Engineered Cellular Vesicles Displaying Glycosylated Nanobodies for Cancer Immunotherapy.

Immune checkpoint blockade targeting the CD47/SIRPα axis represents an alluring avenue for cancer immunotherapy. However, the compromised efficacy and safety concerns in vivo of conventional anti-CD47 antibodies impede their wide clinical applications. Here we introduced a single type of high-mannose glycans into the nanobodies against CD47 (HM-nCD47) and subsequently displayed HM-nCD47 on cellular vesicles (CVs) for enhanced cancer immunotherapy. In this platform, the CVs significantly improved the circulation time of HM-nCD47-CVs, the nCD47 enabled the blockade of the CD47/SIRPα axis, and the HM enhanced recognition of mannose-binding lectin, all synergistically activating the macrophage-mediated antitumor immunity. In both subcutaneous and metastatic murine tumor models, the HM-nCD47-CVs possessed significantly extended half-lives and increased accumulation at the tumor site, resulting in a remarkable macrophage-dependent inhibition of tumor growth, a transcriptomic remodeling of the immune response, and an increase in survival time. By integrating the chemical biology toolbox with cell membrane nanotechnology, the HM-nCD47-CVs represent a new immunotherapeutic platform for cancer and other diseases.

Effect of Treatment with Microarc Oxidation Technology on Tribological Properties of Nonvalve Metal Low-Carbon Steel.

Steel is one of the most widely used alloys because of its excellent properties such as high toughness, good workability, and low cost. However, steel has weak wear resistance, which limits its range of applications and service life. We have used the microarc oxidation (MAO) technique to form an Al2O3 ceramic coating on the surface of nonvalve metal low-carbon steel, which is used to enhance the wear resistance of low-carbon steel. Tribological experiments have shown that the coefficient of friction is reduced by 26.9%, hardness is improved, and wear resistance is enhanced after MAO compared to the substrate. Through a series of characterizations, the wear mechanism of the MAO samples was found to be a complex friction mechanism including abrasive wear, adhesive wear, and friction oxidation. After MAO, the wear resistance of nonvalve metal low-carbon steel is improved. The use of steel can be extended and its service life can be prolonged. This innovative approach provides a viable solution for the development of low-carbon steel coatings.

Myriocin enhances the clearance of M. tuberculosis by macrophages through the activation of PLIN2.

Myriocin is an inhibitor of de novo synthesis of sphingolipids and ceramides. In this research, we showed myriocin could significantly reduce Mtb burden and histopathological inflammation in mice. However, the underlying mechanism remains unclear. RNA-seq analysis revealed a significant increase in gene expression of PLIN2/CD36/CERT1 after myriocin treatment. The reduced bactericidal burden was only reversed after silencing the lipid droplets (LDs) surface protein PLIN2. This suggests that myriocin enhances the ability of macrophages to clear Mtb depending on the PLIN2 gene, which is part of the PPARγ pathway. Indeed, we observed a significant increase in the number of LDs following myriocin treatment.IMPORTANCEMycobacterium tuberculosis has the ability to reprogram host cell lipid metabolism and alter the antimicrobial functions of infected macrophages. The sphingolipids, such as ceramides, are the primary host lipids utilized by the bacteria, making the sphingomyelinase/ceramide system critical in Mtb infections. Surprisingly, the antimicrobial effect of myriocin was found to be independent of its role in reducing ceramides, but instead, it depends on the lipid droplets surface protein PLIN2. Our findings provide a novel mechanism for how myriocin enhances Mtb clearance in macrophages.

Tribological properties of MAO ceramic coatings with annulus array texture on disposable surgical gloves.

Introduction: In recent research, the expansion in the use of Mg alloys for biomedical applications has been approached by modifying their surfaces in conjunction with micro-arc oxidation (MAO) techniques which enhance their abrasion and corrosion resistance. Methods: In this study, combining laser texturing and MAO techniques to produce the dense ceramic coatings with microstructures. On the surface of the AZ31 Mg alloy, a micro-raised annulus array texture has been designed in order to increase the surface friction under liquid lubrication and to improve the operator's grip when holding the tool. For this work, the micro-morphology of the coatings was characterised, and the friction properties of the commonly used scalpel shank material 316 L, the untextured surface and the textured surface were comparatively analysed against disposable surgical gloves. Results and discussion: The results show that the Laser-MAO ceramic coating grows homogenous, the porosity decreases from 14.3% to 7.8%, and the morphology after friction indicates that the coating has good wear resistance. More specifically, the average coefficient of friction (COF) of the three types of gloves coated with Laser-MAO ceramic was higher than that of the 316 L and MAO ceramic coatings under the action of the annulus-integrated texture under the lubrication conditions of physiological saline and defatted sheep blood, which achieved the goal of increasing friction for the purpose of helping to prevent the problem of tool slippage from the hand.

