Comparative Biomechanical Stability of the Fixation of Different Miniplates in Restorative Laminoplasty after Laminectomy: A Finite Element Study.

A novel H-shaped miniplate (HSM) was specifically designed for restorative laminoplasties to restore patients' posterior elements after laminectomies. A validated finite element (FE) model of L2/4 was utilized to create a laminectomy model, as well as three restorative laminoplasty models based on the fixation of different miniplates after a laminectomy (the RL-HSM model, the RL-LSM model, and the RL-THM model). The biomechanical effects of motion and displacement on a laminectomy and restorative laminoplasty with three different shapes for the fixation of miniplates were compared under the same mechanical conditions. This study aimed to validate the biomechanical stability, efficacy, and feasibility of a restorative laminoplasty with the fixation of miniplates post laminectomy. The laminectomy model demonstrated the greatest increase in motion and displacement, especially in axial rotation, followed by extension, flexion, and lateral bending. The restorative laminoplasty was exceptional in preserving the motion and displacement of surgical segments when compared to the intact state. This preservation was particularly evident in lateral bending and flexion/extension, with a slight maintenance efficacy observed in axial rotation. Compared to the laminectomy model, the restorative laminoplasties with the investigated miniplates demonstrated a motion-limiting effect for all directions and resulted in excellent stability levels under axial rotation and flexion/extension. The greatest reduction in motion and displacement was observed in the RL-HSM model, followed by the RL-LSM model and then the RL-THM model. When comparing the fixation of different miniplates in restorative laminoplasties, the HSMs were found to be superior to the LSMs and THMs in maintaining postoperative stability, particularly in axial rotation. The evidence suggests that a restorative laminoplasty with the fixation of miniplates is more effective than a conventional laminectomy due to the biomechanical effects of restoring posterior elements, which helps patients regain motion and limit load displacement responses in the spine after surgery, especially in axial rotation and flexion/extension. Additionally, our evaluation in this research study could benefit from further research and provide a methodological and modeling basis for the design and optimization of restorative laminoplasties.

[Construction and stability analysis of finite element model for spinal canal reconstruction with miniplates fixation].

OBJECTIVE: To establish the finite element model of spinal canal reconstruction and internal fixation,analysis influence of spinal canal reconstruction and internal fixation on spinal stability,and verify the effectiveness and reliability of spinal canal reconstruction and internal fixation in spinal canal surgery. METHODS: A 30-year-old male healthy volunteer with a height of 172 cm and weight of 75 kg was selected and his lumbar CT data were collected to establish a finite element model of normal lumbar L3-L5,and the results were compared with in vitro solid results and published finite element analysis results to verify the validity of the model. They were divided into normal group,laminectomy group and spinal canal reconstruction group according to different treatment methods. Under the same boundary fixation and physiological load conditions,six kinds of activities were performed,including forward bending,backward extension,left bending,right bending,left rotation and right rotation,and the changes of range of motion (ROM) of L3-L4,L4-L5 segments and overall maximum ROM of L3-L5 were analyzed under the six conditions. RESULTS: The ROM displacement range of each segment of the constructed L3-L5 finite element model was consistent with the in vitro solid results and previous literature data,which confirms the validity of the model. In L3-L4,ROM of spinal canal reconstruction group was slightly increased than that of normal group during posterior extension(>5% difference),and ROM of other conditions was similar to that of normal group(<5% difference). ROM in laminectomy group was significantly increase than that in normal group and spinal canal reconstruction group under the condition of flexion,extension,left and right rotation. In L4-L5,ROM in spinal canal reconstruction group was similar to that in normal group(<5% difference),while ROM in laminectomy group was significantly higher than that in normal group and spinal canal reconstruction group(>5% difference). In the overall maximum ROM of L3-L5,spinal canal reconstruction group was only slightly higher than normal group under the condition of posterior extension(>5% difference),while laminectomy was significantly higher than normal group and spinal canal reconstruction group under the condition of anterior flexion,posterior extension,left and right rotation(>5% difference). The changes of each segment ROM and overall ROM of L3-L5 showed laminectomy group>spinal canal reconstruction group>normal group. CONCLUSION: Laminectomy could seriously affect biomechanical stability of the spine,but application of spinal canal reconstruction and internal fixation could effectively reduce ROM displacement of the responsible segment of spine and maintain its biomechanical stability.

