AIM: To establish a simplified method for generating peptide-major histocompatibility complex (MHC) class I tetramers.METHODS: cDNAs encoding the extracellular domain of human lymphocyte antigen (HLA)-A*0201 heavy cha...AIM: To establish a simplified method for generating peptide-major histocompatibility complex (MHC) class I tetramers.METHODS: cDNAs encoding the extracellular domain of human lymphocyte antigen (HLA)-A*0201 heavy chain (A2) and β2-microglobulin (β2m) from total RNA extracted from leukocytes of HLA-A2+ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and β2m proteins were expressed in Escherichia coli strain BL21(DE3) and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain β2m and HLA-A2-restricted peptide antigens. The refolded A2monomers were biotinylated with a commercial biotinylation enzyme (BirA) and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4:1. Flow cytometry was used to confirm the expected tetramer staining of CD8+ T cells.RESULTS: Recombinant genes for HLA-A*0201 heavy chain (A2) fused to a BirA substrate peptide (A2-BSP) and mature β2m from HLA-A2+ donor leukocytes were successfully doned and highly expressed in E. coli. Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of β2m and peptides loaded with either human cytomegalovirus pp65495-503 peptide (NLVPMVATV,NLV; designated as A2-NLV) or influenza virus matrix protein Mp58-66 peptide (GILGFVFTL, GIL; designated as A2-GIL). Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplication as revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8+ T cells from a HLA-A2+ donor,but failed to bind to those from a HLA-A2- donor.CONCLUSION: The procedure is simple and efficient for generating peptide-MHC tetramers.展开更多
The rapid development of electric vehicles and mobile electronic devices is the main driving force to improve advanced high-performance lithium ion batteries(LIBs).The capacity,rate performance and cycle stability of ...The rapid development of electric vehicles and mobile electronic devices is the main driving force to improve advanced high-performance lithium ion batteries(LIBs).The capacity,rate performance and cycle stability of LIBs rely directly on the electrode materials.As far as the development of the advanced LIBs electrode is concerned,the improvement of anode materials is more urgent than the cathode materials.Industrial production of anode materials superior to commercial graphite still faces some challenges.This review sets out the most basic LIBs anode material design.The reaction principles and structural design of carbon materials,various transition metal oxides,silicon and germanium are summarized,and then the progress of other anode materials are analyzed.Due to the rapid development of metal organic frameworks(MOFs)in energy storage and conversion in recent years,the synthesis process and energy storage mechanism of nanostructures derived from MOF precursors are also discussed.From the perspective of novel structural design,the progress of various MOFs-derived materials for alleviating the volume expansion of anode materials is discussed.Finally,challenges for the future development of advanced anode materials for LIBs will be considered.展开更多
Autophagy is closely related to the drug resistance and metastasis in cancer therapy.Nanoparticlemediated co-delivery of combinatorial therapy with small-molecular drugs and nucleic acids is promising to address drug ...Autophagy is closely related to the drug resistance and metastasis in cancer therapy.Nanoparticlemediated co-delivery of combinatorial therapy with small-molecular drugs and nucleic acids is promising to address drug resistance.Here,a drug-delivering-drug(DDD)platform consisting of anti-tumor-drug nanorods as a vehicle for cytosol delivery of nucleic acid(miR-101)with potent autophagic-inhibition activity is reported for combinatorial therapy.The developed 180-nm nanoplatform,with total drug loading of up to 66%,delivers miR-101 to cancer cells,with threefold increase in intracellular level compared to conventional gene carriers and inhibits the autophagy significantly,along with above twofold reduction in LC3II mRNA and approximately fivefold increase in p62 mRNA over the control demonstrated in the results in vivo.And in turn,the delivery of miR-101 potentiates the drug’s ability to kill cancer cells,with a threefold increase in apoptosis over that of chemotherapy alone.The anti-tumor study in vivo indicates the combined therapy that enables a reduction of 80%in tumor volume and>twofold increase in apoptosis than of the single-drug strategy.In summary,via the carrier-free strategy of DDD,this work provides a delivery platform that can be easily customized to overcome drug resistance and facilitates the delivery of combined therapy of small-molecular drugs and nucleic acids.展开更多
A high yield xylanase producing strain, A. usamii L336-23, was screened out from its parent strain A.usamii L336 after microwave irradiation. Its productivity of xylanase activity increased by 35.7% from 21000μ·...A high yield xylanase producing strain, A. usamii L336-23, was screened out from its parent strain A.usamii L336 after microwave irradiation. Its productivity of xylanase activity increased by 35.7% from 21000μ·m1^-1 to 28500μ·m1^-1 and was stable after frequent subcultures and storage for more than two months.The mechanism of microwave mutation was also discussed.展开更多
