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Natural killer (NK) cells are key players in human innate immunity. Cell engager antibody formats that recruit and activate NK cells more effectively have emerged as a promising immunotherapy approach to target cancer cells through more effective antibody-dependent cell-mediated cytotoxicity (ADCC). Monoclonal antibody drugs with ADCC activity have shown clinical benefit and improved outcomes for patients with certain types of cancer. CD16a, a Fc gamma III receptor, is the major component that is responsible for the ADCC activity of NK cells. Screening AvantGen's yeast displayed human antibody libraries led to the isolation of 2 antibody clones, #1A2 and #2-2A2, that selectively recognize both isoforms (F and V) of CD16a on primary NK cells with high affinity, yet minimally (#1A2) or do not (#2-2A2) cross-react with both allelotypes of CD16b (NA1 and NA2) expressed by neutrophils. Epitope mapping studies revealed that they bind to an epitope dependent on residue Y158 of CD16a, since mutation of Y158 to the corresponding CD16b residue H158 completely abolishes binding to CD16a. When formatted as bispecific antibodies targeting CD16a and a tumor-associated antigen (TAA, e.g. CD19), they exhibit specific binding to NK cells and induce potent NK cell activation upon encountering tumor cells, resulting in effective tumor cell killing. Notably, these bispecific antibody engagers stimulate NK cell cytokine release during co-culture with target cells, resulting in target cell cytotoxicity. These anti-CD16a antibody clones are promising candidates for combination with any TAA of interest, offering the potential for novel NK cell engager-based cancer therapeutics that are minimally affected by the high concentrations of human IgG in the circulation.
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Citotoxicidade Celular Dependente de Anticorpos , Células Matadoras Naturais , Receptores de IgG , Humanos , Células Matadoras Naturais/imunologia , Receptores de IgG/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Mapeamento de Epitopos/métodos , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/tratamento farmacológicoRESUMO
Mass spectrometry (MS) imaging of lipids in tissues with high structure specificity is challenging in the effective fragmentation of position-selective structures and the sensitive detection of multiple lipid isomers. Herein, we develop an MS3 imaging method for the simultaneous analysis of phospholipid CâC and sn-position isomers by on-tissue photochemical derivatization, nanospray desorption electrospray ionization (nano-DESI), and a dual-linear ion trap MS system. A novel laser-based sensing probe is developed for the real-time adjustment of the probe-to-surface distance for nano-DESI. This method is validated in mouse brain and kidney sections, showing its capability of sensitive resolving and imaging of the fatty acyl chain composition, the sn-position, and the CâC location of phospholipids in an MS3 scan. MS3 imaging of phospholipids has shown the capability of differentiation of cancerous, fibrosis, and adjacent normal regions in liver cancer tissues.
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Fosfolipídeos , Espectrometria de Massas por Ionização por Electrospray , Camundongos , Animais , Fosfolipídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Isomerismo , Cromatografia Gasosa-Espectrometria de Massas , Diagnóstico por ImagemRESUMO
The opening of high-speed railway (HSR) has accelerated the reorganization and reallocation of regional production elements, and constantly promoted the adjustment and upgrading of industrial structure. The cleaner production effect produced by industrial structure upgrading is of great significance to industrial pollution reduction. As a bridge connecting resources within the city and elements outside the city, the location of HSR stations has become an important factor affecting the structural emission reduction effect. Based on the data of 285 prefecture level and above cities in China from 2004 to 2018, this paper investigates the structural emission reduction effects of HSR opening and the impact of HSR station location on it by employing difference-in-differences (DID) model combined with mediation effect method. The results demonstrate that the opening of HSR has significant structural emission reduction effect, and the upgrading of inter-industry structure and the intra-industry structure are important mechanisms for HSR to achieve industrial emission reduction. The structural emission reduction effect of HSR opening is closely related to the location of HSR stations. With the increase of the distance between HSR station and city center, the industrial structure upgrading effect will continue to weaken, thus inhibiting the exertion of structural emission reduction effect, of which 10 km away from the city center is the optimal site strategy for the HSR service to give full play to the structural emission reduction effect, and exceeding 50 km will be significantly detrimental to its role in promoting industrial structure upgrading.
