Identification of Autophagy-Related Genes Involved in Intervertebral Disc Degeneration by Microarray Data Analysis.

BACKGROUND: Nucleus pulposus cells survive in a hypoxic, acidic, nutrient-poor, and hypotonic microenvironment. Consequently, they maintain low proliferation and undergo autophagy to protect themselves from cellular stress. Therefore, we aimed to identify autophagy-related biomarkers involved in intervertebral disc degeneration pathogenesis. METHODS: Autophagy-related differentially expressed genes were derived from the intersection between the public GSE147383 microarray data set to identify differentially expressed genes and online databases to identify autophagy-related genes. Furthermore, we assessed their biological functions with gene annotation and enrichment analysis in the Metscape portal. Then, the STRING database and Cytoscape software allowed inferring a protein-protein interaction (PPI) network and identifying hub genes. In addition, to predict transcription factors that may regulate the hub genes, we used the GeneMANIA website. Finally, the competing endogenous RNA prediction tools and Cytoscape were also used to construct an mRNA-miRNA-lncRNA network. RESULTS: A total of 123 autophagy-related differentially expressed genes were identified, they were mainly involved in phosphoinositide 3-kinase-Akt signaling, autophagy animal, and apoptosis pathways. Nine were identified as hub genes (PTEN, MYC, CTNNB1, JUN, BECN1, ERBB2, FOXO3, ATM, and FN1) and 36 transcription factors were associated with them. Finally, an autophagy-associated competing endogenous RNA network was constructed based on the 9 hub genes. CONCLUSIONS: Nine hub genes were identified and a network of competing endogenous RNA associated with autophagy was established. They can be used as autophagy-related biomarkers of intervertebral disc degeneration and for further exploration.

[Two different techniques combined with MIS-TLIF in the treatment of degenerative lumbar spondylolisthesis:a case-control study].

OBJECTIVE: To analyze the difference in clinical efficacy of minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) under Quadrant channel system combined with microscope and percutaneous pedicle screw in the treatment of degenerative lumbar spondylolisthesis. METHODS: A total of 114 patients with single-segment degenerative lumbar spondylolisthesis from June 2015 to February 2019, were divided into three groups according to the surgical methods, such as the MIS-TLIF under the microscope surgery group ( microscope group), MIS-TLIF combined with percutaneous pedicle screw technique surgery group(percutaneous group) and posterior lumbar interbody fusion surgery group (open group). In the microscope group, there were 12 males and 26 females, aged from 42 to 83 years with an average of (63.29±9.09) years. In the percutaneous group, there were 16 males and 22 females, aged from 45 to 82 years with an average of (63.37±7.50) years. In the open group, there were 12 males and 26 females, aged from 51 to 82 years with an average of (63.76±8.21) years. The general conditions of operation, such as operation time, intraoperative blood loss, postoperative drainage, length of surgical incision, frequency of intraoperative fluoroscopy and postoperative time of lying in bed were recorded to analyze the differences in surgical related indicators. Visual analogue scale (VAS) of waist and leg pain in preoperative and postoperative period (3 days, 3 months, 6 months and 12 months) were recorded to evaluate pain remission;Oswestry Disability Index(ODI), Japanese Orthopaedic Association (JOA) score were recorded to evaluate the recovery of waist and leg function on preoperative and postoperative 12 months. The lumbar spondylolisthesis rate and intervertebral height at 12 months after operation were recorded to evaluate the reduction of spondylolisthesis. The Siepe intervertebral fusion standard was used to analyze the intervertebral fusion rate at 12 months after operation. RESULTS: ①All 114 patients were followed up more than 1 year, and no complications related to incision infection occurred. In the microscope group, there was 1 case of subcutaneous effusion 8 days after operation. After percutaneous puncture and drainage, waist compression, and then the healing was delayed. In the percutaneous group, 2 cases of paravertebral muscle necrosis occurred on the side of decompression, and the healing was delayed after debridement. In open group, there was 1 case of intraoperative dural tear, which was packed with free adipose tissue during the operation. There was no postoperative cerebrospinal fluid leakage and other related complications.① Compared with microscope group, percutaneous group increased in operation time, intraoperative blood loss, postoperative wound drainage, surgical incision length, intraoperative fluoroscopy times, and postoperative bed rest time. In open group, intraoperative blood loss, postoperative wound drainage, surgical incision length, and postoperative bed rest time increased, but the intraoperative fluoroscopy time decreased. Compared with percutaneous group, the intraoperative blood loss, wound drainage, surgical incision length, and postoperative bed rest time in open group increased, but operative time and the intraoperative fluoroscopy time decreased(P<0.05). ②ODI and JOA scores of the three groups at 12 months after operation were improved compared with those before operation (P<0.05), but there was no significant difference between the three group(P>0.05). ③Compared with microscope group, the VAS of low back pain in percutaneous group increased at 3 days after operation, and VAS of low back pain in open group increased at 3 days, and 12 month after operation. Compared with percutaneous group, the VAS low back pain score of the open group increased at 3 months after operation (P<0.05). ④ The lumbar spondylolisthesis rate of the three groups of patients at 12 months afrer operation was decreased compared with that before operation(P<0.05), and the intervertebral heigh was increased compared with that before operation(P<0.05), however, there was no significant difference among three groups at 12 months afrer operation(P>0.05). ⑤ There was no significant difference between three groups in the lumbar fusion rate at 12 months afrer operation(P>0.05). CONCLUSION: The MIS-TLIF assisted by microscope and the MIS-TLIF combined with percutaneous pedicle screw are safe and effective to treat the degenerative lumbar spondylolisthesis with single-segment, and the MIS-TLIF assisted by microscope may be more invasive, cause less blood loss and achieve better clinical efficacy.