AIM:To investigate whether Z:ZCLA Mongolian gerbils are readily susceptible to infection by human hepatitis E virus(HEV).METHODS:Z:ZCLA Mongolian gerbils were infected with a clinical HEV strain isolated from an acute...AIM:To investigate whether Z:ZCLA Mongolian gerbils are readily susceptible to infection by human hepatitis E virus(HEV).METHODS:Z:ZCLA Mongolian gerbils were infected with a clinical HEV strain isolated from an acutehepati tis E patient,and virus pathogenesis was assessed in this host.Non-infected gerbils served as the control group.Feces samples from gerbils were collected weekly for reverse transcription-nested polymerase chain reaction.Serum anti-HEV Ig G and alanine aminotransferase(ALT) were detected by enzyme linked immunosorbent assay.At sacrifice,each animal's liver,spleen and kidney were collected for histopathologic examination.RESULTS:HEV-infected gerbils showed fatigue,with histopathological changes observed in the liver,spleen and kidney.HEV RNA was detected in fecal samples taken at day 7 after inoculation and the detectable levels lasted out to day 42 after inoculation.Interestingly,ALT levels were only moderately increased in the HEV-infected animals compared with the noninfected control group.CONCLUSION:Z:ZCLA Mongolian gerbils are susceptible to human HEV.展开更多
A new hemA gene encoding 5-aminolevulinate (ALA) synthase was cloned from Agrobacterium ra- diobacter zju-0121. The ALA synthase catalyzes the pyridoxal phosphate-dependent condensation of succinyl coen- zyme A (succi...A new hemA gene encoding 5-aminolevulinate (ALA) synthase was cloned from Agrobacterium ra- diobacter zju-0121. The ALA synthase catalyzes the pyridoxal phosphate-dependent condensation of succinyl coen- zyme A (succinyl-CoA) and glycine to produce ALA. Four plasmids carrying the A, radiobacter hemA gene were transformed into different E. coli strains. The effects of both genetic and physiological factors on the expression of ALA synthase and ALA production were studied. The results indicated that the final intracellular activity of ALA synthase and the production of ALA in different expression systems varied largely. Among them, the recombinant E. coli BL21 (DE3) harboring the expression plasmid pET28-A. R-hemA was the most suitable one. The effects of isopropyl-β-D-thiogalactopyranoside (IPTG) addition time, IPTG concentration, culture temperature and the initial concentration of precursors and glucose on the ALA production were also evaluated. The expressed ALA synthase accounted for about 23.7% of the intracellular soluble protein. The highest specific activity of ALA syn- thase was 13.8nmol·min-1·mg-1 of intracellular soluble protein. In the batch culture of the recombinant E. coli, the extracellular ALA concentration reached 0.9 g·L-1.展开更多
Fermentation experiments to produce validamycins from crude substrates by Streptomyces hygroscopicus were carried out in an external-loop airlift bioreactor (0.0115 m^3 ) with a low ratio of height to diameter of the ...Fermentation experiments to produce validamycins from crude substrates by Streptomyces hygroscopicus were carried out in an external-loop airlift bioreactor (0.0115 m^3 ) with a low ratio of height to diameter of the riser of 2.9 and a ratio of riser to downcomer diameter of 6.6. The influences of gas flow rate and liquid volume on fermentation of validamycins were investigated. Comparisons of validamycin fermentation were made among the external-loop airlift bioreactor, a mechanically stirred tank bioreactor (0.010m^3 ) and shaking flasks. Under the same operation conditions including fermentation medium composition, inoculum ratio and culture temperature, the fermentation time in the external-loop airlift bioreactor (45 h) was shorter than that in the shaking flasks (100 h) and the same as that in the mechanically stirred tank bioreactor. After a total fermentation time of 45 h under optimized operation conditions, average validamycin concentration obtained in the external-loop airlift bioreactor was close to 19630 μg·ml^-1 validamycin concentration in the mechanically stirred tank bioreactor. It was demonstrated that the external-loop airlift bioreactor could substitute for the mechanically stirred tank bioreactor in production of validamycins from crude substrates with dregs by Streptomyces hygroscopicus.展开更多
Background: Von Hippel–Lindau(VHL) syndrome is a dominantly inherited multisystem cancer syndrome caused by a heterozygous mutation in the VHL tumor suppressor gene. Previous studies suggested that similar population...Background: Von Hippel–Lindau(VHL) syndrome is a dominantly inherited multisystem cancer syndrome caused by a heterozygous mutation in the VHL tumor suppressor gene. Previous studies suggested that similar populations of Caucasian and Japanese patients have similar genotype or phenotype characteristics. In this comprehensive study of East Asian patients, we investigated the genetic and clinical characteristics of patients with VHL syndrome.Methods: To create a registry of clinical characteristics and mutations reported in East Asian patients with VHL syndrome, we conducted a comprehensive review of English language and non?English language articles identi?fied through a literature search. Publications in Japanese or Chinese language were read by native speakers of the language, who then performed the data extraction.Results: Of 237 East Asian patients with VHL syndrome, 