Fluid shear stress-induced down-regulation of microRNA-140-5p promotes osteoblast proliferation by targeting VEGFA via the ERK5 pathway.

PURPOSE: Fluid shear stress (FSS) plays a critical role in osteoblast proliferation. However, the role of miRNA in osteoblast proliferation induced by FSS and the possible molecular mechanisms remain to be defined. The aim of the present study was to investigate whether miR-140-5p regulates osteoblast proliferation under FSS and its molecular mechanism. MATERIALS AND METHODS: miR-140-5p expression was measured by qRT-PCR. Western blot was used to measure the expressions of P-ERK1/2, ERK1/2, P-ERK5 and ERK5. The levels of VEGFA, PCNA, CDK4 and Cyclin D1 were identified through qRT-PCR and western blot, respectively. Cell proliferation was detected by CCK-8 assay and EdU labeling assay. Dual-luciferase reporter assay was used to validate the target of miR-140-5p. RESULTS: miR-140-5p was significantly down-regulated when MC3T3-E1 cells were exposed to FSS. We then confirmed that up-regulation of miR-140-5p inhibited and down-regulation of miR-140-5p promoted osteoblast proliferation. In addition, FSS promotes osteoblast proliferation via down-regulating miR-140-5p. Luciferase reporter assay demonstrated that VEGFA is a direct target of miR-140-5p. Furthermore, transfection of mimic-140-5p inhibited the up-regulation of VEGFA protein level induced by FSS, suggesting that FSS regulates VEGFA protein expression via miR-140-5p. Further investigations demonstrated that VEGFA could promote osteoblast proliferation. Lastly, we demonstrated that miR-140-5p regulates osteoblast proliferation and ERK5 activation through VEGFA. CONCLUSIONS: Our study demonstrates that FSS-induced the down-regulation of miR-140-5p promotes osteoblast proliferation through activing VEGFA/ERK5 signaling pathway. These findings may provide a novel mechanism of FSS-induced osteoblast proliferation and offer a new avenue to further investigate osteogenesis induced by mechanical loading.

Integration Host Factor Binds DNA Holliday Junctions.

Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.

Fluid shear stress regulates osteoblast proliferation and apoptosis via the lncRNA TUG1/miR-34a/FGFR1 axis.

LncRNAs and microRNAs play critical roles in osteoblast differentiation and bone formation. However, their exact roles in osteoblasts under fluid shear stress (FSS) and the possible mechanisms remain unclear. The aim of this study was to explore whether and how miR-34a regulates osteoblast proliferation and apoptosis under FSS. In this study, FSS down-regulated miR-34a levels of MC3T3-E1 cells. MiR-34a up-regulation attenuated FSS-induced promotion of proliferation and suppression of apoptosis. Luciferase reporter assay revealed that miR-34a directly targeted FGFR1. Moreover, miR-34a regulated osteoblast proliferation and apoptosis via FGFR1. Further, we validated that lncRNA TUG1 acted as a competing endogenous RNA (ceRNA) to interact with miR-34a and up-regulate FGFR1 protein expression. Furthermore, lncRNA TUG1 could promote proliferation and inhibit apoptosis. Taken together, our study revealed the key role of the lncRNA TUG1/miR-34a/FGFR1 axis in FSS-regulated osteoblast proliferation and apoptosis and may provide potential therapeutic targets for osteoporosis.

The role of fibrinogen in predicting reinfection after DAIR for periprosthetic joint infections.

