Three Undescribed Protoilludane-type Sesquiterpene Aryl Esters from Armillaria gallica.

Three previously undescribed protoilludane-type sesquiterpene aryl esters, armillanals A-C (1-3), along with seven known ones (4-10) were obtained from Armillaria gallica Marxm. & Romagn. Compounds 1 and 2 were a rare class of sesquiterpenes featuring the Δ2(3) and Δ12(13)-protoilludane skeleton. Their structures were established by extensive spectroscopic methods. Based on electronic circular dichroism (ECD) calculations, the absolute configurations of three new compounds (1-3) were determined. The anti-inflammatory activity of compounds 1-10 was screened and compound 3 could dose-dependently decrease the level of lactate dehydrogenase, showing IC50 value of 4.525 µM.

Ion Release and Apatite Formation of Resin Based Pit and Fissure Sealants Containing 45S5 Bioactive Glass.

The purpose of this study was to evaluate a resin based pit and fissure sealant containing 45S5 bioactive glass (BAG) by examining its ion release, pH variation, and apatite-forming properties. To prepare the experimental materials, 45S5 BAG, used as a filler, was incorporated into the light curable resin matrix at concentrations of 0 (control), 12.5, 37.5, and 50.0 wt.%. Ion release, pH variation, and apatite formation (Raman spectrometer and scanning electron microscopy-energy-dispersive X-ray spectrometry measurements) were performed. While no ions were released from the control group, the experimental groups containing 45S5 BAG showed an increased release of Ca and P ions with increasing amounts of 45S5 BAG (p < 0.05). The pH of the experimental group remained high and was significantly different from the control group (p < 0.05). Unlike the control group, it was confirmed that the apatite peak was formed in the 50.0 wt.% BAG group for 90 days, and the apatite layer consisting of Ca and P was deposited on the surface. Thus, a resin based pit and fissure sealant containing 45S5 BAG is a promising material for preventing secondary caries by releasing ions and forming apatite.

Expanding the Structural Diversity of Tubulin-Targeting Agents: Development of Highly Potent Benzimidazoles for Treating Fungal Diseases.

Benzimidazoles, the representative pharmacophore of fungicides, have excellent antifungal potency, but their simple structure and single site of action have hindered their wider application in agriculture. In order to extend the structural diversity of tubulin-targeted benzimidazoles, novel benzimidazole derivatives were prepared by introducing the attractive pyrimidine pharmacophore. 2-((6-(4-(trifluoromethyl)phenoxy)pyrimidin-4-yl)thio)-1H-benzo[d]imidazole (A25) exhibited optimal antifungal activity against Sclerotinia sclerotiorum (S. s.), affording an excellent half-maximal effective concentration (EC50) of 0.158 µg/mL, which was higher than that of the reference agent carbendazim (EC50 = 0.594 µg/mL). Pot experiments revealed that compound A25 (200 µg/mL) had acceptable protective activity (84.7%) and curative activity (78.1%), which were comparable with that of carbendazim (protective activity: 90.8%; curative activity: 69.9%). Molecular docking displayed that multiple hydrogen bonds and π-π interactions could be formed between A25 and ß-tubulin, resulting in a stronger bonding effect than carbendazim. Fluorescence imaging revealed that the structure of intracellular microtubules can be changed significantly after A25 treatment. Overall, these remarkable antifungal profiles of constructed novel benzimidazole derivatives could facilitate the application of novel microtubule-targeting agents.

In vitro antifungal and physicochemical properties of polymerized acrylic resin containing strontium-modified phosphate-based glass.

Acrylic resins are widely used as the main components in removable orthodontic appliances. However, poor oral hygiene and maintenance of orthodontic appliances provide a suitable environment for the growth of pathogenic microorganisms. In this study, strontium-modified phosphate-based glass (Sr-PBG) was added to orthodontic acrylic resin at 0% (control), 3.75%, 7.5%, and 15% by weight to evaluate the surface and physicochemical properties of the novel material and its in vitro antifungal effect against Candida albicans (C. albicans). Surface microhardness and contact angle did not vary between the control and 3.75% Sr-PBG groups (p > 0.05), and the flexural strength was lower in the experimental groups than in the control group (p < 0.05), but no difference was found with Sr-PBG content (p > 0.05). All experimental groups showed an antifungal effect at 24 and 48 h compared to that in the control group (p < 0.05). This study demonstrated that 3.75% Sr-PBG exhibits antifungal effects against C. albicans along with suitable physicochemical properties, which may help to minimize the risk of adverse effects associated with harmful microbial living on removable orthodontic appliances and promote the use of various materials.

