Mammalian Target of Rapamycin 2 (MTOR2) and C-MYC Modulate Glucosamine-6-Phosphate Synthesis in Glioblastoma (GBM) Cells Through Glutamine: Fructose-6-Phosphate Aminotransferase 1 (GFAT1).

Glucose and glutamine are two essential ingredients for cell growth. Glycolysis and glutaminolysis can be linked by glutamine: fructose-6-phosphate aminotransferase (GFAT, composed of GFAT1 and GFAT2) that catalyzes the synthesis of glucosamine-6-phosphate and glutamate by using fructose-6-phosphate and glutamine as substrates. The role of mammalian target of rapamycin (MTOR, composed of MTOR1 and MTOR2) in regulating glycolysis has been explored in human cancer cells. However, whether MTOR can interact with GFAT to regulate glucosamine-6-phosphate is poorly understood. In this study, we report that GFAT1 is essential to maintain the malignant features of GBM cells. And MTOR2 rather than MTOR1 plays a robust role in promoting GFAT1 protein activity, and accelerating the progression of glucosamine-6-phosphate synthesis, which is not controlled by the PI3K/AKT signaling. Intriguingly, high level of glucose or glutamine supply promotes MTOR2 protein activity. In turn, up-regulating glycolytic and glutaminolytic metabolisms block MTOR dimerization, enhancing the release of MTOR2 from the MTOR complex. As a transcriptional factor, C-MYC, directly targeted by MTOR2, promotes the relative mRNA expression level of GFAT1. Notably, our data reveal that GFAT1 immunoreactivity is positively correlated with the malignant grades of glioma patients. Kaplan-Meier assay reveals the correlations between patients' 5-year survival and high GFAT1 protein expression. Taken together, we propose that the MTOR2/C-MYC/GFAT1 axis is responsible for the modulation on the crosstalk between glycolysis and glutaminolysis in GBM cells. Under the condition of accelerated glycolytic and/or glutaminolytic metabolisms, the MTOR2/C-MYC/GFAT1 axis will be up-regulated in GBM cells.

Innovative hydrogel-patch combination for large annulus fibrosus defects: a prospective approach to address herniation recurrence.

BACKGROUND CONTEXT: Large annulus fibrosus (AF) defects often lead to a high rate of reherniation, particularly in the medial AF region, which has limited self-healing capabilities. The increasing prevalence of herniated discs underscores the need for effective repair strategies. PURPOSE: The objectives of this study were to design an AF repair technique to reduce solve the current problems of insufficient mechanical properties and poor sealing capacity. STUDY DESIGN: In vitro biomechanical experiments and finite element analysis. METHODS: The materials used in this study were patches and hydrogels with good biocompatibility and sufficient mechanical properties to withstand loading in the lumbar spine. Five repair techniques were assessed in this study: hydrogel filler (HF), AF patch medial barrier (MB), AF patch medial barrier and hydrogel filler (MB&HF), AF patch medial-lateral barrier (MLB), and AF patch medial-lateral barrier and hydrogel filler (MLB&HF). The repair techniques were subjected to in vitro testing (400 N axial compression and 0-500 N fatigue loading at 5Hz) and finite element analysis (400 N axial compression) to evaluate the effectiveness at repairing large AF defects. The evaluation included repair tightness, spinal stability, and fatigue resistance. RESULTS: From the in vitro testing, the failure load of the repair techniques was in the following order HF MLB >MB&HF >MLB&HF. CONCLUSIONS: The combined use of patches and hydrogels exhibited promising mechanical properties postdiscectomy, providing a promising solution for addressing large AF defects and improving disc stability. CLINICAL SIGNIFICANCE: This study introduces a promising method for repairing large annular fissure (AF) defects after disc herniation, combining patch repair with a hydrogel filler. These techniques hold potential for developing clinical AF repair products to address this challenging issue.

Shape and Size of the Annulus Fibrosus Excision Alters the Biomechanics of the Intervertebral Disc.

STUDY DESIGN: Biomechanical testings and finite element analysis. OBJECTIVES: This study aims to investigate how annulus fibrosus (AF) incision size (RIS, Ratio of incision width to AF height) and shape affect intervertebral disc (IVD) biomechanics. METHODS: A validated finite element model of lumbar spines simulated various incisions in the middle-right posterior region of the AF, with different sizes and shapes. Simulations included axial compression, flexion, extension, bending, and rotation. Parameters assessed included stability, re-herniation, and IVD degeneration by analyzing stress, height, Intradiscal pressure (IDP), and the range of motion (ROM). RESULTS: Incision increased AF stress and ROM under 3 Nm moment, with values rising as RIS increased. RIS exceeding 40% resulted in a 20% AF stress increase during compression and extension, while RIS over 50% led to over 20% AF stress increase during other motions. Incision stress also increased with higher RIS, particularly surpassing 50% RIS. IDP rose across all incision shapes. Endplate stress increased (9.9%-48.9%) with larger incisions, with average increases of 12.8%, 12.7%, 30.5%, and 22.8% for circular, oval, square, and rectangular incisions. Compression and rotation minimally affected NP pressure (<15%), while flexion (19.8%-38.8%) and bending (18.5%-43.9%) had a more pronounced effect. ROM increased with RIS (20.0% ∼ 77.4%), especially with an incision RIS exceeding 40%. CONCLUSIONS: AF injury elevates AF stress, reduces spine stability, heightens degeneration risk with increasing RIS. Reherniation risk rises when RIS exceeds 40%. Circular or oval incisions maintain spine biomechanics better than square or rectangular ones.