Construction of microRNA-messenger networks for human osteosarcoma.

Osteosarcoma is the most common bone tumor in children and young adults. Although the microRNAs (miRNA) expression analyses of osteosarcoma have been performed previously, the construction of miRNA-messenger RNA (mRNA) networks for osteosarcoma is needed. This study aimed to identify osteosarcoma-related miRNAs through analyzing the microarray datasets and to construct the regulatory network of miRNA-mRNA for human osteosarcoma. The datasets were extracted from the Gene Expression Omnibus and the differentially expressed miRNAs were screened through the limma package in Bioconductor. Genes targeted by the differentially expressed miRNAs were screened out by using the Miranda, MirTarget2, PicTar, PITA, and TargetScan databases. The predicted target genes were further analyzed by Gene Ontology and pathway enrichment analysis and a regulatory network of differentially expressed miRNAs and their target osteosarcoma-associated genes was constructed. A total of 36 downregulated miRNAs and 182 upregulated miRNAs were identified in osteosarcoma samples compared with normal samples and 397 target genes for upregulated miRNAs and 222 target genes for downregulated miRNAs were obtained. The enriched pathways for target genes of differentially expressed miRNAs included transcriptional misregulation in cancer, the AMPK signaling pathway, and MAPK signaling pathway. In the regulatory network, has-miR-199a-5p targeted the highest number of genes and nemo-like kinase (NLK) was targeted by five miRNAs (hsa-miR-140-5p, hsa-miR-107, hsa-miR-324-5p, hsa-miR-199a-5p, and hsa-miR-28-5p). The has-miR-324-5p targets NLK, TGFB2, and PPARG. These miRNAs and their target genes may serve as potential therapeutic targets of osteosarcoma.

Isoniazid-loaded chitosan/carbon nanotubes microspheres promote secondary wound healing of bone tuberculosis.

Poor blood circulation makes it difficult for antitubercular drugs to achieve effective bactericidal concentration at tuberculose focus. The residual Mycobacterium tuberculosis around surgical wound would multiply, resulting in nonunion or sinus formation. Carbon nanotubes have strong tissue penetration and can cross many kinds of physiological barriers. Here, we constructed a chitosan/carbon nanotubes nanoparticles to control slow release of isoniazid. Transmission electron microscopy and nanoparticle tracking and analysis results showed that the diameter of chitosan/carbon nanotubes nanoparticles was between 150 and 250 nm. Chitosan/carbon nanotubes nanoparticles significantly prolonged the release time of isoniazid, and the release rate was more uniform, no sudden release was observed. In vitro experiments showed that chitosan/carbon nanotubes nanoparticles did not destroy biological function of isoniazid, but could reduce its cytotoxicity and inflammation. We further constructed animal model of tuberculous ulcer. The results showed that isoniazid/chitosan/carbon nanotubes nanoparticles promoted the healing of tuberculosis ulcer. Compared with isoniazid group and isoniazid/carbon nanotubes group, the area of wounds decreased by 94.6% and 89.8%, respectively. Immunohistochemistry showed that CD3+ and CD4+ T cell number decreased significantly in isoniazid/chitosan/carbon nanotubes group. In conclusion, we constructed a kind of isoniazid/chitosan/carbon nanotubes nanoparticles, which can significantly promote the healing of tuberculosis ulcer. Our study provided an effective way for the treatment of secondary wound healing of bone tuberculosis.