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Poluição Ambiental , Indústrias , China , Cidades , Desenvolvimento EconômicoRESUMO
BACKGROUND AND PURPOSE: Liver failure is associated with psychiatric alterations, partly resulting from the increased brain dopamine levels. We investigated the relationship between increased dopamine levels and mental abnormalities using bile duct ligation (BDL) rats and the mechanism by which liver failure increased dopamine levels in SH-SY5Y cells. Behavioural tests were carried out on day 13 and 27 following BDL, along with measurements of dopamine and metabolites, expressions of enzymes and transporters related to dopamine metabolism, and its transport into the cortex and the hippocampus. SH-SY5Y cells were used to investigate whether NH4 Cl, bile acids and bilirubin affected expression of tyrosine hydroxylase or not. Tyrosine hydroxylase (TH) expression in SH-SY5Y cells co-incubated with bilirubin and signal pathway inhibitors was measured. KEY RESULTS: Open-field test results demonstrated BDL rats showed anxiety-like behaviour, accompanied by increased dopamine levels and expression of TH protein in the cortex. Membrane bound long form (MB)-COMT, slightly but significantly decreased. SH-SY5Y cells indicated that increased bilirubin levels was a factor in inducing TH expression. Both inhibitor of NF-κB pathway BAY 11-7082 and silencing NF-κB p65 reversed bilirubin-induced upregulation of TH protein. NF-κB activator TNF-α increased expression of TH protein. Roles of bilirubin in increases of TH protein expressions and dopamine levels were measured using hyperbilirubinemia rats. Anxiety-like behaviour, was associated with increased dopamine levels and TH protein expressions in hyperbilirubinemia rats. CONCLUSION AND IMPLICATIONS: BDL significantly increased dopamine levels in rat cortex partly due to bilirubin-mediated TH induction. Increased bilirubin induced TH expression via activating NF-κB signalling pathway.
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Falência Hepática , Neuroblastoma , Ratos , Humanos , Animais , Tirosina 3-Mono-Oxigenase/metabolismo , Dopamina/metabolismo , NF-kappa B/metabolismo , Neuroblastoma/metabolismo , Ductos Biliares/cirurgia , Falência Hepática/metabolismo , Hiperbilirrubinemia/metabolismo , Bilirrubina/metabolismo , Bilirrubina/farmacologia , Ligadura , Córtex Cerebral/metabolismo , Fígado/metabolismoRESUMO
Mass spectrometry imaging (MSI) of lipids in biological tissues is useful for correlating molecular distribution with pathological results, which could provide useful information for both biological research and disease diagnosis. It is well understood that the lipidome could not be clearly deciphered without tandem mass spectrometry analysis, but this is challenging to achieve in MSI due to the limitation in sample amount at each image spot. Here we develop a multiplexed MS2 imaging (MS2 I) method that can provide MS2 images for 10 lipid species or more for each sampling spot, providing spatial structural lipidomic information. Coupling with on-tissue photochemical derivatization, imaging of 20 phospholipid C=C location isomers is also realized, showing enhanced molecular images with high definition in structure for mouse brain and human liver cancer tissue sections. Spatially mapped t-distributed stochastic neighbor embedding has also been adopted to visualize the tumor margin with enhancement by structural lipidomic information.
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Fosfolipídeos , Espectrometria de Massas em Tandem , Camundongos , Animais , Humanos , Espectrometria de Massas em Tandem/métodos , Diagnóstico por Imagem , Isomerismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Climate warming is changing plant sexual reproduction, having consequences for species distribution and community dynamics. However, the magnitude and direction of plant reproductive efforts (e.g., number of flowers) and success (e.g., number and mass of fruits or seeds) in response to warming have not been well-characterized. Here, we generated a global dataset of simulated warming experiments, consisting of 477 pairwise comparisons for 164 terrestrial species. We found evidence that warming overall decreased fruit number and increased seed mass, but little evidence that warming influenced flower number, fruit mass, or seed number. The warming effects on seed mass were regulated by the pollination type, and insect-pollinated plants exhibited a stronger response to warming than wind-pollinated plants. We found strong evidence that warming increased the mass of seeds for the nondominant species but no evidence of this for the dominant species. There was no evidence that phylogenetic relatedness explained the effects of warming on plant reproductive effort and success. In addition, the effects of warming on flowering onset negatively related to the responses in terms of the number of fruits and seeds to warming, revealing a cascading effect of plant reproductive development. These findings provide the first quantification of the response of terrestrial plant sexual reproduction to warming and suggest that plants may increase their fitness by producing heavier seeds under a warming climate.
