Salvianolic acid B exerts its anti-tumor immunity by targeting USP2 and reducing the PD-L1 level / 药学学报

With the development of small-molecule immunotherapy drugs, its combination with the programmed cell death ligand 1/programmed cell death protein 1 (PD-L1/PD-1) antibodies would provide a new opportunity for cancer treatment. Therefore, targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity and considered as the next generation of tumor immunotherapy. In the present study, we investigated the anti-tumor role of salvianolic acid B (SAB) by regulating the PD-L1 level in tumors. Changes of total PD-L1 and membrane PD-L1 levels were determined by Western blot, flow cytometry and PD-1/PD-L1 interaction assays. The expression of mRNA level of PD-L1 was detected by real-time PCR. The cytotoxicity of activated peripheral blood mononuclear cell (PBMC) cells toward co-cultured tumor cells was measured by cell impedance assay and crystal violet experiment. Surface plasma resonance technique was used to analyze the direct interaction between SAB and ubiquitin carboxyl-terminal hydrolase 2 (USP2). The antitumor effect of SAB in vivo was examined by C57BL/6 mice bearing MC38 xenograft tumor (all animal experiments were conducted in accordance with the Animal Ethics Committee of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences). Western blot and flow cytometry assay showed that SAB can significantly downregulate the abundance of PD-L1 in RKO and PC3 cells in dose- and time-dependent manner. PD-1/PD-L1 binding assay revealed that SAB reduces the binding of tumor cells to recombinant PD-1 protein. Mechanism studies revealed that SAB can bind directly to USP2 protein and inhibit its activity, thus promote the ubiquitin-proteasome pathway degradation of PD-L1 proteins. In addition, Cell impedance and crystal violet staining indicated that SAB enhances the killing activity of co-cultured PBMC cells toward tumor cells. MC38 tumor transplanted mouse experiments revealed that SAB treatment displayed significant suppression in the growth of MC38 tumor xenografts in C57BL/6 mice with an inhibition rate of 63.2% at 20 mg·kg-1. Our results demonstrate that SAB exerts its anti-tumor activity by direct binding and inhibiting the activity of USP2 and reducing the PD-L1 level. Our study provides an important material basis and scientific basis for the potential application of SAB in tumor immunotherapy drug targeting USP2-PD-L1 axis.

Engineering the plastic degradation enzyme Ple629 from marine consortium to improve its thermal stability / 生物工程学报

Petrochemical-derived polyester plastics such as polyethylene terephthalate (PET) and polybutylene adipate terephthalate (PBAT) have been widely used. However, the difficulty to be degraded in nature (PET) or the long biodegradation cycle (PBAT) resulted in serious environmental pollution. In this connection, treating these plastic wastes properly becomes one of the challenges of environment protection. From the perspective of circular economy, biologically depolymerizing the waste of polyester plastics and reusing the depolymerized products is one of the most promising directions. Recent years have seen many reports on polyester plastics degrading organisms and enzymes. Highly efficient degrading enzymes, especially those with better thermal stability, will be conducive to their application. The mesophilic plastic-degrading enzyme Ple629 from the marine microbial metagenome is capable of degrading PET and PBAT at room temperature, but it cannot tolerate high temperature, which hampers its potential application. On the basis of the three-dimensional structure of Ple629 obtained from our previous study, we identified some sites which might be important for its thermal stability by structural comparison and mutation energy analysis. We carried out transformation design, and performed expression, purification and thermal stability determination of the mutants. The melting temperature (Tm) values of mutants V80C and D226C/S281C were increased by 5.2 ℃ and 6.9 ℃, respectively, and the activity of mutant D226C/S281C was also increased by 1.5 times compared with that of the wild-type enzyme. These results provide useful information for future engineering and application of Ple629 in polyester plastic degradation.

Expression, purification and characterization of a novel bis (hydroxyethyl) terephthalate hydrolase from Hydrogenobacter thermophilus / 生物工程学报

PET (polyethylene terephthalate) is one of the most important petrochemicals that is widely used in mineral water bottles, food and beverage packaging and textile industry. Because of its stability under environmental conditions, the massive amount of PET wastes caused serious environmental pollution. The use of enzymes to depolymerize PET wastes and upcycling is one of the important directions for plastics pollution control, among which the key is the depolymerization efficiency of PET by PET hydrolase. BHET (bis(hydroxyethyl) terephthalate) is the main intermediate of PET hydrolysis, its accumulation can hinder the degradation efficiency of PET hydrolase significantly, and the synergistic use of PET hydrolase and BHET hydrolase can improve the PET hydrolysis efficiency. In this study, a dienolactone hydrolase from Hydrogenobacter thermophilus which can degrade BHET (HtBHETase) was identified. After heterologous expression in Escherichia coli and purification, the enzymatic properties of HtBHETase were studied. HtBHETase shows higher catalytic activity towards esters with short carbon chains such as p-nitrophenol acetate. The optimal pH and temperature of the reaction with BHET were 5.0 and 55 ℃, respectively. HtBHETase exhibited excellent thermostability, and retained over 80% residual activity after treatment at 80 ℃ for 1 hour. These results indicate that HtBHETase has potential in biological PET depolymerization, which may facilitate the enzymatic degradation of PET.

