Investigating the cell membrane localization of PADI4 in breast cancer cells and inhibition of anti-PADI4 monoclonal antibody.

BACKGROUND: Peptidyl arginine deiminase 4 (PADI4) is a post-translational modification enzymecan that converts arginine in protein into citrulline in the presence of calcium ions, which is called citrullination. PADI4 has been reported to be expressed in the cytoplasm and nucleus in a variety of malignant tumors. Based on the GeneCards database and our previous research, it is speculated that PADI4 may also be expressed on the cell membrane. This study aimed to confirm the membrane expression of PADI4 and the effect of anti-PADI4 antibodies on cell membrane PADI4. This may be another mechanism of action of anti-PADI4 monoclonal antibodies in the treatment of breast cancer. METHODS: The subcellular localizations of PADI4 in MDA-MB-231 and MCF-7 breast cancer cells were determined by immunofluorescence, immunoelectron microscopy, and Western blot analysis. The tumor cells were treated with PADI4 antibody, and cell proliferation, migration, colony formation, apoptosis, glycolysis, and epithelial-mesenchymal transition (EMT) were measured as well as the expression of some essential tumor genes. RESULTS: PADI4 was not only localized in the nucleus and cytoplasm of breast cancer cells but was also detected on the cell membrane. Following PADI4 antibody treatment, cell proliferation, migration, colony formation, EMT, and ATP production through glycolysis were decreased, and the mRNA expression of MYC proto-oncogene (MYC), FAT atypical cadherin 1 (FAT1), nuclear factor kappa B subunit 1 (NFκB), and tumor necrosis factor (TNF-α) in breast cancer cells was downregulated, while the mRNA expression of tumor protein p63 (TP63) was upregulated. CONCLUSIONS: PADI4 is expressed on the cell membrane in breast cancer cells. Anti-PADI4 antibodies can affect the biological functions of cell membrane PADI4, including proliferation, migration, apoptosis, and glycolysis, thereby inhibiting tumor progression.

M1-Type Macrophages Secrete TNF-α to Stimulate Vascular Calcification by Upregulating CA1 and CA2 Expression in VSMCs.

Purpose: Vascular calcification is a hallmark of atherosclerosis (AS). We and others confirmed that carbonic anhydrase I (CA1) and CA2 increased expression and catalyzed calcium deposition in atherosclerotic aortas. Macrophages have been demonstrated to be strongly related to AS. This study aimed to clarify how and which macrophage subtypes regulate CA1 and CA2 expression to stimulate aortic calcification. Methods and Results: THP-1 cells were induced to form M0, M1 and M2 macrophage subtypes. These cells and their culture supernatants were separately incubated with human vascular smooth muscle cells (VSMCs). Calcification was strongly increased in VSMCs treated with ß-GP, a chemical inducer of cellular calcification, following incubation with M1 macrophages or their culture supernatants, and was much higher than that in VSMCs treated with ß-GP alone. Meanwhile, the expression of CA1 and CA2, as well as calcification marker genes, including Runx2, BMP-2 and ALP, was increased in VSMCs during this process. TNF-α levels were also increased in the culture medium of M1 macrophages. M0 and M2 macrophages or their supernatants did not significantly stimulate calcification in VSMCs. Following transfection with anti-CA1 or CA2 siRNAs, ß-GP-induced VSMCs showed decreased calcification, but the calcification level was partially increased when those VSMCs were incubated with the supernatants of M1 macrophages, while CA1 and CA2 expression as well as TNF-α levels were also elevated. When VSMCs were treated with TNF-α without ß-GP induction, calcification and the expression of CA1 and CA2 were also significantly increased. Conclusion: The results of this study suggest that M1 macrophages can increase CA1 and CA2 expression to promote atherosclerotic calcification in VSMCs by secreting TNF-α.

Compound 78c exerts a therapeutic effect on collagen-induced arthritis and rheumatoid arthritis.

