Loss of Tet hydroxymethylase activity causes mouse embryonic stem cell differentiation bias and developmental defects.

The TET family is well known for active DNA demethylation and plays important roles in regulating transcription, the epigenome and development. Nevertheless, previous studies using knockdown (KD) or knockout (KO) models to investigate the function of TET have faced challenges in distinguishing its enzymatic and nonenzymatic roles, as well as compensatory effects among TET family members, which has made the understanding of the enzymatic role of TET not accurate enough. To solve this problem, we successfully generated mice catalytically inactive for specific Tet members (Tetm/m). We observed that, compared with the reported KO mice, mutant mice exhibited distinct developmental defects, including growth retardation, sex imbalance, infertility, and perinatal lethality. Notably, Tetm/m mouse embryonic stem cells (mESCs) were successfully established but entered an impaired developmental program, demonstrating extended pluripotency and defects in ectodermal differentiation caused by abnormal DNA methylation. Intriguingly, Tet3, traditionally considered less critical for mESCs due to its lower expression level, had a significant impact on the global hydroxymethylation, gene expression, and differentiation potential of mESCs. Notably, there were common regulatory regions between Tet1 and Tet3 in pluripotency regulation. In summary, our study provides a more accurate reference for the functional mechanism of Tet hydroxymethylase activity in mouse development and ESC pluripotency regulation.

A multi-task generative model for simultaneous post-contrast MR image synthesis and brainstem glioma segmentation.

OBJECTIVES: This study aims to generate post-contrast MR images reducing the exposure of gadolinium-based contrast agents (GBCAs) for brainstem glioma (BSG) detection, simultaneously delineating the BSG lesion, and providing high-resolution contrast information. METHODS: A retrospective cohort of 30 patients diagnosed with brainstem glioma was included. Multi-contrast images, including pre-contrast T1 weighted (pre-T1w), T2 weighted (T2w), arterial spin labeling (ASL) and post-contrast T1w images, were collected. A multi-task generative model was developed to synthesize post-contrast T1w images and simultaneously segment BSG masks from the multi-contrast inputs. Performance evaluation was conducted using peak signal-to-noise ratio (PSNR), structural similarity index (SSIM), and mean absolute error (MAE) metrics. A perceptual study was also undertaken to assess diagnostic quality. RESULTS: The proposed model achieved SSIM of 0.86 ± 0.04, PSNR of 26.33 ± 0.05 and MAE of 57.20 ± 20.50 for post-contrast T1w image synthesis. Automated delineation of the BSG lesions achieved Dice similarity coefficient (DSC) score of 0.88 ± 0.27. CONCLUSIONS: The proposed model can synthesize high-quality post-contrast T1w images and accurately segment the BSG region, yielding satisfactory DSC scores. CLINICAL RELEVANCE STATEMENT: The synthesized post-contrast MR image presented in this study has the potential to reduce the usage of gadolinium-based contrast agents, which may pose risks to patients. Moreover, the automated segmentation method proposed in this paper aids radiologists in accurately identifying the brainstem glioma lesion, facilitating the diagnostic process.

Metallothionein 3 Potentiates Pulmonary Artery Smooth Muscle Cell Proliferation by Promoting Zinc-MTF1-ATG5 Axis-mediated Autophagosome Formation.

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is gradually being adopted as an important direction for the treatment of PH. Metallothioneins (MTs) have been reported to be associated with PH, but the underlying mechanisms are not fully understood. Here, we demonstrated that the expression level of metallothionein 3 (MT3) was significantly increased in pulmonary arterioles from PH patients and chronic hypoxia-induced rat and mouse PH models, as well as in hypoxia-treated human PASMCs. Knockdown of MT3 significantly inhibited the proliferation of human PASMCs by arresting the cell cycle in the G1 phase, while overexpression of MT3 had the opposite effect. Mechanistically, we found that MT3 increased the intracellular zinc (Zn2+) concentration to enhance the transcriptional activity of metal-regulated transcription factor 1 (MTF1), which promoted the expression of autophagy-related gene 5 (ATG5), facilitating autophagosome formation. More importantly, MT3-induced autophagy and proliferation of human PASMCs were largely prevented by knockdown of MTF1 and ATG5. Therefore, in this study, we identified MT3-Zinc-MTF1-ATG5 as a novel pathway that affects PASMC proliferation by regulating autophagosome formation, suggesting that MT3 may be a novel target for the treatment of PH.