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Clima , Frutas , Filogenia , Sementes , Polinização/fisiologia , Reprodução , Plantas , FloresRESUMO
Objective@#To investigate the distribution characteristics and functional genes of cariogenic bacteria in oral microorganisms of patients with type 2 diabetes mellitus and to improve the understanding of the relationship between type 2 diabetes mellitus and dental caries.@*Methods@#The experimental group included 10 patients with type 2 diabetes treated in the Department of Endocrinology, the First Affiliated Hospital of Air Force Medical University. The normal control group included healthy oral subjects without type 2 diabetes in the community population (10 cases). Samples of supragingival plaque from patients with type 2 diabetes mellitus and normal controls were collected and sequenced. Bioinformatics and statistical analysis of cariogenic bacteria such as Streptococcus mutans, Lactobacillus, Actinomyces viscosus and Candida albicans were carried out.@*Results@#There were slightly fewer cariogenic bacteria such as Streptococcus mutans, Lactobacillus, Actinomyces viscosus and Candida albicans in supragingival plaque samples of type 2 diabetic patients than in normal controls, but the difference was not statistically significant (P>0.05). The results of KEGG pathway functional metabolic differences showed that the metabolic pathways of D-arginine and D-ornithine metabolism, biofilm formation-Escherichia coli, carolactam degradation and arginine biosynthesis were more abundant in the T2DM group than in the normal control group, while metabolic pathways such as tyrosine metabolism, selenocompound metabolism and pyruvate metabolism showed the opposite trend. @*Conclusion @#There was no significant difference in the content of cariogenic microorganisms between type 2 diabetic patients and normal control group. The differential metabolic pathways of the functional genes indicated that an increase in the arginine metabolic pathway was beneficial to the maintenance of acid-base balance in the oral microecological environment.
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The root canal systems of maxillary second molar (MSM) variations are complicated, especially the prevalence of fused roots and consequent merged and C-shaped canals, which represent a clinical challenge because canal configurations can be irregular and unpredictable. The purpose of this article was to present 2 cases with a C-shaped configuration diagnosed during root canal retreatment and perform a literature review of this MSM anatomy. Case 1 reports that two palatal root canals fused into a C-shaped configuration that finally formed an apical foramen, which was classified as Type D. Case 2 reflects the fusion of the distobuccal canal and palatal canal into a C-shaped configuration and the configuration was Type C, which was first reported in a case report. Nonsurgical retreatments were proposed and conducted. Evaluation at a 24-month recall revealed that the two patients were symptom-free, and radiographic examination revealed normal periapical tissue. This report serves to remind clinicians of the complexities of the root canal system and that possible anatomic variation should always be anticipated when formulating an effective root canal treatment plan. The use of CBCT imaging coupled with an operative dental microscope will be helpful in locating and identifying supernumerary canals when a preoperative periapical radiograph shows signs of a fused-rooted MSM.
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The construction of new-type urbanization with the theme of innovation, green, and smart development is becoming the endogenous driving force of China's economic transformation and upgrading, and green technological innovation is a key factor in cracking the problems of development motivation and environmental constraints in urbanization construction. This paper investigates the impact of green technology innovation on urbanization based on a panel dataset covering 30 provinces in China from 2005 to 2019. First, we use the entropy method and the super-efficiency DEA method to measure the level of urbanization and green technology innovation, respectively. Moreover, on this basis, we use panel regression model and FGLS model to estimate the direct impact of green technological innovation on urbanization and its three dimensions-population urbanization, industrial urbanization, and ecological urbanization. Then, the mediating effect model is used to further study the indirect impact of green technological innovation on urbanization. The results indicate that green technological innovation is the most effective way to promote the development of new urbanization currently. In addition, green technology innovation can indirectly affect urbanization through the effects of foreign capital, energy consumption and information development, while the effect of industrial structure optimization effects is not significant. Finally, some policy suggestions are discussed to better promote the development of urbanization in China.
