Massively Parallel CRISPR-Cas9 Knockout Screening in Sheep Granulosa Cells for FSH Response Genes.

Follicle-stimulating hormone (FSH) regulates ovarian follicle development through specific gene expression programs. Granulosa cells (GCs) are somatic cells surrounding the oocytes, secreting gonadotropins to regulate ovulation and promote follicular development. By analyzing the effects of different doses of FSH on the proliferation of GCs, we found that adding 10 ng/mL of FSH, as the optimal concentration, could promote the growth of GCs. Furthermore, we have successfully constructed the first CRISPR-Cas9 knockout library targeting the genes on chromosomes 2 and 3 and the X chromosomes of the sheep massively parallel coding gene, as well as an ovarian GCs knockout cell library. For the first time, we have exposed the knockout cell library to a concentration of 10 ng/mL FSH to explore the underlying mechanisms. Through this screening, we have identified 836 positive-negative screening genes that are responsive to FSH, thereby revealing the regulatory mechanisms and screening the functionality of candidate genes. Next, RNA-Seq of control (0 ng/mL), low (10 ng/mL), and high (100 ng/mL) doses of FSH revealed 1708 differentially expressed genes, and combined with 836 genes, we obtained 129 FSH dose-dependent genes with extremely significant differences. This enables us to delve deeper into investigating and identifying the mechanisms by which FSH regulates GCs. More generally, we have discovered new regulatory factors and identified reproductivity-associated major effectors. These findings provide novel research directions for further studies on sheep reproduction.

Application of micro/nanorobot in medicine.

The development of micro/nanorobots and their application in medical treatment holds the promise of revolutionizing disease diagnosis and treatment. In comparison to conventional diagnostic and treatment methods, micro/nanorobots exhibit immense potential due to their small size and the ability to penetrate deep tissues. However, the transition of this technology from the laboratory to clinical applications presents significant challenges. This paper provides a comprehensive review of the research progress in micro/nanorobotics, encompassing biosensors, diagnostics, targeted drug delivery, and minimally invasive surgery. It also addresses the key issues and challenges facing this technology. The fusion of micro/nanorobots with medical treatments is poised to have a profound impact on the future of medicine.

Scanning iron response regulator binding sites using Dap-seq in the Brucella genome.

Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Previous studies suggested that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, in the present study, a Dap-seq approach was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes was identified using qRT-PCR. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), ftrA (BME_RS14525), cation-transporting P-type ATPase (zntA) (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. Furthermore, the iron utilization and growth assay experiments confirmed that rirA was involve in iron metabolism and growth of Brucella. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism, and the rirA was identified as a regulon of Irr and it also plays a role in iron metabolism of Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism.

Targeting Pokemon is a novel strategy to suppress cancer aggressiveness of non-small cell lung cancer: Identification of Pokemon as ideal target for developing anti-NSCLC drugs.

Although it is widely reported that Pokemon acts as an oncogene in the pathogenesis of multiple cancers, but its role and detailed molecular mechanisms in regulating non-small cell lung cancer (NSCLC) progression have not been fully delineated. Here, by performing Real-Time qPCR analysis, we verified that Pokemon was high-expressed in NSCLC tissues and cells, compared to the corresponding normal lung tissues and epithelial cells. Then, the small interfering RNA (siRNA) for Pokemon was transfected into the NSCLC cells to verify its biological functions, and our results suggested that silencing of Pokemon suppressed the malignant phenotypes, including cell viability, mitosis, colony formation, epithelial-mesenchymal transition (EMT), mobility and cancer stem cell (CSC) properties in NSCLC cells. Mechanistically, we confirmed that knockdown of Pokemon decreased the expression levels of phosphorylated Akt (p-Akt), phosphorylated GSK-3ß (p-GSK-3ß) and Snail to inactivate the oncogenic Akt/GSK-3ß/Snail signal pathway, and deletion of Snail also had similar effects to hamper the development of NSCLC. Next, our rescuing experiments validated that Pokemon ablation-induced suppressing effects on NSCLC cell malignancy were all abrogated by overexpressing Snail. Finally, the in vivo experiments confirmed that silencing of Pokemon downregulated Snail to hamper tumorigenesis of NSCLC cells in xenograft tumor-bearing mice models. Taken together, we firstly uncovered the underlying mechanisms by which the Pokemon/Akt/GSK-3ß/Snail signal pathway contributed to the development of NSCLC, and this signal pathway could be potentially used as therapeutic targets for the development of personalized anti-NSCLC drugs.