Antitumor Effect of Anti-c-Myc Aptamer-Based PROTAC for Degradation of the c-Myc Protein.

Targeting "undruggable" targets with intrinsically disordered structures is of great significance for the treatment of disease. The transcription factor c-Myc controls global gene expression and is an attractive therapeutic target for multiple types of cancers. However, due to the lack of defined ligand binding pockets, targeted c-Myc have thus far been unsuccessful. Herein, to address the dilemma of lacking ligands, an efficient and high throughput aptamer screening strategy is established, named polystyrene microwell plate-based systematic evolution of ligands by exponential enrichment (microwell-SELEX), and identify the specific aptamer (MA9C1) against c-Myc. The multifunctional aptamer-based Proteolysis Targeting Chimeras (PROTAC) for proteolysis of the c-Myc (ProMyc) is developed using the aptamer MA9C1 as the ligand. ProMyc not only significantly degrades c-Myc by the ubiquitin-proteasome system, but also reduces the Max protein, synergistically inhibiting c-Myc transcriptional activity. Combination of the artificial cyclization and anti-PD-L1 aptamer (PA1)-based delivery system, circular PA1-ProMyc chimeras achieve tumor regression in the xenograft tumor model, laying a solid foundation for the development of efficacious c-Myc degrader for the clinic. Therefore, this aptamer-based degrader provides an invaluable potential degrader in drug discovery and anti-tumor therapy, offering a promising degrader to overcome the challenge of targeting intractable targets.

Defining a TFAP2C-centered transcription factor network during murine peri-implantation.

Transcription factors (TFs) play important roles in early embryonic development, but factors regulating TF action, relationships in signaling cascade, genome-wide localizations, and impacts on cell fate transitions during this process have not been clearly elucidated. In this study, we used uliCUT&RUN-seq to delineate a TFAP2C-centered regulatory network, showing that it involves promoter-enhancer interactions and regulates TEAD4 and KLF5 function to mediate cell polarization. Notably, we found that maternal retinoic acid metabolism regulates TFAP2C expression and function by inducing the active demethylation of SINEs, indicating that the RARG-TFAP2C-TEAD4/KLF5 axis connects the maternal-to-zygotic transition to polarization. Moreover, we found that both genomic imprinting and SNP-transferred genetic information can influence TF positioning to regulate parental gene expressions in a sophisticated manner. In summary, we propose a ternary model of TF regulation in murine embryonic development with TFAP2C as the core element and metabolic, epigenetic, and genetic information as nodes connecting the pathways.

ADNP modulates SINE B2-derived CTCF-binding sites during blastocyst formation in mice.

CTCF is crucial for chromatin structure and transcription regulation in early embryonic development. However, the kinetics of CTCF chromatin occupation in preimplantation embryos have remained unclear. In this study, we used CUT&RUN technology to investigate CTCF occupancy in mouse preimplantation development. Our findings revealed that CTCF begins binding to the genome prior to zygotic genome activation (ZGA), with a preference for CTCF-anchored chromatin loops. Although the majority of CTCF occupancy is consistently maintained, we identified a specific set of binding sites enriched in the mouse-specific short interspersed element (SINE) family B2 that are restricted to the cleavage stages. Notably, we discovered that the neuroprotective protein ADNP counteracts the stable association of CTCF at SINE B2-derived CTCF-binding sites. Knockout of Adnp in the zygote led to impaired CTCF binding signal recovery, failed deposition of H3K9me3, and transcriptional derepression of SINE B2 during the morula-to-blastocyst transition, which further led to unfaithful cell differentiation in embryos around implantation. Our analysis highlights an ADNP-dependent restriction of CTCF binding during cell differentiation in preimplantation embryos. Furthermore, our findings shed light on the functional importance of transposable elements (TEs) in promoting genetic innovation and actively shaping the early embryo developmental process specific to mammals.

TSP50 Attenuates DSS-Induced Colitis by Regulating TGF-ß Signaling Mediated Maintenance of Intestinal Mucosal Barrier Integrity.

