Substitutability of metal-binding sites in an alcohol dehydrogenase / 生物工程学报

Covalently anchoring of a ligand/metal via polar amino acid side chain(s) is often observed in metalloenzyme, while the substitutability of metal-binding sites remains elusive. In this study, we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme, analyzed the sequence conservation of the three residues Cys37, His59, and Asp150 that bind the zinc ion, and constructed the mutant library. After experimental validation, three out of 224 clones, which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one, were screened out. The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity, which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.

Analysis of point-of-care testing researches in China based on CNKI database / 中华医院管理杂志

Objective:To identify current research hotspots, research trends and future trends of point-of-care testing(POCT) in China, by means of literature review.Methods:The " point-of-care testing" and " POCT" were used as the search terms, to retrieve in CNKI database the literature on point-of-care testing published during 2010-2019 in the method of subject retrieval. CiteSpace software was used to analyze and generate the research institutions and their cooperative networks, the keyword co-occurrence network, and the burst term analysis, for an analysis of the current researches in point-of-care testing in China.Results:A total of 588 literatures were retrieved, and 485 literatures were finalized as research samples, with exclusion of those obviously irrelevant. The research found that the point-of-care testing studies in China focused on portable blood glucose meters, quality control of POCT, comparison of POCT and biochemical analyzer results, and establishment of a POCT quality system. In terms of future research tend, the hotspots fell on " total quality management system of POCT" , " POCT in cardiovascular disease application" , " POCT in battlefield operation or emergency application" .Conclusions:Given the growing number of relevant documents on point-of-care testing, and the expanding scope of researches, regional research centers have yet to be formed.

Elimination of a disulfide bond in the light chain of coagulation factor VIII improves secretion of a BDD-FVIII variant with an engineered inter-chain disulfide / 药学学报

The coagulation VIII factor (FVIII) contains eight pairs of disulfide bonds, which are involved in maintaining its structure and function. It has been demonstrated that the disulfide bond between Cys1899/Cys1903 of the A3 domain in the light chain impedes secretion. In our previous work, an engineered inter-chain disulfide in the B domain-deleted FVIII (BDD-FVIII) promoted heterodimer assembly and secretion of separately expressed heavy and light chains. In this study, we constructed two BDD-FVIII variants, one of which contains an engineered inter-chain disulfide bond (F8C) between Met662 > Cys and Asp1828 > Cys mutations and another contains an endogenous A3 domain with a disrupted disulfide bond from F8C (F8CG) by replacement of Cys1899 and Cys1903 with Gly in F8C. We explored their function and secretion. By transducing F8C and F8CG into HEK293 and COS-7 cells, the formation of disulfide bonds and the secretion and coagulation activity of the two variants in the culture media and their binding affinity for von Willebrand factor (vWF) could be observed. The results show that variants F8C and F8CG are mainly the disulfide bonded heavy and light chain dimer, while the wild type BDD-FVIII (F8) is dominated by the easily dissociated heavy and light chain dimer. The secretion and activity of F8C was significantly higher than that of F8, while the secretion and activity of F8CG was significantly higher than that of F8C. The vWF binding of the two variants is similar to F8. This indicates that the BDD-FVIII variant F8CG may be attractive molecule for protein replacement and as a transgene in gene-therapy strategies. These findings are encouraging for future studies targeting disulfide bond elimination for further enhancement of FVIII secretion.

Effect of Chuankezhi Injection on Prevention and Treatment of H1N1 Influenza Virus in Mice / 中国实验方剂学杂志