Iron metabolism and ferroptosis in diabetic bone loss: from mechanism to therapy.

Osteoporosis, one of the most serious and common complications of diabetes, has affected the quality of life of a large number of people in recent years. Although there are many studies on the mechanism of diabetic osteoporosis, the information is still limited and there is no consensus. Recently, researchers have proven that osteoporosis induced by diabetes mellitus may be connected to an abnormal iron metabolism and ferroptosis inside cells under high glucose situations. However, there are no comprehensive reviews reported. Understanding these mechanisms has important implications for the development and treatment of diabetic osteoporosis. Therefore, this review elaborates on the changes in bones under high glucose conditions, the consequences of an elevated glucose microenvironment on the associated cells, the impact of high glucose conditions on the iron metabolism of the associated cells, and the signaling pathways of the cells that may contribute to diabetic bone loss in the presence of an abnormal iron metabolism. Lastly, we also elucidate and discuss the therapeutic targets of diabetic bone loss with relevant medications which provides some inspiration for its cure.

Digital light processing-bioprinted poly-NAGA-GelMA-based hydrogel lenticule for precise refractive errors correction.

Refractive disorder is the most prevalent cause of visual impairment worldwide. While treatment of refractive errors can bring improvement to quality of life and socio-economic benefits, there is a need for individualization, precision, convenience, and safety with the chosen method. Herein, we propose using pre-designed refractive lenticules based on poly-NAGA-GelMA (PNG) bio-inks photo-initiated by digital light processing (DLP)-bioprinting for correcting refractive errors. DLP-bioprinting allows PNG lenticules to have individualized physical dimensions with precision achievable to 10µm (µm). Material characteristics of PNG lenticules in tests included optical and biomechanical stability, biomimetical swelling and hydrophilic capability, nutritional and visual functionality, supporting its suitability as stromal implants. Cytocompatibility distinguished by morphology and function of corneal epithelial, stromal, and endothelial cells on PNG lenticules suggested firm adhesion, over 90% viability, phenotypic maintenance instead of excessive keratocyte-myofibroblast transformation.In-vitroimmune response analyzed by illumina RNA sequencing in human peripheral blood mononuclear cells indicated that PNG lenticules activated type-2 immunity, facilitating tissue regeneration and suppressing inflammation.In-vivoperformance assessed using intrastromal keratoplasty models in New Zealand white rabbits illustrated that implantation of PNG lenticules maintained stable optical pathway, induced controlled stromal bio-integration and regeneration, avoided complications such as stromal melt, interface scarring, etc, but exerted no adverse effects on the host. Postoperative follow-up examination on intraocular pressure, corneal sensitivity, and tear production remained unaffected by surgery up to 1-month post-implantation of PNG lenticules. DLP-bioprinted PNG lenticule is a bio-safe and functionally effective stromal implants with customizable physical dimensions, providing potential therapeutic strategies in correction of refractive errors.

Crosstalk of necroptosis and pyroptosis defines tumor microenvironment characterization and predicts prognosis in clear cell renal carcinoma.