The integrity of the intestinal mucosal barrier is crucial for protecting the intestinal epithelium against invasion by commensal bacteria and pathogens, thereby combating colitis. The investigation revealed that the absence of TSP50 compromised the integrity of the intestinal mucosal barrier in murine subjects. This disruption facilitated direct contact between intestinal bacteria and the intestinal epithelium, thereby increasing susceptibility to colitis. Mechanistic analysis indicated that TSP50 deficiency in intestinal stem cells (ISCs) triggered aberrant activation of the TGF-ß signaling pathway and impeded the differentiation of goblet cells in mice, leading to impairment of mucosal permeability. By inhibiting the TGF-ß pathway, the functionality of the intestinal mucosal barrier is successfully restored and mitigated colitis in TSP50-deficient mice. In conclusion, TSP50 played a crucial role in maintaining the intestinal mucosal barrier function and exhibited the preventive effect against the development of colitis by regulating the TGF-ß signaling pathway.

Testicular Toxicity in Rats Exposed to AlCl3: a Proteomics Study.

Aluminum contamination is a growing environmental and public health concern, and aluminum testicular toxicity has been reported in male rats; however, the underlying mechanisms of this toxicity are unclear. The objective of this study was to investigate the effects of exposure to aluminum chloride (AlCl3) on alterations in the levels of sex hormones (testosterone [T], luteinizing hormone [LH], and follicle-stimulating hormone [FSH]) and testicular damage. Additionally, the mechanisms of toxicity in the testes of AlCl3-exposed rats were analyzed by proteomics. Three different concentrations of AlCl3 were administered to rats. The results demonstrated a decrease in T, LH, and FSH levels with increasing concentrations of AlCl3 exposure. HE staining results revealed that the spermatogenic cells in the AlCl3-exposed rats were widened, disorganized, or absent, with increased severe tissue destruction at higher concentrations of AlCl3 exposure. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses revealed that differentially expressed proteins (DEPs) after AlCl3 exposure were primarily associated with various metabolic processes, sperm fibrous sheath, calcium-dependent protein binding, oxidative phosphorylation, and ribosomes. Subsequently, DEPs from each group were subjected to protein-protein interaction (PPI) analysis followed by the screening of interactional key DEPs. Western blot experiments validated the proteomics data, revealing the downregulation of sperm-related DEPs (AKAP4, ODF1, and OAZ3) and upregulation of regulatory ribosome-associated protein (UBA52) and mitochondrial ribosomal protein (MRPL32). These findings provide a basis for studying the mechanism of testicular toxicity due to AlCl3 exposure.

Reactive Oxygen Species-Mediated Mitophagy and Cell Apoptosis are Involved in the Toxicity of Aluminum Chloride Exposure in GC-2spd.

Aluminum chloride is an inorganic polymeric coagulant commonly found in daily life and various materials. Although male reproductive toxicity has been associated with AlCl3 exposure, the underlying mechanism remains unclear. This study aimed to examine the impact of AlCl3 exposure on mitophagy and mitochondrial apoptosis in testicular tissue and mouse spermatocytes. Reactive oxygen species (ROS) and ATP levels were measured in GC-2spd after AlCl3 exposure using a multifunctional enzyme labeler. The changes in mitochondrial membrane potential (MMP) and TUNEL were observed through confocal laser microscopy, and the expression of proteins associated with mitophagy and apoptosis was analyzed using Western blot. Our results demonstrated that AlCl3 exposure disrupted mitophagy and increased apoptosis-related protein expression in testicular tissues. In the in vitro experiments, AlCl3 exposure induced ROS production, suppressed cell viability and ATP production, and caused a decrease in MMP, leading to mitophagy and cell apoptosis in GC-2spd cells. Intervention with N-acetylcysteine (NAC) reduced ROS production and partially restored mitochondrial function, thereby reversing the resulting mitophagy and cell apoptosis. Our findings provide evidence that ROS-mediated mitophagy and cell apoptosis play a crucial role in the toxicity of AlCl3 exposure in GC-2spd. These results contribute to the understanding of male reproductive toxicity caused by AlCl3 exposure and offer a foundation for future research in this area.

Copper exposure induces ovarian granulosa cell apoptosis by activating the caspase-dependent apoptosis signaling pathway and corresponding changes in microRNA patterns.