Near-Barrierless CO Oxidation Using Phosphotungstic Acid-Supported Single-Atom Catalysts.

Efficient CO oxidation at ambient or low temperatures is essential for environmental purification and selective CO oxidation in H2, yet achieving this remains a challenge with current methodologies. In this research, we extensively evaluated the catalytic performance of phosphotungstic acid (PTA)-supported 11 M1/PTA single-atom catalysts (SACs) using density functional theory calculations across both gas phase and 12 common solvents. The Rh1/PTA, Pd1/PTA, and Pt1/PTA systems exhibit moderate CO adsorption energies, facilitating the feasibility of oxygen vacancy formation. Remarkably, the Pd1/PTA and Pt1/PTA catalysts exhibited negligible energy barriers and demonstrated exceptionally high catalytic rates, with values reaching up to (1 × 1010)11, markedly exceeding the threshold for room temperature reactions, set at 6.55 × 108. This phenomenon is attributed to a transition from the high-energy barrier processes of oxygen dissociation in O2 and N-O bond dissociation in N2O to the more efficient dissociation of H2O2. Orbital analysis and charge variations at metal sites throughout the reaction process provide deeper insights into the role of the three metal catalytic sites in CO activation. Our findings not only reveal key aspects of SACs in facilitating CO oxidation at low temperatures but also provide valuable insights for future catalytic reaction mechanism studies and environmental applications.

Sustainable Production of Biofuels from Biomass Feedstocks Using Modified Montmorillonite Catalysts.

The rampant exploitation of fossil fuels has led to the significant energy scarcity and environmental disruption, affecting the sound momentum of development and progress of human civilization. To build a closed-loop anthropogenic carbon cycle, development of biofuels employing sustainable biomass feedstocks stands at the forefront of advancing carbon neutrality, yet its widespread adoption is mainly hampered by the high production costs. Montmorillonite, however, has garnered considerable attention serving as an efficient heterogeneous catalyst of ideal economic feasibility for biofuel production, primarily due to its affordability, accessibility, stability, and excellent plasticity. Up to now, nevertheless, it has merely received finite concerns and interests in production of various biofuels using montmorillonite-based catalysts. There is no timely and comprehensive review that addresses this latest relevant progress. This review fills the gap by providing a systematically review and summary in controllable synthesis, performance enhancement, and applications related to different kinds of biofuels including biodiesel, biohydrogenated diesel, levulinate, γ-valerolactone, 5-ethoxymethylfurfural, gaseous biofuels (CO, H2), and cycloalkane, by using montmorillonite catalysts and its modified forms. Particularly, this review critically depicts the design strategies for montmorillonite, illustrates the relevant reaction mechanisms, and assesses their economic viability, realizing sustainable biofuels production via efficient biomass valorization.

Clinical management of extra­adrenal myelolipoma in the central nervous system: A case report.

Extra-adrenal myelolipoma (EAM) is a rare benign tumor composed of mature adipose and hematopoietic tissues. Its etiology remains to be elucidated and there are few case reports describing the clinical features and treatment of EAMs in the central nervous system. The present study presented our experience and practice in the clinical management of a case of EAM in the right frontal region. A 56-year-old woman was found to have a space-occupying right frontal lesion on computed tomography (CT) of the head. Unenhanced magnetic resonance imaging (MRI) showed a lesion of ~1.5x1.2 cm. Enhanced whole abdominal CT showed a right presacral mass, 2.0 cm in diameter, with clear margins. The postoperative histopathological findings showed mainly mature adipose tissue mixed with extramedullary hematopoietic components. This confirmed the diagnosis of a (bone) marrow lipoma. Myelolipoma of the central nervous system is extremely rare. to the best of the authors' knowledge, only two cases of intracranial myelolipoma have been reported, and the present study introduced the first case in a Chinese patient reported in English. However, when CT shows high density and MRI shows mixed density in the tumor area even without enhancement, the possibility of myelolipoma should be considered in the differential diagnosis.