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Invenções , Urbanização , China , Desenvolvimento Econômico , IndústriasRESUMO
A high proportion of massive patients with hepatocellular carcinoma (HCC) are not amenable for surgical resection at initial diagnosis, owing to insufficient future liver remnant (FLR) or an inadequate surgical margin. For such patients, portal vein embolization (PVE) is an essential approach to allow liver hypertrophy and prepare for subsequent surgery. However, the conversion resection rate of PVE only is unsatisfactory because of tumor progression while awaiting liver hypertrophy. We report here a successfully treated case of primary massive HCC, where surgical resection was completed after PVE and multimodality therapy, comprising hepatic artery infusion chemotherapy (HAIC), Lenvatinib plus Sintilimab. A pathologic complete response was achieved. This case demonstrates for the first time that combined PVE with multimodality therapy appears to be safe and effective for massive, potentially resectable HCC and can produce deep pathological remission in a primary tumor.
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The prediction of electrochemical performance is the basis for long-term service of all-solid-state-battery (ASSB) regarding the time-aging of solid polymer electrolytes. To get insight into the influence mechanism of electrolyte aging on cell fading, we have established a continuum model for quantitatively analyzing the capacity evolution of the lithium battery during the time-aging process. The simulations have unveiled the phenomenon of electrolyte-aging-induced capacity degradation. The effects of discharge rate, operating temperature, and lithium-salt concentration in the electrolyte, as well as the electrolyte thickness, have also been explored in detail. The results have shown that capacity loss of ASSB is controlled by the decrease in the contact area of the electrolyte/electrode interface at the initial aging stage and is subsequently dominated by the mobilities of lithium-ion across the aging electrolyte. Moreover, reducing the discharge rate or increasing the operating temperature can weaken this cell deterioration. Besides, the thinner electrolyte film with acceptable lithium salt content benefits the durability of the ASSB. It has also been found that the negative effect of the aging electrolytes can be relieved if the electrolyte conductivity is kept being above a critical value under the storage and using conditions.
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Antibodies represent powerful tools to examine signal transduction pathways. Here, we present a strategy integrating multiple state-of-the-art methods to produce, validate, and utilize antibodies. Focusing on understudied synaptic proteins, we generated 137 recombinant antibodies. We used yeast display antibody libraries from the B cells of immunized rabbits, followed by FACS sorting under stringent conditions to identify high affinity antibodies. The antibodies were validated by high-throughput functional screening, and genome editing. Next, we explored the temporal dynamics of signaling in single cells. A subset of antibodies targeting opioid receptors were used to examine the effect of treatment with opiates that have played central roles in the worsening of the 'opioid epidemic.' We show that morphine and fentanyl exhibit differential temporal dynamics of receptor phosphorylation. In summary, high-throughput approaches can lead to the identification of antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling.
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Anticorpos/farmacologia , Especificidade de Anticorpos , Ensaios de Triagem em Larga Escala , Proteína Quinase C/antagonistas & inibidores , Receptores Opioides mu/antagonistas & inibidores , Sinapses/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Animais , Anticorpos/imunologia , Linhagem Celular Tumoral , Ativação Enzimática , Fentanila/farmacologia , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Morfina/farmacologia , Fosforilação , Proteína Quinase C/imunologia , Proteína Quinase C/metabolismo , Coelhos , Receptores Opioides mu/imunologia , Receptores Opioides mu/metabolismo , Transdução de Sinais , Sinapses/imunologia , Sinapses/metabolismo , Fatores de TempoRESUMO
The emergence of COVID-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. Currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of COVID-19 are needed. We identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the SARS-CoV-2 spike protein receptor binding domain that neutralized the virus in vitro . Administration of the lead antibody clone to Syrian hamsters challenged with SARS-CoV-2 significantly reduced viral load and histopathology score in the lungs. Moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. The use of this antibody could provide an important therapy for treatment of COVID-19 patients.
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Over the past 40 years since China's reform and opening up, the industrial structure has undergone tremendous changes. The rapid development of the economy has been accompanied by a surge in carbon emissions. How to achieve a win-win situation for economic growth and carbon emissions reduction has aroused widespread concern from all sectors of society. Here, this paper discusses the dynamic relationship of industrial structure upgrading, economic growth, and carbon emission reduction. Results show that there is a long-term equilibrium relationship among industrial structure upgrading, economic growth, and carbon emissions. In the short term, when the three variables deviate from the long-term equilibrium state, the non-equilibrium state will be pulled back to equilibrium with the adjustment strength of - 0.0633, - 0.0097, and 0.0013. Carbon emission reduction promotes industrial structure upgrading. Industrial structure upgrading has a greater positive impact on economic growth. Industrial structure upgrading and economic growth have a negative impact on carbon emissions, thereby promoting emission reduction. And at the 10% significance level, there is a one-way Granger causality from carbon emissions to industrial structure upgrading, economic growth can cause one-way changes in carbon emissions, and industrial structure upgrading is a one-way Granger cause of economic growth. Finally, several carbon emission reduction policies are proposed promote industrial restructuring and sustainable economic development.