Environmental copper (Cu) contamination is a complex worldwide public health problem. However, information on the effects of Cu pollution on human reproduction is limited. Although our previous studies have indicated that Cu exposure disrupts ovarian folliculogenesis, the underlying mechanism needs to be further explored. In this study, human luteinized ovarian granulosa cells and a rat animal model were used to investigate whether Cu exposure affects ovarian follicle development by inducing apoptosis and to elucidate the possible mechanisms. The results showed that Cu exposure from weaning to sexual maturity significantly decreased the proportion of preantral follicles but increased the proportion of atretic follicles (P < 0.05). In addition, 6 mg/kg Cu increased the proportion of antral follicles, while 12 and 25 mg/kg Cu decreased it (P < 0.05). We also found that 6 mg/kg Cu exposure inhibited apoptosis of ovarian granulosa cells, while 12 and 25 mg/kg Cu promoted apoptosis (P < 0.05). Experiments on primary human luteinized ovarian granulosa cells suggested that higher levels of Cu exposure induced a significant increase in the mRNA levels of Bcl2 Bax , Fas, Caspase8, and Caspase3 (P < 0.05), and the protein levels of BAX, BCL2, CASPASE3, CASPASE8, CLE-CASPASE3, CLE-CASPASE8 and BAX/BCL2 were also increased (P < 0.05). miRNA chip analyses identified a total of 95 upregulated and 10 downregulated miRNAs in human luteinized granulosa cells exposed to Cu. Hsa-miR-19b-3p, hsa-miR-19a-3p, miR-548ar-3p, hsa-miR-652-5p, and hsa-miR-29b-5p were decreased after Cu exposure (P < 0.05). Additionally, the level of hsa-miR-144-5p was increased (P < 0.05). Together, our results reveal that Cu exposure induces abnormal ovarian folliculogenesis by inducing ovarian granulosa cell apoptosis, which is triggered by the caspase-dependent apoptosis signaling pathway, and that miRNAs may be involved in this process.

Tectorigenin targets PKACα to promote GLUT4 expression in skeletal muscle and improve insulin resistance in vitro and in vivo.

The decreased expression and dysfunction of glucose transporter 4 (GLUT4), the insulin-responsive glucose transporter, are closely related to the occurrence of insulin resistance (IR). To improve the expression of GLUT4 may represent a promising strategy to prevent and treat IR and type 2 diabetes (T2DM). Here, we demonstrate that the natural compound tectorigenin (TG) enhances GLUT4 expression, glucose uptake and insulin responsiveness via activating AMP-activated protein kinase (AMPK)/myocyte enhancer factor 2 (MEF2) signaling in both normal and IR skeletal muscle cells and tissues. Accordingly, prophylactic and therapeutic uses of TG can significantly ameliorate IR and hyperglycemia in T2DM mice. Mechanistically, we identify protein kinase A catalytic subunit α (PKACα) as the target of TG to increase GLUT4 expression and TG-PKACα binding promotes the dissociation of PKACα from the regulatory subunits, leading to the activation of PKA/AMPK signaling. PKACα knockdown in local quadriceps muscles almost completely abolished the therapeutic effects of TG on IR and T2DM, as well as the enhancement on AMPK signaling and GLUT4 expression in skeletal muscle. This study supports TG as a new drug candidate to treat IR and its related diseases, but also enriches our knowledge of PKA signaling in glucose metabolism in skeletal muscle.

Transcriptome sequencing identifies prognostic genes involved in gastric adenocarcinoma.