Advances in poly(ethylene terephthalate) hydrolases / 生物工程学报

Plastics have brought invaluable convenience to human life since it was firstly synthesized in the last century. However, the stable polymer structure of plastics led to the continuous accumulation of plastic wastes, which poses serious threats to the ecological environment and human health. Poly(ethylene terephthalate) (PET) is the most widely produced polyester plastics. Recent researches on PET hydrolases have shown great potential of enzymatic degradation and recycling of plastics. Meanwhile, the biodegradation pathway of PET has become a reference model for the biodegradation of other plastics. This review summarizes the sources of PET hydrolases and their degradation capacity, degradation mechanism of PET by the most representative PET hydrolase-IsPETase, and recently reported highly efficient degrading enzymes through enzyme engineering. The advances of PET hydrolases may facilitate the research on the degradation mechanism of PET and further exploration and engineering of efficient PET degradation enzymes.

Structure-guided engineering for improving the thermal stability of zearalenone hydrolase / 生物工程学报

Zearalenone is one of the most widely polluted Fusarium toxins in the world, seriously endangering livestock and human health. Zearalenone hydrolase (ZHD) derived from Clonostachys rosea can effectively degrade zearalenone. However, the high temperature environment in feed processing hampers the application of this enzyme. Structure-based rational design may provide guidance for engineering the thermal stability of enzymes. In this paper, we used the multiple structure alignment (MSTA) to screen the structural flexibility regions of ZHD. Subsequently, a candidate mutation library was constructed by sequence conservation scoring and conformational free energy calculation, from which 9 single point mutations based on residues 136 and 220 were obtained. The experiments showed that the thermal melting temperature (Tm) of the 9 mutants increased by 0.4-5.6 ℃. The S220R and S220W mutants showed the best thermal stability, the Tm of which increased by 5.6 ℃ and 4.0 ℃ compared to that of the wild type. Moreover, the thermal half-inactivation time at 45 ℃ were 15.4 times and 3.1 times longer, and the relative activities were 70.6% and 57.3% of the wild type. Molecular dynamics simulation analysis showed that the interaction force at and around the mutation site was enhanced, contributing to the improved thermal stability of ZHD. The probability of 220-K130 hydrogen bond of the mutants S220R and S220W increased by 37.1% and 19.3%, and the probability of K130-D223 salt bridge increased by 30.1% and 12.5%, respectively. This work demonstrated the feasibility of thermal stability engineering strategy where the structural and sequence alignment as well as free energy calculation of natural enzymes were integrated, and obtained ZHD variants with enhanced thermal stability, which may facilitate the industrial application of ZHD.

Detection methods for polyethylene terephthalate degrading enzymes: a review / 生物工程学报

Polyethylene terephthalate (PET) is one of the most widely used synthetic polyester. It poses serious threat to terrestrial, aquatic ecosystems and human health since it is difficult to be broken down and deposited in the environment. The biodegradation based on enzymatic catalysis offers a sustainable method for recycling PET. A number of PET hydrolases have been discovered in the last 20 years, and protein engineering has increased their degradation capabilities. However, no PET hydrolases that are practical for widespread industrial use have been identified. Screening of PET hydrolase using conventional detection techniques is laborious and inefficient process. Effective detection techniques are required to promote the commercialization of PET hydrolases. Using efficient detection techniques to screen potent industrial enzymes is essential for supporting the widespread industrial implementation of PET hydrolases. To define PET hydrolase, scientists have created a number of analytical techniques recently. The detection techniques that can be used to screen PET hydrolase, including high performance liquid chromatography, ultraviolet absorption spectrometric, and fluorescence activated droplet sorting method, are summarized in this study along with their potential applications.