154 unique kindreds were identified for analysis. Analyzed by kindred, missense mutations were the most common(40.9%, 63/154), followed by large/complete deletions(32.5%, 50/154) and nonsense mutations(11.7%, 18/154). Compared with a previously reported study of both East Asian and non?East Asian patients, we found several key differences. First, missense and frameshift mutations in the VHL gene occurred less commonly in our population of East Asian patients(40.9% vs. 52.0%; P = 0.012 and 8.4% vs. 13.0%; P < 0.001, respectively). Second, large/complete deletions were more common in our population of East Asian patients(32.5% vs. 10.5%; P < 0.001). Third, phenotypically, we observed that, in our population of East Asian patients with VHL syndrome, the incidence of retinal capillary hemangioblastoma was lower, whereas the incidence of renal cell carcinoma was higher.Conclusions: Evidence suggests that the genotypic and phenotypic characteristics of East Asian patients with VHL syndrome differ from other populations. This should be considered when making screening recommendations for VHL syndrome in Asia.展开更多
Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The ef...Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid pro- ductivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.展开更多
Neuropeptide Y (NPY) is one of the most important orexigenic agents in central regulation of feeding behavior, body weight and energy homeostasis in domestic chickens. To examine differences in the hypothalamic NPY be...Neuropeptide Y (NPY) is one of the most important orexigenic agents in central regulation of feeding behavior, body weight and energy homeostasis in domestic chickens. To examine differences in the hypothalamic NPY between layer-type and meat-type of chickens, which are two divergent kinds of the domestic chickens in feeding behavior and body weight, we detected mRNA levels of NPY in hypothalamic infundibular nucleus (IN), paraventricular nucleus (PVN) and lateral hypothalamic area (LHA) of these two types of chickens using one-step real time RT-PCR. The meat-type chicken had more food daily (about 1.7 folds) and greater body weights (about 1.5 folds) and brain weights than the layer-type chicken at the age of 14 d. In the meat-type of chicken, NPY mRNA levels of the IN and PVN were significantly greater than those of the LHA, and were not significantly different between the IN and PVN. However, in the layer-type of chicken, NPY mRNA levels were significantly greater in the IN than those in the LHA and PVN, and were not significantly different between the PVN and LHA. In all these hypothalamic regions, the layer-type of chicken had significantly higher NPY mRNA levels than the meat-type chicken did. These results suggest the expression of NPY in the hypothalamus has a type-dependent pattern in domestic chickens.展开更多
Background:For patients with nasopharyngeal carcinoma(NPC) who undergo re-irradiation with intensity-modulated radiotherapy(IMRT),lethal nasopharyngeal necrosis(LNN) is a severe late adverse event.The purpose of this ...Background:For patients with nasopharyngeal carcinoma(NPC) who undergo re-irradiation with intensity-modulated radiotherapy(IMRT),lethal nasopharyngeal necrosis(LNN) is a severe late adverse event.The purpose of this study was to identify risk factors for LNN and develop a model to predict LNN after radical re-irradiation with IMRT in patients with recurrent NPC.Methods:Patients who underwent radical re-irradiation with IMRT for locally recurrent NPC between March 2001 and December 2011 and who had no evidence of distant metastasis were included in this study.Clinical characteristics,including recurrent carcinoma conditions and dosimetric features,were evaluated as candidate risk factors for LNN.Logistic regression analysis was used to identify independent risk factors and construct the predictive scoring model.Results:Among 228 patients enrolled in this study,204 were at risk of developing LNN based on risk analysis.Of the 204 patients treated,31(15.2%) developed LNN.Logistic regression analysis showed that female sex(P = 0.008),necrosis before re-irradiation(P = 0.008),accumulated total prescription dose to the gross tumor volume(GTV) ≥ 145.5 Gy(P = 0.043),and recurrent tumor volume >25.38 cm3(P = 0.009) were independent risk factors for LNN.A model to predict LNN was then constructed that included these four independent risk factors.Conclusions:A model that includes sex,necrosis before re-irradiation,accumulated total prescription dose to GTV,and recurrent tumor volume can effectively predict the risk of developing LNN in NPC patients who undergo radical re-irradiation with IMRT.展开更多
A species of mushroom, Termitomyces albuminosus, was cultured in liquid medium for production of chromium-enriched mycelium. The influence of chromium (Ⅲ ) on mycelial growth of T. albuminosus was investigated. An op...A species of mushroom, Termitomyces albuminosus, was cultured in liquid medium for production of chromium-enriched mycelium. The influence of chromium (Ⅲ ) on mycelial growth of T. albuminosus was investigated. An optimum medium composed of 5.6g/L yeast extract, 51.6g/L hydrolyzed rice, 2g/L KH2PO4, and 20mg/L chromium(Ⅲ ) with initial pH of 4.5 was obtained by using method of central composite design (CCD). After incubation of 84h, the maximal biomass of chromium-enriched mycelia reached 24.23g DMW(dried mycelial weight)/L with 272μg/g DMW chromium content in 500mL flasks containing 100mL medium with an inoculum of 8% on a shaker of 100r/min under an optimized cultivation condition at 28℃.展开更多