Isolation of Limosilactobacillus reuteri Strain with Anti-porcine Epidemic Diarrhea Virus from Swine Feces.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea diseases in piglets, which has brought huge economic losses to the pig industry. As the dominant Lactobacillus species in the piglet intestine, the antiviral effect of Limosilactobacillus reuteri (L. reuteri) has been reported. Nine L. reuteri strains were isolated and identified from swine feces in this study. The CCK-8 assay examined the anti-PEDV potential of their cell-free supernatant (CFS). Among the nine L. reuteri isolates examined, LRC8 had a higher inhibition rate to PEDV than the other strains. Thus, the biological properties of the LRC8 strain, such as growth ability, acid production ability, acid and bile salt tolerance, and adhesion to IPEC-J2 cells, were evaluated. Besides, the anti-PEDV activity of LRC8-CFS (LRC8 metabolites, LRM) was assessed using plaque reduction assays, indirect immunofluorescence assays, RT-qPCR, and western blotting. The mRNA relative expression levels of inflammatory factors including IL-1ß, IL-6, IL-8, MCP1, and TNF-α were determined by RT-qPCR. The results showed that the LRC8 strain grew well, was resistant to acid, tolerated bile salts, and adhered strongly to IPEC-J2 cells. In addition, treatment with its CFS (LRM) dramatically downregulated the mRNA expression levels of inflammatory cytokines, and in the Vero cell culture, prophylactic, therapeutic, competitive, and direct-inhibitory actions were seen against PEDV. Finally, we explored the anti-PEDV effects of the LRC8 strain in piglets and found that the LRC8 strain effectively relieved the clinical symptoms and intestinal damage of piglets infected by PEDV. To sum up, we found a L. reuteri strain with an anti-PEDV effect.

MAFB Promotes Cancer Stemness and Tumorigenesis in Osteosarcoma through a Sox9-Mediated Positive Feedback Loop.

Despite the fact that osteosarcoma is one of the most common primary bone malignancies with poor prognosis, the mechanism behind the pathogenesis of osteosarcoma is only partially known. Here we characterized differentially expressed genes by extensive analysis of several publicly available gene expression profile datasets and identified musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) as a key transcriptional regulator in osteosarcoma progression. MAFB was highly expressed in tumor tissues and required for proliferation and tumorigenicity of osteosarcoma cells. MAFB expression was elevated in osteosarcoma stem cells to maintain their self-renewal potential in vitro and in vivo through upregulation of stem cell regulator Sox9 at the transcriptional level. Sox9 in turn activated MAFB expression via direct recognition of its sequence binding enrichment motif on the MAFB locus, thereby forming a positive feedback regulatory loop. Sox9-mediated feedback activation of MAFB was pivotal to tumorsphere-forming and tumor-initiating capacities of osteosarcoma stem cells. Moreover, expression of MAFB and Sox9 was highly correlated in osteosarcoma and associated with disease progression. Combined detection of both MAFB and Sox9 represented a promising prognostic biomarker that stratified a subset of patients with osteosarcoma with shortest overall survival. Taken together, these findings reveal a MAFB-Sox9 reciprocal regulatory axis driving cancer stemness and malignancy in osteosarcoma and identify novel molecular targets that might be therapeutically applicable in clinical settings. SIGNIFICANCE: Transcription factors MAFB and Sox9 form a positive feedback loop to maintain cell stemness and tumor growth in vitro and in vivo, revealing a potential target pathway for therapeutic intervention in osteosarcoma.

Structure and characterization of Aspergillus fumigatus lipase B with a unique, oversized regulatory subdomain.