Leche materna deslactosada con la enzima beta galactosidasa para lactantes intolerantes / Leite materno sem lactose com a enzima beta-galactosidase para lactentes intolerantes / Lactose-free breast milk with the enzyme beta galactosidase for intolerant infants

Introducción: los bebés deben beneficiarse de la leche materna, incluso cuando presentan intolerancia a la lactosa. Por esto, se debe recurrir a la obtención de leche materna deslactosada. Objetivo: analizar el efecto de la enzima beta galactosidasa en la hidrólisis de la lactosa de leche materna madura para bebés clínicamente diagnosticados con intolerancia a la lactosa. Materiales y método: estudio exploratorio, descriptivo y explicativo. El contenido de lactosa se cuantificó desde el inicio hasta el final del tratamiento, controlando temperatura, tiempos y cantidad de enzima ß-galactosidasa adicionada en la leche materna. Se recolectaron 1000 ml de leche materna, obtenidos del Banco de Leche del Hospital General de Medellín (Antioquia, Colombia). Resultados: las muestras donadas se encontraban pasteurizadas y posteriormente fueron sometidas a la acción de la enzima lactasa. Se cuantificó el contenido de lactosa sin la enzima, reportando en promedio 6,34 mg/100 ml ± 0,23. El mayor aporte de lactosa obtenido posterior a la exposición a la enzima (30 minutos) fue de 6,07 mg/ml ± 0,35 (correspondiente a 95 % del contenido inicial), finalizando con un aporte de 0,35 % a una concentración de 0,4 % tras 24 horas, porcentaje que representa 95 % de la hidrólisis total en la leche materna. Conclusiones: en todas las muestras analizadas de diferentes madres se pudo obtener leche materna con bajas concentraciones de lactosa tras 24 horas de haber sido sometidas a la acción de ß-galactosidasa. Lo anterior se establece como una alternativa para los bebés intolerantes a la lactosa, que permitiría no privarlos de todos los beneficios que ofrece este alimento.

Introduction: Babies should benefit from breast milk, even when they are lactose intolerant. For this reason, parents should resort to obtaining lactose-free breast milk. Objective: To examine the effect of the enzyme ß-galactosidase on the hydrolysis of lactose in mature breast milk for babies clinically diagnosed with lactose intolerance. Materials and method: Exploratory, descriptive, and explanatory study. The lactose content was quantified from the beginning to the end of the treatment, controlling variables such as temperature, times, and the amount of ß-galactosidase enzyme added in breast milk. A total of 1000 ml of breast milk were obtained from the milk bank at Hospital General de Medellín (Antioquia, Colombia). Results: Donated samples were first pasteurized and subsequently subjected to the action of the enzyme lactase. The lactose content without the enzyme was quantified, reporting an average of 6.34 mg/100 mL±0.23. The highest contribution of lactose obtained after exposure to the enzyme was 6.07 mg/mL±0.35 (corresponding to 95% of the initial content), at 30 minutes, ending with a contribution of 0.35% at a concentration of 0.4% in 24 hours, percentage that represents 95% of total hydrolysis in breast milk. Conclusions: In all the examined samples from different mothers, it was possible to obtain breast milk with low concentrations of lactose 24 hours after these were exposed to the action of ß-galactosidase. This becomes an alternative for feeding lactose intolerant babies and not deprive them from all the benefits offered by breast milk.

Introdução: os bebês se devem beneficiar do leite materno, mesmo quando tenham intolerância à lactose, razão pela qual se deve recorrer à obtenção de leite materno sem lactose. Objetivo: analisar o efeito da enzima beta-galactosidase na hidrólise da lactose no leite materno maduro para bebês diagnosticados clinicamente com intolerância à lactose. Materiais e método: estudo exploratório, descritivo, explicativo. O teor de lactose foi quantificado do início ao fim do tratamento; temperatura, tempos e quantidade de enzima beta-galactosidase adicionada no leite materno foram controlados; foram coletados 1000ml de leite materno, obtidos no Banco de Leite do Hospital General de Medellín (Antioquia, Colômbia). VResultados: as amostras doadas foram pasteurizadas e posteriormente submetidas à ação da enzima lactase. O teor de lactose sem a enzima foi quantificado, relatando uma média de 6,34mg/100ml±0,23. A maior contribuição de lactose obtida após a exposição à enzima foi de 6,07mg/ml±0,35 (correspondendo a 95% do conteúdo inicial) em 30 minutos, finalizando com uma contribuição de 0,35% na concentração de 0,4% em 24 horas, percentual que representa 95% da hidrólise total no leite materno. Conclusões: em todas as amostras analisadas de diferentes mães, foi possível obter leite materno com baixas concentrações de lactose 24 horas após ser submetido à ação da beta galactosidase, como alternativa para bebês intolerantes à lactose e não os privar de todos os outros benefícios oferecidos por esse alimento ideal.