Pyroptosis and necroptosis are two recently identified forms of immunogenic cell death in the tumor microenvironment (TME), indicating a crucial involvement in tumor metastasis. However, the characteristics of necroptosis and pyroptosis that define tumor microenvironment and prognosis in ccRCC patients remain unknown. We systematically investigated the transcriptional variation and expression patterns of Necroptosis and Pyroptosis related genes (NPRGs). After screening the necroptosis-pyroptosis clusters, the potential functional annotation for clusters was explored by GSVA enrichment analysis. The Necroptosis-Pyroptosis Genes (NPG) scores were used for the prognosis model construction and validation. Then, the correlations of NPG score with clinical features, cancer stem cell (CSC) index, tumor mutation burden (TMB), TME, and Immune Checkpoint Genes (ICGs) were also individually explored to evaluate the prognosis predictive values in ccRCC. Microarray screenings identified 27 upregulated and 1 downregulated NPRGs. Ten overall survival associated NPRGs were filtered to construct the NPG prognostic model indicating a better prognostic signature for ccRCC patients with lower NPG scores (P< 0.001), which was verified using the external cohort. Univariate and multivariate analyses along with Kaplan-Meier survival analysis demonstrated that NPG score prognostic model could be applied as an independent prognostic factor, and AUC values of nomogram from 1- to 5- year overall survival with good agreement in calibration plots suggested that the proposed prognostic signature possessed good predictive capabilities in ccRCC. A high-/sNPG score is proven to be connected with tumor growth and immune-related biological processes, according to enriched GO, KEGG, and GSEA analyses. Comparing patients with a high-NPG score to those with a low-NPG score revealed significant differences in clinical characteristics, growth and recurrence of malignancies (CSC index), TME cell infiltration, and immunotherapeutic response (P< 0.005), potentially making the NPG score multifunctional in the clinical therapeutic setting. Furthermore, AIM2, CASP4, GSDMB, NOD2, and RBCK1 were also found to be highly expressed in ccRCC cell lines and tumor tissues, and GASP4 and GSDMB promote ccRCC cells' proliferation, migration, and invasion. This study firstly suggests that targeting the NPG score feature for TME characterization may lend novel insights into its clinical applications in the prognostic prediction of ccRCC.

MicroRNA-503 Exacerbates Myocardial Ischemia/Reperfusion Injury via Inhibiting PI3K/Akt- and STAT3-Dependent Prosurvival Signaling Pathways.

Acute myocardial infarction is a leading cause of death worldwide, while restoration of blood flow to previously ischemic myocardium may lead to ischemia/reperfusion (I/R) injury. Accumulated evidence shows that microRNAs play important roles in cardiovascular diseases. However, the potential role of microRNA-503 (miR-503) in myocardial I/R injury is little known. Thus, this study is aimed at determining whether and how miR-503 affects myocardial I/R injury in vivo and in vitro. A mouse model of myocardial I/R injury and H9c2 cell model of hypoxia/reoxygenation (H/R) injury were established. The postischemic cardiac miR-503 was downregulated in vivo and in vitro. Mechanistically, PI3K p85 and Bcl-2 are miR-503 targets. The post-ischemic cardiac PI3K p85 protein level was decreased in vivo. Agomir-503 treatment exacerbated H/R-induced injuries manifested as decreased cell viability, increased lactate dehydrogenase activity, and cell apoptosis. Agomir-503 treatment reduced cell viability under normoxia as well and reduced both PI3K p85 and Bcl-2 protein levels under either normoxia or H/R condition. It reduced phosphorylation of Stat3 (p-Stat3-Y705) and Akt (T450) in cells subjected to H/R. In contrast, Antagomir-503 treatment attenuated H/R injury and increased p-Stat3 (Y705) under normoxia and increased p-Akt (T450) under either normoxia or H/R condition. It is concluded that miR-503 exacerbated I/R injury via inactivation of PI3K/Akt and STAT3 pathways and may become a therapeutic target in preventing myocardial I/R injury.

Decreased expression of miR-195 mediated by hypermethylation promotes osteosarcoma.