Association between lactate metabolism­related molecules and venous thromboembolism: A study based on bioinformatics and an in vitro model.

Venous thromboembolism (VTE) is characterized by a high recurrence rate and adverse consequences, including high mortality. Damage to vascular endothelial cells (VECs) serves a key role in VTE and lactate (LA) metabolism is associated with VEC damage. However, the pathogenesis of VTE and the role of lactate metabolism-related molecules (LMRMs) remain unclear. Based on the GSE48000 dataset, the present study identified differentially expressed (DE-)LMRMs between healthy individuals and those with VTE. Thereafter, LMRMs were used to establish four machine learning models, namely, the random forest, support vector machine and generalized linear model (GLM) and eXtreme gradient boosting. To verify disease prediction efficiency of the models, nomograms, calibration curves, decision curve analyses and external datasets were used. The optimal machine learning model was used to predict genes involved in disease and an in vitro oxygen-glucose deprivation (OGD) model was used to detect the survival rate, LA levels and LMRM expression levels of VECs. A total of four DE-LMRMs, solute carrier family 16 member 1 (SLC16A1), SLC16A7, SLC16A8 and SLC5A12 were obtained and GLM was identified as the best performing model based on its ability to predict differential expression of the embigin, lactate dehydrogenase B, SLC16A1, SLC5A12 and SLC16A8 genes. Additionally, SLC16A1, SLC16A7 and SLC16A8 served key roles in VTE and the OGD model demonstrated a significant decrease in VEC survival rate as well as a significant increase and decrease in intracellular LA and SLC16A1 expression levels in VECs, respectively. Thus, LMRMs may be involved in VTE pathogenesis and be used to build accurate VTE prediction models. Further, it was hypothesized that the observed increase in intracellular LA levels in VECS was associated with the decrease in SLC16A1 expression. Therefore, SLC16A1 expression may be an essential target for VTE treatment.

Effects and regulatory pathway of proopinmelanocortin on feeding habit domestication in mandarin fish.

Proopiomelanocortin (POMC) is a hormone precursor, and has been reported to participate in domestication. However, its effects on feeding habit domestication in fish are poorly understood. Mandarin fish (Siniperca chuatsi) feeds solely on live prey fish since first-feeding. In the present study, the high expression of pomc in mandarin fish, both the pomc siRNA and MC4R inhibitor treatments increased the success rate of domestication from live prey fish to dead prey fish and food intake of dead prey fish, suggesting the role of pomc on the special feeding habit of live prey fish in mandarin fish. In addition, one c-fos binding site was identified in the region that from -1053 bp to -931 bp upstream of the transcription start site of pomc, and this region exhibited positive promoter activity. The mandarin fish brain cells treated with c-fos siRNA displayed suppressed pomc mRNA expression, indicating that c-fos positively regulated pomc expression. Furthermore, the mRNA expression of c-fos was higher in the mandarin fish which were more difficult to domesticate. The results of ChIP assay and inhibitor treatment confirmed that the activation of c-fos gene by histone H3K4me3 was catalyzed by Setd1b in mandarin fish. Three open peaks were found at the upstream regulatory region of setd1b by ATAC-seq, and the mRNA expression of setd1b was higher in the mandarin fish which were more difficult to domesticate. These results indicated that Setd1b could methylate histone H3K4 to activate the c-fos transcription, maintaining the high expression of pomc, which might contribute to the special feeding habit of mandarin fish.

Random Traffic Flow Simulation of Heavy Vehicles Based on R-Vine Copula Model and Improved Latin Hypercube Sampling Method.