[Expressions of p-c-jun and cyclinD1 between vascular smooth muscle cells and endothelial cells exposured to cigarette smoke extract].

OBJECTIVE: To investigate the cell viabilities of vascular smooth muscle cells and vascular endothelial cells stimulated by cigarette smoke extract(CSE) . METHODS: The CSE was prepared by smoke-bubbled phosphate buffered saline(PBS) generation.After culturing cells with different concentrations of CSE, we used the cell counting kit-8 to determine the cell viability.The expression levels of c-jun and cyclinD1 were analyzed through Western blot.The c-jun plasmid was transfected to detect the change of cyclinD1 expression. RESULTS: The smooth muscle cell viability increased when the CSE concentration ranged 0.625%-10%, whereas the endothelial cells viability decreased when exposed to the CSE concentration. After exposure to CSE for 48 hours, there was no difference in c-jun expression between toxin group and PBS group;however, the expression of p-c-jun in the smooth muscle cells significantly increased in the toxin groups than in the PBS group(P<0.05) and the expression of p-c-jun in the vascular endothelial cells significantly decreased(P<0.05) . The level of cyclinD1 significantly increased after exposed to CSE, and its expression level also increased in respond to the c-jun overexpression. CONCLUSION: CSE can enhance the proliferation of vascular smooth muscle cells and decrease in the activity of endothelial cells proliferation, which may be explained by the phosphorylation of c-jun and the expression of cyclinD1.

Biomechanical evaluation of a novel intervertebral disc repair technique for large box-shaped ruptures.

Objective: The purpose of this study was to analyze the feasibility of repairing a ruptured intervertebral disc using a patch secured to the inner surface of the annulus fibrosus (AF). Different material properties and geometries for the patch were evaluated. Methods: Using finite element analysis, this study created a large box-shaped rupture in the posterior-lateral region of the AF and then repaired it with a circular and square inner patch. The elastic modulus of the patches ranged from 1 to 50 MPa to determine the effect on the nucleus pulposus (NP) pressure, vertical displacement, disc bulge, AF stress, segmental range of motion (ROM), patch stress, and suture stress. The results were compared against the intact spine to determine the most suitable shape and properties for the repair patch. Results: The intervertebral height and ROM of the repaired lumbar spine was similar to the intact spine and was independent of the patch material properties and geometry. The patches with a modulus of 2-3 MPa resulted in an NP pressure and AF stresses closest to the healthy disc, and produced minimal contact pressure on the cleft surfaces and minimal stress on the suture and patch of all models. Circular patches caused lower NP pressure, AF stress and patch stress than the square patch, but also caused greater stress on the suture. Conclusion: A circular patch with an elastic modulus of 2-3 MPa secured to the inner region of the ruptured annulus fibrosus was able to immediately close the rupture and maintain an NP pressure and AF stress similar to the intact intervertebral disc. This patch had the lowest risk of complications and produced the greatest restorative effect of all patches simulated in this study.

Seroprevalence of porcine cytomegalovirus and sapovirus infection in pigs in Hunan province, China.

The seroprevalence of porcine cytomegalovirus (PCMV) and sapovirus (SaV) infections in pigs was investigated in Hunan province, China, between May 2005 and October 2010. A total of 500 pig serum samples collected from 10 representative administrative regions in Hunan province were evaluated for antibodies against PCMV and SaV using enzyme-linked immunosorbent assay (ELISA). The overall seroprevalence of porcine cytomegalovirus and sapovirus in pigs was 96.40% (482/500) and 63.40% (317/500), and the seropositivity of 10 herds we surveyed varied, ranging from 94.74% to 98.48% and 56.36% to 72.50%, respectively. The highest prevalence was found in breeding sows (96.67% for PCMV and 83.33% for SaVs). The results of the present survey indicated that infections with porcine cytomegalovirus and sapovirus are highly prevalent in pigs in Hunan province, China.

RT-PCR test for detecting porcine sapovirus in weanling piglets in Hunan Province, China.