Osteosarcoma (OS) is the most common type of primary malignant bone tumor. The early lung metastasis of osteosarcoma is one of the main factors of poor prognosis. Therefore, searching for new targets and new mechanisms of osteosarcoma metastasis is essential for the prevention and treatment of osteosarcoma. Our previous studies suggested that fatty acid synthase (FASN) was an oncogene and promoted osteosarcoma. In addition, it is reported that the expression of miR-195 was negatively correlated with osteosarcoma. Aberrant DNA methylation can reversely regulate the expression of miRNAs. However, whether miR-195 could target FASN in osteosarcoma and whether ectopic DNA methylation is the upstream regulatory mechanism of miR-195 in metastasis of osteosarcoma are not fully studied. The expressions were detected by qPCR and western blot, and methylation level was determined by methylation-specific PCR. Luciferase reporter assay, MTT, wound healing, and Transwell assay were used. We found that the expression of miR-195 was low in osteosarcoma. The methylation of miR-195 was high. miR-195 targeted and decreased the expression of FASN. In osteosarcoma, miR-195 inhibited cell proliferation, cell migration, and invasion. The methylation of miR-195 was related to decreased miR-195, it might promote osteosarcoma.

Advances in 3D bioprinting technology for functional corneal reconstruction and regeneration.

Corneal transplantation constitutes one of the major treatments in severe cases of corneal diseases. The lack of cornea donors as well as other limitations of corneal transplantation necessitate the development of artificial corneal substitutes. Biosynthetic cornea model using 3D printing technique is promising to generate artificial corneal structure that can resemble the structure of the native human cornea and is applicable for regenerative medicine. Research on bioprinting artificial cornea has raised interest into the wide range of materials and cells that can be utilized as bioinks for optimal clarity, biocompatibility, and tectonic strength. With continued advances in biomaterials science and printing technology, it is believed that bioprinted cornea will eventually achieve a level of clinical functionality and practicality as to replace donated corneal tissues, with their associated limitations such as limited or unsteady supply, and possible infectious disease transmission. Here, we review the literature on bioprinting strategies, 3D corneal modelling, material options, and cellularization strategies in relation to keratoprosthesis design. The progress, limitations and expectations of recent cases of 3D bioprinting of artifial cornea are discussed. An outlook on the rise of 3D bioprinting in corneal reconstruction and regeneration is provided.

Predicting asymptomatic neurosyphilis using peripheral blood indicators.

BACKGROUND: The high misdiagnosis rate of asymptomatic neurosyphilis (ANS) has long challenged infectious disease clinicians. We aim to develop a model for diagnosing ANS in asymptomatic syphilis (AS) patients without CSF indicators. RESULTS: 277 AS patients with HIV-negative and underwent lumbar puncture were enrolled in this horizontal study.The area under the curve for predicting ANS by CSF leukocytes and protein was 0.643 and 0.675 [95% CI, 0.583-0.699VS.0.616-0.729]. Through LRM, the AUC increased to 0.806 [95% CI, 0.732-0.832], and the Youden's index was 0.430. If the score is ≤ 0.159, ANS can be excluded with a predictive value of 92.9%; we can identify ANS while the score is over 0.819, with a predictive value of 91.7% and a specificity of 99.25%. This study showed that the LRM can diagnose ANS in AS patients effectively. CONCLUSION: Given a large number of misdiagnosis ANS patients and CSF results' insufficiency, the model is more practical. Our research will help clinicians track suspected syphilis, especially those who cannot accept the CSF test.

Are the Chinese Moving toward a Healthy Diet? Evidence from Macro Data from 1961 to 2017.