BACKGROUND: Fibrinogen (FIB) has been found to be a promising marker in diagnosing periprosthetic joint infection (PJI), however, the value of FIB in predicting reinfection of PJI is unknown. The purpose of this study was to evaluate the value of FIB in predicting reinfection after debridement, antibiotics, and implant retention (DAIR) for PJI. METHODS: We retrospectively analyzed the clinical data of patients who were diagnosed with PJI and underwent DAIR from 2013 to 2019. The levels of the FIB, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) were measured before DAIR. After DAIR, patients were followed and reinfections were identified. For both acute and chronic PJI, the predictive value of FIB was evaluated by calculating the sensitivity, specificity, and area under the curve (AUC) of the receiver operating characteristic curve (ROC), and was compared with traditional inflammatory markers including ESR and CRP. RESULTS: The expression of FIB differed between patients reinfected and those not reinfected in both acute and chronic PJI (p < 0.05). In patients who underwent DAIR for acute PJI, the sensitivity and specificity of FIB were 81.82 and 83.33%, respectively, which were significantly higher than that of CRP (sensitivity, 72.73%; specificity, 50%; p < 0.05), while the specificity was higher than that of ESR (specificity, 41.67%; p < 0.05). In patients who underwent DAIR for chronic PJI, the sensitivity and specificity of FIB were 80.00 and 66.66%, respectively, which were significantly higher than that of CRP (sensitivity, 53.33%; specificity, 66.66%; p < 0.05) and ESR (sensitivity was 66.00%; specificity, 16.66%; p < 0.05). The ROC curves showed that FIB demonstrated the highest AUC among the biomarkers in both acute and chronic PJI. CONCLUSION: FIB is a promising indicator in predicting reinfection after DAIR for both acute and chronic PJI, and it seems to perform better than ESR and CRP.

Effect of palm oil consumption on plasma lipid concentrations related to cardiovascular disease: a systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: The precise association between palm oil consumption and lipid-related cardiovascular disease risk remains unclear. A systematic review was thus performed to assess whether palm oil consumption has a negative effect on plasma lipid-related cardiovascular disease marker levels. METHODS AND STUDY DESIGN: In June 2018, the electronic bibliographic databases PubMed, EMBASE (Ovid), the Cochrane Library (Ovid) and the Chinese National Knowledge Infrastructure were searched and a total of 11 eligible dietary intervention articles involving 961 volunteers were selected. Both random and fixed effect models were used to calculate pooled weighted mean differences (WMD). RESULTS: A total of 11 articles involving 547 participants met the inclusion criteria. The pooled analysis revealed that palm oil increased the concentration of high-density lipoprotein cholesterol (WMD: 0.15 mmol/L; p<0.00001). Palm oil consumption had no significant effects on blood total cholesterol (WMD: -0.01 mmol/L; p=0.82) and LDL-c (WMD: -0.05mmol/L; p=0.10) and triglyceride concentrations (WMD: 0.00 mmol/L; p=0.96), relative to the effects of unsaturated fatty acid consumption. Subgroup analyses revealed that palm oil has a beneficial effect on High-density lipoprotein cholesterol levels when more than 30% of total dietary energy was constituted by fat. CONCLUSIONS: This review revealed that palm oil does not induce increases in cardiovascular disease risk risk-related biomarkers relative to unsaturated fatty acids. Furthermore, larger-scale samples of human dietary intervention trials are required to increase the accuracy of meta-analyses.

Defining the Role of Extrachromosomal DNA Amplifications in Medulloblastoma.

Circular extrachromosomal DNA (ecDNA), a common mechanism of oncogene amplification, has been identified as a major contributor to intratumoral heterogeneity and patient outcomes. In a recent publication in Nature Genetics, Chapman and colleagues further explored the role of ecDNA in the context of medulloblastoma. Using whole-genome sequencing, they found that 18% of the patients carry ecDNA amplification across a 468 medulloblastoma patient cohort. The presence of ecDNA was associated with worse survival. Single-cell FISH imaging and multiomic sequencing revealed that ecDNA copy number displayed a cell-to-cell variability within the sample, contributing to tumor heterogeneity. Furthermore, through sequencing and CRISPRi experiments, the authors uncovered frequent enhancer rewiring events on ecDNA that drive proliferation.

A novel lncRNA GM15416 regulates osteoblast apoptosis and differentiation through the c-Fos/Fas axis and mitigates osteoporosis.

Osteoporosis (OP) is a common systemic bone disorder, and the programmed cell death of osteoblasts is closely linked to the development of osteoporosis. Previous studies have shown that c-fos can cause osteoblast apoptosis. Furthermore, it has been demonstrated that long non-coding RNA (lncRNA) plays a pervasive role in regulating the biology of osteoblasts. Nevertheless, the precise role and mechanism of long non-coding RNA (lncRNA) in relation to c-Fos at the transcriptional level in osteoblast cell death remain uncertain. Compared with normal osteoblasts, serum deprivation resulted in significant upregulation of the transcription factor c-Fos and apoptosis-related Fas proteins in osteoblasts. In addition, the expression of lncRNA GM15416 related to c-Fos was significantly increased. The results showed that overexpression of c-Fos leads to an increase in downstream Fas protein, which subsequently leads to osteoblast apoptosis and hinders osteogenesis. On the contrary, a decrease in lncRNA GM15416 expression leads to a decrease in c-Fos/Fas expression, which hinders osteoblast apoptosis and promotes osteogenesis. Our results suggest that lncRNA GM15416 exerts inhibitory effects on osteoblast apoptosis and acts as a preventive factor against osteoporosis. As a result, GM15416 emerges as an important lncRNA associated with osteoporosis and holds potential as a future therapeutic target.