OBJECTIVES: Many studies have found that CD38 expression is increased in rheumatoid arthritis (RA), an autoimmune disease in which immune tolerance is dysregulated. Inhibition of CD38 expression or activity can significantly alleviate collagen-induced arthritis (CIA), a well-known animal model used for the study of RA. This study aimed to confirm the therapeutic effect of 78c, a specific inhibitor of CD38, and the role of CD38+ NK cells in immune imbalance in RA. METHODS: CIA mice were injected with 78c to observe the therapeutic effect. CD38+ NK cells were extracted from human peripheral blood and treated with 78c. The pretreated NK cells were co-cultured with CD4+ T cells. RESULTS: We found that 78c significantly suppressed joint inflammation; reduced the levels of B cells, IL-6 and TNF-α; and increased the levels of IL-10, energy metabolism and spontaneous movement in CIA mice. 78c treatment also increased Treg cell numbers and decreased the Th1/Th2 ratio in the CIA model animals. Moreover, the proportion of CD38+ NK cells was increased in the CIA mice and significantly decreased following 78c treatment. Human CD4+ T cells that were co-cultured with 78c-pretreated CD38+ NK cells differentiated into more Treg cells and had lower Th17/Treg and Th1/Th2 ratios than CD4+ T cells co-cultured with CD38+ NK cells without the pretreatment. Transcriptomic analyses demonstrated that 78c changed expression pro les in CD38+ NK cells. CONCLUSIONS: These results suggested that 78c could be used for the treatment of RA and CIA as it alleviates the inhibitory effect of CD38+ NK cells on CD4+ T cell differentiation to Treg cells to restore immune balance.

Children parents attitude and demand towards family based child sexual abuse prevention education / 中国学校卫生

Objective@#To investigate the attitudes and demands of parents of children in Luzhou towards family based child sexual abuse prevention education.@*Methods@#A self administered anonymous questionnaire survey was conducted among parents of children in Luzhou City who were selected from stratified cluster sampling. Multiple linear regression model was used to analyze parents attitudes.@*Results@#Parents attitude towards prevention of sexual assault education was positive (average score 16.70± 3.67 ). The results of multiple regression analysis showed that parents of only child ( β =0.30), parents who participated in related activities ( β =1.28), communicated with relatives and friends ( β =0.81), and lived in urban areas ( β =0.49) had more positive attitudes. In terms of parental factors, average annual family income higher than 100 000 yuan ( β =0.39), mothers of young children ( β =0.88), and parents with a high level of knowledge about sexual assault prevention education ( β =0.98), the mother being a teacher or a medical staff ( β =0.52), and educational background of the mother being high school/secondary school ( β =1.03), college/undergraduate or above ( β =1.42) were associated with more positive attitudes( P <0.01). The results of demand analysis showed that parents had high demand for child s self protection (96.86%).@*Conclusion@#Parents of young children in Luzhou City show generally positive attitude and high demand towards family based sexual abuse prevention. Knowledge training and publicity regarding child sexual abuse should be improved for children who had siblings, from rural and township areas, and whose parents with low educational background.

Anti-PADI4 antibody suppresses breast cancer by repressing the citrullinated fibronectin in the tumor microenvironment.

BACKGROUND: PADI4, an enzyme catalyzing arginine residues to citrulline residues, is highly expressed in malignant tumors. This study prepared a monoclonal anti-human PADI4 antibody and investigated the therapeutic effect of the antibody on breast tumors and the functional mechanism. METHODS: After treatment with PADI4 antibody, the changes in tumor-bearing mice were examined by PET-CT, pathological assays, biochemical tests, routine blood tests, cytokine assays and metabolic assays. We used PADI4 recombinant protein to catalyze fibronectin (Fn) and then used citrullinated fibronectin (Cit-Fn) to culture MDA-MB-231 cells. We also treated Cit-Fn cultured cells with PADI4 antibody. The cultured cells were examined using cell proliferation, apoptosis, colony formation, migration and glycolic ATP production. Citrullination in the tumor tissues and peripheral blood was measured using Western blotting and ELISA, respectively. RESULTS: Following PADI4 antibody treatment, tumor growth was significantly suppressed, and the number of apoptotic cells in tumor tissues was increased. The citrullination level in peripheral blood and tumor tissues was decreased, EMT-related gene expression in tumors was also decreased, and the spontaneous movement of tumor-bearing mice was increased following treatment. Following antibody treatment, the serum concentrations of IL-10, IL-12p70, IL-23, ALT and AST were significantly decreased. MDA-MB-231 cells treated with Cit-Fn showed increased cell proliferation, cell migration, colony formation and glycolytic ATP production and decreased apoptosis. The growth and migration of MDA-MB-231 cells were reduced following PADI4 antibody treatment, and PADI4 antibody inhibited the citrullination of fibronectin in vitro. CONCLUSIONS: The PADI4 antibody had a therapeutic effect on breast tumors by inhibiting the citrullination of fibronectin to change the tumor tissue microenvironment. PADI4 antibody is a potential means for tumor treatment.