The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, S...The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 °C was 8.5 and the optimum temperature at pH 8.5 was 55 °C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.展开更多
Teicoplanin is one of the macrocyclic glycopeptide antibiotics, which is active against Gram-positive bacteria, and has attracted a lot of attention in the field of chiral separation recently. In this work, the partit...Teicoplanin is one of the macrocyclic glycopeptide antibiotics, which is active against Gram-positive bacteria, and has attracted a lot of attention in the field of chiral separation recently. In this work, the partition coefficients and extraction ratio of teicoplanin in three different solvent systems were studied: conventional extraction, reactive extraction and reverse micelle extraction. With conventional solvent extraction, n-butanol demonstrated high partition coefficient for teicoplanin, but low extraction ratio because of its high solubility in water. Reactive extraction of teicoplanin showed the highest partition coefficient with almost 100% recovery in organic phase when tri-n-octylmethyl ammonium chloride (TOMAC) was used as extractant and pH value was above 5.0. A reverse micelle system, consisted of isooctane +10 mmol-L^-1 TOMAC -t-1% n-octanol, also offered high separation factor for teicoplanin. The results are beneficial for the design of teicoplanin separation and purification process.展开更多
BACKGROUND: Studies have shown that both salviae miltiorrhizae and ligustrazine can promoteprotein expression of nerve growth factor (NGF) and regeneration of peripheral nerve.OBJECTIVE: To verify the effect of salvia...BACKGROUND: Studies have shown that both salviae miltiorrhizae and ligustrazine can promoteprotein expression of nerve growth factor (NGF) and regeneration of peripheral nerve.OBJECTIVE: To verify the effect of salviae miltiorrhizae and ligustrazine hydrochloride injection onaxonal regeneration and NGF protein expression in a rat model of sciatic nerve injury.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at theLaboratory of Traditional Chinese Medicine and the Institute of Bioengineering of Jinan Universityfrom July to December 2008.MATERIALS: Salviae miltiorrhizae and ligustrazine hydrochloride injection (containing 20 mgsalviae miltiorrhizae and 100 mg ligustrazine per 100 mL injection) was provided by Guizhou BaitePharmaceutical, China; salviae miltiorrhizae and ligustrazine decoctions (containing 1 g raw drugper 1 mL decoction) were provided by Guangzhou Baiyunshan Factory for Traditional ChineseMedicine, China; rabbit-anti-rat NGF monoclonal antibody was provided by Beijing BiosynthesisBiotechnology, China.METHODS: A total of 80 healthy, male, Sprague Dawley rats were used to establish a sciatic nerveinjury model via neurotomy, and were then randomly assigned to 4 groups: salviae miltiorrhizae andligustrazine hydrochloride injection group (intraperitoneal injection of 35 mL/kg per day salviaemiltiorrhizae and ligustrazine hydrochloride injection), salviae miltiorrhizae group (intragastricperfusion of 2 mL salviae miltiorrhizae), ligustrazine group (intragastric perfusion of 2 mLligustrazine), and model group (intraperitoneal injection of 35 mL/kg per day saline), with 20 rats ineach group. Thereafter, rats in each group were then divided into 4 subgroups according to varyingtime points of 1,2, 4, and 8 weeks post-surgery, with 5 rats in each subgroup.MAIN OUTCOME MEASURES: Axons were quantified using chromotrope 2R-brilliant green andsilver staining combined with image analysis to calculate the axonal regeneration rate; NGFexpression was detected using immunohistochemistry and Western blot analysis; toe interspacewas measured by behavior at 4 and 8 weeks.RESULTS: With increasing time after sciatic nerve injury, the axonal regeneration rate, NGF proteinexpression, and toe interspace gradually increased. At 4 and 8 weeks post-surgery, axonalregeneration rate and NGF protein expression were significantly increased in the injured tissue ofthe salviae miltiorrhizae and ligustrazine hydrochloride injection, salviae miltiorrhizae, andligustrazine groups, compared with the model group (P < 0.05 or P < 0.01), and toe interspace wasremarkably enlarged (P < 0.05 or P < 0.01), especially in the salviae miltiorrhizae and ligustrazinehydrochloride injection group.CONCLUSION: Salviae miltiorrhizae and ligustrazine hydrochloride injection promoted axonalregeneration and NGF protein expression in the injured sciatic nerve, and also enhancedneurofunctional recovery. Its effect was superior to salviae miltiorrhizae or ligustrazine alone.展开更多
[Objective] This study aimed to explore immature embryo culture of Lagerstroemia indica and investigate the appropriate conditions for growth and differentiation. [Method] Immature embryos of L. indica were employed a...[Objective] This study aimed to explore immature embryo culture of Lagerstroemia indica and investigate the appropriate conditions for growth and differentiation. [Method] Immature embryos of L. indica were employed as the explants for germination induction to establish aseptic lines. Based on that,the effects of different hormone levels and culture conditions on immature embryo culture of L. indica were analyzed. [Result] Peeled immature embryos of L. indica were germinated easily,leading to a germination rate of 100%. The optimal initial medium was MS + BA0. 5+ NAA0. 1+ sucrose 3. 0% + agar 0. 7%; the optimal shoot induction medium was MS + BA0. 5+ NAA0. 1+ sucrose 3. 0% + agar 0. 7% + coconut milk 10%; the optimal rooting medium was MS + BA0. 5+ IBA0. 1+sucrose 3. 0% + agar 0. 7% + coconut milk 10%. [Conclusion] This study provided a technical reference for subsequent optimized breeding of L. indica.展开更多