Identification of α-L-fucosidase (ALFuc) of Blastocystis sp. subtypes ST1, ST2 and ST3

ABSTRACT Blastocystis sp. is a common intestinal microorganism. The α-L-fucosidase (ALFuc) is an enzyme long associated with the colonization of the gut microbiota. However, this enzyme has not been experimentally identified in Blastocystis cultures. The objective of the present study was to identify ALFuc in supernatants of axenic cultures of Blastocystis subtype (ST)1 ATCC-50177 and ATCC-50610 and to compare predicted ALFuc proteins of alfuc genes in sequenced STs1-3 isolates in human Blastocystis carriers. Excretion/secretion (Es/p) and cell lysate proteins were obtained by processing Blastocystis ATCC cultures and submitting them to SDS-PAGE and immunoblotting. In addition, 18 fecal samples from symptomatic Blastocystis human carriers were analyzed by sequencing of amplification products for subtyping. A complete identification of the alfuc gene and phylogenetic analysis were performed. Immunoblotting showed that the amplified band corresponding to ALFuc (~51 kDa) was recognized only in the ES/p. Furthermore, prediction analysis of ALFuc 3D structures revealed that the domain α-L-fucosidase and the GH29 family's catalytic sites were conserved; interestingly, the galactose-binding domain was recognized only in ST1 and ST2. The phylogenetic inferences of ALFuc showed that STs1-3 were clearly identifiable and grouped into specific clusters. Our results show, for the first time through experimental data that ALFuc is a secretion product of Blastocystis sp., which could have a relevant role during intestinal colonization; however, further studies are required to clarify this condition. Furthermore, the alfuc gene is a promising candidate for a phylogenetic marker, as it shows a conserved classification with the SSU-rDNA gene.

Structure-guided discovery of potent and oral soluble epoxide hydrolase inhibitors for the treatment of neuropathic pain

Soluble epoxide hydrolase (sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases. A new series of memantyl urea derivatives as potent sEH inhibitors was obtained using our previous reported compound 4 as lead compound. A preferential modification of piperidinyl to 3-carbamoyl piperidinyl was identified for this series via structure-based rational drug design. Compound A20 exhibited moderate percentage plasma protein binding (88.6%) and better metabolic stability in vitro. After oral administration, the bioavailability of A20 was 28.6%. Acute toxicity test showed that A20 was well tolerated and there was no adverse event encountered at dose of 6.0 g/kg. Inhibitor A20 also displayed robust analgesic effect in vivo and dose-dependently attenuated neuropathic pain in rat model induced by spared nerve injury, which was better than gabapentin and sEH inhibitor (±)-EC-5026. In one word, the oral administration of A20 significantly alleviated pain and improved the health status of the rats, demonstrating that A20 was a promising candidate to be further evaluated for the treatment of neuropathic pain.

Understanding the physiological functions of the host xenobiotic-sensing nuclear receptors PXR and CAR on the gut microbiome using genetically modified mice

Pharmacological activation of the xenobiotic-sensing nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) is well-known to increase drug metabolism and reduce inflammation. Little is known regarding their physiological functions on the gut microbiome. In this study, we discovered bivalent hormetic functions of PXR/CAR modulating the richness of the gut microbiome using genetically engineered mice. The absence of PXR or CAR increased microbial richness, and absence of both receptors synergistically increased microbial richness. PXR and CAR deficiency increased the pro-inflammatory bacteria Helicobacteraceae and Helicobacter. Deficiency in both PXR and CAR increased the relative abundance of Lactobacillus, which has bile salt hydrolase activity, corresponding to decreased primary taurine-conjugated bile acids (BAs) in feces, which may lead to higher internal burden of taurine and unconjugated BAs, both of which are linked to inflammation, oxidative stress, and cytotoxicity. The basal effect of PXR/CAR on the gut microbiome was distinct from pharmacological and toxicological activation of these receptors. Common PXR/CAR-targeted bacteria were identified, the majority of which were suppressed by these receptors. hPXR-TG mice had a distinct microbial profile as compared to wild-type mice. This study is the first to unveil the basal functions of PXR and CAR on the gut microbiome.

