Modification of nitrilase based on computer screening and efficient biosynthesis of 4-cyanobenzoic acid.

4-cyanobenzoic acid serves as a crucial intermediate for the synthesis of various high-value organic compounds. The enzymatic hydrolysis of terephthalonitrile to produce 4-cyanobenzoic acid using nitrilase offers the advantages of a simple reaction pathway, environmental friendliness, and easy product separation. In order to efficiently develop nitrilases that meet industrial production requirements, the virtual screening method used in the study is established and mature. From a total of 371 amino acids in the nitrilase AfNIT, which exhibits activity in terephthalonitrile hydrolysis, three candidate sites (F168, S192, and T201) were identified, and a "small and accurate" mutant library was constructed. The triple mutant F168V/T201N/S192F was screened from this small mutant library with a specific activity of 227.3 U mg-1 , which was 3.8 times higher than that of the wild-type AfNIT. Using the whole-cell biocatalyst containing the mutant F168V/T201N/S192F, terephthalonitrile was successfully hydrolyzed at a concentration of 150 g L-1 to produce 4-cyanobenzoic acid with a final yield of 170.3 g L-1 and a conversion rate of 98.7%. The obtained nitrilase mutant F168V/T201N/S192F in this study can be effectively applied in the biomanufacturing of 4-cyanobenzoic acid using terephthalonitrile as a substrate. Furthermore, the results also demonstrate the significant improvement in predictive accuracy achieved through the latest AI-assisted computer simulation methods. This approach represents a promising and feasible new technological pathway for assisting enzyme engineering research, laying a theoretical foundation for other related studies.

Antidepressant effect of venlafaxine in chronic unpredictable stress: Evidence of the involvement of key enzymes responsible for monoamine neurotransmitter synthesis and metabolism.

A number of studies have linked abnormalities in the function of the serotonergic and noradrenergic systems to the pathophysiology of depression. It has been reported that selective serotonin reuptake inhibitors promote the expression of tryptophan hydroxylase (TPH), which is involved in the synthesis of serotonin. However, limited evidence of TPH alteration has been found in selective serotonin and noradrenaline reuptake inhibitors (SNRIs), and more key enzymes need to be investigated. The aim of the present study was to determine whether venlafaxine (VLX; a classical SNRI) regulates TPH and other key enzymes responsible for the synthesis and metabolism of monoaminergic transmitters in rats with chronic unpredictable stress (CUS). The present results suggested that CUS­exposed rats exhibited decreased locomotor activity in the open­field test and increased immobility time in the forced swim test, as compared with the controls. Pretreatment with VLX (20 mg/kg) significantly increased locomotor activity and reduced immobility time in the CUS­exposed rats. In addition, VLX (20 mg/kg) treatment prevented the CUS­induced reduction in tyrosine hydroxylase and TPH expression in the cortex and hippocampus. Furthermore, VLX alleviated the CUS­induced oxidative stress in the serum, cortex and hippocampus. However, VLX administration did not have an effect on indoleamine­2,3­dioxygenase overexpression in the hippocampus. It was therefore concluded that the regulation of abnormalities in the synthesis and metabolism of monoaminergic transmitters may be associated with the antidepressant effects of VLX, suggesting that multimodal pharmacological treatments can efficiently treat depression.

Finite element analysis for blood accumulation in intracerebral hemorrhage.

Biomechanical methods may provide a novel way to understand blood accumulation in intracerebral hemorrhage (ICH). The current study presents the results of a biomechanical analysis of blood accumulation in ICH using a finite element analysis, with an emphasis on the pressure exerted by the mass effect of blood in early ICH. A two-dimensional finite model of the human brain parenchyma and the human ventricular system was developed and analyzed under two preloading conditions. The material properties of the human parenchyma were derived from previous reports. Ogden's theory was applied to describe the stress-strain association in soft tissue. The results of the present study indicated that maximal stress was located at the two ends of the hemorrhage cavity, with the majority of stresses distributed on the zone surrounding the bleed. The two load environments demonstrated similar stress distributions. The loads put on the detached edges were not less than the intracranial pressure (ICP) when the stress threshold was reached. The results of the present study suggest that the direction of blood accumulation can be determined by the shape of the initial blood mass. Mechanical factors (blood pressure and ICP) did not serve a definitive role in preventing blood from accumulating in the early stages of ICH. The present study may aid in understanding the effects of mechanical factors in blood accumulation and hemostasis in patients with early ICH.