The rationality of heavy vehicle models is crucial to the structural safety assessment of bridges. To establish a realistic heavy vehicle traffic flow model, this study proposes a heavy vehicle random traffic flow simulation method that fully considers the vehicle weight correlation based on the measured weigh-in-motion data. First, a probability model of the key parameters in the actual traffic flow is established. Then, a random traffic flow simulation of heavy vehicles is realized using the R-vine Copula model and improved Latin hypercube sampling (LHS) method. Finally, the load effect is calculated using a calculation example to explore the necessity of considering the vehicle weight correlation. The results indicate that the vehicle weight of each model is significantly correlated. Compared to the Monte Carlo method, the improved LHS method better considers the correlation between high-dimensional variables. Furthermore, considering the vehicle weight correlation using the R-vine Copula model, the random traffic flow generated by the Monte Carlo sampling method ignores the correlation between parameters, leading to a weaker load effect. Therefore, the improved LHS method is preferred.

Small Molecule-Activated O-GlcNAcase for Spatiotemporal Removal of O-GlcNAc in Live Cells.

The nutrient sensor O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification found on thousands of nucleocytoplasmic proteins. O-GlcNAc levels in cells dynamically respond to environmental cues in a temporal and spatial manner, leading to altered signal transduction and functional effects. The spatiotemporal regulation of O-GlcNAc levels would accelerate functional interrogation of O-GlcNAc and manipulation of cell behaviors for desired outcomes. Here, we report a strategy for spatiotemporal reduction of O-GlcNAc in live cells by designing an O-GlcNAcase (OGA) fused to an intein triggered by 4-hydroxytamoxifen (4-HT). After rational protein engineering and optimization, we identified an OGA-intein variant whose deglycosidase activity can be triggered in the desired subcellular compartments by 4-HT in a time- and dose-dependent manner. Finally, we demonstrated that 4-HT activation of the OGA-intein fusion can likewise potentiate inhibitory effects in breast cancer cells by virtue of the reduction of O-GlcNAc. The spatiotemporal control of O-GlcNAc through the chemically activatable OGA-intein fusion will facilitate the manipulation and functional understanding of O-GlcNAc in live cells.

Kruppel-like Factors 3 Regulates Migration and Invasion of Gastric Cancer Cells Through NF-κB Pathway.

Context: The poorly understood regulatory mechanisms impede gastric cancer therapy. Kruppel-like factors (KLFs) are associated with the development of various tumors, The studies on the role of the KLF transcription factor 13 (KLF13) in gastric cancer progression haven't been studied. Objective: The current study aimed to investigate the role of KLF13 in the migration and invasion of gastric cancer and the regulatory mechanism of KLF13 in gastric cancer progression. Design: The research team performed a laboratory study. Setting: The study took place at the Zengcheng District People's Hospital of Guangzhou in Zengcheng, China. Participants: In addition to using normal gastric cells, GES1, and seven gastric cancer cell lines, the research team compared the fresh, gastric cancer tissues (T) and paired, adjacent, noncancerous gastric tissues (ANT) from eight patients undergoing surgical resection at the hospital. The research team also downloaded the data for 33 gastric cancer tissues and adjacent, normal gastric tissues from the Cancer Genome Atlas' TCGA database. Intervention: The research team used: (1) short hairpin RNAs (shRNAs) to knock down KLF13, (2) wound healing and transwell invasion analyses to determine the effects of KLF13 on the migration and invasion of gastric cancer, and (3) a Luciferase reporter assay to determine the effects of KLF13 on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity. Results: KLF13 was upregulated in gastric cancer cells and tissues, and the patients with a high KLF13 expression had poor outcome. Downregulation of KLF13 significantly inhibited the migration and invasion of gastric cancer cells. Mechanistically, downregulation of KLF13 significantly inhibited NF-κB activity, and its targets such as: (1) snail family transcriptional repressor 1 (SNAI1 or Snail), (2) snail family transcriptional repressor 2 (SNAI2 or Slug), (3) zinc finger e-box binding homeobox 1 (ZEB1), (4) Smad interacting protein 1 (Sip1), (5) twist family basic helix-loop-helix (BHLH) transcription factor (Twist), (6) matrix metallopeptidase 2 (MMP2), and (7) MMP9. Tumor necrosis factor alpha (TNF-α) can activate NF-κB. Treating with TNF-α can reverse the effects of KLF13 downregulation on migration and invasion, confirming that KLF13 promotes the migration and invasion of gastric cancer cells through activating the NF-κB pathway. Conclusions: KLF13 promoted the migration and invasion of gastric cancer cells through activating the NF-κB pathway, providing a new target for gastric cancer therapy.