Role of CD155 in hepatocyte apoptosis and liver fibrosis induced by Echinococcus multilocularis infection in mice / 国际生物医学工程杂志

Objective:To investigate the role of CD155 in hepatocyte apoptosis and liver fibrosis in mice infected with Echinococcus multilocularis. Methods:Thirty-six female C57BL/6 mice were randomly divided into a sham surgery group and a model group, with 18 mice in each group. Mice in the model group were injected with protoscolex via the portal vein to create an animal model of E. multilocularis infection. Mice in the sham surgery group were injected with the same amount of saline. The mice were sacrificed at 1 month, 3 months, and 6 months after modeling, and liver samples were collected. Hepatic pathological changes were observed by hematoxylin-eosin staining. Liver fibrosis was detected by Sirius red staining, and expression of Caspase-3 and CD155 in hepatocytes was detected by immunohistochemical staining. The correlation between CD155 expression in hepatocytes and Caspase-3 and liver fibrosis levels were analyzed by Person. Results:There were obvious lesions in the liver of the model group accompanied by severe liver fibrosis. Compared with the sham surgery group, the expression of CD155 and Caspase-3 in mouse hepatocytes at different stages in the model group was significantly increased, and the differences were statistically significant ( P<0.05). The model group's liver fibrosis level was significantly higher at different stages than the sham surgery group, with statistical significance ( P<0.05). In addition, correlation analysis showed that expression of CD155 in hepatocytes was positively correlated with the expression of Caspase-3 ( r=0.956 8; P<0.001; 95% CI: 0.885 5-0.984 1) and that expression of CD155 in hepatocytes was positively correlated with the degree of liver fibrosis( r=0.853 9; P<0.001; 95% CI: 0.643 7-0.944 3). Conclusions:CD155 expression was significantly up-regulated in mouse hepatocytes infected with E. multilocularis at different stages, which was positively correlated with the degree of hepatocyte apoptosis and liver fibrosis, suggesting that CD155 may be involved in the process of hepatocyte apoptosis and liver fibrosis caused by E. multilocularis infection.

Curcumin improves cardiac fibrosis by inhibiting endothelial-mesenchymal transition through NRF2-DDAH-ADMA-NO pathway / 中国中药杂志

The present study analyzed the correlations between curcumin(Cur), nuclear factor E2 related factor 2(NRF2)-dimethylarginine dimethylaminohydrolase(DDAH)-asymmetric dimethylarginine(ADMA)-nitric oxide(NO) pathway, and endothelial-mesenchymal transition(EndMT) based on SD rats with cardiac fibrosis, and explored the effect and mechanism of Cur in resisting cardiac fibrosis to provide an in-depth theoretical basis for its clinical application in the treatment of heart failure. The cardiac fibrosis model was induced by subcutaneous injection of isoprenaline(Iso) in rats. Thirty-two rats were randomly divided into a control group, a model group, a low-dose Cur group(100 mg·kg~(-1)·d~(-1)), and a high-dose Cur group(200 mg·kg~(-1)·d~(-1)), with eight in each group. After 21 days of treatment, cardiac function was detected by echocardiography, degree of cardiac fibrosis by Masson staining, expression of CD31 and α-SMA by pathological staining, expression of VE-cadherin, vimentin, NRF2, and DDAH by Western blot, and ADMA level by HPLC. Compared with the model group, the Cur groups showed alleviated cardiac fibrosis, accompanied by increased CD31 and VE-cadherin expression and decreased α-SMA and vimentin expression, indicating relieved EndMT. Additionally, DDAH and NRF2 levels were elevated and ADMA and NO expression declined. Cur improves cardiac fibrosis by inhibiting EndMT presumedly through the NRF2-DDAH-ADMA-NO pathway.

Effects of deleting peptidoglycan hydrolase genes on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease / 生物工程学报

In order to explore the effect of peptidoglycan hydrolase on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease, five peptidoglycan hydrolase genes (lytC, lytD, lytE, lytF and lytG) of B. amyloliquefaciens TCCC111018 were knocked out individually. The viable cell counts of the bacteria and their alkaline protease activities before and after gene deletion were determined. The viable cell counts of the knockout mutants BA ΔlytC and BA ΔlytE achieved 1.67×106 CFU/mL and 1.44×106 CFU/mL respectively after cultivation for 60 h, which were 32.5% and 14.3% higher than that of the control strain BA Δupp. Their alkaline protease activities reached 20 264 U/mL and 17 265 U/mL, respectively, which were 43.1% and 27.3% higher than that of the control strain. The results showed that deleting some of the peptidoglycan hydrolase genes effectively maintained the viable cell counts of bacteria and increased the activity of extracellular enzymes, which may provide a new idea for optimization of the microbial host for production of industrial enzymes.