The haplotype-resolved genome of diploid Chrysanthemum indicum unveils new acacetin synthases genes and their evolutionary history.

Acacetin, a flavonoid compound, possesses a wide range of pharmacological effects, including antimicrobial, immune regulation, and anticancer effects. Some key steps in its biosynthetic pathway were largely unknown in flowering plants. Here, we present the first haplotype-resolved genome of Chrysanthemum indicum, whose dried flowers contain abundant flavonoids and have been utilized as traditional Chinese medicine. Various phylogenetic analyses revealed almost equal proportion of three tree topologies among three Chrysanthemum species (C. indicum, C. nankingense, and C. lavandulifolium), indicating that frequent gene flow among Chrysanthemum species or incomplete lineage sorting due to rapid speciation might contribute to conflict topologies. The expanded gene families in C. indicum were associated with oxidative functions. Through comprehensive candidate gene screening, we identified five flavonoid O-methyltransferase (FOMT) candidates, which were highly expressed in flowers and whose expressional levels were significantly correlated with the content of acacetin. Further experiments validated two FOMTs (CI02A009970 and CI03A006662) were capable of catalyzing the conversion of apigenin into acacetin, and these two genes are possibly responsible acacetin accumulation in disc florets and young leaves, respectively. Furthermore, combined analyses of ancestral chromosome reconstruction and phylogenetic trees revealed the distinct evolutionary fates of the two validated FOMT genes. Our study provides new insights into the biosynthetic pathway of flavonoid compounds in the Asteraceae family and offers a model for tracing the origin and evolutionary routes of single genes. These findings will facilitate in vitro biosynthetic production of flavonoid compounds through cellular and metabolic engineering and expedite molecular breeding of C. indicum cultivars.

Synthesis of organic-inorganic hybrid nanocomposites modified by catalase-like catalytic sites for the controlling of kiwifruit bacterial canker.

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. Actinidiae (Psa), is one of the most important diseases in kiwifruit, creating huge economic losses to kiwifruit-growing countries around the world. Metal-based nanomaterials offer a promising alternative strategy to combat plant diseases induced by bacterial infection. However, it is still challenging to design highly active nanomaterials for controlling kiwifruit bacterial canker. Here, a novel multifunctional nanocomposite (ZnO@PDA-Mn) is designed that integrates the antibacterial activity of zinc oxide nanoparticles (ZnO NPs) with the plant reactive oxygen species scavenging ability of catalase (CAT) enzyme-like active sites through introducing manganese modified polydopamine (PDA) coating. The results reveal that ZnO@PDA-Mn nanocomposites can efficiently catalyze the conversion of H2O2 to O2 and H2O to achieve excellent CAT-like activity. In vitro experiments demonstrate that ZnO@PDA-Mn nanocomposites maintain the antibacterial activity of ZnO NPs and induce significant damage to bacterial cell membranes. Importantly, ZnO@PDA-Mn nanocomposites display outstanding curative and protective efficiencies of 47.7% and 53.8% at a dose of 200 µg mL-1 against Psa in vivo, which are superior to those of zinc thiozole (20.6% and 8.8%) and ZnO (38.7% and 33.8%). The nanocomposites offer improved in vivo control efficacy through direct bactericidal effects and decreasing oxidative damage in plants induced by bacterial infection. Our research underscores the potential of nanocomposites containing CAT-like active sites in plant protection, offering a promising strategy for sustainable disease management in agriculture.

High accuracy model for HBsAg loss based on longitudinal trajectories of serum qHBsAg throughout long-term antiviral therapy.