Objective: To evaluate the effect of Chuankezhi injection on mouse model of pneumonia induced by influenza A (H1N1) FM1 strain. Method: ICR mice were randomly divided into normal group, model group, tamiflu control group (27.5 mg·kg-1·d-1) and Chuankezhi injection group (1.5 mL·kg-1·d-1). In the death protection experiment, mice were infected with 2×half lethal dose (LD50) of influenza virus FM1.The Chuankezhi injection was given once a day for 4 days. The number of death animal within 14 days was counted. The mortality and the death protection rate were calculated. In the treatment experiment, mice were infected with 0.8×LD50 of influenza virus, and the Chuankezhi injection was given once a day for 4 days. On the 5th day after the infection, the levels of interleukin-8 (IL-8) in lung, prostaglandin E2 (PGE2) and vasopressin (AVP) in brain were tested by enzyme-linked immunosorbent assay (ELISA). The viral load of influenza virus in lung was tested by Real-time PCR. In the pre-treatment experiment, mice were given Chuankezhi injection once a day for 5 days. 1 hour after the last treatment, mice were infected with 0.8×LD50 influenza virus. 4 days after the infection, the lung index, spleen index, thymus index, and viral load in lung tissue were calculated. Result: Compared with normal group, the IL-8, PGE2 content, lung index and viral load in the lung tissue of model group were significantly increased (P2, and the viral load of influenza(PPPPConclusion: Chuankezhi injection could effectively prevent the mouse model of pneumonia induced by influenza A (H1N1) virus. The mechanism might be related to the reduction of inflammation and inhibiting viral replication.

Animal Models of EV71 and CA16 Hand-foot-mouth Disease and Characteristics of Viral Infection / 中国实验方剂学杂志

Hand-foot-mouth disease (HFMD) is a common infectious disease caused by enterovirus in children. It has a high incidence and can cause fatal complications such as pulmonary edema, myocarditis and aseptic meningitis, seriously threatening the health of children. At present, some core problems such as the pathogenesis of disease, the relationship between different genotypes of pathogenic viruses, the pharmacodynamic evaluation methods, and the antiviral mechanism of drugs are still unclear. The construction of disease animal models with simulation performance of human exposure is the key to solve the above problems. Researchers both at home and abroad have established a variety of animal models for HFMD, of which enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are most common and most widely used. Both EV71 and CA16 are enterovirus A in picornavirus family, so they have similarities in terms of pathogenicity, infection and replication characteristics, clinical symptoms caused by infection and immune response, but also have significant differences in age of susceptibility, method of infection, as well as neurotoxicity, clinical symptoms and signs, and degree of tissue and organ damage. Therefore, researchers shall select and establish proper animal models based on actual conditions, which is critical to the reliability of the results. In this paper, the different types of HFMD animal models established by EV71 and CA16 viruses were reviewed, especially on the species strains, virus strain types, infection methods, and characteristics of viral infections in each model, and the characteristics and clinical symptoms of HFMD induced by EV71 and CA16 were also investigated to provide reference for related research.

Establishment and practice of a point-of-care testing management system in hospitals / 中华医院管理杂志

With the market-oriented application of new technologies and the development of various subjects in hospitals, more and more point-of-care test(POCT) devices appear in medical institutions, as an important method for rapid clinical acquisition of test results. As introduced by the authors, the hospital since 2017, established based on the hospital′s total quality management system, the POCT management committee, and built the hospital′s POCT quality management system. Thanks to its efforts, the hospital has fully standardized the POCT management from equipment procurement, test, maintenance, personnel training and examination, thus further ensuring medical quality.

Immunogenicity of quadrivalence recombinant human papillomavirus vaccine (6, 11, 16 and 18 types) (Hansenulapolymorpha): results from phaseⅠ clinical trial / 中华微生物学和免疫学杂志

Objective@#To make a preliminary assessment on the immunogenicity of a quadrivalence recombinant human papillomavirus (HPV) vaccine (6, 11, 16 and 18 types) (Hansenulapolymorpha) in healthy women aged 18-45 years in phaseⅠclinical study.@*Methods@#It was a single-center, double-blind, randomized, placebo-controlled phaseⅠ clinical study. Women aged 18-45 years were randomized (2∶1) to receive HPV vaccine (n=60) or placebo control (n=30) at months 0, 2 and 6. Antibodies against HPV6/11/16/18 were detected by pseudovirus-based neutralisation assay in serum samples collected at 0 d, 180 d and 210 d. Seroconversion rates and geometric mean titres (GMT) of antibodies against the four types of antigens were calculated.@*Results@#Seroconversion rates of the vaccination group at 180 d (before the third dose) and 210 d (one month after the third dose) were generally similar and between 85%-100% for all types of antibodies. The GMT of antibodies at one month after the last dose improved significantly compared with those before immunization.@*Conclusions@#These results showed that the HPV vaccine had good immunogenicity in the population of healthy women aged 18-45 years. Higher antibody titers were elicited by the vaccine compare with the tites before the first dose and in the placebo control group.