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Carbono , Desenvolvimento Econômico , Carbono/análise , Dióxido de Carbono/análise , China , IndústriasRESUMO
Previous studies have indicated that inhibition of type I interferon production may be an important reason for porcine reproductive and respiratory syndrome virus (PRRSV) to achieve immune escape, revealing the mechanism of inhibiting the production of type I interferon will help design novel strategies for controlling PRRS. Here, we found that PRRSV infection upregulated the expression of miR-382-5p, which in turn inhibited polyI:C-induced the production of type I interferon by targeting heat shock protein 60 (HSP60), thus facilitating PRRSV replication in MARC-145 cells. Furthermore, we found that HSP60 could interact with mitochondrial antiviral signaling protein (MAVS), an important signal transduction protein for inducing production of type I interferon, and promote polyI:C-mediated the production of type I interferon in a MAVS-dependent manner. Finally, we also found that HSP60 could inhibit PRRSV replication in a MAVS-dependent manner, which indicated that HSP60 was a novel antiviral protein against PRRSV replication. In conclusion, the study demonstrated that miR-382-5p was upregulated during PRRSV infection and may promote PRRSV replication by negatively regulating the production of type I interferon, which also indicated that miR-382-5p and HSP60 might be the potential therapeutic targets for anti-PRRSV.
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Chaperonina 60/metabolismo , Interferon Tipo I/metabolismo , MicroRNAs/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Animais , Linhagem Celular , Chaperonina 60/genética , Cricetinae , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Interferon Tipo I/genética , MicroRNAs/genética , Plasmídeos/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Interferência de RNA , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
The emergence of COVID-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. Currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of COVID-19 are needed. We identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the SARS-CoV-2 spike protein receptor binding domain that neutralized the virus in vitro. Administration of the lead antibody clone to Syrian hamsters challenged with SARS-CoV-2 significantly reduced viral load and histopathology score in the lungs. Moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. The use of this antibody could provide an important therapy for treatment of COVID-19 patients.
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Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Tratamento Farmacológico da COVID-19 , COVID-19 , Imunoglobulina G , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , COVID-19/sangue , COVID-19/imunologia , Chlorocebus aethiops , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Masculino , Mesocricetus , Índice de Gravidade de Doença , Células Vero , Carga Viral/efeitos dos fármacos , Carga Viral/imunologiaRESUMO
Glycoprotein (GP) 2a was a minor structural protein of porcine reproductive and respiratory syndrome virus (PRRSV) and was one of crucial proteins for PRRSV to bind cell receptor, which indicated that there were neutralizing epitopes on GP2a. In the present work, we used mouse anti-GP2a41-208aa serum and one GP2a41-208aa specific monoclonal antibody (McAb) to identify B-cell epitopes of GP2a by peptide-based ELISA. A liner B-cell epitope F194PTPGSRPKLHDFQQ208 was identified. However, the results of virus neutralization experiment showed that the McAb could not reduce the titers of PRRSV, which indicated that the identified epitope was not the neutralizing epitope of PRRSV. While the amino acid sequence of this epitope was conserved in North American (type 2) PRRSV, which suggested that this epitope might be diagnostic potential for type 2 PRRSV strains. In conclusion, our present work identified a new epitope on GP2a and this epitope might be diagnostic potential for type 2 PRRSV strains.