基金Supported by the National Natural Science Foundation of China, No. 30230350 and No. 30371651Major State Basic Research Development Program of China, 973 Program, No. G2000057006
文摘AIM: To establish a simplified method for generating peptide-major histocompatibility complex (MHC) class I tetramers.METHODS: cDNAs encoding the extracellular domain of human lymphocyte antigen (HLA)-A*0201 heavy chain (A2) and β2-microglobulin (β2m) from total RNA extracted from leukocytes of HLA-A2+ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and β2m proteins were expressed in Escherichia coli strain BL21(DE3) and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain β2m and HLA-A2-restricted peptide antigens. The refolded A2monomers were biotinylated with a commercial biotinylation enzyme (BirA) and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4:1. Flow cytometry was used to confirm the expected tetramer staining of CD8+ T cells.RESULTS: Recombinant genes for HLA-A*0201 heavy chain (A2) fused to a BirA substrate peptide (A2-BSP) and mature β2m from HLA-A2+ donor leukocytes were successfully doned and highly expressed in E. coli. Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of β2m and peptides loaded with either human cytomegalovirus pp65495-503 peptide (NLVPMVATV,NLV; designated as A2-NLV) or influenza virus matrix protein Mp58-66 peptide (GILGFVFTL, GIL; designated as A2-GIL). Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplication as revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8+ T cells from a HLA-A2+ donor,but failed to bind to those from a HLA-A2- donor.CONCLUSION: The procedure is simple and efficient for generating peptide-MHC tetramers.
基金financial support from the National Natural Science Foundation of China(81671737)the support from‘Sponsored by Shanghai Pujiang Program’(18PJD020)the Interdisciplinary Program of Shanghai Jiao Tong University(YG2019QNB31)。
文摘The rapid development of electric vehicles and mobile electronic devices is the main driving force to improve advanced high-performance lithium ion batteries(LIBs).The capacity,rate performance and cycle stability of LIBs rely directly on the electrode materials.As far as the development of the advanced LIBs electrode is concerned,the improvement of anode materials is more urgent than the cathode materials.Industrial production of anode materials superior to commercial graphite still faces some challenges.This review sets out the most basic LIBs anode material design.The reaction principles and structural design of carbon materials,various transition metal oxides,silicon and germanium are summarized,and then the progress of other anode materials are analyzed.Due to the rapid development of metal organic frameworks(MOFs)in energy storage and conversion in recent years,the synthesis process and energy storage mechanism of nanostructures derived from MOF precursors are also discussed.From the perspective of novel structural design,the progress of various MOFs-derived materials for alleviating the volume expansion of anode materials is discussed.Finally,challenges for the future development of advanced anode materials for LIBs will be considered.
基金supported by Grants from the National Natural Science Foundation of China(Nos.81872823,81871477)the Ministry of Science and Technology of China(2017ZX09101001-004)+1 种基金the Double First-Class(CPU2018PZQ13)of CPUthe Key Members of the Outstanding Young Teacher of Jiangsu Qing Lan Project(2016).
文摘Autophagy is closely related to the drug resistance and metastasis in cancer therapy.Nanoparticlemediated co-delivery of combinatorial therapy with small-molecular drugs and nucleic acids is promising to address drug resistance.Here,a drug-delivering-drug(DDD)platform consisting of anti-tumor-drug nanorods as a vehicle for cytosol delivery of nucleic acid(miR-101)with potent autophagic-inhibition activity is reported for combinatorial therapy.The developed 180-nm nanoplatform,with total drug loading of up to 66%,delivers miR-101 to cancer cells,with threefold increase in intracellular level compared to conventional gene carriers and inhibits the autophagy significantly,along with above twofold reduction in LC3II mRNA and approximately fivefold increase in p62 mRNA over the control demonstrated in the results in vivo.And in turn,the delivery of miR-101 potentiates the drug’s ability to kill cancer cells,with a threefold increase in apoptosis over that of chemotherapy alone.The anti-tumor study in vivo indicates the combined therapy that enables a reduction of 80%in tumor volume and>twofold increase in apoptosis than of the single-drug strategy.In summary,via the carrier-free strategy of DDD,this work provides a delivery platform that can be easily customized to overcome drug resistance and facilitates the delivery of combined therapy of small-molecular drugs and nucleic acids.