Structure-function relationships of industrial enzymes / 生物工程学报

Industrial enzymes are the "chip" of modern bio-industries, supporting tens- and hundreds-fold of downstream industries development. Elucidating the relationships between enzyme structures and functions is fundamental for industrial applications. Recently, with the advanced developments of protein crystallization and computational simulation technologies, the structure-function relationships have been extensively studied, making the rational design and de novo design become possible. This paper reviews the progress of structure-function relationships of industrial enzymes and applications, and address future developments.

Protein engineering: from directed evolution to computational design / 生物工程学报

By constructing mutant libraries and utilizing high-throughput screening methods, directed evolution has emerged as the most popular strategy for protein design nowadays. In the past decade, taking advantages of computer performance and algorithms, computer-assisted protein design has rapidly developed and become a powerful method of protein engineering. Based on the simulation of protein structure and calculation of energy function, computational design can alter the substrate specificity and improve the thermostability of enzymes, as well as de novo design of artificial enzymes with expected functions. Recently, machine learning and other artificial intelligence technologies have also been applied to computational protein engineering, resulting in a series of remarkable applications. Along the lines of protein engineering, this paper reviews the progress and applications of computer-assisted protein design, and current trends and outlooks of the development.

Immunogenicity of quadrivalence recombinant human papillomavirus vaccine ( 6, 11, 16 and 18 types) ( Hansenulapolymorpha):results from phaseⅠ clinical trial / 中华微生物学和免疫学杂志

Objective To make a preliminary assessment on the immunogenicity of a quadriva-lence recombinant human papillomavirus (HPV) vaccine (6, 11, 16 and 18 types) (Hansenulapolymor-pha) in healthy women aged 18-45 years in phaseⅠclinical study. Methods It was a single-center, doub-le-blind, randomized, placebo-controlled phaseⅠclinical study. Women aged 18-45 years were randomized (2 : 1) to receive HPV vaccine (n=60) or placebo control (n=30) at months 0, 2 and 6. Antibodies against HPV6/11/16/18 were detected by pseudovirus-based neutralisation assay in serum samples collected at 0 d, 180 d and 210 d. Seroconversion rates and geometric mean titres ( GMT) of antibodies against the four types of antigens were calculated. Results Seroconversion rates of the vaccination group at 180 d ( be-fore the third dose) and 210 d ( one month after the third dose) were generally similar and between 85％-100％ for all types of antibodies. The GMT of antibodies at one month after the last dose improved signifi-cantly compared with those before immunization. Conclusions These results showed that the HPV vaccine had good immunogenicity in the population of healthy women aged 18-45 years. Higher antibody titers were elicited by the vaccine compare with the tites before the first dose and in the placebo control group.

Recent advances in directed evolution / 生物工程学报

Screening is the bottleneck of directed evolution. In order to address this problem, a series of novel semi-rational designed strategies have been developed based on combinatorial active-site saturation test and iterative saturation mutagenesis, including single code saturation mutagenesis, double code saturation mutagenesis and triple code saturation mutagenesis. By creation of "small and smart" high qualified mutant libraries and combinatorial mutagenesis of specific sites, these new strategies have been successfully applied in multiparameter optimization, e.g. stereo/regioselectivity and activity. This review summarized recent advances in directed evolution and its applications in biocatalysis field.