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Epitopos de Linfócito B/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência Conservada , Mapeamento de Epitopos , Camundongos , Alinhamento de Sequência , Proteínas do Envelope Viral/químicaRESUMO
The urban wastewater treatment industry produces a large amount of excess activated sludge which is mainly composed of microbial biomass and costly to be disposed. In this research, a comprehensive utilization of activated sludge was developed by sequentially extracting hydrolytic enzymes and polyhydroxyalkanoates (PHAs), and the residue was used to prepare water-retaining organic fertilizer. The sludge was extracted with fourfold H2O-containing 1% Triton X-100 with the yield of 66.7% protease activity. The enzyme solution was precipitated in 80% acetone and vacuum dried at 40°C at the dried enzyme yield of 2.4 g/kg wet sludge. The enzyme product contains collagenase, lipase, amylase, and cellulase activities, which are good compound enzymes to feed. The PHAs were extracted with 30% sodium hypoclorite:chloroform (1:3). The PHA solution was decolored and dried, and pure white PHAs were obtained at the yield of 70.1 g/kg wet sludge. The residue was used to prepare water-retaining organic fertilizer at the optimal condition. The fertilizer absorbs 131.3-fold distilled water and had good performance in water retention and can effectively slow down the loss of soil moisture when added into soil. This work provides a simple and practical approach for comprehensive utilizing activated sludge with significant economic benefits.
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Fertilizantes , Peptídeo Hidrolases/isolamento & purificação , Poli-Hidroxialcanoatos/química , Polímeros/química , Esgotos/microbiologia , Fertilizantes/análise , Hidrólise , Peptídeo Hidrolases/química , Poli-Hidroxialcanoatos/isolamento & purificação , Água/químicaRESUMO
Nattokinase is an important fibrinolytic enzyme with therapeutic applications for cardiovascular diseases. The full-length and mature nattokinase genes were cloned from Bacillus subtilis var. natto and expressed in pQE30 vector in Escherichia coli. The full-length gene expressed low nattokinase activity in the intracellular soluble and the medium fractions. The mature gene expressed low soluble nattokinase activity and large amount insoluble protein in inclusion bodies without enzyme activity. Large amount of refolding solutions (RSs) at different pH values were screening and RS-10 and RS-11 at pH 9 were selected to refold nattokinase inclusion bodies. The recombinant cells were lysed with 0.1mg/mL lysozyme and ultrasonic treatment. After centrifugation, the pellete was washed twice with 20mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100 to purify the inclusion bodies. The inclusion bodies were dissolved in water at pH 12.0 and refolded with RS-10. The refolded proteins showed 42.8IU/mg and 79.3IU/mg fibrinolytic activity by the traditional dilution method (20-fold dilution into RS-10) and the directly mixing the protein solution with equal volume RS-10, respectively, compared to the 52.0IU/mg of total water-soluble proteins from B. subtilis var. natto. This work demonstrated that the inclusion body of recombinant nattokinase expressed in E. coli could be simply refolded to the natural enzyme activity level by directly mixing the protein solution with equal volume refolding solution.
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Escherichia coli/genética , Corpos de Inclusão/metabolismo , Subtilisinas/genética , Subtilisinas/metabolismo , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Fibrinolíticos/metabolismo , Corpos de Inclusão/química , Redobramento de Proteína , Subtilisinas/químicaRESUMO
OBJECTIVE: To use three-dimensional (3D) printed craniocerebral models to guide neurosurgery and design the best operative route preoperatively. METHODS: Computed tomography, magnetic resonance imaging, computed tomography angiography, and functional magnetic resonance images of the patients were collected as needed, reconstructed to form multicolor 3D craniocerebral images, and printed to form solid 3D models. The hollow aneurysm model was printed with rubberlike material; craniocerebral models were printed with resin or gypsum. RESULTS: The 3D printed hollow aneurysm model was highly representative of what was observed during the surgery. The model had realistic texture and elasticity and was used for preoperative simulation of aneurysm clipping for clip selection, which was the same as was used during the surgery. The craniocerebral aneurysm model clearly showed the spatial relation between the aneurysm and surrounding tissues, which can be used to select the best surgical approach in the preoperative simulation, to evaluate the necessity of drilling the anterior clinoid process, and to determine the feasibility of using a contralateral approach. The craniocerebral tumor and anatomic model showed the spatial relation between tumor and intracranial vasculatures, tractus pyramidalis, and functional areas, which was helpful 1) when selecting the optimal surgical approach to avoid damage to brain function, 2) for learning the functional anatomy of the craniocerebral structure, and 3) for preoperative selection of surgical spaces in the sellar region. CONCLUSIONS: 3D printing provides neurosurgeons with solid craniocerebral models that can be observed and operated on directly and effectively, which further improves the accuracy of neurosurgeries.