A novel mechanism of Vildagliptin in regulating bone metabolism and mitigating osteoporosis.

Osteoporosis has become a global social problem with the tendency toward the aging population. The challenge in managing osteoporosis is to develop new anti-osteoporosis drugs that target bone anabolism. The purpose of this study was to uncover the novel mechanism of Vildagliptin on bone metabolism. We revealed that Vildagliptin significantly promoted osteogenic differentiation of precursor osteoblasts and bone marrow mesenchymal stem cells (BMSCs). At the same time, it significantly enhanced the polarization of RAW264.7 macrophages to the M2 type and the secretion of osteogenic factors BMP2 and TGF-ß1. This was confirmed by the increased osteogenic differentiation observed in the osteoblast-RAW264.7 co-culture system. Moreover, Vildagliptin significantly enhanced the transformation of BMSCs into the osteogenic morphology in the osteoblast-BMSC co-culture system. Finally, Vildagliptin also inhibited osteoclastic differentiation of RAW 264.7 cells. The potential mechanism underlying these effects involved targeting the GAS6/AXL/ERK5 pathway. In the in vivo study, Vildagliptin significantly alleviated postmenopausal osteoporosis in ovariectomized mice. These findings represent the first comprehensive revelation of the regulatory effect of Vildagliptin on bone metabolism. Specifically, Vildagliptin demonstrates the ability to promote bone anabolism and inhibit bone resorption by simultaneously targeting osteoblasts, BMSCs, and osteoclasts. The bone-protective effects of Vildagliptin were further confirmed in a postmenopausal osteoporosis model. The clinical significance of this study lies in laying a theoretical foundation for bone protection therapy in type-2 diabetes patients with compromised bone conditions or postmenopausal osteoporosis.

Unraveling Key m6A Modification Regulators Signatures in Postmenopausal Osteoporosis through Bioinformatics and Experimental Verification.

OBJECTIVE: Bone marrow mesenchymal stem cells (BMSCs) show significant potential for osteogenic differentiation. However, the underlying mechanisms of osteogenic capability in osteoporosis-derived BMSCs (OP-BMSCs) remain unclear. This study aims to explore the impact of YTHDF3 (YTH N6-methyladenosine RNA binding protein 3) on the osteogenic traits of OP-BMSCs and identify potential therapeutic targets to boost their bone formation ability. METHODS: We examined microarray datasets (GSE35956 and GSE35958) from the Gene Expression Omnibus (GEO) to identify potential m6A regulators in osteoporosis (OP). Employing differential, protein interaction, and machine learning analyses, we pinpointed critical hub genes linked to OP. We further probed the relationship between these genes and OP using single-cell analysis, immune infiltration assessment, and Mendelian randomization. Our in vivo and in vitro experiments validated the expression and functionality of the key hub gene. RESULTS: Differential analysis revealed seven key hub genes related to OP, with YTHDF3 as a central player, supported by protein interaction analysis and machine learning methodologies. Subsequent single-cell, immune infiltration, and Mendelian randomization studies consistently validated YTHDF3's significant link to osteoporosis. YTHDF3 levels are significantly reduced in femoral head tissue from postmenopausal osteoporosis (PMOP) patients and femoral bone tissue from PMOP mice. Additionally, silencing YTHDF3 in OP-BMSCs substantially impedes their proliferation and differentiation. CONCLUSION: YTHDF3 may be implicated in the pathogenesis of OP by regulating the proliferation and osteogenic differentiation of OP-BMSCs.

METTL3-mediated m6A modification of SOX4 regulates osteoblast proliferation and differentiation via YTHDF3 recognition.