Ultrasound-guided Transversus Abdominis Plane Block Improves Postoperative Analgesia and Early Recovery in Patients Undergoing Retroperitoneoscopic Urologic Surgeries: A Randomized Controlled Double-blinded Trial / 中国医学科学杂志(英文版)

<strong>Objective</strong> To evaluate the effects of ultrasound-guided transversus abdominis plane (TAP) block on postoperative analgesia and early recovery in patients undergoing retroperitoneoscopic urologic surgeries. <strong>Methods</strong> This was a randomized, controlled, double-blinded trial. Eligible patients scheduled for retroperitoneoscopic urologic surgeries were randomly assigned to two groups. Group TAP received ultrasound-guided TAP block with 0.5% ropivacaine 20 ml at 30 minutes before surgery, and Group C received TAP sham block with normal saline. All patients received retroperitoneoscopic urologic surgeries under general anesthesia. The primary outcome was the severity of pain after surgery. Secondary outcomes included opioids consumption, analgesics, postoperative nausea and vomiting, time to Foley catheter removal and to passage of flatus, length of post-anesthesia care unit stay and hospital stay. <strong>Results</strong> Eighty patients completed the study, forty cases in each group. Compared to the Group C, the Group TAP had lower visual analogue scale pain scores within two postoperative days (all P<0.05). They also had less consumption of intraoperative fentanyl (2.0±0.5 vs. 3.8±0.7 μg/kg, P<0.05), reduced incidence of postoperative rescue analgesic usage (12.5% vs. 45.0%, P<0.05), and lower incidence of postoperative nausea and vomiting within postoperative 48 hours (12.5% vs. 25.0%, P<0.05) when compared to the Group C. In addition, Group TAP had a shortened post-anesthesia care unit stay (25±8 vs. 49±12 minutes, P<0.05), and a greater proportion of patients discharged within postoperative three days (57.5% vs. 35.0%, P<0.05). <strong>Conclusion</strong> Preoperative ultrasound-guided TAP block is an effective technique to improve postoperative analgesia and early recovery in patients undergoing retroperitoneoscopic urologic surgeries.

Ultrasound-guided Transversus Abdominis Plane Block Improves Postoperative Analgesia and Early Recovery in Patients Undergoing Retroperitoneoscopic Urologic Surgeries: A Randomized Controlled Double-blinded Trial / 中国医学科学杂志(英文版)

Objective To evaluate the effects of ultrasound-guided transversus abdominis plane (TAP) block on postoperative analgesia and early recovery in patients undergoing retroperitoneoscopic urologic surgeries. Methods This was a randomized, controlled, double-blinded trial. Eligible patients scheduled for retroperitoneoscopic urologic surgeries were randomly assigned to two groups. Group TAP received ultrasound-guided TAP block with 0.5% ropivacaine 20 ml at 30 minutes before surgery, and Group C received TAP sham block with normal saline. All patients received retroperitoneoscopic urologic surgeries under general anesthesia. The primary outcome was the severity of pain after surgery. Secondary outcomes included opioids consumption, analgesics, postoperative nausea and vomiting, time to Foley catheter removal and to passage of flatus, length of post-anesthesia care unit stay and hospital stay. Results Eighty patients completed the study, forty cases in each group. Compared to the Group C, the Group TAP had lower visual analogue scale pain scores within two postoperative days (all P<0.05). They also had less consumption of intraoperative fentanyl (2.0±0.5 vs. 3.8±0.7 μg/kg, P<0.05), reduced incidence of postoperative rescue analgesic usage (12.5% vs. 45.0%, P<0.05), and lower incidence of postoperative nausea and vomiting within postoperative 48 hours (12.5% vs. 25.0%, P<0.05) when compared to the Group C. In addition, Group TAP had a shortened post-anesthesia care unit stay (25±8 vs. 49±12 minutes, P<0.05), and a greater proportion of patients discharged within postoperative three days (57.5% vs. 35.0%, P<0.05). Conclusion Preoperative ultrasound-guided TAP block is an effective technique to improve postoperative analgesia and early recovery in patients undergoing retroperitoneoscopic urologic surgeries.

