Objective Infertility affects approximately one-sixth of the people of childbearing age worldwide,causing not only economic burdens of treatment for families with fertility problems but also psychological stress for patients and presenting challenges to societal and economic development.Premature ovarian insufficiency(POI)refers to the loss of ovarian function in women before the age of 40 due to the depletion of follicles or decreased quality of remaining follicles,constituting a significant cause of female infertility.In recent years,with the help of the rapid development in genetic sequencing technology,it has been demonstrated that genetic factors play a crucial role in the onset of POI.Among the population suffering from POI,genetic studies have revealed that genes involved in processes such as meiosis,DNA damage repair,and mitosis account for approximately 37.4% of all pathogenic and potentially pathogenic genes identified.FA complementation group M(FANCM)is a group of genes involved in the damage repair of DNA interstrand crosslinks(ICLs),including FANCA-FANCW.Abnormalities in the FANCM genes are associated with female infertility and FANCM gene knockout mice also exhibit phenotypes similar to those of POI.During the genetic screening of POI patients,this study identified a suspicious variant in FANCM.This study aims to explore the pathogenic mechanisms of the FANCM genes of the FA pathway and their variants in the development of POI.We hope to help shed light on potential diagnostic and therapeutic strategies for the affected individuals.Methods One POI patient was included in the study.The inclusion criteria for POI patients were as follows:women under 40 years old exhibiting two or more instances of basal serum follicle-stimulating hormone levels>25 IU/L(with a minimum interval of 4 weeks inbetween tests),alongside clinical symptoms of menstrual disorders,normal chromosomal karyotype analysis results,and exclusion of other known diseases that can lead to ovarian dysfunction.We conducted whole-exome sequencing for the POI patient and identified pathogenic genes by classifying variants according to the standards and guidelines established by the American College of Medical Genetics and Genomics(ACMG).Subsequently,the identified variants were validated through Sanger sequencing and subjected to bioinformatics analysis.Plasmids containing wild-type and mutant FANCM genes were constructed and introduced into 293T cells.The 293T cells transfected with wild-type and mutant human FANCM plasmids and pEGFP-C1 empty vector plasmids were designated as the EGFP FANCM-WT group,the EGFP FANCM-MUT group,and the EGFP group,respectively.To validate the production of truncated proteins,cell proteins were extracted 48 hours post-transfection from the three groups and confirmed using GFP antibody.In order to investigate the impact on DNA damage repair,immunofluorescence experiments were conducted 48 hours post-transfection in the EGFP FANCM-WT group and the EGFP FANCM-MUT group to examine whether the variant affected FANCM's ability to localize on chromatin.Mitomycin C was used to induce ICLs damage in vitro in both the EGFP FANCM-WT group and the EGFP FANCM-MUT group,which was followed by verification of its effect on ICLs damage repair using γ-H2AX antibody.Results In a POI patient from a consanguineous family,we identified a homozygous variant in the FANCM gene,c.1152-1155del:p.Leu386Valfs*10.The patient presented with primary infertility,experiencing irregular menstruation since menarche at the age of 16.Hormonal evaluation revealed an FSH level of 26.79 IU/L and an anti-Müllerian hormone(AMH)level of 0.07 ng/mL.Vaginal ultrasound indicated unsatisfactory visualization of the ovaries on both sides and uterine dysplasia.The patient's parents were a consanguineous couple,with the mother having regular menstrual cycles.The patient had two sisters,one of whom passed away due to osteosarcoma,while the other exhibited irregular menstruation,had been diagnosed with ovarian insufficiency,and remained childless.Bioinformatics analysis revealed a deletion of four nucleotides(c.1152-1155del)in the exon 6 of the patient's FANCM gene.This variant resulted in a frameshift at codon 386,introducing a premature stop codon at codon 396,which ultimately led to the production of a truncated protein consisting of 395 amino acids.In vitro experiments demonstrated that this variant led to the production of a truncated FANCM protein of approximately 43 kDa and caused a defect in its nuclear localization,with the protein being present only in the cytoplasm.Following treatment with mitomycin C,there was a significant increase in γ-H2AX levels in 293T cells transfected with the mutant plasmid(P<0.01),indicating a statistically significant impairment of DNA damage repair capability caused by this variant.Conclusions The homozygous variant in the FANCM gene,c.1152-1155del:p.Leu386Valfs*10,results in the production of a truncated FANCM protein.This truncation leads to the loss of its interaction site with the MHF1-MHF2 complex,preventing its entry into the nucleus and the subsequent recognition of DNA damage.Consequently,the localization of the FA core complex on chromatin is disrupted,impeding the normal activation of the FA pathway and reducing the cell's ability to repair damaged ICLs.By disrupting the rapid proliferation and meiotic division processes of primordial germ cells,the reserve of oocytes is depleted,thereby triggering premature ovarian insufficiency in females.

In recent years,nano-silver base inorganic antibacterial agents have been widely used in medicinal and biological fields.The recent research progress on nano-silver base inorganic antibacterial agents used in denture resin base,antibacterial effects,biocompatibility and the influence of the agents on the mechanical properties and color of the resin are introduced.

OBJECTIVEThis study further explores the stromal cell-derived factor-1 (SDF-1)/chemokine receptor 4 (CXCR4) signaling axis mechanism in temporomandibular joint osteoarthritis (OA) by detecting the changes in CXCR4, interleukin (IL)-6, and collagen X expression in the ATDC5 cell line stimulated by the cyclic tensile strain and SDF-1.