Gastric adenocarcinoma (GAC) is one of the world's most lethal malignant tumors. It has been established that the occurrence and progression of GAC are linked to molecular changes. However, the pathogenesis mechanism of GAC remains unclear. In this study, we sequenced 6 pairs of GAC tumor tissues and adjacent normal tissues and collected GAC gene expression profile data from the TCGA database. Analysis of this data revealed 465 differentially expressed genes (DEGs), of which 246 were upregulated and 219 were downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that DEGs were observably enriched in ECM-receptor interaction, protein digestion and absorption, and gastric acid secretion pathways. Six key genes (MATN3, COL1A1, COL5A2, P4HA3, SERPINE1 and VCAN) associated with poor GAC prognosis were screened from the protein‒protein interaction (PPI) network by survival analysis, and P4HA3 and MATN3 have rarely been reported to be associated with GAC. We further analyzed the function of P4HA3 in the GAC cell line SGC-7901 by RT‒qPCR, MTT, flow cytometry, colony formation, wound healing, Transwell and western blot assays. We found that P4HA3 was upregulated in the SGC-7901 cell line versus normal control cells. The outcomes of the loss-of-function assay illustrated that P4HA3 significantly enhanced the ability of GAC cells to proliferate and migrate. This study provides a new basis for the selection of prognostic markers and therapeutic targets for GAC.

Matrine alleviates depressive-like behaviors via modulating microbiota-gut-brain axis in CUMS-induced mice.

BACKGROUND: The realization of the "microbiota-gut-brain" axis plays a critical role in neuropsychiatric disorders, particularly depression, is advancing rapidly. Matrine is a natural bioactive compound, which has been found to possess potential antidepressant effect. However, the underlying mechanisms of regulation of the "microbiota-gut-brain" axis in the treatment of depression by oral matrine remain elusive. METHODS: Its antidepressant effects were initially evaluated by behavioral tests and relative levels of monoamine neurotransmitters, and matrine has been observed to attenuate the depression-like behavior and increase neurotransmitter content in CUMS-induced mice. Subsequently, studies from the "gut" to "brain" were conducted, including detection of the composition of gut microbiota by 16S rRNA sequencing; the metabolomics detection of gut metabolites and the analysis of differential metabolic pathways; the assessment of relative levels of diamine oxidase, lipopolysaccharide, pro-inflammatory cytokines, and brain-derived neurotrophic factor (BDNF) by ELISA kits or immunofluorescence. RESULTS: Matrine could regulate the disturbance of gut microbiota and metabolites, restore intestinal permeability, and reduce intestinal inflammation, thereby reducing the levels of pro-inflammatory cytokines in peripheral blood circulation and brain regions, and ultimately increase the levels of BDNF in brain. CONCLUSION: Matrine may ameliorate CUMS-induced depression in mice by modulating the "microbiota-gut-brain" axis.

STS1 and STS2 Phosphatase Inhibitor Baicalein Enhances the Expansion of Hematopoietic and Progenitor Stem Cells and Alleviates 5-Fluorouracil-Induced Myelosuppression.

STS1 and STS2, as the protein phosphatases that dephosphorylate FLT3 and cKIT, negatively regulate the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs). To obtain the small molecule inhibitors of STS1/STS2 phosphatase activity used to expand HSPCs both in vitro and in vivo, we establish an in vitro phosphatase assay using the recombinant proteins of the STS1/STS2 histidine phosphatase (HP) domain, by which we screened out baicalein (BC) as one of the effective inhibitors targeting STS1 and STS2. Then, we further demonstrate the direct binding of BC with STS1/STS2 using molecular docking and capillary electrophoresis and verify that BC can restore the phosphorylation of FLT3 and cKIT from STS1/STS2 inhibition. In a short-term in vitro culture, BC promotes profound expansion and enhances the colony-forming capacity of both human and mouse HSPCs along with the elevation of phospho-FLT3 and phospho-cKIT levels. Likewise, in vivo administration with BC significantly increases the proportions of short-term hematopoietic stem cells (ST-HSCs), multipotent progenitors (MPPs) and especially long-term HSCs (LT-HSCs) in healthy mouse bone marrow and increases the numbers of colony-forming units (CFU) formed by HSPCs as well. More importantly, pre-administration of BC significantly enhances the survival of mice with lethal 5-fluorouracil (5-FU) injection due to the alleviation of 5-FU-induced myelosuppression, as evidenced by the recovery of bone marrow histologic injury, the increased proportions of LT-HSCs, ST-HSCs and MPPs, and enhanced colony-forming capacity. Collectively, our study not only suggests BC as one of the small molecule candidates to stimulate HSPC expansion both in vitro and in vivo when needed in either physiologic or pathologic conditions, but also supports STS1/STS2 as potential therapeutic drug targets for HSPC expansion and hematopoietic injury recovery.