基金Natural Science Foundation of Zhejiang Province(No.395186)
文摘A high yield xylanase producing strain, A. usamii L336-23, was screened out from its parent strain A.usamii L336 after microwave irradiation. Its productivity of xylanase activity increased by 35.7% from 21000μ·m1^-1 to 28500μ·m1^-1 and was stable after frequent subcultures and storage for more than two months.The mechanism of microwave mutation was also discussed.
基金Supported by Science Technology Department of Zhejiang Province No.2011F20015Health and Family Planning Commission of Zhejiang Province,No.XKQ-010001
文摘AIM:To investigate whether Z:ZCLA Mongolian gerbils are readily susceptible to infection by human hepatitis E virus(HEV).METHODS:Z:ZCLA Mongolian gerbils were infected with a clinical HEV strain isolated from an acutehepati tis E patient,and virus pathogenesis was assessed in this host.Non-infected gerbils served as the control group.Feces samples from gerbils were collected weekly for reverse transcription-nested polymerase chain reaction.Serum anti-HEV Ig G and alanine aminotransferase(ALT) were detected by enzyme linked immunosorbent assay.At sacrifice,each animal's liver,spleen and kidney were collected for histopathologic examination.RESULTS:HEV-infected gerbils showed fatigue,with histopathological changes observed in the liver,spleen and kidney.HEV RNA was detected in fecal samples taken at day 7 after inoculation and the detectable levels lasted out to day 42 after inoculation.Interestingly,ALT levels were only moderately increased in the HEV-infected animals compared with the noninfected control group.CONCLUSION:Z:ZCLA Mongolian gerbils are susceptible to human HEV.
基金Supported by the National Natural Science Foundation of China (No. 20306026)
文摘A new hemA gene encoding 5-aminolevulinate (ALA) synthase was cloned from Agrobacterium ra- diobacter zju-0121. The ALA synthase catalyzes the pyridoxal phosphate-dependent condensation of succinyl coen- zyme A (succinyl-CoA) and glycine to produce ALA. Four plasmids carrying the A, radiobacter hemA gene were transformed into different E. coli strains. The effects of both genetic and physiological factors on the expression of ALA synthase and ALA production were studied. The results indicated that the final intracellular activity of ALA synthase and the production of ALA in different expression systems varied largely. Among them, the recombinant E. coli BL21 (DE3) harboring the expression plasmid pET28-A. R-hemA was the most suitable one. The effects of isopropyl-β-D-thiogalactopyranoside (IPTG) addition time, IPTG concentration, culture temperature and the initial concentration of precursors and glucose on the ALA production were also evaluated. The expressed ALA synthase accounted for about 23.7% of the intracellular soluble protein. The highest specific activity of ALA syn- thase was 13.8nmol·min-1·mg-1 of intracellular soluble protein. In the batch culture of the recombinant E. coli, the extracellular ALA concentration reached 0.9 g·L-1.
基金Supported by the Natural Science Foundation of Zhejiang Province (No. ZB0106)the National Natural Science Foundation of China (No. 20176055).
文摘Fermentation experiments to produce validamycins from crude substrates by Streptomyces hygroscopicus were carried out in an external-loop airlift bioreactor (0.0115 m^3 ) with a low ratio of height to diameter of the riser of 2.9 and a ratio of riser to downcomer diameter of 6.6. The influences of gas flow rate and liquid volume on fermentation of validamycins were investigated. Comparisons of validamycin fermentation were made among the external-loop airlift bioreactor, a mechanically stirred tank bioreactor (0.010m^3 ) and shaking flasks. Under the same operation conditions including fermentation medium composition, inoculum ratio and culture temperature, the fermentation time in the external-loop airlift bioreactor (45 h) was shorter than that in the shaking flasks (100 h) and the same as that in the mechanically stirred tank bioreactor. After a total fermentation time of 45 h under optimized operation conditions, average validamycin concentration obtained in the external-loop airlift bioreactor was close to 19630 μg·ml^-1 validamycin concentration in the mechanically stirred tank bioreactor. It was demonstrated that the external-loop airlift bioreactor could substitute for the mechanically stirred tank bioreactor in production of validamycins from crude substrates with dregs by Streptomyces hygroscopicus.
文摘Background: Von Hippel–Lindau(VHL) syndrome is a dominantly inherited multisystem cancer syndrome caused by a heterozygous mutation in the VHL tumor suppressor gene. Previous studies suggested that similar populations of Caucasian and Japanese patients have similar genotype or phenotype characteristics. In this comprehensive study of East Asian patients, we investigated the genetic and clinical characteristics of patients with VHL syndrome.Methods: To create a registry of clinical characteristics and mutations reported in East Asian patients with VHL syndrome, we conducted a comprehensive review of English language and non?English language articles identi?fied through a literature search. Publications in Japanese or Chinese language were read by native speakers of the language, who then performed the data extraction.Results: Of 237 East Asian patients with VHL syndrome, 154 unique kindreds were identified for analysis. Analyzed by kindred, missense mutations were the most common(40.9%, 63/154), followed by large/complete deletions(32.5%, 50/154) and nonsense mutations(11.7%, 18/154). Compared with a previously reported study of both East Asian and non?East Asian patients, we found several key differences. First, missense and frameshift mutations in the VHL gene occurred less commonly in our population of East Asian patients(40.9% vs. 52.0%; P = 0.012 and 8.4% vs. 13.0%; P < 0.001, respectively). Second, large/complete deletions were more common in our population of East Asian patients(32.5% vs. 10.5%; P < 0.001). Third, phenotypically, we observed that, in our population of East Asian patients with VHL syndrome, the incidence of retinal capillary hemangioblastoma was lower, whereas the incidence of renal cell carcinoma was higher.Conclusions: Evidence suggests that the genotypic and phenotypic characteristics of East Asian patients with VHL syndrome differ from other populations. This should be considered when making screening recommendations for VHL syndrome in Asia.