Strong association of glaucoma with atherosclerosis.

Carbonic anhydrases (CAs) catalyze the synthesis of HCO3- from H2O and CO2. The dysfunction of CAs leads to aqueous humor secretion and high intraocular pressure to cause glaucoma pathogenesis. Methazolamide (MTZ), a CA inhibitor, can effectively treat glaucoma by reducing aqueous humor secretion. We previously reported that carbonic anhydrase I (CA1), a CA family member, was highly expressed in atherosclerotic tissues of the aorta and stimulated atherosclerosis (AS) by promoting calcification. MTZ showed therapeutic and preventive effects on AS in a mouse model. The above findings suggest a relationship between AS and glaucoma. This study explored the possible association between AS prevalence and glaucoma prevalence and the therapeutic effect of MTZ on AS by analyzing medical records. Among 10,751 patients with a primary diagnosis of glaucoma, 699 (6.5%) were also diagnosed with AS. However, the incidences of AS in patients with keratitis and scleritis, which are also ophthalmic diseases, were 2.5% (206/8383 patients) and 3.5% (46/1308 patients), respectively. In the population without ophthalmic records, the AS prevalence was only 1.9% (99,416/5,168,481 patients) (all p values between each group were below 0.001). Among 152,425 patients with a primary diagnosis of AS, 1245 (0.82%) were also diagnosed with glaucoma. Among 199,782 patients with a primary diagnosis of hypertension (excluding AS), 1149 (0.57%) were diagnosed with glaucoma, and among 5,313,433 patients without AS or hypertension, 9513 (0.18%) were diagnosed with glaucoma (all p values between each group were below 0.001). Additionally, among 14 patients who suffered from both AS and glaucoma and were treated with MTZ to cure their glaucoma, 9 of them showed reduced low-density lipoprotein (LDL) levels, the main index of AS, within 3 months after medication use (2.81 ± 0.61 mmol/L vs. 2.38 ± 0.58 mmol/L, p = 0.039). The above findings demonstrated a strong relation between AS and glaucoma and suggested that AS patients with glaucoma were more likely to suffer from angle-closure glaucoma.

Fucoidan affects oral squamous cell carcinoma cell functions in vitro by regulating FLNA-derived circular RNA.

Oral squamous cell carcinoma (OSCC) is one of the most common cancer types, with a high annual incidence. Although chemotherapy contributes to suppressing OSCC tumorigenesis, the available treatments result in poor prognosis because of local recurrence and regional lymph node metastasis. Thus, it is necessary to discover novel and safe drugs with greater effectiveness and fewer side effects. Fucoidan is a component of the cell wall of brown seaweed that has been shown to produce a wide range of biological activities. The present study aimed to investigate the effectiveness of fucoidan in treating OSCC. In in vitro studies, we found that fucoidan inhibited OSCC growth and suppressed migration and invasion of OSCC cells. In addition, the potential interaction between fucoidan and filamin A (FLNA)-derived circular RNA (circFLNA) was predicted using bioinformatics databases and then confirmed in OSCC samples and cell lines. Indeed, fucoidan increased the expression of circFLNA in OSCC cell lines. Furthermore, both fucoidan and circFLNA could mediate the expression of key proteins related to cell growth, apoptosis, migration, and invasion. In conclusion, our research demonstrated that fucoidan might be considered as a potential natural drug in the treatment of OSCC patients by targeting circFLNA.

Amino Acid Conjugated Anthraquinones from the Marine-Derived Fungus Penicillium sp. SCSIO sof101.

Emodacidamides A-H (1-8), natural products featuring anthraquinone-amino acid conjugates, have been isolated from a marine-derived fungus, Penicillium sp. SCSIO sof101, together with known anthraquinones 9 and 10. The planar structures of 1-8 were elucidated using a combination of NMR spectroscopy and mass spectrometry. The absolute configurations of the amino acid residues were confirmed using Marfey's method and chiral-phase HPLC analyses. Additionally, isolates were evaluated for possible immunomodulatory and cytotoxic activities. Emodacidamides A (1), C (3), D (4), and E (5) inhibited interleukin-2 secretion from Jurkat cells with IC50 values of 4.1, 5.1, 12, and 5.4 µM, respectively.