FRMD3 inhibits the growth and metastasis of breast cancer through the ubiquitination-mediated degradation of vimentin and subsequent impairment of focal adhesion.

Recurrence and metastasis are the main causes of breast cancer (BRCA)-related death and remain a challenge for treatment. In-depth research on the molecular mechanisms underlying BRCA progression has been an important basis for developing precise biomarkers and therapy targets for early prediction and treatment of progressed BRCA. Herein, we identified FERM domain-containing protein 3 (FRMD3) as a novel potent BRCA tumor suppressor which is significantly downregulated in BRCA clinical tissue and cell lines, and low FRMD3 expression has been closely associated with progressive BRCA and shortened survival time in BRCA patients. Overexpression and knockdown experiments have revealed that FRMD3 significantly inhibits BRCA cell proliferation, migration, and invasion in vitro and suppresses BRCA xenograft growth and metastasis in vivo as well. Mechanistically, FRMD3 can interact with vimentin and ubiquitin protein ligase E3A(UBE3A) to induce the polyubiquitin-mediated proteasomal degradation of vimentin, which subsequently downregulates focal adhesion complex proteins and pro-cancerous signaling activation, thereby resulting in cytoskeletal rearrangement and defects in cell morphology and focal adhesion. Further evidence has confirmed that FRMD3-mediated vimentin degradation accounts for the anti-proliferation and anti-metastasis effects of FRMD3 on BRCA. Moreover, the N-terminal ubiquitin-like domain of FRMD3 has been identified as responsible for FRMD3-vimentin interaction through binding the head domain of vimentin and the truncated FRMD3 with the deletion of ubiquitin-like domain almost completely loses the anti-BRCA effects. Taken together, our study indicates significant potential for the use of FRMD3 as a novel prognosis biomarker and a therapeutic target of BRCA and provides an additional mechanism underlying the degradation of vimentin and BRCA progression.

[Study of the association of lncRNA-GAS5 gene polymorphisms with systemic lupus erythematosus in Guangxi population].

OBJECTIVE: To assess the association of rs55829688 and rs75315904 polymorphisms of the lncRNA-GAS5 gene with susceptibility to systemic lupus erythematosus (SLE) in Guangxi population. METHODS: Peripheral venous blood samples were collected from the SLE group and control group. Following extraction of genomic DNA, SNPscan and Sanger sequencing were carried out to determine the genotypes for the rs55829688 and rs75315904 loci of the lncRNA-GAS5 gene. RESULTS: No difference was found between the two groups with regard to the genotypic frequencies for rs55829688 and rs75315904 (P > 0.05). However, the frequencies of C allele of rs55829688 between the two groups was significantly different (P < 0.05). In the SLE group, the frequencies of C allele and CT+CC genotype for rs55829688 among SLE patients with nephritis were significantly lower than those of SLE patients without nephritis (P < 0.05). In addition, haplotype analysis showed that the frequency of rs55829688 C/rs75315904 A allele in the SLE group was lower than that of the control group (P < 0.05). CONCLUSION: In Guangxi population, the carrier status of rs55829688 C allele of the lncRNA-GAS5 gene may reduce the risk of SLE and its complicated nephritis, and the rs55829688 C/rs75315904 A haplotype may reduce the risk for SLE.

Clinical Research Progress of Small Molecule Compounds Targeting Nrf2 for Treating Inflammation-Related Diseases.

Studies have found that inflammation is a symptom of various diseases, such as coronavirus disease 2019 (COVID-19) and rheumatoid arthritis (RA); it is also the source of other diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), lupus erythematosus (LE), and liver damage. Nrf2 (nuclear factor erythroid 2-related factor 2) is an important multifunctional transcription factor in cells and plays a central regulatory role in cellular defense mechanisms. In recent years, several studies have found a strong association between the activation of Nrf2 and the fight against inflammation-related diseases. A number of small molecule compounds targeting Nrf2 have entered clinical research. This article reviews the research status of small molecule compounds that are in clinical trials for the treatment of COVID-19, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, lupus erythematosus, and liver injury.

Discovery of Phenolic Glycoside from Hyssopus cuspidatus Attenuates LPS-Induced Inflammatory Responses by Inhibition of iNOS and COX-2 Expression through Suppression of NF-κB Activation.