文摘Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid pro- ductivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.
基金Project supported by the Natural Science Foundation of ZhejiangProvince (No. Y306220)the Scientific Research Startup Fund ofZhejiang Academy of Agricultural Sciences, China
文摘Neuropeptide Y (NPY) is one of the most important orexigenic agents in central regulation of feeding behavior, body weight and energy homeostasis in domestic chickens. To examine differences in the hypothalamic NPY between layer-type and meat-type of chickens, which are two divergent kinds of the domestic chickens in feeding behavior and body weight, we detected mRNA levels of NPY in hypothalamic infundibular nucleus (IN), paraventricular nucleus (PVN) and lateral hypothalamic area (LHA) of these two types of chickens using one-step real time RT-PCR. The meat-type chicken had more food daily (about 1.7 folds) and greater body weights (about 1.5 folds) and brain weights than the layer-type chicken at the age of 14 d. In the meat-type of chicken, NPY mRNA levels of the IN and PVN were significantly greater than those of the LHA, and were not significantly different between the IN and PVN. However, in the layer-type of chicken, NPY mRNA levels were significantly greater in the IN than those in the LHA and PVN, and were not significantly different between the PVN and LHA. In all these hypothalamic regions, the layer-type of chicken had significantly higher NPY mRNA levels than the meat-type chicken did. These results suggest the expression of NPY in the hypothalamus has a type-dependent pattern in domestic chickens.
基金supported by the National Natural Science Foundation of China(No.81472525 and 81572665)the Science and Technology Planning Project of Guangdong Province,China(No.2014A050503033)
文摘Background:For patients with nasopharyngeal carcinoma(NPC) who undergo re-irradiation with intensity-modulated radiotherapy(IMRT),lethal nasopharyngeal necrosis(LNN) is a severe late adverse event.The purpose of this study was to identify risk factors for LNN and develop a model to predict LNN after radical re-irradiation with IMRT in patients with recurrent NPC.Methods:Patients who underwent radical re-irradiation with IMRT for locally recurrent NPC between March 2001 and December 2011 and who had no evidence of distant metastasis were included in this study.Clinical characteristics,including recurrent carcinoma conditions and dosimetric features,were evaluated as candidate risk factors for LNN.Logistic regression analysis was used to identify independent risk factors and construct the predictive scoring model.Results:Among 228 patients enrolled in this study,204 were at risk of developing LNN based on risk analysis.Of the 204 patients treated,31(15.2%) developed LNN.Logistic regression analysis showed that female sex(P = 0.008),necrosis before re-irradiation(P = 0.008),accumulated total prescription dose to the gross tumor volume(GTV) ≥ 145.5 Gy(P = 0.043),and recurrent tumor volume >25.38 cm3(P = 0.009) were independent risk factors for LNN.A model to predict LNN was then constructed that included these four independent risk factors.Conclusions:A model that includes sex,necrosis before re-irradiation,accumulated total prescription dose to GTV,and recurrent tumor volume can effectively predict the risk of developing LNN in NPC patients who undergo radical re-irradiation with IMRT.
文摘A species of mushroom, Termitomyces albuminosus, was cultured in liquid medium for production of chromium-enriched mycelium. The influence of chromium (Ⅲ ) on mycelial growth of T. albuminosus was investigated. An optimum medium composed of 5.6g/L yeast extract, 51.6g/L hydrolyzed rice, 2g/L KH2PO4, and 20mg/L chromium(Ⅲ ) with initial pH of 4.5 was obtained by using method of central composite design (CCD). After incubation of 84h, the maximal biomass of chromium-enriched mycelia reached 24.23g DMW(dried mycelial weight)/L with 272μg/g DMW chromium content in 500mL flasks containing 100mL medium with an inoculum of 8% on a shaker of 100r/min under an optimized cultivation condition at 28℃.
文摘The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 °C was 8.5 and the optimum temperature at pH 8.5 was 55 °C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.