N6-methyladenosine (m6A), the most prevalent internal modification in mRNA, is related to the pathogenesis of osteoporosis (OP). Although methyltransferase Like-3 (METTL3), an m6A transferase, has been shown to mitigate OP progression, the mechanisms of METTL3-mediated m6A modification in osteoblast function remain unclear. Here, fluid shear stress (FSS) induced osteoblast proliferation and differentiation, resulting in elevated levels of METTL3 expression and m6A modification. Through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and Transcriptomic RNA Sequencing (RNA-seq), SRY (Sex Determining Region Y)-box 4 (SOX4) was screened as a target of METTL3, whose m6A-modified coding sequence (CDS) regions exhibited binding affinity towards METTL3. Further functional experiments demonstrated that knockdown of METTL3 and SOX4 hampered osteogenesis, and METTL3 knockdown compromised SOX4 mRNA stability. Via RNA immunoprecipitation (RIP) assays, we further confirmed the direct interaction between METTL3 and SOX4. YTH N6-Methyladenosine RNA Binding Protein 3 (YTHDF3) was identified as the m6A reader responsible for modulating SOX4 mRNA and protein levels by affecting its degradation. Furthermore, in vivo experiments demonstrated that bone loss in an ovariectomized (OVX) mouse model was reversed through the overexpression of SOX4 mediated by adeno-associated virus serotype 2 (AAV2). In conclusion, our research demonstrates that METTL3-mediated m6A modification of SOX4 plays a crucial role in regulating osteoblast proliferation and differentiation through its recognition by YTHDF3. Our research confirms METTL3-m6A-SOX4-YTHDF3 as an essential axis and potential mechanism in OP.

Combinatorial Design of Hydrolytically Degradable, Bone-like Biocomposites Based on PHEMA and Hydroxyapatite.

With advantages such as design flexibility in modifying degradation, surface chemistry, and topography, synthetic bone-graft substitutes are increasingly demanded in orthopedic tissue engineering to meet various requirements in the growing numbers of cases of skeletal impairment worldwide. Using a combinatorial approach, we developed a series of biocompatible, hydrolytically degradable, elastomeric, bone-like biocomposites, comprising 60 wt% poly(2-hydroxyethyl methacrylate-co-methacrylic acid), poly(HEMA-co-MA), and 40 wt% bioceramic hydroxyapatite (HA). Hydrolytic degradation of the biocomposites is rendered by a degradable macromer/crosslinker, dimethacrylated poly(lactide-b-ethylene glycol-b-lactide), which first degrades to break up 3-D hydrogel networks, followed by dissolution of linear pHEMA macromolecules and bioceramic particles. Swelling and degradation were examined at Hank's balanced salt solution at 37 °C in a 12-week period of time. The degradation is strongly modulated by altering the concentration of the co-monomer of methacrylic acid and of the macromer, and chain length/molecular weight of the macromer. 95% weight loss in mass is achieved after degradation for 12 weeks in a composition consisting of HEMA/MA/Macromer = 0/60/40, while 90% weight loss is seen after degradation only for 4 weeks in a composition composed of HEMA/MA/Macromer = 27/13/60 using a longer chain macromer. For compositions without a co-monomer, only about 14% is achieved in weight loss after 12-week degradation. These novel biomaterials offer numerous possibilities as drug delivery carriers and bone grafts particularly for low and medium load-bearing applications.

A novel ceRNA regulatory network involving the long noncoding NEAT1, miRNA-466f-3p and its mRNA target in osteoblast autophagy and osteoporosis.