OBJECTIVE: Hepatitis B surface antigen (HBsAg) loss is the optimal outcome for patients with chronic hepatitis B (CHB) but this rarely occurs with currently approved therapies. We aimed to develop and validate a prognostic model for HBsAg loss on treatment using longitudinal data from a large, prospectively followed, nationwide cohort. DESIGN: CHB patients receiving nucleos(t)ide analogues as antiviral treatment were enrolled from 50 centres in China. Quantitative HBsAg (qHBsAg) testing was prospectively performed biannually per protocol. Longitudinal discriminant analysis algorithm was used to estimate the incidence of HBsAg loss, by integrating clinical data of each patient collected during follow-up. RESULTS: In total, 6792 CHB patients who had initiated antiviral treatment 41.3 (IQR 7.6-107.6) months before enrolment and had median qHBsAg 2.9 (IQR 2.3-3.3) log10IU/mL at entry were analysed. With a median follow-up of 65.6 (IQR 51.5-84.7) months, the 5-year cumulative incidence of HBsAg loss was 2.4%. A prediction model integrating all qHBsAg values of each patient during follow-up, designated GOLDEN model, was developed and validated. The AUCs of GOLDEN model were 0.981 (95% CI 0.974 to 0.987) and 0.979 (95% CI 0.974 to 0.983) in the training and external validation sets, respectively, and were significantly better than those of a single qHBsAg measurement. GOLDEN model identified 8.5%-10.4% of patients with a high probability of HBsAg loss (5-year cumulative incidence: 17.0%-29.1%) and was able to exclude 89.6%-91.5% of patients whose incidence of HBsAg loss is 0. Moreover, the GOLDEN model consistently showed excellent performance among various subgroups. CONCLUSION: The novel GOLDEN model, based on longitudinal qHBsAg data, accurately predicts HBsAg clearance, provides reliable estimates of functional hepatitis B virus (HBV) cure and may have the potential to stratify different subsets of patients for novel anti-HBV therapies.

Engineering a biomimicking strategy for discovering nonivamide-based quorum-sensing inhibitors for controlling bacterial infection.

The overuse of antibiotics over an extended period has led to increasing antibiotic resistance in pathogenic bacteria, culminating in what is now considered a global health crisis. To tackle the escalating disaster caused by multidrug-resistant pathogens, the development of new bactericides with new action mechanism is highly necessary. In this study, using a biomimicking strategy, a series of new nonivamide derivatives that feature an isopropanolamine moiety [the structurally similar to the diffusible signal factor (DSF) of Xanthomonas spp.] were prepared for serving as potential quorum-sensing inhibitors (QSIs). After screening and investigation of their rationalizing structure-activity relationships (SARs), compound A26 was discovered as the most optimal active molecule, with EC50 values of 9.91 and 7.04 µg mL-1 against Xanthomonas oryzae pv oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac). A docking study showed that compound A26 exhibited robust interactions with Glu A: 161 of RpfF, which was strongly evidenced by fluorescence titration assay (KA value for Xoo RpfF-A26 = 104.8709 M-1). Furthermore, various bioassays showed that compound A26 could inhibit various bacterial virulence factors, including biofilm formation, extracellular polysaccharides (EPS), extracellular enzyme activity, DSF production, and swimming motility. In addition, in vivo anti-Xoo results showed that compound A26 had excellent control efficiency (curative activity: 43.55 %; protective activity: 42.56 %), surpassing that of bismerthiazol and thiodiazole copper by approximately 8.0%-37.3 %. Overall, our findings highlight a new paradigm wherein nonivamide derivatives exhibit potential in combating pathogen resistance issues by inhibiting bacterial quorum sensing systems though attributing to their new molecular skeleton, novel mechanisms of action, and non-toxic features.

Hematopoietic stem cell division is governed by distinct RUNX1 binding partners.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

SLC40A1-related hemochromatosis associated with a p.Y333H mutation in mainland China: a pedigree report and literature review.

