Effect of Xileisan temperature-sensitive gels on endothelial nitric oxide synthase, vascular endothelial growth factor A and tumor necrosis factor-α expression in rats with bleeding internal hemorrhoids / 预防医学

Objective@#To investigate the effect of Xileisan temperature-sensitive gels on endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor A (VEGF-A) and tumor necrosis factor-α (TNF-α) expression in rats with bleeding internal hemorrhoids, so as to provide insights into the illustration of the pathogenesis of internal hemorrhoid hemorrhage. @*Methods@#Thirty six-week-old SPF-graded rats of the SD strain were randomly divided into the normal group, model group and Xileisan temperature-sensitive gel group, of 10 rats in each group (half male and half female). Cotton balls were soaked with 0.16 mL of croton oil mixture and then inserted into the anus of rats in the model group and Xileisan temperature-sensitive gel group for 10 s. After 6 h when the rectal mucosa tissues presented remarkable swelling, the perianal mucosa was rubbed repeatedly with a rough glass rod until the glass rod was bloody. Following successful modeling, rats in the Xileisan temperature-sensitive gel group was given rectal administration of Xileisan temperature-sensitive gel at a dose of 0.5 mL/d, while animals in the normal group and model group were given rectal administration of the blank gel at the same dose. Following administration for 7 successive days, rats were sacrificed, and the hemorrhoids tissues were collected for pathological examinations. The eNOS, VEGF-A and TNF-α expression was determined using immunohistochemistry and compared among groups.@*Results@#Compared with the normal group, the rat hemorrhoids mucosa showed inflammatory changes in the model group, with submucosal congestion and edema, blood vessel congestion and dilation, and visible new blood vessels, and remarkable improvements were seen in the hemorrhoid mucosal inflammation in the Xileisan temperature-sensitive gel group. There were significant differences in the integrated option density (IOD) of eNOS and VEGF-A expression in rat hemorrhoids tissues among the three groups (P<0.05), and no gender-specific differences were seen (P>0.05). The IOD values of eNOS (45.84±13.66) and VEGF-A expression (45.89±9.06) were higher in rat hemorrhoids tissues in the model group than in the normal group (23.11±5.64 and 27.91±11.65) and the Xileisan temperature-sensitive gel group (27.41±8.89 and 33.44±6.20) (P<0.05), while no significant differences were detected in the IOD of TNF-α expression in rat hemorrhoids tissues among the three groups (P>0.05).@*Conclusion@#Xileisan temperature-sensitive gel may alleviate inflammation and internal hemorrhoids hemorrhage through inhibiting eNOS and VEGF-A expression in rat hemorrhoids tissues.

Recent advances in ultrasound-controlled fluorescence technology for deep tissue optical imaging / 药物分析学报

Fluorescence imaging is a noninvasive and dynamic real-time imaging technique;however,it exhibits poor spatial resolution in centimeter-deep tissues because biological tissues are highly scattering media for optical radiation.The recently developed ultrasound-controlled fluorescence(UCF)imaging is a novel imaging technique that can overcome this bottleneck.Previous studies suggest that the effective contrast agent and sensitive imaging system are the two pivotal factors for generating high-resolution UCF images ex vivo and/or in vivo.Here,this review highlights the recent advances(2015-2020)in the design and synthesis of contrast agents and the improvement of imaging systems to realize high-resolution UCF imaging of deep tissues.The imaging performances of various UCF systems,including the signal-to-noise ratio,imaging resolution,and imaging depth,are specifically discussed.In addition,the challenges and prospects are highlighted.With continuously increasing research interest in this field and emerging multidisciplinary applications,UCF imaging with higher spatial resolution and larger imaging depth may be developed shortly,which is expected to have a far-reaching impact on disease surveillance and/or therapy.

Intranasal temperature-sensitive hydrogels of cannabidiol inclusion complex for the treatment of post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is a psychiatric disease that seriously affects brain function. Currently, selective serotonin reuptake inhibitors (SSRIs) are used to treat PTSD clinically but have decreased efficiency and increased side effects. In this study, nasal cannabidiol inclusion complex temperature-sensitive hydrogels (CBD TSGs) were prepared and evaluated to treat PTSD. Mice model of PTSD was established with conditional fear box. CBD TSGs could significantly improve the spontaneous behavior, exploratory spirit and alleviate tension in open field box, relieve anxiety and tension in elevated plus maze, and reduce the freezing time. Hematoxylin and eosin and c-FOS immunohistochemistry slides showed that the main injured brain areas in PTSD were the prefrontal cortex, amygdala, and hippocampus CA1. CBD TSGs could reduce the level of tumor necrosis factor-