Anterior cervical discectomy and fusion with zero-profile versus stand-alone cages for two-level cervical spondylosis: A retrospective cohort study.

Objective: To assess the mid-long-term clinical and radiological outcomes of zero-profile (ZP) compared with stand-alone (ST) cages for two-level anterior cervical discectomy and fusion (ACDF). Methods: We included 77 patients (39 women and 38 men) who underwent two-level ACDF between May 5, 2016, and May 5, 2020, and who were followed up for at least 1 year. The subjects were divided into the ST (n = 38) and ZP (n = 39) group. For the evaluation of functional status, Japanese Orthopedic Association (JOA), Neck Disability Index (NDI), and Visual Analogue Scale (VAS) scores were used. Additionally, radiological outcomes and procedure complications were observed at final follow-up. Results: Both groups had excellent clinical outcomes at the final follow-up. There were no significant intergroup (ZP vs. ST) differences in the fusion rate (91.02% vs. 90.79%, P > 0.05) and postoperative dysphagia (15.4% vs. 2.6%, P = 0.108). However, the disc height at the final follow-up in the ZP group was higher than that in the ST group (6.86 ± 0.84 vs. 6.17 ± 1.03, P = 0.002). The ZP group accomplished a lower loss of cervical lordosis (18.46 ± 4.78 vs. 16.55 ± 4.36, P = 0.071), but without reaching statistical significance. Conclusion: ACDF with either ZP or ST cages turns out to be a dependable strategy for two-level ACDF in terms of clinical results. However, compared with the ST, the ZP cage may achieve a significantly lower loss of disc height.

Preparation and performance study of a reactive polyurethane hot-melt adhesive/CS-Fe3O4 magnetic nanocomposite film/fabric.

Magnetic nanoparticles are attracting significant attention for their wide application as biomaterials and magnetic storage materials. As an environmentally friendly adhesive, reactive polyurethane hot-melt adhesive (PUR) is a biocompatible polymer with a wide range of applications. In this paper, chitosan (CS)-surface-modified magnetic Fe3O4 nanoparticles were synthesized by the sol-gel method. Surface modification of the Fe3O4 nanoparticles with CS enhanced their mechanical properties in PUR. The nanoparticles were characterized by Fourier transform infrared (FTIR) and X-ray diffraction (XRD) analyses, while their surface morphology was elucidated using scanning electron microscopy (SEM) and projection electron microscopy (TEM) techniques. Subsequently, PUR/CS-Fe3O4 magnetic nanocomposite films were prepared using an in situ method, wherein different amounts of CS-surface-modified magnetic Fe3O4 nanoparticles were doped into the PUR and coated on the films. The thermal, UV resistance and mechanical properties of the PUR/CS-Fe3O4 magnetic nanocomposite films were investigated by TGA, UV spectrometer and tensile testing. CS-Fe3O4 nanoparticles were successfully prepared using the sol-gel method and CS to modify the surface of the Fe3O4 nanoparticles. The results show that the mechanical properties and UV resistance of PUR/CS-Fe3O4 magnetic nanocomposites are improved by almost 50%, so the constructed PUR/CS-Fe3O4 magnetic nanocomposites have good UV-resistant properties and mechanical properties. The as-synthesized CS-Fe3O4 magnetic nanocomposites show great potential for application to mechanical and textile development.

Preparation and characterization of polyethylene glycol/chitosan composite water-based wound healing lubricant.

The proportion of pregnant women giving birth through cesarean section is increasing annually worldwide. However, post-cesarean section scar diverticulum is a common condition that occurs and requires better surgical strategies than the methods currently used. We hypothesized that using biological lubricant topically on the incision area during C-section could be an option to minimize the scar. This water-based polyethylene glycol (PEG)/chitosan (CS) composite lubricant was prepared via ultrasonic blending. The product was characterized using scanning electron microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and Raman spectroscopy. The thermal stability of the materials and their heat absorption and release during heating were analyzed using thermogravimetric analysis and differential scanning calorimetry. Tribological tests proved that the PEG/CS composite had a better lubrication effect than either the PEG or CS lubricant alone, and the cell viability experiments verified that it had good biocompatibility. Finally, application of the composite lubricant onto the backs of mice modeling full-thickness skin incisions further confirmed that the product improved both the re-epithelialization and the collagen levels of the wounded skin. In conclusion, we expect our newly formulated PEG/CS composite lubricant to be useful not only for managing post-cesarean section scar diverticulum but also for healing skin wounds in general.