BACKGROUND: Haemochromatosis is a genetic disease characterized by the excessive deposition of iron in various tissues and organs, eventually results in organ damage including cirrhosis, diabetes, cardiomyopathy, etc. SLC40A1-related haemochromatosis is associated with gain-of-function mutations in the SLC40A1 gene, which encodes ferroportin. While sporadic reports of this condition exist in mainland China, the understanding of the phenotype and genetic pattern associated with the SLC40A1 p.Y333H mutation remains incomplete. CASE PRESENTATION: We report a pedigree with heterozygous p.Y333H mutation in Chinese Han population. The proband is a 64-year-old man complaining of persistent abnormality of liver enzyme levels for 1 year, with a history of knee joint pain, diabetes and skin pigmentation. He displayed markedly elevated serum ferritin level and transferrin saturation. Magnetic resonance imaging showed iron deposition in the liver, spleen, and pancreas, along with cirrhosis and splenomegaly. Whole exome sequencing identified a heterozygous allelic variant c.997T > C (p.Y333H). Genetic screening of family members identified four first-degree relatives and three second-degree relatives having the same mutation. Additional cases with this mutation from two published studies were included. Among the probands and screened relatives, all eight males aged over 30 y had ferritin level > 1000 µg/L, transferrin saturation > 90%. Four patients with organ damage in the present study received therapeutic phlebotomy, alleviating clinical symptoms and improving in transferrin saturation and serum ferritin. CONCLUSIONS: This study reports the largest pedigree with heterozygous SLC40A1 p.Y333H mutation in the Chinese population to date. In Chinese families, males over 30 years old with hemochromatosis due to SLC40A1 p.Y333H mutation exhibit severe iron overload phenotypes.

A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma.

Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this TGFb Induced Enhancer driving green fluorescent protein (TIE:EGFP). TIE:EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of advanced melanomas. Single-cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response while up-regulating a novel set of chronic TGFb target genes. ChIP-sequencing demonstrated that AP-1 factor binding is required for activation of chronic TGFb response. Overexpression of SATB2, a chromatin remodeler associated with tumor spreading, showed activation of TGFb signaling in early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages localize to TIE:EGFP+ regions and preferentially phagocytose TIE:EGFP+ melanoma cells compared to TIE:EGFP- melanoma cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.

Melatonin activates Nrf2/HO-1 signalling pathway to antagonizes oxidative stress-induced injury via melatonin receptor 1 (MT1) in cryopreserved mice ovarian tissue.

Our previous research has shown that melatonin (MLT) can reduce cryopreserved ovarian damage in mice. Yet, the molecular mechanism of MLT protection is still unclear. Some studies have shown that melatonin receptor 1 (MT1) is very important for animal reproductive system. To evaluate whether MLT exerts its protective effect on cryopreserved mice ovarian tissue via MT1, we added antagonist of MT1/MT2 (Luzindor) or antagonist of MT2 (4P-PDOT) to the freezing solution, followed by cryopreservation and thawing of ovarian tissue. The levels of total superoxide dismutase (T-SOD), catalase (CAT), nitric oxide (NO) and malondialdehyde (MDA) were detected. Besides, by using RT-PCR and Western blotting, the expression of Bcl-2, Bax and Nrf2/HO-1 signalling pathway-related proteins was detected. These findings demonstrated that compared with the melatonin group, the addition of Luzindor increased apoptosis, NO and MDA activities, decreased CAT and T-SOD activities and inhibited Nrf2/HO-1 signalling pathway. In conclusion, melatonin can play a protective role in cryopreserved ovarian tissue of mice through MT1 receptor.

Identification of novel 4-substituted 7H-pyrrolo[2,3-d]pyrimidine derivatives as new FtsZ inhibitors: Bioactivity evaluation and computational simulation.

Bacterial infections and the consequent outburst of bactericide-resistance issues are fatal menace to both global health and agricultural produce. Hence, it is crucial to explore candidate bactericides with new mechanisms of action. The filamenting temperature-sensitive mutant Z (FtsZ) protein has been recognized as a new promising and effective target for new bactericide discovery. Hence, using a scaffold-hopping strategy, we designed new 7H-pyrrolo[2,3-d]pyrimidine derivatives, evaluated their antibacterial activities, and investigated their structure-activity relationships. Among them, compound B6 exhibited the optimal in vitro bioactivity (EC50 = 4.65 µg/mL) against Xanthomonas oryzae pv. oryzae (Xoo), which was superior to the references (bismerthiazol [BT], EC50 = 48.67 µg/mL; thiodiazole copper [TC], EC50 = 98.57 µg/mL]. Furthermore, the potency of compound B6 in targeting FtsZ was validated by GTPase activity assay, FtsZ self-assembly observation, fluorescence titration, Fourier-transform infrared spectroscopy (FT-IR) assay, molecular dynamics simulations, and morphological observation. The GTPase activity assay showed that the final IC50 value of compound B6 against XooFtsZ was 235.0 µM. Interestingly, the GTPase activity results indicated that the B6-XooFtsZ complex has an excellent binding constant (KA = 103.24 M-1). Overall, the antibacterial behavior suggests that B6 can interact with XooFtsZ and inhibit its GTPase activity, leading to bacterial cell elongation and even death. In addition, compound B6 showed acceptable anti-Xoo activity in vivo and low toxicity, and also demonstrated a favorable pharmacokinetic profile predicted by ADMET analysis. Our findings provide new chemotypes for the development of FtsZ inhibitors as well as insights into their underlying mechanisms of action.

