Objective To study the CT features and clinical value of cerebral ischemia infarction. Methods the CT feature of 106 diabetes ischemia infarction were analyzed. Results The patients with is-chemia brain white matter change (35 cases),small area cerebral ischemia infarction (85 cases),great area cerebral ischemia infarction ( 12 cases ),cerebral hemorrhage (6 cases). Conclusions Cerebral ischemia infarction of diabetes mellitus mainly as small area multiple cerebral ischemia infarction,ischemia cerebral white matter lesion located at the area of base ganglion thalamencephalon and cerebellum,termly CT cerebral examination can diagnosis and instruct treatment to the complication of cerebral ischemia apoplexy of type 2 diabetes mellitus.

OBJECTIVE:To prepare urapidil osmotic pump tablet(OPT) characterized by 24 h constant drug release in vitro.METHODS:OPT of urapidil was prepared using NaCl and low or high moleculan weight PEO(Mr 4?106、2?105) as core,CA and PEG-400 as the coating material.Similarity factor was used to evaluate formulation of osmotic pump tablets.The drug release mechanism was investigated as well.RESULTS:The optimal core formulation consisted of urapidil 60 mg,NaCl 190 mg,PEO(Mr 4?106) 90 mg,PEO(Mr 2?105) 90 mg.The drug was released from OPT at controlled rate 24 h.CONCLUSION:The preparation of osmotic pump tablets was simple and characterized by zero-order release.

Objective To evaluate the effects of Ro20?1724 on cognitive dysfunction induced by repetitive ketamine anesthesia in immature rats. Methods Thirty?two Sprague?Dawley rats of both sex, aged 21 days, weighing 45-55 g, were randomly divided into 4 groups ( n=8 each ) using a random number table: control group ( group C ) , ketamine group ( group K ) , ketamine+Ro20?1724 group ( group K+R) , and ketamine+anhydrous alcohol group ( group K+A) . In K, K+R and K+E groups, 70 mg∕kg ketamine was intraperitoneally injected once a day for 7 consecutive days. The equal volume of normal saline was given instead in group C. Two days after the rats were fed a common diet, 0.5 mg∕kg Ro20?1724 and the equal volume of anhydrous alcohol were injected in K+R and K+E groups, respectively, and the equal volume of normal saline was given instead in K and C groups, once a day for 7 consecutive days. Morris water maze test was used to test cognitive function, and the escape latency and frequency of crossing the original platform were recorded. The rats were sacrificed after the end of behavior tests, and hippocampi were removed to detect the content of brain?derived neurotrophic factor ( BDNF) in CA1 region using enzyme?linked immunosorbent assay. Results Compared with group C, the escape latency was significantly prolonged on 1st-4th days in K and K+E groups, the escape latency was prolonged on 3rd-4th days in K+R group, and the frequency of crossing the original platform and content of BDNF in CA1 region were decreased in K, K+R and K+E groups. Compared with K group, the escape latency was significantly shortened, and the frequency of crossing the original platform was increased on 3rd-4th days, and the content of BDNF in CA1 region was increased in K+R group, and no significant changes were found in the parameters mentioned above in K+E group. Conclusion Ro20?1724 can improve cognitive dysfunction induced by repetitive ketamine anesthesia in immature rats, and enhanced production of BDNF is involved in the mechanism.

Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.
Glutamate Decarboxylase ; metabolism ; Half-Life ; Industrial Microbiology ; Lactobacillus brevis ; enzymology ; Mutagenesis, Site-Directed ; Mutation ; Temperature

Objective To compare the real-time dissolutions of 3 kinds of Simvastatin tablets used in our hospital, and provide reference information for clinical applications. Methods The dissolution rate of simvastatin tablets was deter-mined by the Chinese Pharmacopoeia 2010, and the HPLC method was used to determine the simvastatin content at the wavelength of 238 nm and the Weibull’s equation was adopted to fit the dissolution curves. The shape parameters (m) were performed statistical analysis; Using the similarity f2 as the index, compared the dissolution curve between tablets and the preparation. Results The dissolution of simvastatin tablets manufactured by the 3 manufaccturer was no less than 90% in 30 min, which comply with the national specifications. Compared with the dissolution curve of 6 tablets from the same batch, the homogenicity of samples from A was better, with poor homogenicity of tablets from B or C. There’s significant difference in the shape parameter m of tablets from B and C (P< 0.05), and there’s no significant difference in m of tablets from C and A(P>0.05). Conclusion The in vitro dissolution of the simvastatin tablets are de-termined with single point test procedures. The dissolution of the tablets from 3 manufactures comply with national specifications. However, there’s significant difference in real time dissolution of national tablets. It’s necessary to im-prove the quality of national tablets. Cautions should be taken while use in clinical practices.

