Focal adhesion kinase(FAK),a non-receptor protein tyrosine kinase,is siting at the cross road of numerous intra-cellular signal transduction.The activated FAK has been implicated in a diverse array of cellular behavious.Recent research on FAK and fibrosis show that FAK plays an important role in fibrosis disease,so the targeting of FAK may be a novel therapeutic strategy.

ObjectiveTo study the relationship between EphA2 and laryngeal carcinoma angionegesis.MethodsImmunohistochemical staining was used to determine the expression of EphA2 and CD34 in 20 normal squamous epithelium tissues and 50 laryngeal carcinoma.The relationship between EphA2 expression and Microvascular density was analyzed bg Spearman correlation and linear regression.ResultsThe MVD in laryngeal carcinoma (54.89 ± 13.67) was significantly higher than that in normal squamous epithelial tissues ( 17.15 ± 5.21 ) ; The EphA2 in laryngeal carcinoma(4.56 ± 1.38) was significantly higher than that in normal squamous epithelial tissues ( 2.49 ± 1.23 ) ( t =16.721,5.847,all P ＜ 0.05 ).There was significant positive linear correlation between EphA2 and MVD in laryngeal squamous cell carcinoma(P ＜ 0.01 ).ConclusionEphA2 may correlate with angionegesis in laryngeal squamous cell carcinoma.

Objective To investigate the expression and correlation of cell cycle protein-D1 and Ki-67 in laryngeal cancer tis-sues.Methods Immunohistochemical streptavidin-perosidase ( SP) staining method was used to detect the expression of Cyclin D 1 and Ki-67 proteins in laryngeal squamous cell carcinoma tissues and their correlation between Cyclin D 1 and Ki-67 was analyzed .Re-sults Among 56 cases of Cyclin D1 positive over-expressed laryngeal squamous cell carcinoma tissues , 41 cases of Ki-67 had positive over-expression.Among 51 cases of Ki-67 low-expressed tissues, 35 cases of Cyclin D1 had low-expression.Spearman rank correlation test showed that expressions of Cyclin D 1 and Ki-67 in laryngeal squamous cell carcinoma tissues had a significant positive correlation ( rs =0.620, P <0.01).Conclusions Cyclin D1 and Ki-67 expressions in laryngeal squamous cell carcinoma have a significantly positive correlation .

Objective:To assess the impact of caudal regional anesthesia on complications after hypospadias repair with tubularised incised plate urethroplasty (TIP).Methods:A total of 125 cases with hypospadias undergoing TIP surgery from June 2017 to June 2019 at Beijing Children′s Hospital, Capital Medical University, were reviewed, aged 12-75 months, American Society of Anesthesiologists Ⅰ orⅡ grade.Totally, 86 cases had distal and 39 cases suffered from proximal hypospadias.Caudal anesthetics were used in 42 cases (caudal anesthesia group) and general anesthetics were used in 83 cases (general anesthesia group). All cases were repaired by TIP procedure.The children with urethral fistula and urethral stricture were followed up for 6 months, and multivariate statistical analyses were performed.Results:There were 11 cases of urethral fistula after hypospadias surgery, with 8 cases (9.64%)in the general anesthesia group and 3 cases (7.14%) in the caudal anesthesia group.There were no significant differences between the 2 groups ( χ2=0.223, P=0.636), and 12 cases of urethral stricture, with 8 cases(9.64%) in the general anesthesia group and 4 cases(9.52%) in the caudal anesthesia group.There were no significant differences between the 2 groups ( χ2=0.001, P=0.984). Based on multivariable Logistic regression, urethral fistula was associated with proximal hypospadias ( OR=0.036, 95% CI: 0.003-0.511, P=0.014), and the width of glans( OR=0.469, 95% CI: 0.220-0.998, P=0.049). Urethral stricture was correlated with proximal hypospadias( OR=0.004, 95% CI: 0.000-0.146, P=0.002), the width of urethral plate( OR=0.004, 95% CI: 0.000-0.422, P=0.020), and the duration of catheter( OR=72.976, 95% CI: 1.802-2 594.790, P=0.023). Conclusion:Caudal regional anesthesia appears to have no impacts on urethral fistula and stricture after hypospadias repair.