Fungal lipases are efficient and environment-friendly biocatalysts for many industrially relevant processes. One of the most widely applied lipases in the manufacturing industry is Candida antarctica lipase B (CALB). Here, we report the biochemical and structural characterization of a novel CALB-like lipase from an important human pathogen-Aspergillus fumigatus (AFLB), which has high sn-1,3-specificity toward triolein. AFLB crystal structure displays a CALB-like catalytic domain and hosts a unique tightly closed 'lid' domain that contains a disulfide bridge, as well as an extra N-terminal subdomain composed of residues 1-128 (including the helix α1-α5 located above the active site). To gain insight into the function of this novel lid and N-terminal subdomain, we constructed and characterized a series of mutants in these two domains. Deleting the protruding bulk lid's residues, replacing the bulk and tight lid with a small and loose lid from CALB, or breaking the disulfide bridge increased the affinity of CALB for glyceride substrates and improved its catalytic activity, along with the loss of enzyme fold stability and thermostability. N-terminal truncation mutants revealed that the N-terminal peptide (residues 1-59) is a strong inhibitor of AFLB binding to lipid films. This peptide thus limits AFLB's penetration power and specific activity, revealing a unique enzyme activity regulatory mechanism. Our findings on the functional and structural properties of AFLB provide a better understanding of the functions of the CALB-like lipases and pave the way for its future protein engineering. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6IDY.

Biocontrol activity of recombinant aspartic protease from Trichoderma harzianum against pathogenic fungi.

The use of cell wall degrading enzymes of Trichoderma is a promising alternative for improving food storage. The aspartic protease P6281 secreted by the fungus Trichoderma harzianum plays an important role in mycoparasitism on phytopathogenic fungi. In this study, recombinant P6281 (rP6281) expressed in Pichia pastoris showed high activity of 321.8 U/mL. Maximum activity was observed at pH 2.5 and 40 °C, and the enzyme was stable in the pH range of 2.5-6.0. rP6281 significantly inhibited spore germination and growth of plant and animal pathogenic fungi such as Botrytis cinerea, Mucor circinelloides, Aspergillus fumigatus, Aspergillus flavus, Rhizoctonia solani, and Candida albicans. Transmission electron microscopy revealed that rP6281 efficiently damages the cell wall of Botrytis cinerea. In addition, the protease significantly inhibited the development of grey mold that causes rotting of apple, orange, and cucumber, indicating that rP6281 may be developed as an effective anti-mold agent for fruit storage.

A novel pH-stable, endoglucanase (JqCel5A) isolated from a salt-lake microorganism, Jonesia quinghaiensis

Background: Endoglucanase, one of three type cellulases, can randomly cleave internal p-1,4-linkages in cellulose polymers. Thus, it could be applied in agricultural and industrial processes. Results: A novel endoglucanase gene (JqCel5A) was cloned from Jonesia quinghaiensis and functionally expressed in Escherichia coli Rosetta (DE3). It contained 1722 bp and encoded a 573-residue polypeptide consisting of a catalytic domain of glycoside hydrolase family 5 (GH5) and a type 2 carbohydrate-binding module (CBM2), together with a predicted molecular mass of 61.79 kD. The purified JqCel5A displayed maximum activity at 55°C and pH 7.0, with 21.7 U/mg, 26.19 U/mg and 4.81 U/mg towards the substrate carboxymethyl cellulose, barley glucan and filter paper, respectively. Interestingly, JqCel5A exhibited high pH stability over a broad pH range of pH (3-11), and had good tolerance to a wide variety of deleterious chemicals including heavy metals and detergent. The catalytic mechanism of JqCel5A was also investigated by site mutagenesis and homology-modeling in this study. Conclusions: It was believed that these properties might make JqCel5A to be potentially used in the suitable industrial catalytic condition, which has a broad pH fluctuation and/or chemical disturbance.

Improving the activity of the endoglucanase, Cel8M from Escherichia coli by error-prone PCR.

Endoglucanase is a key enzyme involved in cellulose hydrolysis and can be used in multiple industrial fields. In this study, we used error-prone PCR to engineer the endoglucanase, Cel8M, from Escherichia coli. The Cel8M belongs to the glycoside hydrolase family 8 and shows 99% identity with the reported endoglucanase from E. coli K12. Through screening of approximately 10,000Cel8M variants, two variants, Cel8ME15 and Cel8ME18, respectively showing 1.42 fold and 1.61 fold increased activities, were obtained. Through sequence analysis, it was found that Cel8ME15 had two mutations, with the residues Ala9 and Glu353 respectively substituting the residues Val9 and Lys353 of Cel8M; while Cel8ME18 had one mutation with the residue Ser117 replacing the residue Gly117 of Cel8M. Based on the analysis of the predicted 3D structure of Cel8M, it was suggested that changes of K353E and G117S might directly affect the substrate binding affinity and therefore contribute to the improved activities of Cel8ME15 and Cel8ME18. Based on all the results we had, it is believed that this study should provide a useful reference for the future engineering of other endoglucanases from glycoside hydrolase family 8.