Objective:To study the safety and efficacy of combining portal vein resection and reconstruction (PVR) with resection of perihilar cholangiocarcinoma (PHC).Methods:A total of 104 patients with PHC who underwent hepatectomies for either biliary resection alone or biliary resection combined with PVR from October 2006 to December 2019 at the Department of Hepatopancreatobiliary, Ningbo Medical Center of Lihuili Hospital entered into this study. There were 63 males and 41 females, with the age of (64.4±10.4) years. The control group consisted of 75 patients who underwent biliary resection alone, while the PVR group consisted 29 patients with biliary resection combined with PVR. The patient characteristics and the follow-up outcomes of the two groups were analyzed and compared. Survival analyses were performed using the Kaplan Meier method with the log-rank test.Results:Wedge resection of portal vein, side to side anastomosis in 2 cases, segmental resection and end to end anastomosis in 27 cases. The time taken for PVR and portal vein resection were (12.7±2.9)(range 8 to 18)min and (20.7±7.3)(range 8 to 38) mm, respectively. The estimated blood loss for the PVR group was significantly more than the control group [ M( Q1, Q3)] 800.0 (600.0, 1 500.0) ml vs. 600.0(500.0, 1 000.0) ml ( P<0.05). Based on postoperative pathological studies, the proportion of lymph node metastasis was significantly higher in the PVR group than the control group (58.6% vs. 32.0%, P<0.05). Clavien-Dindo grade Ⅲ and above complications were 30.7%(23/75) and 34.5%(10/29) in the control and PVR groups, respectively ( P>0.05). The re-operation and postoperative 90 days mortality rates were 9.3%(7/75) and 2.7%(2/75) in the control group, compared with 3.4%(1/29) and 0 in the PVR group, respectively (both P>0.05). The 1-, 3- and 5-year survival rates were 81.1%, 44.8% and 36.4% respectively for the control group and 78.1%, 35.9% and 31.4% for the PVR group (χ 2=0.33, P=0.570). Conclusion:When compared to biliary resection alone, biliary resection combined with PVR did not significantly increase postoperative complication and mortality rates, but with comparable long-term survival outcomes. Combined biliary resection with PVR was safe and improved the resection rate in selected patients with locally advanced PHC.

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t_1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T¹⁰₅₀) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.
Transaminases/chemistry* ; Molecular Docking Simulation ; Amines/chemistry* ; Pyruvic Acid/metabolism* ; Enzyme Stability

Chiral amines are important building blocks for the synthesis of pharmaceutical products and fine chemicals. Highly stereoselective synthesis of chiral amines compounds through asymmetric amination has attracted more and more attention. ω-transaminases (ω-TAs) are a promising class of natural biocatalysts which provide an efficient and environment-friendly access to production of chiral amines with stringent enantioselectivity and excellent catalytic efficiency. Compared with (S)-ω-TA, the research focused on (R)-ω-TA was relatively less. However, increasing demand for chiral (R)-amines as pharmaceutical intermediates has rendered industrial applications of (R)-ω-TA more attractive. Improving the thermostability of (R)-ω-TA with potential biotechnological application will facilitate the preparation of chiral amines. In this study, the dynamic surface loop with higher B-factor from Aspergillus terreus (R)-ω-TA was predicted by two computer softwares (PyMOL and YASARA). Then mutant enzymes were obtained by deleting amino acid residues of a dynamic surface loop using site-directed mutagenesis. The results showed that the best two mutants R131del and P132-E133del improved thermostability by 2.6 ℃ and 0.9 ℃ in T₅₀¹⁰ (41.1 ℃ and 39.4 ℃, respectively), and 2.2-fold and 1.5-fold in half-life (t1/2) at 40 ℃ (15.0 min and 10.0 min, respectively), compared to that of wild type. Furtherly, the thermostability mechanism of the mutant enzymes was investigated by molecular dynamics (MD) simulation and intermolecular interaction analysis. R131del in the loop region has lower root mean square fluctuation (RMSF) than the wild type at 400 K for 10 ns, and mutant enzyme P132-E133del increases four hydrogen bonds in the loop region. In this study, we obtain two stability-increased mutants of (R)-ω-TA from A. terreus by deleting its dynamic surface loop and also provide methodological guidance for the use of rational design to enhance the thermal stability of other enzymes.

Glutamate decarboxylase, a unique pyridoxal 5'-phosphate-dependent enzyme, catalyzes α-decarboxylation of L-glutamate to γ-aminobutyrate. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, proline was introduced at 13 different positions in glutamate decarboxylase by using the design strategy of homologous sequence alignment between Thermococcus kodakarensis and Lactobacillus brevis CGMCC No.1306. A mutant enzyme G364P with higher thermostability was obtained. Compared to the wild type, thermostability of the mutant G364P was significantly improved, the half-life time (t1/2) at 55 °C and the semi-inactivation temperature (T₅₀ ¹⁵) of the mutant G364P increased 19.4 min and 5.3 °C, respectively, while kcat/Km of the mutant enzyme remained nearly unchanged. Further analysis of their thermostability by molecular dynamics simulations were performed. The root mean square deviation of G364P and root mean square fluctuation in the loop region including G364 were lower than the wild type at 313 K for 10 ns, and G364P increased one hydrophobic interaction in the loop region. It proves that mutation of flexible 364-Gly to rigid proline endows glutamate decarboxylase with enhanced thermostability.
Glutamate Decarboxylase ; Glutamic Acid ; Lactobacillus brevis ; Molecular Dynamics Simulation ; Proline

Vision formation is classically based on projections from retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Neurons in the mouse V1 are tuned to light stimuli. Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single light-stimulated neuron in V1 remain unknown. In our study, in vivo calcium imaging and whole-cell electrophysiological patch-clamp recording were utilized to identify 53 individual cells from layer 2/3 of V1 as light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of each cell after functional tests were aspirated in vivo through a patch-clamp pipette for mRNA sequencing. Moreover, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in a live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with a high expression of genes related to transmission regulation, such as Rtn4r and Rgs7, and genes involved in membrane transport, such as Na/K ATPase and NMDA-type glutamatergic receptors, preferentially responded to light stimulation. Furthermore, an antagonist that blocks Rtn4r signals could inactivate the neuronal responses to light stimulation in live mice. In conclusion, our findings of the vivo-seq analysis indicate the key role of the strength of synaptic transmission possesses neurons in V1 of light sensory.