Osteoporosis (OP) is a systemic metabolic disorder characterized by a reduction in bone tissue volume. LncRNAs have been reported to act as regulators of several human diseases. Specifically, lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in proliferation, differentiation and apoptosis in osteoclasts and bone marrow mesenchymal stem cells and regulates the occurrence and development of OP. However, the relationship between NEAT1 and osteoblast autophagy and its mechanism are still unclear. Western blotting of LC3 and P62 was used to evaluate the effect of fluid shear stress (FSS) on autophagy in MC3T3-E1 osteoblasts. Total transcriptome sequencing and bioinformatics analyses were performed on osteoblasts loaded with and without FSS. qPCR was performed to examine the expression of NEAT1 in OP bone tissues and osteoblasts. The RNA-FISH was performed to study the localization of lncRNA NEAT1 and miR-466f-3p in MC3T3-E1 osteoblasts. In vitro, western blotting, transmission electron microscopy (TEM), immunofluorescence (IF) staining and qPCR were performed to verify the biological functions of NEAT1, miR-466f-3p and HK2. Subsequently, we conducted bioinformatics analysis and dual luciferase reporter assays to identify the relationships among NEAT1, miR-466f-3p and HK2. Additionally, rescue assays were conducted on osteoblasts to clarify the regulatory network of the NEAT1/miR-466f-3p/HK2 signalling pathway. In vivo, the OVX mouse model was used to investigate the effects of si-NEAT1 on autophagy in OP mice. The distal femur and serum were collected for further micro-CT analysis, blood biochemistry, and haematoxylin-eosin and Alizarin red staining (ARS). Immunohistochemistry (IHC) was performed to assess the protein expression of LC3 and HK2. NEAT1 expression was upregulated in OP tissues and osteoblast lines exposed to FSS. Knockdown of NEAT1 inhibited autophagy in vitro and in vivo. Further studies demonstrated that NEAT1 positively regulated HK2 expression via its competing endogenous RNA effects on miR-466f-3p. Moreover, we found the NEAT1/miR-466f-3p/HK2 axis regulated autophagy in osteoblasts. Knocking down NEAT1 inhibited autophagy in osteoblasts via the miR-466f-3p/HK2 signalling pathway, which may provide new ideas for novel molecular therapeutic targets of postmenopausal OP. KEY MESSAGES: • Fluid shear stress (FSS) can promote autophagy of osteoblast and performed transcriptome sequencing. • NEAT1 is overexpressed in osteoporosis and could regulate osteoblast cells autophagy. • Knockdown of lncRNA NEAT1 inhibited osteoblast cells autophagy by sponging miRNA-466f-3p and targeting HK2 in osteoporosis.

Mangiferin promotes osteogenic differentiation and alleviates osteoporosis in the ovariectomized mouse via the AXL/ERK5 pathway.

Mangiferin is a xanthone glucoside extracted from multiple plants, which has been shown to inhibit bone resorption and alleviate osteoporosis. However, the effect of purified Mangiferin on osteoporosis and its specific mechanisms is unknown. This study aimed to explore whether Mangiferin can promote osteogenic differentiation and alleviate osteoporosis in ovariectomized (OVX) mice and explore the potential mechanisms. Different concentrations and durations of Mangiferin were applied to MC3T3-E1 cells. The optimal concentration and duration of Mangiferin were determined by evaluating the cell viability via cell count kit-8 (CCK-8). The gene and protein expressions of AXL, ERK5, and osteogenic differentiation markers, including BMP2, Collagen1, OPN, Osterix, and Runx2, were detected using western blotting, qRT-PCR, immunofluorescence, and flow cytometry. Mangiferin was administered to OVX mice, and the severity of osteoporosis was evaluated by H and E staining, immunohistochemistry (IHC), microscopic computed-tomography (micro-CT) scanning, western blotting, and immunofluorescence of bone tissue. We found that Mangiferin promoted osteogenic differentiation in a dose-dependent manner at concentrations less than 30 µM. The 30 µM Mangiferin significantly upregulated the expression of AXL, ERK5, and osteogenic differentiation, including the ALP activity, percentage of alizarin red, and the levels of osteogenic differentiation markers. However, these expression levels decreased when AXL was knocked down in MC3T3-E1 cells and it could not be rescued by Mangiferin. Mangiferin relieved osteoporosis in OVX mice without causing severe organ damage. This study concluded that Mangiferin promoted osteogenic differentiation of MC3T3-E1 cells and alleviated osteoporosis in OVX mice. The potential mechanism was via the AXL/ERK5 pathway.

Diagnostic Value of Next-Generation Sequencing in Periprosthetic Joint Infection: A Systematic Review.

