ethanol (ETOH) extracts, were investigated on the growth of C. difficile RT001 and its toxigenic cell-free supernatant (Tox-S) induced inflammation and apoptosis. Methods: Phytochemical analysis of extracts was performed by HPLC and GC analysis. The antimicrobial properties of extracts were evaluated against C. difficile RT001. Cell viability and cytotoxicity of Caco-2 and Vero cells treated with various concentrations of extracts and Tox-S were examined by MTT assay and microscopy, respectively. Anti-inflammatory and anti-apoptotic effects of extracts were assessed in Tox-S stimulated Caco-2 cells by RT-qPCR. Results: Analysis of the phytochemical profile of extracts revealed that the main component identified in both extracts was chlorogenic acid. Both extracts displayed significant antimicrobial activity against C. difficile RT001. Moreover, both extracts at concentration 50 µg/mL had no significant effect on cell viability compared to untreated cells. Pre-treatment of cells with extracts (50 µg/mL) significantly reduced the percentage of Vero cells rounding induced by Tox-S. Also, both pre-treatment and co-treatment of Tox-S stimulated Caco-2 cells with extracts significantly downregulated the gene expression level of IL-8, IL-1β, TNF-α, TGF-β, iNOS, Bax, caspase-9 and caspase-3 and upregulated the expression level of Bcl-2. Conclusion: The results of the present study for the first time demonstrate the antimicrobial activity and protective effects of A. millefolium extracts on inflammatory response and apoptosis induced by Tox-S from C. difficile RT001 clinical strain in vitro. Further research is needed to evaluate the potential application of A. millefolium extracts as supplementary medicine for CDI prevention and treatment in clinical setting. [ABSTRACT FROM AUTHOR]

Background and Objectives: Mesenchymal stem cells hold promise for tissue regeneration, given their robust growth and versatile differentiation capabilities. An analysis of bone marrow-sourced mesenchymal stem cell proliferation showed that 17β-estradiol could enhance their growth. This study aims to investigate the influence of 17β-estradiol on the shape, survival, osteogenic differentiation, and mineralization of human mesenchymal stem cells. Materials and Methods: Spheroids made from human gingiva-derived stem cells were cultivated with varying concentrations of 17β-estradiol: 0, 0.01, 0.1, 1, and 10 nM. Morphology was assessed on days 1, 3, and 5. The live/dead kit assay was employed on day 3 for qualitative cell viability, while cell counting kit-8 was used for quantitative viability assessments on days 1, 3, and 5. To evaluate the osteogenic differentiation of the spheroids, a real-time polymerase chain reaction assessed the expressions of RUNX2 and COL1A1 on day 7. Results: The stem cells formed cohesive spheroids, and the inclusion of 17β-estradiol did not noticeably alter their shape. The spheroid diameter remained consistent across concentrations of 0, 0.01, 0.1, 1, and 10 nM of 17β-estradiol. However, cellular viability was boosted with the addition of 1 and 10 nM of 17β-estradiol. The highest expression levels for RUNX2 and COL1A1 were observed with the introduction of 17β-estradiol at 0.1 nM. Conclusions: In conclusion, from the results obtained, it can be inferred that 17β-estradiol can be utilized for differentiating stem cell spheroids. Furthermore, the localized and controlled use, potentially through localized delivery systems or biomaterials, can be an area of active research. While 17β-estradiol holds promise for enhancing stem cell applications, any clinical use requires a thorough understanding of its mechanisms, careful control of its dosage and delivery, and extensive testing to ensure safety and efficacy. [ABSTRACT FROM AUTHOR]