AIM: To investigate the effect of FAK - related non - kinase ( FRNK) on the expression of membrane - type matrix metalloproteinase -1 ( MT1 - MMP) in hepatic stellate cells ( HSC). METHODS:FRNK were trans-fected into HSCs by cationic liposome method. The protein levels of FRNK in HSC were assayed by Western blotting. The levels of MT1 - MMP were determined by RT - PCR for mRNA and by Western blotting for protein, respectively. RESULTS: The up -regulated expression of FRNK protein was observed and it was at 48 h after transfection that the FRNK protein content was the highest ( P < 0.05 ). The expressions of MT1 - MMP mRNA and protein were also up - regulated by the transfection of FRNK, and it was at 48 h after transfection that the MT1 - MMP protein content was significantly increased. CONCLUSION: The mRNA and protein of FRNK were over - expressed in HSC transfected with the gene of FRNK. The inhibitory effect of FRNK on the collagen synthesis in HSC may be through the up - regulation of MT1 - MMP.

We investigated serotype and resistance of Salmonella during pig slaughtering in Shandong Province,China,providing basic data for the risk assessment of Salmonella and for guiding the clinical medication.We used rapid classification kit to identify Salmonella serotype,adopted broth microdilution method to detect the resistance of 13 kinds drugs belong to 8 categories.Result showed that the identified 9 kinds of serotype were mainly S.derby and S.typhimurium.The resistance to 13 kinds drugs of 298 Salmonella were different.The higher percentage of tetracycline drugs as Doxycycline(DOX) and Tetracycline(TE) were 97.99％ and 80.20％,respectively,which was most sensitive to Colistine E.The resistant rate was only 2.01 ％,following by Amoxicillin/Clavulanic acid and Ofloxacin which were 2.35 ％ and 4.03％,and the multiple resistant rate was 81.88％.TE-DOX was the regnant drug-resistant spectrum.In conclusion,the predominant serotype of Salmonella in links of pig slaughtering in Shandong Province is S.derby,and resistance is different to the different drugs.The drug resistance of different slaughter links exist significant differences,multiple drug resistance is serious,and drug-resistant spectrum are varied.

Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.
Citric Acid Cycle ; Corynebacterium glutamicum/metabolism* ; Glutamic Acid/metabolism* ; Metabolic Engineering ; Metabolic Networks and Pathways/genetics*

L-glutamic acid is the world's largest bulk amino acid product that is widely used in the food, pharmaceutical and chemical industries. Using Corynebacterium glutamicum G01 as the starting strain, the fermentation by-product alanine content was firstly reduced by knocking out the gene encoding alanine aminotransferase (alaT), a major by-product related to alanine synthesis. Secondly, since the α-ketoglutarate node carbon flow plays an important role in glutamate synthesis, the ribosome-binding site (RBS) sequence optimization was used to reduce the activity of α-ketoglutarate dehydrogenase and enhance the glutamate anabolic flow. The endogenous conversion of α-ketoglutarate to glutamate was also enhanced by screening different glutamate dehydrogenase. Subsequently, the glutamate transporter was rationally desgined to improve the glutamate efflux capacity. Finally, the fermentation conditions of the strain constructed using the above strategy were optimized in 5 L fermenters by a gradient temperature increase combined with a batch replenishment strategy. The glutamic acid production reached (135.33±4.68) g/L, which was 41.2% higher than that of the original strain (96.53±2.32) g/L. The yield was 55.8%, which was 11.6% higher than that of the original strain (44.2%). The combined strategy improved the titer and the yield of glutamic acid, which provides a reference for the metabolic modification of glutamic acid producing strains.
Glutamic Acid ; Corynebacterium glutamicum/genetics* ; Ketoglutaric Acids ; Metabolic Engineering ; Alanine