Cytocompatibility of pH-sensitive, chitosan-coated Fe3O4 nanoparticles in gynecological cells.

Nanoparticles that contact human cells without damaging basic human tissues are becoming more widely used in medicine. Efficient delivery to the intracellular target cell or compartment through the cell membrane must be achieved with minimal cytotoxicity to healthy cells. Fe3O4 nanoparticles have been widely used in biomedical research for their magnetic, non-toxic, and biocompatible properties. However, the effects of Fe3O4 nanoparticles coated with chitosan (CS) on gynecological cells are unclear. In this study, the Fe3O4 nanoparticles were coated with CS to enhance their cytocompatibility and dispersion in water. These CS-Fe3O4 nanoparticles were taken up by gynecological cells and did not affect cell viability in vitro. They have greater cytocompatibility in acidic environments than normal Fe3O4 nanoparticles and have the potential for drug delivery into gynecological cells.

Curcumin attenuates vascular calcification via the exosomal miR-92b-3p/KLF4 axis.

Vascular calcification (VC) is the most widespread pathological change in diseases of the vascular system. However, we do not have a good understanding of the molecular mechanisms and effective therapeutic approaches for VC. Curcumin (CUR) is a natural polyphenolic compound that has hypolipidemic, anti-inflammatory, and antioxidant effects on the cardiovascular system. Exosomes are known to have extensive miRNAs for intercellular regulation. This study investigated whether CUR attenuates VC by affecting the secretion of exosomal miRNAs. Calcification models were established in vivo and in vitro using vitamin D3 and ß-glycerophosphate, respectively. Appropriate therapeutic concentrations of CUR were detected on vascular smooth muscle cells (VSMCs) using a cell counting kit 8. Exosomes were extracted by super speed centrifugation from the supernatant of cultured VSMCs and identified by transmission electron microscopy and particle size analysis. Functional and phenotypic experiments were performed in vitro to verify the effects of CUR and exosomes secreted by VSMCs treated with CUR on calcified VSMCs. Compared with the calcified control group, both CUR and exosomes secreted by VSMCs after CUR intervention attenuated calcification in VSMCs. Real-Time quantitative PCR (RT-qPCR) experiments showed that miR-92b-3p, which is important for alleviating VC, was expressed highly in both VSMCs and exosomes after CUR intervention. The mimic miR-92b-3p significantly decreased the expression of transcription factor KLF4 and osteogenic factor RUNX2 in VSMCs, while the inhibitor miR-92b-3p had the opposite effect. Based on bioinformatics databases and dual luciferase experiments, the prospective target of miR-92b-3p was determined to be KLF4. Both mRNA and protein of RUNX2 were decreased and increased in VSMCs by inhibiting and overexpressing of KLF4, respectively. In addition, in the rat calcification models, CUR attenuated vitamin D3-induced VC by increasing miR-92b-3p expression and decreasing KLF4 expression in the aorta. In conclusion, our study suggests that CUR attenuates vascular calcification via the exosomal miR-92b-3p/KLF4 axis.

Lysine Deprivation Regulates Npy Expression via GCN2 Signaling Pathway in Mandarin Fish (Siniperca chuatsi).

Regulation of food intake is associated with nutrient-sensing systems and the expression of appetite neuropeptides. Nutrient-sensing systems generate the capacity to sense nutrient availability to maintain energy and metabolism homeostasis. Appetite neuropeptides are prominent factors that are essential for regulating the appetite to adapt energy status. However, the link between the expression of appetite neuropeptides and nutrient-sensing systems remains debatable in carnivorous fish. Here, with intracerebroventricular (ICV) administration of six essential amino acids (lysine, methionine, tryptophan, arginine, phenylalanine, or threonine) performed in mandarin fish (Siniperca chuatsi), we found that lysine and methionine are the feeding-stimulating amino acids other than the reported valine, and found a key appetite neuropeptide, neuropeptide Y (NPY), mainly contributes to the regulatory role of the essential amino acids on food intake. With the brain cells of mandarin fish cultured in essential amino acid deleted medium (lysine, methionine, histidine, valine, or leucine), we showed that only lysine deprivation activated the general control nonderepressible 2 (GCN2) signaling pathway, elevated α subunit of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation, increased activating transcription factor 4 (ATF4) protein expression, and finally induced transcription of npy. Furthermore, pharmacological inhibition of GCN2 and eIF2α phosphorylation signaling by GCN2iB or ISRIB, effectively blocked the transcriptional induction of npy in lysine deprivation. Overall, these findings could provide a better understanding of the GCN2 signaling pathway involved in food intake control by amino acids.