Recent advances in the use of extracellular vesicles from adipose-derived stem cells for regenerative medical therapeutics.

Adipose-derived stem cells (ADSCs) are a subset of mesenchymal stem cells (MSCs) isolated from adipose tissue. They possess remarkable properties, including multipotency, self-renewal, and easy clinical availability. ADSCs are also capable of promoting tissue regeneration through the secretion of various cytokines, factors, and extracellular vesicles (EVs). ADSC-derived EVs (ADSC-EVs) act as intercellular signaling mediators that encapsulate a range of biomolecules. These EVs have been found to mediate the therapeutic activities of donor cells by promoting the proliferation and migration of effector cells, facilitating angiogenesis, modulating immunity, and performing other specific functions in different tissues. Compared to the donor cells themselves, ADSC-EVs offer advantages such as fewer safety concerns and more convenient transportation and storage for clinical application. As a result, these EVs have received significant attention as cell-free therapeutic agents with potential future application in regenerative medicine. In this review, we focus on recent research progress regarding regenerative medical use of ADSC-EVs across various medical conditions, including wound healing, chronic limb ischemia, angiogenesis, myocardial infarction, diabetic nephropathy, fat graft survival, bone regeneration, cartilage regeneration, tendinopathy and tendon healing, peripheral nerve regeneration, and acute lung injury, among others. We also discuss the underlying mechanisms responsible for inducing these therapeutic effects. We believe that deciphering the biological properties, therapeutic effects, and underlying mechanisms associated with ADSC-EVs will provide a foundation for developing a novel therapeutic approach in regenerative medicine.

Hydrated Calcium Silicate in Resin Composites for Prevention of Secondary Caries.

INTRODUCTION AND AIMS: The gaps at the margins of restorative composite resin can increase as the carious process occurs underneath the materials, causing further demineralization along the tooth cavity wall. The aim of this study was to evaluate the effects of restorative resin composite containing hydrated calcium silicate (hCS) filler on enamel protection against demineralization by simulating microleakage between the test material and teeth in a cariogenic environment. METHODS: The experimental resin composites were composed of 70 wt.% filler, which was mixed with a glass filler and hCS in a weight ratio of 70.0% glass (hCS 0), 17.5% hCS + 52.5% glass (hCS 17.5), 35.0% hCS + 35.0% glass (hCS 35.0), and 52.5% hCS + 17.5% glass (hCS 52.5). A light-cured experimental resin composite disk was positioned over a polished bovine enamel disk, separated by a 30-µm gap, and immersed in artificial saliva with pH 4.0 for 15, 30, and 60 days. After the immersion period, the enamel disk was separated from the resin composite disk and evaluated using a microhardness tester, atomic force microscopy, and polarized light microscopy. The opposing sides of the enamel and resin composite disks were observed using scanning electron microscopy/energy dispersive X-ray spectrometry. RESULTS: The enamel surface showed a significant increase in microhardness, decreased roughness, and remineralization layer as the proportion of hCS increased (P < .05). In the scanning electron microscopy image, the enamel surface with hCS 35.0 and 52.5 after all experimental immersion periods, showed a pattern similar to that of a sound tooth. CONCLUSIONS: The results demonstrated that increasing the hCS filler level of restorative resin composites significantly decreased enamel demineralization. CLINICAL RELEVANCE: Hydrated calcium silicate laced restorative resin composites may be a promising dental biomaterial for protecting teeth against demineralization and preventing secondary caries around restorations.