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGAD^{S24R/D88R/Y309K} was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and Lpgad^{S24R/D88R/Y309K} genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD₆₀₀) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.
Glutamate Decarboxylase/genetics* ; Lactobacillus plantarum/genetics* ; Catalysis ; gamma-Aminobutyric Acid ; Hydrogen-Ion Concentration ; Glutamic Acid

Background A large amount of iron deposition in the brain can cause neuronal damage by inducing oxidative stress, neuroinflammation, and abnormal mitochondrial function. In addition, iron deposition is also reported to be closely related to the pathogenesis of Alzheimer's disease (AD). The neurofibrillary tangles aggregated by tau hyperphosphorylation are one of the important pathological features of AD. Objective To investigate potential effect of exogenous trivalent iron ions on neuronal activity in human neuroblastoma (SH-SY5Y) cells and tau hyperphosphorylation and aggregation. Methods SH-SY5Y cells were treated with ferric chloride (FeCl₃) at four concentrations (10, 100, 200, and 400 mg·L⁻¹). Cell survival rate was then detected by CCK8 assay. Intracellular iron content was determined prussian blue (Perl's) by iron staining after 24 h exposure to FeCl₃ at 10 or 200 mg·L⁻¹. Transfection of tau-P301L plasmid was conducted to construct an AD-like cell model for tau overexpression. The differences in the expression of the phosphorylated tau (p-tau) protein in SH-SY5Y cells and SH-SY5Y cells with tau overexpression were detected by Western blotting after 24 h exposure to FeCl₃ at 10 and 200 mg·L⁻¹. After dilution with phosphate buffered saline (PBS), FeCl₃, human tauR3, and FeCl₃ + tauR3 were incubated at 37℃, and the fluorescence intensity reflecting tau aggregation level was measured by thioflavin T(ThT) method at 12, 24, 36, 48, 60, 72, 84, and 96 h, respectively. Meanwhile, after 96 h coincubation of FeCl₃ and tauR3, the fibers formed by tau aggregation were observed under a transmission electron microscope (TEM). Results After 24 h of FeCl₃ exposure, the cell survival rate of SH-SY5Y cells among all groups was statistically different (F=8.63, P<0.01). The cell survival rates in the 200 and 400 mg·L⁻¹ groups were 80.1% and 68.7% of the control group, respectively (P<0.05). Compared with the control group, the nuclei of the 200 mg·L⁻¹ FeCl₃ group were mainly yellowish-brown after iron staining and the positive cell rate was up-regulated by 12.9% (P<0.01). After 24 h of FeCl₃ exposure , the p-tau (Ser396) protein expression was statistically different among all groups (F=11.6, P<0.01). Compared with the control group, the p-tau protein expression level of SH-SY5Y cells in the 200 mg·L⁻¹ group was up-regulated by 72.7% (P<0.01). After FeCl₃-treated SH-SY5Y cells with tau overexpression for 24 h, the p-tau (Ser396) protein expression was statistically different among all groups (F=27.8, P<0.01). Compared with the tau group, the p-tau (Ser396) protein expression level of SH-SY5Y cells in the tau + 200 mg·L⁻¹ group was up-regulated by 44.6% (P<0.05). Compared with the tauR3 group, the fluorescence intensities in the 84 and 96 h tauR3 + FeCl₃ groups were up-regulated by 49.9% and 53.7% (P<0.01) respectively. After 96 h of coincubation, compared with the tauR3 group, FeCl₃ + tauR3 aggravated tau aggregation and formed fiber deposition under TEM. Conclusion Exogenous trivalent iron ions may inhibit SH-SY5Y cell viability, promote the phosphorylation of tau in SH-SY5Y cells transfected with tau-P301L plasmid, and aggravate tauR3 aggregation and fiber production.