METHODSInsulin-transferrin-selenium (ITS) was used to induce ATDC5 cells to differentiate into chondrocyte-like cells. After three weeks, the cells were divided into two groups: those with and without cyclic tensile strain. These groups were further divided into the negative control and SDF-1 groups. Strain force of 20% was applied. After 12 h, the total proteins were extracted from cells of the four groups, and Western blot analysis was used to detect the changes in CXCR4, IL-6, and collagen X expression.

RESULTSSDF-1 could enhance CXCR4, IL-6, and collagen X expressions in the chondrocytes, and 20% tensile strain force could further upregulate the three factors.

CONCLUSIONUnder abnormal tensile force, SDF-1 can upregulate its specific receptor CXCR4, thus increasing its-binding efficiency and resulting in the activation of the SDF-1/CXCR4 axis. This condition enhances the expressions of IL-6 and other inflammatory factors and directly damages to cartilage tissue. Such damage directly promotes chondrocyte hypertrophy, which enhances collagen X expression.

Cell Differentiation ; Cell Line ; Chemokine CXCL12 ; Collagen ; Humans ; Interleukin-6 ; Receptors, CXCR4 ; Signal Transduction ; Stromal Cells

Objective This study further explores the stromal cell-derived factor-1 (SDF-1)/chemokine receptor 4 (CXCR4) signaling axis mechanism in temporomandibular joint osteoarthritis (OA) by detecting the changes in CXCR4, interleukin (IL)-6, and collagen X expression in the ATDC5 cell line stimulated by the cyclic tensile strain and SDF-1. Methods Insulin-transferrin- selenium(ITS) was used to induce ATDC5 cells to differentiate into chondrocyte-like cells. After three weeks, the cells were divided into two groups: those with and without cyclic tensile strain. These groups were further divided into the negative control and SDF-1 groups. Strain force of 20% was applied. After 12 h, the total proteins were extracted from cells of the four groups, and Western blot analysis was used to detect the changes in CXCR4, IL-6, and collagen X expression. Results SDF-1 could enhance CXCR4, IL-6, and collagen X expressions in the chondrocytes, and 20% tensile strain force could further upregulate the three factors. Conclusion Under abnormal tensile force, SDF-1 can upregulate its specific receptor CXCR4, thus increasing its binding efficiency and resulting in the activation of the SDF-1/CXCR4 axis. This condition enhances the expressions of IL-6 and other inflammatory factors and directly damages to cartilage tissue. Such damage directly promotes chondrocyte hypertrophy, which enhances collagen X expression.

OBJECTIVETo investigate the effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on the stability of resin-dentin bonds against pH cycling.

METHODSResin-bonded dentin specimens were prepared following manufacturers' instructions, and randomly divided into 3 groups. Among them, 2 groups experienced pH cycling, in which specimens applied CPP-ACP or distilled and deionized water (DDW) on the bonding interface, respectively. Microtensile bond strength (muTBS) testing, failure mode analysis, micromorphological and nanoleakage evaluation of bonding interface and elemental analysis within hybrid layer were performed after 15 days pH cycling. The other group was tested immediately after specimens' preparation without pH cycling.

RESULTSNo significant differences were found in muTBS between no pH cycling and pH cycling/CPP-ACP group. Their muTBS were both significantly higher than that of pH cycling/DDW group (P < 0.05). Mixed fractures were the most prevalent failure mode. The quality of hybrid layer in pH cycling/CPP-ACP group was better than that of pH cycling/DDW group, and the nanoleakage was also less severe. Comparing with pH cycling/DDW group, the atomic percentages of Ca in the other two groups were both significantly higher, while those of Ag were statistically lower (P < 0.05).

CONCLUSIONLocal application of CPP-ACP can promote the stability of resin-dentin bonding interface against pH cycling and prolong bonding degeneration.

Calcium Phosphates ; Caseins ; Dental Bonding ; Dentin ; Dentin-Bonding Agents ; Humans ; Phosphopeptides ; Resin Cements

OBJECTIVE: To investigate the feasibility of a novel molecular probe of Zn-DPA-PSS794 to monitor the efficiency of doxorubicin to ovarian cancer and compare with Cy5.5-annexin V. METHODS: Efficiency of doxorubicin to OVCAR-8 cells in vitro was measured by MTT assay and flow cytometry. The in vivo studies were performed on an OVCAR-8 xenograft tumor model. Mice were divided into a control group and a treatment group. Each group was divided into 2 subgroups, DPA and annexin V. In the treatment group, the mice were treated with doxorubicin for 2 doses. All mice were performed optical imaging by Zn-DPA-PSS794 or Cy5.5-annexin V, respectively and then sacrificed. The tumor was separated and stained by HE. The expression of caspase-3 protein was measured by Western blot. RESULTS: The IC50 of doxorubicin to OVCAR-8 was 6 μmol/L. The percentage of apoptosis and dead cells was 35% after doxorubicin treatment. In the optical image, photons accumulated in the tumor either by Zn-DPA-PSS794 or Cy 5.5-annexin V in the treatment group. That was negative in the control group. The fluorescence intensity had significant difference between the 2 groups(P<0.001). The nuclei were big and stained with deep color after the cells were stained with HE. The caspase-3 expression was high in the treatment group, while it was low in the control group. CONCLUSION Zn-DPA-PSS794 as a probe used by optical imaging can monitor the efficiency of doxorubicin to OVCAR-8 xenograft tumor, which is similar to Cy5.5-annexin V.
Animals ; Apoptosis ; drug effects ; Carbocyanines ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Female ; Fluorescent Dyes ; Humans ; Infrared Rays ; Mice ; Mice, Nude ; Molecular Imaging ; Organometallic Compounds ; Ovarian Neoplasms ; pathology ; Picolines ; Spectroscopy, Near-Infrared ; methods