The change in diet structure is one of the critical features of social transformation, and diet structure is directly related to human health. In China, with rapid economic development, changes in the diet structure of the population have begun and are proceeding at a fairly rapid rate. In order to reveal how the Chinese diet is approaching or deviating from the nutritional goal, a novel index, NDBI (National Dietary Balance Index), is developed in this study to investigate the Chinese diet from 1961 to 2017 at a national level. The results show that the Chinese diet has transitioned from the under-intake stage to the over-intake stage. Before the 1980s, Chinese people ate all foods inadequately except staple foods; after the 1980s, the issue of under-intake began to fade, and the intake of meats even became excessive. The intake of staple foods is always excessive during this period. Currently, the Chinese diet is still unhealthy because of the inadequate intake of dairy products and the excessive intake of staple foods and meats. By evaluating diet structure on a national level, this study can help people to better understand how the Chinese diet deviated from the nutritional goal and provides information for policymakers intervening in China's food consumption.

Prevent action of magnoflorine with hyaluronic acid gel from cartilage degeneration in anterior cruciate ligament transection induced osteoarthritis.

According to the Chinese medicine, magnoflorine exerted significant anti-inflammatory effects and potentially promoted synthesis of proteoglycans in chondrocytes to reverse the progression of rheumatoid arthritis. However, the latent beneficial effect of magnoflorine for the treatment of traumatic osteoarthritis (OA) is still unknown. Therefore, we aim to demonstrate the efficacy of magnoflorine combined with HA-gel in attenuating cartilage degeneration in anterior cruciate ligament transection (ACLT) induced OA rat model. We found that the histological results showed the elevated cartilage matrix, chondrogenic signals and chondroprogenitor cells in HA-gel + magnoflorine treatment. HA-gel + magnoflorine treatment resulted in a decreased modified Mankin's score, and a higher volume ratio of hyaline cartilage (HC)/calcified cartilage (CC) and HC/Sum (whole cartilage), compared to ACLT and HA-gel groups. Furthermore, both the volume ratios of HC/Sum and HC/CC were negatively correlated with modified Mankin's scores. Finally, HA-gel + magnoflorine could significantly increase the BV/TV, Tb.Th, and decrease the Tb.Pf, Po(tot), Conn.Dn and Tb.Sp. In vitro, 50 µg/ml magnoflorine treatment could significantly increase the viability, S-phase, migration rate and chondrogenesis of chondroprogenitor cells. There were significant downregulations of MAPK/NF-κB signaling, and upregulations of chondrogenic signals in 50 µg/ml magnoflorine treatment. There were significant downregulations of proinflammatory cytokines and upregulation of IL-10 in HA-gel + magnoflorine treated group. Therefore, our study elucidated a protective effect of HA-gel + magnoflorine on attenuating cartilage degradation and maintaining SCB stabilization in ACLT induced OA.

A novel mechanical parameter to quantify the microarchitecture effect on apparent modulus of trabecular bone: A computational analysis of ineffective bone mass.