基金Project supported by the National Natural Science Foundation of China (No. 30400290) and the National Basic Research Program(973) of China (No. 2002CB111403)
文摘Teicoplanin is one of the macrocyclic glycopeptide antibiotics, which is active against Gram-positive bacteria, and has attracted a lot of attention in the field of chiral separation recently. In this work, the partition coefficients and extraction ratio of teicoplanin in three different solvent systems were studied: conventional extraction, reactive extraction and reverse micelle extraction. With conventional solvent extraction, n-butanol demonstrated high partition coefficient for teicoplanin, but low extraction ratio because of its high solubility in water. Reactive extraction of teicoplanin showed the highest partition coefficient with almost 100% recovery in organic phase when tri-n-octylmethyl ammonium chloride (TOMAC) was used as extractant and pH value was above 5.0. A reverse micelle system, consisted of isooctane +10 mmol-L^-1 TOMAC -t-1% n-octanol, also offered high separation factor for teicoplanin. The results are beneficial for the design of teicoplanin separation and purification process.
基金Supported by: the Natural Science Foundation of Guangdong province, No. 5300544High-Tech Research and Development Program of Guangdong Province, No. 2009B030801238+3 种基金2006B35602009the Grants from Guangdong Province Administration of Traditional Chinese Medicine, No. 2008092 1060114the Science and Technology Foundation of Guangzhou,No.2009Z1-E091
文摘BACKGROUND: Studies have shown that both salviae miltiorrhizae and ligustrazine can promoteprotein expression of nerve growth factor (NGF) and regeneration of peripheral nerve.OBJECTIVE: To verify the effect of salviae miltiorrhizae and ligustrazine hydrochloride injection onaxonal regeneration and NGF protein expression in a rat model of sciatic nerve injury.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at theLaboratory of Traditional Chinese Medicine and the Institute of Bioengineering of Jinan Universityfrom July to December 2008.MATERIALS: Salviae miltiorrhizae and ligustrazine hydrochloride injection (containing 20 mgsalviae miltiorrhizae and 100 mg ligustrazine per 100 mL injection) was provided by Guizhou BaitePharmaceutical, China; salviae miltiorrhizae and ligustrazine decoctions (containing 1 g raw drugper 1 mL decoction) were provided by Guangzhou Baiyunshan Factory for Traditional ChineseMedicine, China; rabbit-anti-rat NGF monoclonal antibody was provided by Beijing BiosynthesisBiotechnology, China.METHODS: A total of 80 healthy, male, Sprague Dawley rats were used to establish a sciatic nerveinjury model via neurotomy, and were then randomly assigned to 4 groups: salviae miltiorrhizae andligustrazine hydrochloride injection group (intraperitoneal injection of 35 mL/kg per day salviaemiltiorrhizae and ligustrazine hydrochloride injection), salviae miltiorrhizae group (intragastricperfusion of 2 mL salviae miltiorrhizae), ligustrazine group (intragastric perfusion of 2 mLligustrazine), and model group (intraperitoneal injection of 35 mL/kg per day saline), with 20 rats ineach group. Thereafter, rats in each group were then divided into 4 subgroups according to varyingtime points of 1,2, 4, and 8 weeks post-surgery, with 5 rats in each subgroup.MAIN OUTCOME MEASURES: Axons were quantified using chromotrope 2R-brilliant green andsilver staining combined with image analysis to calculate the axonal regeneration rate; NGFexpression was detected using immunohistochemistry and Western blot analysis; toe interspacewas measured by behavior at 4 and 8 weeks.RESULTS: With increasing time after sciatic nerve injury, the axonal regeneration rate, NGF proteinexpression, and toe interspace gradually increased. At 4 and 8 weeks post-surgery, axonalregeneration rate and NGF protein expression were significantly increased in the injured tissue ofthe salviae miltiorrhizae and ligustrazine hydrochloride injection, salviae miltiorrhizae, andligustrazine groups, compared with the model group (P < 0.05 or P < 0.01), and toe interspace wasremarkably enlarged (P < 0.05 or P < 0.01), especially in the salviae miltiorrhizae and ligustrazinehydrochloride injection group.CONCLUSION: Salviae miltiorrhizae and ligustrazine hydrochloride injection promoted axonalregeneration and NGF protein expression in the injured sciatic nerve, and also enhancedneurofunctional recovery. Its effect was superior to salviae miltiorrhizae or ligustrazine alone.
文摘[Objective] This study aimed to explore immature embryo culture of Lagerstroemia indica and investigate the appropriate conditions for growth and differentiation. [Method] Immature embryos of L. indica were employed as the explants for germination induction to establish aseptic lines. Based on that,the effects of different hormone levels and culture conditions on immature embryo culture of L. indica were analyzed. [Result] Peeled immature embryos of L. indica were germinated easily,leading to a germination rate of 100%. The optimal initial medium was MS + BA0. 5+ NAA0. 1+ sucrose 3. 0% + agar 0. 7%; the optimal shoot induction medium was MS + BA0. 5+ NAA0. 1+ sucrose 3. 0% + agar 0. 7% + coconut milk 10%; the optimal rooting medium was MS + BA0. 5+ IBA0. 1+sucrose 3. 0% + agar 0. 7% + coconut milk 10%. [Conclusion] This study provided a technical reference for subsequent optimized breeding of L. indica.