Next-generation sequencing (NGS) has developed rapidly in the last decade and is emerging as a promising diagnostic tool for periprosthetic joint infection (PJI). However, its diagnostic value for PJI is still uncertain. This systematic review aimed to explore the diagnostic value of NGS for PJI and verify its accuracy for culture-negative PJI patients. We conducted this systematic review in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Medline, Embase, and Cochrane Library were searched to identify diagnostic technique studies evaluating the accuracy of NGS in the diagnosis of PJI. The diagnostic sensitivity, specificity, and positive and negative predictive values were estimated for each article. The detection rate of NGS for culture-negative PJI patients or PJI patients with antibiotic administration history was also calculated. Of the 87 identified citations, nine studies met the inclusion criteria. The diagnostic sensitivities and specificities of NGS ranged from 63% to 96% and 73% to 100%, respectively. The positive and negative predictive values ranged from 71% to 100% and 74% to 95%, respectively. The detection rate of NGS for culture-negative PJI patients in six studies was higher than 50% (range from 82% to 100%), while in three studies it was lower than 50% (range from 9% to 31%). Also, the detection rate of NGS for PJIs with antibiotic administration history ranged from 74.05% to 92.31%. In conclusion, this systematic review suggests that NGS may have the potential to be a new tool for the diagnosis of PJI and should be considered to be added to the portfolio of diagnostic procedures. Furthermore, NGS showed a favorable diagnostic accuracy for culture-negative PJI patients or PJI patients with antibiotic administration history. However, due to the small sample sizes of studies and substantial heterogeneity among the included studies, more research is needed to confirm or disprove these findings.

Research on Void Dynamics during In Situ Consolidation of CF/High-Performance Thermoplastic Composite.

Automated fiber placement (AFP) in situ consolidation of continuous CF/high-performance thermoplastic composite is the key technology for efficient and low-cost manufacturing of large thermoplastic composites. However, the void in the in situ composite is difficult to eliminate because of the high pressure and the short consolidation time; the void content percentage consequently is the important defect that determines the performance of the thermoplastic composite parts. In this paper, based on the two-dimensional Newtonian fluid extrusion flow model, the void dynamics model and boundary conditions were established. The changes of the void content percentage were predicted by the cyclic iteration method. It was found that the void content percentage increased gradually along the direction of the layers' thickness. With the increasing of the laying speed, the void content percentage increased gradually. With the increasing of the pressure of the roller, the void content percentage gradually decreased. When the AFP speed was 11 m/min and the pressure of the compaction roller reached 2000 N, the void content percentage of the layers fell below 2%. It was verified by the AFP test that the measured results of the layers' thickness were in good agreement with the predicted results of the model, and the test results of the void content percentage were basically equivalent to the predicted results at different AFP speeds, which indicates that the kinetic model established in this paper is representative to predict the void content percentage. According to the metallographic observation, it was also found that the repeated pressure of the roller was helpful to reduce the void content percentage.

Live-Cell Imaging Shows Uneven Segregation of Extrachromosomal DNA Elements and Transcriptionally Active Extrachromosomal DNA Hubs in Cancer.

Oncogenic extrachromosomal DNA elements (ecDNA) play an important role in tumor evolution, but our understanding of ecDNA biology is limited. We determined the distribution of single-cell ecDNA copy number across patient tissues and cell line models and observed how cell-to-cell ecDNA frequency varies greatly. The exceptional intratumoral heterogeneity of ecDNA suggested ecDNA-specific replication and propagation mechanisms. To evaluate the transfer of ecDNA genetic material from parental to offspring cells during mitosis, we established the CRISPR-based ecTag method. ecTag leverages ecDNA-specific breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying ecTag during mitosis revealed disjointed ecDNA inheritance patterns, enabling rapid ecDNA accumulation in individual cells. After mitosis, ecDNAs clustered into ecDNA hubs, and ecDNA hubs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and shed new light on mechanisms through which ecDNAs contribute to oncogenesis. SIGNIFICANCE: ecDNAs are vehicles for oncogene amplification. The circular nature of ecDNA affords unique properties, such as mobility and ecDNA-specific replication and segregation behavior. We uncovered fundamental ecDNA properties by tracking ecDNAs in live cells, highlighting uneven and random segregation and ecDNA hubs that drive cargo gene transcription.See related commentary by Henssen, p. 293.This article is highlighted in the In This Issue feature, p. 275.

Mechanosensory and mechanotransductive processes mediated by ion channels in articular chondrocytes: Potential therapeutic targets for osteoarthritis.