BACKGROUND: One of the characteristics of osteoporotic bone is the deterioration of trabecular microarchitecture. Previous studies have shown microarchitecture alone can vary the apparent modulus of trabecular bone significantly independent of bone volume fraction (BV/TV) from morphological and topological perspectives. However, modulus is a mechanical quantity and there is a lack of mechanical explanatory parameters. This study aims to propose a novel mechanical parameter to quantify the microarchitecture effect on the apparent modulus of trabecular bone. MATERIALS AND METHODS: Fourteen human female cadaveric vertebrae were scanned with a dual-energy X-ray (DXA) equipment followed by a micro-CT (µCT) system at 18 µm isotropic resolution. Four trabecular bone specimens (3.46 × 3.46 × 3.46 mm) were obtained from each vertebral body and converted to voxel-based micro finite element (µFE) models. The apparent modulus (E) of the µFE model was computed using a linear micro finite element analysis (µFEA). The normalized apparent modulus (E*) was computed as E divided by BV/TV. The relationship between E and BV/TV was analyzed by linear, power-law and exponential regressions. Linear regression was performed between E* and BV/TV. Ineffective bone mass (InBM) was defined as the bone mass with a negligible contribution to the load-resistance and represented by elements with von Mises stress less than a certain stress threshold. InBM was quantified as the low von Mises stress ratio (LSVMR), which is the ratio of the number of InBM elements to the total number of elements in the µFE model. An incremental search technique with coarse and fine search intervals of 10 and 1 MPa, respectively, was adopted to determine the stress threshold for calculating LSVMR of the µFE model. Correlation between E* and LSVMR was analyzed using linear and power-law models for each stress threshold. The threshold producing the highest coefficient of determination (R2) in the correlation between E* and LSVMR was taken as the optimal stress threshold for calculating LSVMR. Linear regression was performed between E and LSVMR. Multiple linear regression of E against both BV/TV and LSVMR was further analyzed. RESULTS: E significantly (p < .001) correlates to BV/TV whereas E* has no significant (p = .75) correlation with BV/TV. Incremental search suggests 59 MPa to be the optimal stress threshold for calculating LSVMR. BV/TV alone can explain 59% of the variation in E using power-law regression model (E = 2254.64BV/TV1.04, R2 = 0.59, p < .001). LSVMR alone can explain 48% of the variation in E using linear regression model (E = 1696.4-1647.1LSVMR, R2 = 0.48, p < .001). With these two predictors taken into consideration, 95% of the variation in E can be explained in a multiple linear regression model (E = 1364.89 + 2184.37BV/TV - 1605.38LSVMR, adjusted R2 = 0.95, p < .001). CONCLUSION: LSVMR can be adopted as the mechanical parameter to quantify the microarchitecture effect on the apparent modulus of trabecular bone.

DDR1 promotes breast tumor growth by suppressing antitumor immunity.

Breast cancer is the second leading cause of cancer­associated mortality among women worldwide. Triple­negative breast cancer (TNBC) accounts for 15­20% of all breast cancers and is defined by its aggressive nature and limited treatment options. Therefore, there is an urgent need to develop effective therapies for TNBC in order to improve breast cancer outcomes, as targeted therapies have done in other subtypes of breast cancer. Discoidin domain receptor tyrosine kinase 1 (DDR1) is activated by collagens, which are important components of the tumor stroma; therefore, DDR1 may serve a critical role in the communication between tumor cells and the tumor microenvironment. The aim of the present study was to determine how tumor DDR1 regulated tumor growth by affecting tumor infiltrated T cells. First, the DDR1 expression levels from a cohort of patients with breast cancer were analyzed. The results revealed that there were higher levels of DDR1 expression in tumor tissues compared with adjacent normal tissues. Overexpression of DDR1 in 4T1 cells promoted tumor growth in vivo, while knockout of DDR1 in EMT6 cells decreased tumor growth in vivo. In addition, it was revealed that DDR1 regulated tumor growth by modulating tumor infiltrating T cells, CD4+ and CD8+. Furthermore, inhibition of DDR1 by neutralizing antibodies decreased breast cancer growth in vivo. To the best of our knowledge, the results of the present study demonstrated for the first time that DDR1 expressed on the tumor cells promoted breast tumor growth by suppressing antitumor immunity. The present findings indicated that DDR1 may not only have a critical role in the progression of breast cancer, but may also serve as a potential therapeutic target for breast cancer, particularly TNBC.

Precisely Controlled Delivery of Abaloparatide through Injectable Hydrogel to Promote Bone Regeneration.

Side-effects from allograft, limited bone stock, and site morbidity from autograft are the major challenges to traditional bone defect treatments. With the advance of tissue engineering, hydrogel injection therapy is introduced as an alternative treatment. Therapeutic drugs and growth factors can be carried by hydrogels and delivered to patients. Abaloparatide, as an analog of human recombinant parathyroid hormone protein (PTHrp) and an alternative to teriparatide, has been considered as a drug for treating postmenopausal osteoporosis since 2017. Since only limited cases of receiving abaloparatide with polymeric scaffolds have been reported, the effects of abaloparatide on pre-osteoblast MC3T3-E1 are investigated in this study. It is found that in vitro abaloparatide treatment can promote pre-osteoblast MC3T3-E1 cells' viability, differentiation, and mineralization significantly. For the drug delivery system, 3D porous structure of the methacrylated gelatin (GelMA) hydrogel is found effective for prolonging the release of abaloparatide (more than 10 days). Therefore, injectable photo-crosslinked GelMA hydrogel is used in this study to prolong the release of abaloparatide and to promote healing of defected bones in rats. Overall, data collected in this study show no contradiction and imply that Abaloparatide-loaded GelMA hydrogel is effective in stimulating bone regeneration.