Topical anesthetic effects of compound lidocaine cream coated endotracheal tube to prevent cough and agitation during extubation in thyroidectomy under general anesthesia / 基础医学与临床

Objective To evaluate the topical anesthetic effects of compound lidocaine cream ( CLC) coated endo-tracheal tube on the prevention of cough/agitation during extubation in thyroidectomy under general anesthesia . Methods 42 patients scheduled for thyroidectomy in Peking Union Medical College Hospital were continuously en -rolled in this prospective double-blinded randomized controlled trial .Patients were randomized into two groups . Group L were intubated with endotracheal tube coated with CLC , while the control group , group C with liquid par-affin .The end point of study was spontaneous or induced cough /agitation during extubation .Hemodynamic parame-ters and the consumption of opioids were recorded .Patients were followed for major complications during their stay in hospital .Results Spontaneous cough/agitation rate was lower in group L than group C ( 15% vs 65%, P<0.01 );so was induced cough/agitation (35%vs 90%, P<0.001 );the value of SBP ×HR was lower in group L at 1 min after extubation ( P<0.05 ) .Conclusions The topical anesthetic effects of CLC coated endotracheal tube could prevent cough and agitation during extubation in thyroidectomy under general anesthesia .

A retrospective study on the diagnosis and treatment of vocal folds white lesions / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To analyze the pathogenesis and clinical features of the vocal fold white lesions, and to investigate the principles of the diagnosis and treatment.</p><p><b>METHODS</b>One hundred and fifty-six cases of vocal fold white lesions encountered between January 2006 and December 2013 were analyzed. All patients had bilateral-vocal folds whitening under laryngoscopic examination, and all the patients had been observed over three months. There were 135 males and 21 females in the study, the age ranged from 18 to 60 years, with the mean age of (38.8±8.1) years old. All patients had a history of vocal abuse, with moderate-severe degree of voice disorder.</p><p><b>RESULTS</b>After a month of voice rest and suitable treatment, the result of reexamination showed that the symptoms faded away in 81 cases (51.9%), with the voice recuperated; the range of white lesion was reduced with the voice improving in 71 cases (45.4%); the range of white lesion was not obviously changed in 4 cases (2.6%), the pathological examination in these 4 cases showed moderate-severe dysplasia in two cases, and the surgical treatment was applied for them. Two months later, the reexamination showed 26 male cases still had vocal fold white lesion with various degree; and three months later, only 7 male cases retained mild vocal fold white lesion.</p><p><b>CONCLUSION</b>Histopathologically, not all vocal fold white lesions were laryngeal precancerous lesions, and not all of these patients should be treated surgically.</p>

In vivo pharmacokinetics of strychnos alkaloids in rats after ig administration / 中草药

Objective: To evaluate the in vivo pharmacokinetics of strychnos alkaloids [brucine, total alkaloids in Strychni Semen (TASS), and optimal total alkaloids in Strychni Semen (OTASS)] by ig administration to rats. Methods: The rats were ig administered with strychnos alkaloids and their blood samples were taken. Huperzine A was used as an internal standard. The brucine in plasma of rats was detected by HPLC method. The compartment model was fitted and the pharmacokinetic parameters were calculated by DAS1.0 program. Results: The pharmacokinetic analysis showed that brucine behaved as a two-compartment model in the three solutions after ig administration in rats. Compared with brucine monomer administration, the solution of TASS and OTASS could obviously increase the absorption of brucine in vivo, but other pharmacokinetic parameters had no significant difference. Conclusion: After ig administration with strychnos alkaloids, the other alkaloids in Strychni Semen could promote the absorption of brucine.

Aiming at tumors and their microenvironments: Targeted strategies of mesenchymal stem cell-mediated genetic therapy / 肿瘤

The early detection of some cancer types combined with the advent of specific drugs has increased median survival of numerous cancer patients. However, many drugs with poor pharmacokinetics rapidly lose activity when delivered systemically and may even not reach the tumors. Based on tropism for tumors, genetic-modified mesenchymal stem cells provide a promising cellular vehicle for drug deliveries. During rapid progression of cancer disease, an activated stromal microenvironment is produced to facilitate tumor development. As a result, it appears a potential chance to modify the cancer therapy in a way, aiming at both the tumor cells and their microenvironments. Copyright © 2013 by TUMOR.

Leucine zippers improves protein splicing-mediated coagulation factor VIII gene delivery by dual-vector system / 药学学报

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.

Trans-splicing of Cys mutated coagulation factor VIII / 药学学报

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.

Enhancing effect of deoxynivalenol-mediated GRP78 down-regulation on heavy chain secretion and bioactivity of two-chain FVIII gene co-transfected cells / 药学学报

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.