Peptide-Conjugated Gold Nanoprobe: Intrinsic Nanozyme-Linked Immunsorbant Assay of Integrin Expression Level on Cell Membrane.

Precisely quantifying the membrane protein expression level on cell surfaces is of vital importance for early cancer diagnosis and efficient treatment. We demonstrate that gold nanoparticle bioconjugated by a rationally designed peptide as nanoprobe possesses selective labeling and accurate quantification capacity of integrin GPIIb/IIIa on the human erythroleukemia cell line. Through selective recognition and marking of integrin, two-photon photoluminescence of the nanoprobe is exploited for direct observation of protein spatial distribution on cell membrane. More importantly, utilizing intrinsic enzyme-like catalysis property of the nanoprobe, the expression level of integrin on human erythroleukemia cells can be quantitatively counted in an amplified and reliable colorimetric assay without cell lysis and protein extraction process. In addition, the analysis of the correlation between the gold nanoparticle and the membrane protein via relevant inductively coupled plasma mass spectrometry measurement verifies the reliability of the new analytical method. It is anticipated that this facile and efficient strategy holds a great promise for a rapid, precise, and reliable quantification of interested functional membrane proteins on the cell surface.

Development of magnetic molecularly imprinted polymers based on carbon nanotubes - application for trace analysis of pyrethroids in fruit matrices.

The sensitive and efficient magnetic molecularly imprinted polymers (MMIPs) were successfully synthesized using carbon nanotubes as matrix and Fe3O4 particles as magnetic ingredient. Tetraethyl orthosilicate was used as modification material of the carbon nanotubes. Cyhalothrin, methacrylic acid and ethylene glycol dimethacrylate were used as template molecule, functional monomer and cross-linker, respectively. Azo-isobutyronitrile and polyvinylpyrrolidone were used as initiator and dispersant, respectively. The MMIPs were used for the separation of pyrethroids including beta-cyfluthrin, cyhalothrin, cyphenothrin and permethrin in fruit samples followed by high performance liquid chromatography analysis. The polymers were characterized with Fourier transform infrared spectrometry, Brunauer-Emmett-Teller method, transmission electron microscopy and a physical property measurement system. The isothermal absorption experiment, kinetics absorption experiment and selectivity of MMIPs were studied in detail. Scatchard analysis revealed that two kinds of different binding sites existed in MMIPs. The maximum adsorption capacities of two binding sites were 65.21 and 189.83mgg(-1), and dissociation constants were 7.11 and 30.40µgmL(-1), respectively. The kinetic property of MMIPs was well fitted to the second-order equation. The selectivity experiment indicated that MMIPs had higher selectivity toward cyhalothrin and its structural analogs than reference compound. The feasibility of detecting pyrethroids from real samples was testified in spiked fruit samples with different concentrations (0.025, 0.25 and 2.5mgkg(-1)). The LODs of beta-cyfluthrin, cyhalothrin, cyphenothrin and permethrin were 0.0072, 0.0035, 0.0062 and 0.0068mgkg(-1), respectively. Precisions of intra-day and inter-day ranging from 2.6% to 4.3% and 4.2% to 5.6% were obtained, respectively. This method was applied to determine pyrethroids in different fruit samples including apple, pear, orange, grape and peach, and satisfied recoveries (82.4-101.7%) were obtained.

[Genetic polymorphism of penta e locus in four Chinese nationalities].

To study the genetic polymorphism of Penta E locus in four Chinese nationalities using home made reagent kits, DNA samples were obtained from about 400 unrelated peoples of four different Chinese nationalities. As a result, we found 20 alleles in the four nationalities with frequencies ranging from 0.0048 to 0.2396. The genotype frequencies of Penta E locus met Hardy-Weinberg equilibrium. It proved that Penta E locus was a high polymorphic STR genetic marker and was valuable for forensic science.