Anoikis resistant mediated by FASN promoted growth and metastasis of osteosarcoma.

The pulmonary metastasis of osteosarcoma (OS) occurs commonly, which resulted from anoikis resistant (AR) of tumor cells as reported by previous studies, but the exact roles of AR in osteosarcoma were not fully studied. Our previous investigations showed fatty acid synthase (FASN) was relating to clinical features of patients with OS. In this study, we aim to explore the functions of FASN in the AR OS cells in vitro and in vivo and study the downstream effectors of FASN. In the present study, we used our established cell model to study the AR. We revealed that AR promoted cell proliferation and migration as determined by colony formation assay and transwell assay. In addition, AR assisted tumor growth in vivo. In the AR cells, the expression of FASN was higher. Thus, we constructed lentiviruses to silence or overexpress FASN in four cell lines to study functions of FASN. Silence of FASN reduced cell colonies and migration while overexpression of FASN increased colonies and migration in suspended cells. Loss of functions of FASN induced cell apoptosis in suspended OS cells while gain of function of FASN suppressed apoptosis as determined by flow cytometry. We found the levels of p-ERK1/2 and Bcl-xL declined when FASN was silenced while they increased when FASN was overexpressed. In addition, results showed that the levels of FASN and its potential related molecules (p-ERK1/2 and Bcl-xL) increased in 143B-AR and MG-63-AR cells. In vivo study showed that inhibition of FASN decreased pulmonary metastasis of OS. In conclusion, we showed that anoikis resistant and FASN as two interactional factors facilitated the progress of osteosarcoma.

Strontium inhibits osteoclastogenesis by enhancing LRP6 and ß-catenin-mediated OPG targeted by miR-181d-5p.

Strontium is a drug with the bone formation and anti-resorption effects on bone. The underlying mechanisms for the dual effect of strontium on bone metabolism, especially for the anti-resorption effects remain unknown. Thus, we aim to investigate the mechanisms of effects of strontium on osteoclastogenesis. Firstly, we found that strontium decreased the levels of important biomarkers of receptor activator of nuclear factor kappa-B ligand (RANKL) which induced osteoclast differentiation, indicating that strontium might directly inhibit osteoclast differentiation. Next, we revealed that strontium enhanced Low Density Lipoprotein Receptor-Related Protein 6 (LRP6)/ß-catenin/osteoprotegerin (OPG) signaling pathway in MC3T3-E1 cells. The signaling pathway may negatively regulate osteoclastogenesis. Thus, strontium indirectly inhibited RANKL induced osteoclast differentiation. Finally, we revealed that OPG was targeted by miR-181d-5p as determined by luciferase reporter assay and downregulated by miR-181d-5p at both mRNA and protein levels as determined by western blot.

A Novel Dnmt3a1 Transcript Inhibits Adipogenesis.