[Evaluation on contribution rate of each component total salvianolic acids and characterization of apparent oil/water partition coefficient].

The difference between three representative components of total salvianolic acids in pharmacodynamic activity were compared by three different pharmacological experiments: HUVECs oxidative damage experiment, 4 items of blood coagulation in vitro experiment in rabbits and experimental myocardial ischemia in rats. And the effects of contribution rate of each component were calculated by multi index comprehensive evaluation method based on CRITIC weights. The contribution rates of salvianolic acid B, rosmarinic acid and Danshensu were 28.85%, 30.11%, 41.04%. Apparent oil/water partition coefficient of each representative components of total salvianolic acids in n-octyl alcohol-buffer was tested and the total salvianolic acid components were characterized based on a combination of the approach of self-defined weighting coefficient with effects of contribution rate. Apparent oil/water partition coefficient of total salvianolic acids was 0.32, 1.06, 0.89, 0.98, 0.90, 0.13, 0.02, 0.20, 0.56 when in octanol-water/pH 1.2 dilute hydrochloric acid solution/ pH 2.0, 2.5, 5.0, 5.8, 6.8, 7.4, 7.8 phosphate buffer solution. It provides a certain reference for the characterization of components.

Histone acetylation and expression of mono-aminergic transmitters synthetases involved in CUS-induced depressive rats.

Histone acetylation has been linked to depression, the etiology of which involves many factors such as genetics, environments, and epigenetics. The aim of the present study was to investigate whether it was associated with epigenetic histone modification and gene expression of enzymes responsible for the biosynthesis of norepinephrine and serotonin in rat depression model induced by chronic unpredictable stress (CUS). Eight-week-old male Sprague-Dawley rats were exposed to CUS over 28 days. It was shown that the CUS-induced rats displayed remarked anxiety- and depression-like behavior with weakened locomotor activity in open field test and prolonged immobility in forced swimming test. Western blot revealed that CUS led to significant decrease in acetylation of H3 at Lysine 9 (K9) and H4 at Lysine 12 (K12) with obviously increasing histone deacetylases 5 (HDAC5) expression in hippocampus of CUS-induced rats. Meanwhile, there was an obviously decreased expression of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) both at protein and mRNA levels. Administration of sodium valproate (VPA), a histone deacetylase 5 (HDAC5) inhibitor, not only significantly relieved the anxiety- and depression-like behaviors of CUS-induced rats but also clearly blunted decrease of H3(K9) and H4(K12) acetylation and expression of TH and TPH, and prevented increase of HDAC5 expression. The results indicate that there exists possible interrelation between TH and TPH gene expression and epigenetic histone acetylation in CUS-induced depressive rats, which at least partly contributes to the etiology of depression.

[Polybasic research on the biopharmaceutical characteristics of 20 (S)-protopanaxadiol].

In this study, the biopharmaceutical properties of 20 (S)-protopanaxadiol (PPD) were studied. Firstly, the equilibrium solubility and apparent oil/water partition coefficient of PPD were used to predict the absorption in vivo. Meanwhile the membrane permeability and absorption window were studied by Caco-2 cell model and single-pass intestinal perfusion model. Furthermore, the bioavailability and metabolism were combined to study the absorption properties and metabolic properties in vivo. All of them were used to provide theoretical and practical foundation for designing PPD preparation. The results showed that PPD is poorly water-soluble, and the equilibrium solubility in water is only 35.24 mg x L(-1). The oil-water partition coefficient is 46.21 (logP = 1.66). By Caco-2 cell model, the results showed PPD uptake in general, and it also has efflux. By in situ intestinal perfusion model, the results showed that the absorption of PPD in the intestine is good, and the effective permeability coefficient were duodenum > jejunum > ileum > colon. The oral bioavailability of PPD was 29.39%. It was not well. Metabolic studies showed PPD in vivo presented a wide spread metabolism. So the main factors that restricted oral bioavailability of PPD were the poor solubility and first-pass effect.

A novel drug-phospholipid complex enriched with micelles: preparation and evaluation in vitro and in vivo.