The prevalence of porcine sapovirus infection in weanling pigs was investigated in Hunan Province, China, between August 2006 and October 2007. A total of 153 diarrheic fecal samples from ten intensive pig farms from ten representative administrative regions in Hunan province were examined for porcine sapoviruses using RT-PCR. Twenty-two of 153 (14.37 %) samples were found to contain porcine sapoviruses. Phylogenetic analysis showed that all the porcine sapovirus isolates in Hunan Province belonged to the porcine sapovirus genogroup III. The results of the present investigation have implications for the control of porcine sapovirus infection in pigs in Hunan Province, China.

Biomechanical evaluation of a short-rod technique for lumbar fixation surgery.

Objective: The purpose of this study was to analyze the stability and instrument-related complications associated with fixation of the lumbar spine using the Short-Rod (SR) technique. Methods: Using finite element analysis, this study assessed the stability of a bilateral lumbar fixation system when inserting the pedicle screws at angles of 10°, 15°, and 20° to the endplate in the sagittal plane. Using the most stable construct with a screw angle, the model was then assessed with different rod lengths of 25, 30, 35, and 45 mm. The optimal screw inclination angle and rod length were incorporated into the SR model and compared against traditional parallel screw insertion (pedicle screws in parallel to the endplate, PPS) in terms of the stability and risk of instrument-related complications. The following parameters were evaluated using the validated L4-L5 lumbar finite element model: axial stiffness, range of motion (ROM), stress on the endplate and facet joint, von-Mises stress on the contact surface between the screw and rod (CSSR), and screw displacement. Results: The results showed that the SR model with a 15° screw inclination angle and 35 mm rod length was superior in terms of construct stability and risk of complications. Compared to the PPS model, the SR model had lower stiffness, lower ROM, less screw displacement, and lower stress on the facet cartilage, the CSSR, and screws. However, the SR model also suffered more stress on the endplate in flexion and lateral bending. Conclusion: The SR technique with a 15° screw inclination and 35 mm rod length offers good lumbar stability with a low risk of instrument-related complications.

[Establishment of a targeting protein for Xenopus kinesin-like protein 2 C' terminal SBP-3 x Flag tagged HCT 116 colorectal cancer cell model].

OBJECTIVE: To develop a targeting protein for Xenopus kinesin-like protein 2 (TPX2) C' terminal SBP-3 x Flag-tagged HCT 116 cell model. METHODS: Homologous arms were amplified by polymerase chain reaction (PCR), and then the adeno-associated virus (AAV) -targeting vector of TPX2 was constructed. HCT 116 cells were targeted after the viruses were packaged. Positive cell clones with neomycin resistance gene were obtained by G418 and PCR screening. Finally, the neomycin gene cassette was excised after the targeted clones were infected with adenovirus expressing Cre-recombinase, and the TPX2 C' terminal SBP and 3 x Flag endogenous double-tagged HCT 116 cells were obtained by PCR screening. RESULTS: Two positive cell clones with neomycin resistance gene were obtained by PCR screening. The positive clones with neomycin resistance gene excised were obtained by Cre adenovirus infection, and the knock-in of SBP-3 x Flag gene was verified by Western blot analysis. CONCLUSION: The TPX2 C' terminal SBP-3 x Flag tagged HCT 116 cell model was successfully established.

[Microbial Communities and Sludge Specific Resistance in Two SBRs Treating Leachate].

The relationship between microbial populations and sludge filtration performance was studied when active sludge was used to treat the leachate from municipal solid waste incineration plants. Two SBRs (SBR1 and SBR2) were operated at the same conditions, except that SBR1 was exposed to the sunlight and SBR2 was in the dark. To identify the difference in microbial populations in two reactors, high-throughput sequencing method was used. On the 50th day, the fungi abundance in SBR2 was higher than in SBR1. Phylum Rozellomycota became the dominant fungi in SBR1, whose relative abundance was 83.71%. Phylum Basidiomycota and Genus Trichosporon became only dominant fungi in SBR2, whose relative abundances were 99.84% and 99.78%, respectively. Bacterial abundance in SBR1 was higher than in SBR2. In SBR1, Thauera was the major bacterial genus, whose relative abundance was 39.35%. In SBR2, Planktosalinus, Thauera, and Ottowia were the major bacterial genera, whose relative abundances were 16.84%, 16.23%, and 12.55%. Rotifers and other predatory metazoan were detected on the 30th-50th days in SBR1 and sludge specific resistance began to decline on the 35th day. Filamentous fungi bulking caused by Trichosporon resulted in a continuous increase in sludge specific resistance of SBR2. The dominant microbial communities (especially fungi) and sludge specific resistances in SBR1 and SBR2 were very different because of the effect of sunlight. Therefore, sunlight plays an important role on microbial communities and sludge characteristics.