DNA (cytosine-5)-methyltransferase 3a (Dnmt3a) is an enzyme that catalyzes the transfer of methyl groups to specific CpG forms in DNA. In mammals, two variant transcripts of Dnmt3a have been successfully identified. To the best of our knowledge, no Dnmt3a transcripts in an avian have been successfully identified. This study was performed to detect different transcripts of Dnmt3a in chickens and to examine whether a novel Dnmt3a transcript named Dnmt3a1 may regulate adipogenesis. In addition to cloning, sequencing, transcript detection, and expression studies, a novel Dnmt3a1 transcript overexpression and knockdown were conducted to explore the potential role of Dnmt3a1 in preadipocyte proliferation and the early stage of adipocyte differentiation. In chicken abdominal fat tissue, we detected a novel Dnmt3a1 transcript that differs from Dnmt3a by lacking 23 amino acids at the exon-1/exon-2 border. Dnmt3a1 mRNA was ubiquitously expressed in a variety of tissues or cells and highly expressed in chicken adipose tissue/cells. The expression of Dnmt3a1 was regulated under different physiological conditions including aging, fasting, and high-fat diet. In addition, overexpression of Dnmt3a1 significantly decreased preadipocyte proliferation and induced cell-cycle arrest while its inhibition increased cell proliferation and S-phase cells. Furthermore, the overexpression of Dnmt3a1 significantly upregulated the mRNA level of cell-cycle-related genes, such as CDKN1A, CDKN1B, CCNB3, CCND2, CCNG2, CDKN2B, and CDK9, or the protein level of CDKN1A, CDKN1B, and CCNG2. Conversely, the knockdown of Dnmt3a1 by siRNA had the opposite effects. Moreover, during early adipocyte differentiation, the overexpression of Dnmt3a1 significantly decreased the mRNA and the protein levels of PPAR-γ, C/EBP-α, ADIPOR1, and STAT3, and the mRNA levels of FAS, LEPR, LPL, PRKAB2, and ATGL. In contrast, their expression was significantly increased after the knockdown of Dnmt3a1. Taken together, we identified a novel transcript of Dnmt3a, and it played a potential role in adipogenesis.

Magnoflorine with hyaluronic acid gel promotes subchondral bone regeneration and attenuates cartilage degeneration in early osteoarthritis.

OBJECTIVE: To investigate efficacy of Chinese medicine magnoflorine combined with hyaluronic acid (HA)-gel in promoting subchondral bone (SCB) regeneration and attenuating cartilage degeneration in early osteoarthritis (OA). METHODS: MC3T3-E1 under magnoflorine treatment was assayed by XTT to determine cell viability. Cell proliferation was reflected through cell cycle. Osteoblast mineralization was stained by Alizarin Red. Standardized bone canal of 1 mm in diameter and 4 mm in depth was made on tibial medial plateau of 4-month-old Dunkin-Hartley spontaneous knee OA guinea pigs. Guinea pigs (n = 5/group) were treated once intra-bone canal injection of 2 µl HA-gel, 2 µl HA-gel+50 ng magnoflorine and null (Defect) respectively, except sham group. The left hind limbs were harvested for µCT scan and histopathological staining 2-month post-surgery. RESULTS: 25 µg/ml magnoflorine treatment significantly increased cell viability, S-phase and mineralization of MC3T3-E1 cells. In vivo, HA-gel + magnoflorine treatment significantly altered SCB microstructure; changes included increase in bone volume fraction (BV/TV), trabecular number (Tb.N), connectivity density (Conn.Dn), and decrease in degree of anisotropy (DA), which implied trabecular bone regeneration. Treatment also resulted in a decrease in modified Mankin's scores, and an increase in volume ratio of hyaline cartilage (HC)/calcified cartilage (CC) and fractal dimension (FD, roughness indicator of osteochondral conjunction), compared to Defect and HA groups. Furthermore, FD was positively associated with volume ratio of HC/CC and negatively associated with modified Mankin's scores. Finally, histological results showed that due to a faster regeneration of SCB with the HA-gel + magnoflorine treatment, the reduction of cartilage matrix and the decreased expression of chondrogenic signals were attenuated. CONCLUSION: Our study elucidated the potential benefits of HA-gel + magnoflorine in promoting SCB regeneration and revealed a protective effect of stimulating recovery of the SCB integrity on attenuating cartilage degradation to prevent OA progression.