Articular cartilage consists of an extracellular matrix including many proteins as well as embedded chondrocytes. Articular cartilage formation and function are influenced by mechanical forces. Hind limb unloading or simulated microgravity causes articular cartilage loss, suggesting the importance of the healthy mechanical environment in articular cartilage homeostasis and implying a significant role of appropriate mechanical stimulation in articular cartilage degeneration. Mechanosensitive ion channels participate in regulating the metabolism of articular chondrocytes, including matrix protein production and extracellular matrix synthesis. Mechanical stimuli, including fluid shear stress, stretch, compression and cell swelling and decreased mechanical conditions (such as simulated microgravity) can alter the membrane potential and regulate the metabolism of articular chondrocytes via transmembrane ion channel-induced ionic fluxes. This process includes Ca2+ influx and the resulting mobilization of Ca2+ that is due to massive released Ca2+ from stores, intracellular cation efflux and extracellular cation influx. This review brings together published information on mechanosensitive ion channels, such as stretch-activated channels (SACs), voltage-gated Ca2+ channels (VGCCs), large conductance Ca2+-activated K+ channels (BKCa channels), Ca2+-activated K+ channels (SKCa channels), voltage-activated H+ channels (VAHCs), acid sensing ion channels (ASICs), transient receptor potential (TRP) family channels, and piezo1/2 channels. Data based on epithelial sodium channels (ENaCs), purinergic receptors and N-methyl-d-aspartate (NMDA) receptors are also included. These channels mediate mechanoelectrical physiological processes essential for converting physical force signals into biological signals. The primary channel-mediated effects and signaling pathways regulated by these mechanosensitive ion channels can influence the progression of osteoarthritis during the mechanosensory and mechanoadaptive process of articular chondrocytes.

Single-cell multimodal glioma analyses identify epigenetic regulators of cellular plasticity and environmental stress response.

Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to therapeutic resistance. We integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes and bulk multi-omic profiles across 11 adult IDH mutant or IDH wild-type gliomas to delineate sources of intratumoral heterogeneity. We showed that local DNA methylation disorder is associated with cell-cell DNA methylation differences, is elevated in more aggressive tumors, links with transcriptional disruption and is altered during the environmental stress response. Glioma cells under in vitro hypoxic and irradiation stress increased local DNA methylation disorder and shifted cell states. We identified a positive association between genetic and epigenetic instability that was supported in bulk longitudinally collected DNA methylation data. Increased DNA methylation disorder associated with accelerated disease progression and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work identified an epigenetically facilitated adaptive stress response process and highlights the importance of epigenetic heterogeneity in shaping therapeutic outcomes.

Three-dimensional biomimetic mineralization of dense hydrogel templates.

An electric-current-assisted method was used to mineralize dense hydrogels and create hydroxyapatite/hydrogel composites with unique hierarchical structures. The microstructure of the final material can be controlled by the mineralization technique and the chemistry of the organic matrix. A hydroxyapatite/hydrogel composite was obtained with a large inorganic content (approximately 60% of the weight of the organics). After being heated to 1050 degrees C, the sintered inorganic phase has a very uniformly distributed porosity and its Brunauer-Emmett-Teller (BET) surface area is 0.68 m(2)/g.

Genomics Analysis of L-DOPA Exposure in Drosophila sechellia.

Drosophila sechellia is a dietary specialist fruit fly that evolved from a generalist ancestor to specialize on the toxic fruit of Morinda citrifolia This species pair has been the subject of numerous studies where the goal has largely been to determine the genetic basis of adaptations associated with host specialization. Because one of the most striking features of M. citrifolia fruit is the production of toxic volatile compounds that kill insects, most genomic studies in D. sechellia to date have focused on gene expression responses to the toxic compounds in its food. In this study, we aim to identify new genes important for host specialization by profiling gene expression response to 3,4-dihydroxyphenylalanine (L-DOPA). Recent work found it to be highly abundant in M. citrifolia, critical for reproductive success of D. sechellia, and supplementation of diet with the downstream pathway product dopamine can influence toxin resistance phenotypes in related species. Here we used a combination of functional genetics and genomics techniques to identify new genes that are important for D. sechellia ecological adaptation to this new niche. We show that L-DOPA exposure can affect toxin resistance phenotypes, identify genes with plastic responses to L-DOPA exposure, and functionally test an identified candidate gene. We found that knock-down of Esterase 6 (Est6) in a heterologous species alters toxin resistance suggesting Est6 may play an important role in D. sechellia host specialization.