The Structure and Enzyme Characteristics of a Recombinant Leucine Aminopeptidase rLap1 from Aspergillus sojae and Its Application in Debittering.

A leucine aminopeptidase Lap1 was cloned from Aspergillus sojae GIM3.30. The truncated Lap1 without a signal peptide was over-expressed in P. pastoris, and the enzymatic characteristics of recombinant Lap1 (rLap1) were tested. The rLap1 was about 36.7 kDa with an optimal pH 8.0 and optimal temperature 50 °C for substrate Leu-p-nitroanilide and it sustained 50 % activity after 1 h incubation at 50 °C. The activity of rLap1 was significantly inhibited by EDTA, whereas Co(2+), Mn(2+), and Ca(2+) ions, but not Zn(2+) ions, restored its activity. rLap1 showed the highest activity against Arg-pNA and then Leu-, Lys-, Met-, and Phe-pNA. The 3D structure of rLap1 showed it had a conserved functional charge/dipole complex and a hydrogen bond network of Zn2-D179-S228-Q177-D229-S158 around its active center. An acidic Asp residue was found at the bottom of the substrate binding pocket, which explains its preference for basic N-terminal amino acid substrates such as Arg and Lys. rLap1 improved the degree of hydrolysis of casein and soy protein hydrolysates and also decreased their bitterness, indicating its potential utility in food production.

The flavonoid ampelopsin inhibited cell growth and induced apoptosis and G0/G1 arrest in human osteosarcoma MG-63 cells in vitro.

Ampelopsin (AMP), a novel flavonoid, has been shown to effectively inhibit the proliferation and induce apoptosis of some prostate cancer and breast cancer cell lines. Whether AMP has chemopreventive effects on the cell growth and apoptosis of human osteosarcoma MG-63 cells remains unknown. In the present study, MG-63 cells were exposed to different concentrations (0, 25, 50, 75, 100 µmol/L) of AMP for 24, 48, 72 and 96 h and then the cell viability was measured by CCK-8 assay. The AMP-induced apoptotic cells were identified by Hochest33258 staining and quantified by Annexin V-FITC/PI double staining using flowcytometry (FCM). The effect of ampelopsin (AMP) on cell cycle was evaluated using PI staining with FCM. The protein levels of cyclin A, CDK2 and p21(CIP1) were measured by Western blotting. The cell viability was reduced in a time- and dose-dependent manner after exposure to AMP atarangeof 20-100 µmol/L. For the treatment of AMP, increases of apoptotic index and rate were observed in MG-63 cells. The AMP blocked cells in the G0/G1 phase of the cell cycle. Furthermore, AMP increased p21(CIP1) expression but decreased cyclin A and CDK2 expression after AMP exposure. AMP inhibited cell growth and induced apoptosis and G0/G1 phase arrest in MG-63 cells in vitro, with the potential mechanism of the negative regulation of cell cycle-related protein.

Enzymatic characteristics of a recombinant neutral protease I (rNpI) from Aspergillus oryzae expressed in Pichia pastoris.

A truncated neutral protease I (NpI) from Aspergillus oryzae 3.042 was expressed in Pichia pastoris with a high enzyme yield of 43101 U/mL. Its optimum pH was about 8.0, and it was stable in the pH range of 5.0-9.0. Its optimum temperature was about 55 °C and retained >90% activity at 50 °C for 120 min. Recombinant NpI (rNpI) was inhibited by Cu(2+) and EDTA. Eight cleavage sites of rNpI in oxidized insulin B-chain were determined by mass spectrometry, and five of them had high hydrophobic amino acid affinity, which makes it efficient in producing antihypertensive peptide IPP from ß-casein and a potential debittering agent. The high degree of hydrolysis (DH) of rNpI to soybean protein (8.8%) and peanut protein (11.1%) compared to papain and alcalase makes it a good candidate in the processing of oil industry byproducts. The mutagenesis of H(429), H(433), and E(453) in the deduced zinc-binding motif confirmed rNpI as a gluzincin. All of these results show the great potential of rNpI to be used in the protein hydrolysis industry.