Overexpression of microRNA-145 enhanced docetaxel sensitivity in breast cancer cells via inactivation of protein kinase B gamma-mediated phosphoinositide 3-kinase -protein kinase B pathway.

Chemoresistance is a major challenge for the treatment of breast cancer (BC). Previous studies showed that miR-145 level decreases in chemoresistant BC tissues. Nevertheless, the biological function of miR-145 on docetaxel resistance of BC cells remains unclear, which is what our research attempted to clarify. RT-qPCR analyzed miR-145 level, and cell viability and colony formation assays assessed the impact of miR-145 on docetaxel resistance. Molecular mechanisms of miR-145-mediated docetaxel sensitivity were examined by Luciferase reporter assay and Western Blot assessed the function of AKT3 and PI3K/AKT signaling. Our research found that miR-145 expression presented significant downregulation in docetaxel-resistant BC cells. Meanwhile, miR-145 overexpression facilitated the docetaxel sensitivity of BC cells in vivo and in vitro, while the miR-145 inhibitor decreased the sensitivity of BC cells to docetaxel. We also observed that miR-145 inhibited docetaxel resistance mainly via downregulation of the AKT3 expression and further inhibited PI3K/AKT pathway. To conclude, this research provides a novel strategy for improving chemosensitivity through the newly identified miR-145-AKT3/PI3K-AKT signaling pathway in BC.

Role Mismatch in Medical Decision-Making Participation Is Associated with Anxiety and Depression in Family Members of Patients in the Intensive Care Unit.

This study aimed to investigate the mismatch between the preferred and actual roles in the medical decision-making of intensive care unit (ICU) patients' family members and the relationship between the role mismatch of family members' decisions and anxiety and depression syndromes. A total of 223 family members of ICU patients in the Affiliated Hospital of Jiangnan University in China were enrolled. The simple Chinese version of the Control Preference Scale was used to complete the surveys to assess the preferred and actual roles, and anxiety and depression syndromes were measured using the Generalized Anxiety Disorder-7 scale and Patient Health Questionnaire-9, respectively. For the preferred and actual roles, the active role rates were 16.1% and 8.1%, the cooperative role rates were 49.3% and 31.4%, and the passive role rates were 34.5% and 60.5%, respectively. The incidence of mismatch was 43.0% between the preferred and actual roles, and the consistency between their preferred and actual decision-making roles was poor (kappa = 0.309, P < 0.001). Family members with mismatched decision-making roles had significantly higher incidence rates of anxiety (90.6% vs. 57.5%, P < 0.001) and depression (86.5% vs. 63.0%, P < 0.001). Logistic regression analysis revealed that mismatches in decision-making roles remained independently associated with these outcomes after adjustment for family members' sociodemographic features. The results of the present study demonstrate that the preferred role of ICU patients' family members is mainly cooperative, and the actual role is mainly passive. The mismatch between the preferred and actual roles is associated with anxiety and depression among the ICU patients' family members.

Identification of potential biomarkers of vascular calcification using bioinformatics analysis and validation in vivo.

Background: Vascular calcification (VC) is the most widespread pathological change in diseases of the vascular system. However, we know poorly about the molecular mechanisms and effective therapeutic approaches of VC. Methods: The VC dataset, GSE146638, was downloaded from the Gene Expression Omnibus (GEO) database. Using the edgeR package to screen Differentially expressed genes (DEGs). Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were used to find pathways affecting VC. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed on the DEGs. Meanwhile, using the String database and Cytoscape software to construct protein-protein interaction (PPI) networks and identify hub genes with the highest module scores. Correlation analysis was performed for hub genes. Receiver operating characteristic (ROC) curves, expression level analysis, GSEA, and subcellular localization were performed for each hub gene. Expression of hub genes in normal and calcified vascular tissues was verified by quantitative reverse transcription PCR (RT-qPCR) and immunohistochemistry (IHC) experiments. The hub gene-related miRNA-mRNA and TF-mRNA networks were constructed and functionally enriched for analysis. Finally, the DGIdb database was utilized to search for alternative drugs targeting VC hub genes. Results: By comparing the genes with normal vessels, there were 64 DEGs in mildly calcified vessels and 650 DEGs in severely calcified vessels. Spp1, Sost, Col1a1, Fn1, and Ibsp were central in the progression of the entire VC by the MCODE plug-in. These hub genes are primarily enriched in ossification, extracellular matrix, and ECM-receptor interactions. Expression level results showed that Spp1, Sost, Ibsp, and Fn1 were significantly highly expressed in VC, and Col1a1 was incredibly low. RT-qPCR and IHC validation results were consistent with bioinformatic analysis. We found multiple pathways of hub genes acting in VC and identified 16 targeting drugs. Conclusions: This study perfected the molecular regulatory mechanism of VC. Our results indicated that Spp1, Sost, Col1a1, Fn1, and Ibsp could be potential novel biomarkers for VC and promising therapeutic targets.