The Expression and Functional Roles of Epoxide Hydrolase 2 in Hepatocellular Carcinoma / 中国生物化学与分子生物学报

The expression, function and prognostic significance of epoxide hydrolase 2 (EPHX2) in hepatocellular carcinoma (HCC) were comprehensively analyzed through collecting HCC tissues and public database. The GEO and MitoCarta databases were used to identify the mitochondria-related differentially expressed genes (DEGs) in HCC. The Cancer Genome Atlas (TCGA) database was applied to analyze the expression levels of DEGs in HCC, including EPHX2 and its co-expressed genes. The R package was applied to draw the Kaplan-Meier survival curve and gene function enrichment analysis. The STRING database and GSEA software were used to analyze the protein-protein interaction (PPI) network and gene set enrichment analysis. qPCR and GEO database were applied to verify the expression level of EPHX2 in HCC. In the present study, a total of 15 mitochondria-related DEGs were identified in HCC. The expression of EPHX2 in HCC was significantly decreased compared to the normal liver tissues (P < 0. 01). The expression of EPHX2 was related to gender, tumor stage and grade in HCC, but not associated with age, T stage, et al in HCC. Moreover, compared with the patients with lower expression of EPHX2, patients with higher expression of EPHX2 had a better prognosis. EPHX2 was associated with fatty acid degradation. In addition, PPI results indicated that HAO1, AGXT, ACOX1, GSTκ1, SCP-2, CAT, CYP2C8, CYP2C9, CYP2B6, and CYP2J2 were co-expressed with EPXH2 in HCC. Furthermore, GSEA results showed that the group with lower expression of EPHX2 was positively correlated with the gene set of liver cancer cell proliferation and liver cancer recurrence. qPCR and GEO database results verified that the expression of EPHX2 was significantly decreased in HCC. The expression of EPHX2 was decreased in HCC, strongly suggesting that EPHX2 might function as a tumor suppressor gene in HCC. However, the potential mechanism of EPHX2 in HCC needs to be further verified.

LXYL-P1-2 immobilized on magnetic nanoparticles and its potential application in paclitaxel production

BACKGROUND: LXYL-P1-2 is the first reported glycoside hydrolase that can catalyze the transformation of 7-b-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT) by removing the D-xylosyl group at the C7 position. Successful synthesis of paclitaxel by one-pot method combining the LXYL-P1-2 and 10- deacetylbaccatin III-10-b-O-acetyltransferase (DBAT) using XDT as a precursor, making LXYL-P1-2 a highly promising enzyme for the industrial production of paclitaxel. The aim of this study was to investigate the catalytic potential of LXYL-P1-2 stabilized on magnetic nanoparticles, the surface of which was modified by Ni2+-immobilized cross-linked Fe3O4@Histidine. RESULTS: The diameter of matrix was 20­40 nm. The Km value of the immobilized LXYL-P1-2 catalyzing XDT (0.145 mM) was lower than that of the free enzyme (0.452 mM), and the kcat/Km value of immobilized enzyme (12.952 mM s 1 ) was higher than the free form (8.622 mM s 1 ). The immobilized form maintained 50% of its original activity after 15 cycles of reuse. In addition, the stability of immobilized LXYL-P1-2, maintained 84.67% of its initial activity, improved in comparison with free form after 30 d storage at 4 C. CONCLUSIONS: This investigation not only provides an effective procedure for biocatalytic production of DT, but also gives an insight into the application of magnetic material immobilization technology.

Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake, and investigations into its prebiotic activity

BACKGROUND: Manno-oligosaccharides (MOS) is known as a kind of prebiotics. Mannanase plays a key role for the degradation of mannan to produce MOS. In this study, the mannanases of glycoside hydrolase (GH) families 5 Man5HJ14 and GH26 ManAJB13 were employed to prepare MOS from locust bean gum (LBG) and palm kernel cake (PKC). The prebiotic activity and utilization of MOS were assessed in vitro using the probiotic Lactobacillus plantarum strain. RESULTS: Galactomannan from LBG was converted to MOS ranging in size from mannose up to mannoheptose by Man5HJ14 and ManAJB13. Mannoheptose was got from the hydrolysates produced by Man5HJ14, which mannohexaose was obtained from LBG hydrolyzed by ManAJB13. However, the same components of MOS ranging in size from mannose up to mannotetrose were observed between PKC hydrolyzed by the mannanases mentioned above. MOS stability was not affected by high-temperature and high-pressure condition at their natural pH. Based on in vitro growth study, all MOS from LBG and PKC was effective in promoting the growth of L. plantarum CICC 24202, with the strain preferring to use mannose to mannotriose, rather than above mannotetrose. CONCLUSIONS: The effect of mannanases and mannan difference on MOS composition was studied. All of MOS hydrolysates showed the stability in adversity condition and prebiotic activity of L. plantarum, which would have potential application in the biotechnological applications.