It seems quite necessary to obtain effective substances from natural products against inflammatory response (IR) as there are presently clinical problems regarding accompanying side effects and lowered quality of life. This work aimed to investigate the abilities of hyssopuside (HY), a novel phenolic glycoside isolated from Hyssopus cuspidatus (H. cuspidatus), against IR in lipopolysaccharide (LPS)-induced RAW 264.7 cells and mouse peritoneal macrophages. The results indicated that HY could reduce nitric oxide (NO) production and inhibit the production and secretion of pro-inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) in LPS-stimulated macrophages. Moreover, data from the immunofluorescence study showed that HY suppressed nuclear translocation of nuclear factor-kappa B (NF-κB) upon LPS induction. The Western blot results suggested that HY reversed the LPS-induced degradation of IκB (inhibitor of NF-κB), which is normally required for the activation of NF-κB. Meanwhile, the overexpression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) diminished significantly with the presence of HY in response to LPS stimulation. On the other hand, HY had a negligible impact on the activation of mitogen-activated protein kinase (MAPK) pathways. Moreover, an in silico study of HY against four essential proteins/enzymes revealed that COX-2 was the most efficient enzyme for the interaction, and binding of residues Phe179, Asn351, and Ser424 with HY played crucial roles in the observed activity. The structure analysis indicated the typical characterizations with phenylethanoid glycoside contributed to the anti-inflammatory effects of HY. These results indicated that HY manipulated its anti-inflammatory effects mainly through blocking the NF-κB signal transduction pathways. Collectively, we believe that HY could be a potential alternative phenolic agent for alleviating excessive inflammation in many inflammation-associated diseases.

Ursolic Acid Targets Glucosyltransferase and Inhibits Its Activity to Prevent Streptococcus mutans Biofilm Formation.

Streptococcus mutans (S. mutans), the prime pathogen of dental caries, can secrete glucosyltransferases (GTFs) to synthesize extracellular polysaccharides (EPSs), which are the virulence determinants of cariogenic biofilms. Ursolic acid, a type of pentacyclic triterpene natural compound, has shown potential antibiofilm effects on S. mutans. To investigate the mechanisms of ursolic acid-mediated inhibition of S. mutans biofilm formation, we first demonstrated that ursolic acid could decrease the viability and structural integrity of biofilms, as evidenced by XTT, crystal violet, and live/dead staining assays. Then, we further revealed that ursolic acid could compete with the inherent substrate to occupy the catalytic center of GTFs to inhibit EPS formation, and this was confirmed by GTF activity assays, computer simulations, site-directed mutagenesis, and capillary electrophoresis (CE). In conclusion, ursolic acid can decrease bacterial viability and prevent S. mutans biofilm formation by binding and inhibiting the activity of GTFs.

Discovery and Verification of Key Liver Cancer Genes and Alternative Splicing Events Based on Second-Generation Sequencing Data Analysis.

Hepatocellular carcinoma (HCC) is the most common malignant liver disease in the world. Existing screening and early diagnosis methods are not highly sensitive for HCC, and patients are likely to develop the disease to the middle and advanced stages before being diagnosed. Therefore, finding new and efficient diagnosis and treatment methods has become an urgent problem. We aimed at finding and verifying new liver cancer markers by combining informatics analysis with experimental exploration to provide new ideas and methods for the diagnosis and treatment of clinical liver cancer. We used two different bioinformatic pipelines to analyze sequencing data of clinical liver cancer samples and identify differentially expressed genes and key variants after combining them with The Cancer Genome Atlas sequencing data. Then, we explored the functions and mechanisms of the key variants to identify potential liver cancer markers. Through bioinformatic analysis of sequencing data, 139 differentially expressed genes were found, including 53 upregulated genes and 86 downregulated genes. Through enrichment and alternative splicing event analysis of sequencing data, we found nine key variants with exon skipping events. Metallothionein 1E (MT1E)-203 was found to be a key variant that influenced cell proliferation through the p53 cell cycle pathway through cell viability and proliferation assays, and MT1E-203 lost the ability to bind two zinc ions due to exon skipping according to the structure prediction of MT1E-203. MT1E-203 is a potential biomarker for HCC and may play an important role in the diagnosis and treatment of HCC.