MiR-7a-5p Attenuates Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis by Targeting VDAC1.

MicroRNA-7a-5p (miR-7a-5p) is closely related to apoptosis and plays an important role in ischemia/reperfusion (I/R) injury. Whether miR-7a-5p is involved in hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis is unknown. Therefore, this study aims to evaluate the role of miR-7a-5p in cardiomyocyte H9C2 cells in response to H/R stimulation. The results of RT-qPCR demonstrated that the expression level of miR-7a-5p was significantly down-regulated in H/R-treated H9C2 cells. MTT assay revealed that the cell viability was notably decreased in H/R group. Flow cytometric analysis found that the ratio of apoptotic cells was increased markedly following H/R. Enforced miR-7a-5p expression increased cell viability and decreased the apoptotic rate. Western blot analysis revealed that the expressions of pro-apoptotic proteins cleaved caspase-3 and Bax were down-regulated, while the expression of anti-apoptotic protein Bcl-2 was up-regulated in H/R-treated H9C2 cells transfected with miR-7a-5p mimic. On the contrary, miR-7a-5p downexpressing promoted apoptosis in H/R-treated H9C2 cells. Furthermore, the bioinformatics prediction manifested voltage-dependent anion channel 1 (VDAC1) was a potential target for miR-7a-5p, and dual-luciferase reporter assay confirmed that miR-7a-5p targeted VDAC1 3' untranslated regions, which leads to the repressed expressions of VDAC1 mRNA and protein. Knockdown of VDAC1 potentiated the protective effects of miR-7a-5p against H/R-induced cell injury. In conclusion, our results demonstrated that miR-7a-5p is involved in H/R-induced cardiomyocyte apoptosis through targeting VDAC1. MiR-7a-5p/VDAC1 axis might be utilized as hopeful biomarkers to reveal the potential mechanism of myocardial I/R injury.

Identification of potential biomarkers for diagnosis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a high mortality rate owing to its complexity. Identification of abnormally expressed genes in HCC tissues compared to those in normal liver tissues is a viable strategy for investigating the mechanisms of HCC tumorigenesis and progression as a means of developing novel treatments. A significant advantage of the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) is that the data therein were collected from different independent researchers and may be integrated, allowing for a more robust data analysis. Accordingly, in the present study, the gene expression profiles for HCC and control samples were downloaded from the GEO and TCGA. Functional enrichment analysis was performed using a Metascape dataset, and a protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/proteins (STRING) online database. The prognostic value of mRNA for HCC was assessed using the Kaplan-Meier Plotter, a public online tool. A gene mRNA heatmap and DNA amplification numbers were obtained from cBioPortal. A total of 2,553 upregulated genes were identified. Functional enrichment analysis revealed that these differentially expressed genes (DEGs) were mainly accumulated in metabolism of RNA and the cell cycle. Considering the complexity and heterogeneity of the molecular alterations in HCC, multiple genes for the prognostication of patients with HCC are more reliable than a single gene. Thus, the PPI network and univariate Cox regression analysis were applied to screen candidate genes (small nuclear ribonucleoprotein polypeptide B and B1, nucleoporin 37, Rac GTPase activating protein 1, kinesin family member 20A, minichromosome maintenance 10 replication initiation factor, ubiquitin conjugating enzyme E2 C and hyaluronan mediated motility receptor) that are associated with the overall survival and progression-free survival of patients with HCC. In conclusion, the present study identified a set of genes that are associated with overall survival and progression-free survival of patients with HCC, providing valuable information for the prognosis of HCC.