Relationship between ubiquitin C-terminal hydrolase-L1, neuroglobinB and neurological prognosis in patients with coma after cardiopulmonary resuscitation / 中国综合临床

Objective:To investigate the relationship between serum ubiquitin C-terminal hydrolase-L1 (UCH-L1) and neuroglobin levels and the prognosis of neurological function in coma patients after cardiopulmonary resuscitation, and to analyze their value in predicting the prognosis of patients.Methods:From February 2018 to June 2020, 45 comatose patients admitted to the Chengdu Third People′s Hospital of Sichuan Province after successful cardiopulmonary resuscitation were prospectively selected as the coma group, and 62 patients admitted to the emergency intensive care unit during the same period after successful cardiopulmonary resuscitation with consciousness recovered within 24 hours were selected as the control group.Serum UCH-L1 and neuroglobin levels were detected within 24 hours after admission.Glasgow coma Scale (GCS)and cerebral performance category(CPC)were used to evaluate coma severity and neurological prognosis.Spearman rank correlation analyzed the correlation between UCH-L1 and neuroglobin levels and GCS and CPC scores.Logistic regression analyzed the factors affecting the prognosis of neurological function in coma patients after cardiopulmonary resuscitation.Receiver operating characteristic curve (ROC) was used to analyze the value of UCH-L1 and neuroglobin in predicting the prognosis of neurological function in coma patients after successful cardiopulmonary resuscitation.Results:In coma group, serum UCH-L1((0.63±0.21) μg/L) and the concentration of neuroglobin ((89.34±21.35) mg/L) was higher than that in the control group ((0.27±0.08) μg/L, (32.13±9.21) mg/L), the difference was statistically significant( t=12.338, 18.846; all P<0.001). The levels of UCH-L1 and neuroglobin in mild, medium and severe coma groups were increased in turn, and the differences between the groups were statistically significant( F=86.430, 26.958; all P<0.001). The serum levels of UCH-L1((0.72±0.06) μg/L)and neuroglobin ((100.35±5.79) mg/L)in the group with poor neurological prognosis were higher than those in the group with good neurological prognosis((0.52±0.08) μg/L, (75.58±6.91) mg/L), and the differences between the groups were statistically significant( t=9.585, 13.086; all P<0.001). UCH-L1 and neuroglobin were negatively correlated with GCS score(rs=-0.685, -0.669; all P<0.001), and positively correlated with CPC score (rs=0.688, 0.670; all P<0.001). Multivariate Logistic regression analysis showed that low GCS score( OR=0.552, 95% CI: 0.392-0.776, P<0.001), high UCH-L1 ( OR=1.881, 95% CI: 1.276-2.773, P<0.001)and neuroglobin( OR=1.677, 95% CI: 1.206-2.331, P=0.001)were independently associated with poor neurological outcomes in coma patients after cardiopulmonary resuscitation .The AUC of combining UCH-L1 and neuroglobin in predicting poor neurological outcomes in coma patients after cardiopulmonary resuscitation was 0.954, which was higher than that of UCH-L1 and neuroglobin alone (0.821, 0.790) ( Z=2.351, 2.649; all P<0.05). Conclusion:After successful cardiopulmonary resuscitation, the levels of UCH-L1 and neuroglobin in coma patients are increased.High levels of UCH-L1 and neuroglobin are associated with coma severity and neurological dysfunction, which can be used as a potential biological indicator for prognosis evaluation of neurological function.

Computation-aided design of the flexible region of zearalenone hydrolase improves its thermal stability / 生物工程学报

The zearalenone hydrolase (ZHD101) derived from Clonostachys rosea can effectively degrade the mycotoxin zearalenone (ZEN) present in grain by-products and feed. However, the low thermal stability of ZHD101 hampers its applications. High throughput screening of variants using spectrophotometer is challenging because the reaction of hydrolyzing ZEN does not change absorbance. In this study, we used ZHD101 as a model enzyme to perform computation-aided design followed by experimental verification. By comparing the molecular dynamics simulation trajectories of ZHD101 at different temperatures, 32 flexible sites were selected. 608 saturated mutations were introduced into the 32 flexible sites virtually, from which 12 virtual mutants were screened according to the position specific score and enzyme conformation free energy calculation. Three of the mutants N156F, S194T and T259F showed an increase in thermal melting temperature (ΔTm>4 °C), and their enzyme activities were similar to or even higher than that of the wild type (relative enzyme activity 95.8%, 131.6% and 169.0%, respectively). Molecular dynamics simulation analysis showed that the possible mechanisms leading to the improved thermal stability were NH-π force, salt bridge rearrangement, and hole filling on the molecular surface. The three mutants were combined iteratively, and the combination of N156F/S194T showed the highest thermal stability (ΔTm=6.7 °C). This work demonstrated the feasibility of engineering the flexible region to improve enzyme performance by combining virtual computational mutations with experimental verification.