Mixed micelles are widely used to increase solubility and bioavailability of poorly soluble drugs. One promising antitumor drug candidate is 20(S)-protopanaxadiol (PPD), although its clinical application is limited by low water solubility and poor bioavailability after oral administration. In this study, we developed mixed micelles consisting of PPD-phospholipid complexes and Labrasol(®) and evaluated their potential for oral PPD absorption. Micelles were prepared using a solvent-evaporation method, and their physicochemical properties, including particle size, zeta potential, morphology, crystal type, drug loading, drug entrapment efficiency, and solubility, were characterized. Furthermore, in vitro release was investigated using the dialysis method, and transport and bioavailability of the mixed micelles were investigated through a Caco-2 cell monolayer and in vivo absorption studies performed in rats. Compared with the solubility of free PPD (3 µg/mL), the solubility of PPD in the prepared mixed micelles was 192.41 ± 1.13 µg/mL in water at room temperature. The in vitro release profiles showed a significant difference between the more rapid release of free PPD and the slower and more sustained release of the mixed micelles. At the end of a 4-hour transport study using Caco-2 cells, the apical-to-basolateral apparent permeability coefficients (P(app)) increased from (1.12 ± 0.21) × 10(6) cm/s to (1.78 ± 0.16) × 10(6) cm/s, while the basolateral-to-apical P(app) decreased from (2.42 ± 0.16) × 10(6) cm/s to (2.12 ± 0.32) × 10(6). In this pharmacokinetic study, compared with the bioavailability of free PPD (area under the curve [AUC](0-∞)), the bioavailability of PPD from the micelles (AUC(0-∞)) increased by approximately 216.36%. These results suggest that novel mixed micelles can significantly increase solubility, enhance absorption, and improve bioavailability. Thus, these prepared micelles might be potential carriers for oral PPD delivery in antitumor therapies.

[Study on dosage form design for improving oral bioavailability of traditional Chinese medicines].

Both chemical drugs and traditional Chinese medicines have the problem of low bioavailability. However, as traditional Chinese medicines are a multi-component complex, their dosage forms are required to be designed in line with their characteristics, in order to improve the bioavailability of traditional Chinese medicines. Traditional Chinese medicines are mostly prepared into pill, powder, paste, elixir and decoction, but with such drawbacks as high administration dose and poor efficacy. With the process of modernization of traditional Chinese medicines, new-type preparations have be developed and made outstanding achievements. However, they fail to make an organic integration between traditional Chinese medicine theories and modern preparation theories. Characteristics of traditional Chinese medicines are required to be taken into account during the development of traditional Chinese medicines. In the article, multi-component preparation technology was adopted to establish a multi-component drug release system of traditional Chinese medicines on the basis of multiple components of traditional Chinese medicines.

[Establishment of modern multi-component sustained-release preparations of oral traditional Chinese medicines].

Traditional Chinese medicines have a long history, with a large quantity of efficient traditional Chinese medicines and prescriptions. However, the vast majority of pharmaceutical dose forms remain common preparations, with very few efficient, long-lasting and low-dose preparations. The sustain-release preparation allows sustained drug release in a longer period of time, maintains blood drug concentration, reduces the toxic effect and medication frequency, and improves medication compliance. Unlike monomer drugs, the material base of traditional Chinese medicine and compounds is multi-component, instead of single or several active monomers. Therefore, under the guidance of the Chinese medicine theories, modern multi-component sustained-release preparations were developed for oral traditional Chinese medicines, with the aim of finally improving the clinical efficacy of traditional Chinese medicines.

Association of promoter polymorphism of apolipoprotein E gene with cerebral vasospasm after spontaneous SAH.

Cerebral vasospasm (CV) is the main complication of spontaneous subarachnoid hemorrhage (SAH), affecting clinical outcome of patients with SAH. Accumulating evidence indicates that apolipoprotein E (apoE protein, APOE gene) gene polymorphism is associated with prognosis of patients with SAH. The current study aimed to investigate the association of promoter polymorphism of APOE with CV in patients with SAH. One hundred and one patients with spontaneous SAH were involved in this study. Venous blood samples were collected to identify the promoter genotype of APOE by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). CV was judged by transcranial Doppler sonography (TCD) combined with patients' condition. Associations of APOE promoter polymorphism with CV after SAH were analyzed by χ(2) test, uni- and multivariate logistic regression analyses. In 101 patients, 42 of 87 patients (48.3%) with promoter -219T allele showed CV, which was significantly different from those with -219G allele (23/61, 37.7%, P=0.04). Uni- and multivariate logistic regression analyses also showed that promoter -219T was a risk factor to predispose CV after SAH. However, there was no significant association between promoter -491A/T (rs#449647) or -427C/T (rs#769446) polymorphisms and SAH induced CV (P>0.05). Our finding suggests that patients with APOE -219T promoter are apt to CV after spontaneous SAH.