Expression and biological role of the neutral cholesterol ester hydrolase 1 gene in liver cancer tissue and cell lines / 临床肝胆病杂志

ObjectiveTo investigate the expression of the neutral cholesterol ester hydrolase 1 (NCEH1) gene in liver cancer tissue and human hepatoma cell lines and the effect of NCEH1 gene knockdown on the proliferation, apoptosis, invasion, and metastasis abilities of human hepatoma SMMC-7721 cells. MethodsLiver cancer tissue samples and adjacent tissue samples were collected from 32 patients with liver cancer who underwent surgical treatment in Guangzhou Red Cross Hospital Affiliated to Jinan University from January 2013 to June 2019, and quantitative real-time PCR was used to measure the relative expression level of the NCEH1 gene. Gene expression data of liver cancer samples up to September 2020 were downloaded from the ICGC database, and R software was used to analyze the data and obtain the expression level of the NCEH1 gene in each sample. The paired Wilcoxon signed-rank test and the Wilcoxon rank-sum test were used to investigate the differences between liver cancer tissue and adjacent tissue. Quantitative real-time PCR was used to measure the expression level of the NCEH1 gene in human hepatoma SMMC-7721, Bel-7402, HepG2, and Hep3B cells and normal human HL7702 liver cells. The lentivirus-mediated small interfering RNA (siRNA) technique was used to establish a human hepatoma SMMC-7721 cell line with NCEH1 gene knockdown, and the cells were divided into NCEH1 knockdown group (KD group) and negative control group (NC group); quantitative real-time PCR was used to measure the knockdown efficiency of the NCEH1 gene, and then MTT assay, flow cytometry with Annexin V-APC single staining, wound healing assay, Transwell assay, and Transwell chamber invasion assay were used to measure the proliferation, apoptosis, metastasis, and invasion abilities of SMMC-7721 cells in both groups. The t-test was used for statistical analysis of data between the two groups. ResultsThe mean expression level of the NCEH1 gene in liver cancer tissue was significantly higher than that in adjacent tissue (specimens from our hospital: Z=2.263, P=0.024; ICGC database: U=18 768, P＜0.001). SMMC-7721 cell line with moderate potential of invasion and metastasis had the highest expression level of the NCEH1 gene, followed by BEL-7402 and HepG2 cell lines with low potential of invasion and metastasis, and Hep3B cell line without the potential of invasion and metastasis had the lowest expression level. The KD group had a significantly lower expression level of the NCEH1 gene than the NC group (t=11.578, P=0000 3), and the knockdown efficiency of the NCEH1 gene was as high as 74.0%. Compared with the NC group, the KD group had a significant reduction in cell growth rate, a significant increase in apoptosis rate, and significant reductions in migration rate and the number of metastatic and invasive cells (t=32.100, 27.303, 9.51, 38.123, and 22.331, all P＜0.001). Conclusion There is a significant increase in the expression of the NCEH1 gene in liver cancer tissue and cell lines, and the NCEH1 gene can promote the growth, proliferation, invasion, and metastasis of hepatoma cells and inhibit their apoptosis, suggesting that it may be a potential therapeutic target for liver cancer.

Clinical significance of EPHX2 deregulation in prostate cancer / 亚洲男科学杂志(英文版)

The arachidonic acid (AA) metabolic pathway participates in various physiological processes as well as in the development of malignancies. We analyzed genomic alterations in AA metabolic enzymes in the Cancer Genome Atlas (TCGA) prostate cancer (PCa) dataset and found that the gene encoding soluble epoxide hydrolase (EPHX2) is frequently deleted in PCa. EPHX2 mRNA and protein expression in PCa was examined in multiple datasets by differential gene expression analysis and in a tissue microarray by immunohistochemistry. The expression data were analyzed in conjunction with clinicopathological variables. Both the mRNA and protein expression levels of EPHX2 were significantly decreased in tumors compared with normal prostate tissues and were inversely correlated with the Gleason grade and disease-free survival time. Furthermore, EPHX2 mRNA expression was significantly decreased in metastatic and recurrent PCa compared with localized and primary PCa, respectively. In addition, EPHX2 protein expression correlated negatively with Ki67 expression. In conclusion, EPHX2 deregulation is significantly correlated with the clinical characteristics of PCa progression and may serve as a prognostic marker for PCa.