Application of environmentally sensitive hydrogels in drug delivery / 药学学报

Environmentally sensitive hydrogels are a novel formulation that has developed rapidly in recent years. It could form semi-solid with good adhesion in the topical sites based on different physiological environments. Its long local retention time is conducive for sustained drug release, and the preparation process is relatively simple and easy to realize industrialization. This review summarized the categories, commonly used polymer, and different administration routes based on the recently published literatures. According to different response factors, it can be divided into temperature, pH, ion, light, and multiple sensitive hydrogels, among which temperature-sensitive hydrogels are the most common. The most commonly used polymers include chitosan, poly N-isopropyl acrylamide, and poloxamer. There are different administration routes for environmentally sensitive hydrogels, such as transdermal, ophthalmic, nasal, oral, vaginal, rectal, injection, etc. Environmentally sensitive hydrogels have broad prospects in clinical application.

Application of temperature-sensitive chitosan hydrogel encapsulated exosomes in ischemic diseases / 中国组织工程研究

BACKGROUND: Exosomes derived from mesenchymal stem cells can reduce myocardial ischemia and reperfusion injury, but there are some problems such as short half-life, fast clearance and low targeting. OBJECTIVE: To modify and encapsulate exosomes with temperature-sensitive chitosan hydrogel to increase the retention rate of exosomes in the body, and to achieve better therapeutic effect. METHODS: Cell transfection method was used to knock down piR823 in exosomes derived from umbilical cord mesenchymal stem cells, and the effect of knockdown of piR823 exosomes on the proliferation and apoptosis of C2C12 cells was detected. Chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel was prepared and mixed directly with exosomes to prepare chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel encapsulated with exosomes. The gel-forming properties, rheology and in vitro sustained release properties of the hydrogel after encapsulation of exosomes were tested. Thirty C57BL/6J mice were taken to establish hind limb ischemia models, and randomly divided into five groups. The gastrocnemius of group A was injected with PBS; group B was injected with chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel; and group C was injected with exosomes-encapsulated chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel; group D was injected with exosomes; group E was injected with exosomes knocking down piR823. Limb function and recovery, blood flow, grip strength, exercise endurance and muscle regeneration were detected in each group. RESULTS AND CONCLUSION: (1) Exosomes knocking down piR823 inhibited the proliferation of C2C12 cells; normal exosomes inhibited hydrogen peroxide-induced apoptosis of C2C12 cells. The inhibitory effect of exosomes on apoptosis was weakened after piR823 was knocked down. (2) The hydrogel encapsulating exosomes had gel-forming properties, but the gel-forming time was shortened, and it could slowly and continuously release exosomes for more than 30 days. (3) After 28 days of hindlimb ischemia, the blood flow recovery of the left limb in group C was better than that in groups B and D (P < 0.05); it in group D was better than group E (P < 0.05); the grip strength, endurance, running time and distance of group C were better than those of groups D and B (P < 0.05), and above indexes in group D were better than in group E (P < 0.05). Muscle regeneration was better in group C than in groups B and D (P < 0.05), and it in group D was better than group E (P < 0.05). (4) The results showed that encapsulation of exosomes by chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel prolonged the residence time of exosomes in vivo, significantly enhanced blood perfusion and recovery of tissue function after ischemia, and the treatment effect was more significant.

Preparation of warangalone-loaded liposomes and its inhibitory effect on breast cancer cells / 南方医科大学学报

OBJECTIVE@#To prepare warangalone-loaded thermosensitive liposomes (WLTSL) and evaluate its inhibitory effect on breast cancer cells .@*METHODS@#MTT assay was used to assess the changes in proliferation of 3 breast cancer cell lines (MDA-MB-231, MCF7, and SKBR3) following treatment with warangalone, soy isoflavone and genistein. Colony-forming assay and wound healing assay was used to assess colony forming activity and migration of MDA-MB-231 cells treated with warangalone. The effect of warangalone on the expression of MMP2 and MMP9 in MDA-MB-231 cells was examined with Western blotting. The thermosensitive liposomes (TSL) and WLTSL were prepared using a thin film hydration method, and the morphology, size, encapsulation efficiency and stability of the prepared liposomes were characterized using transmission electron microscopy, dynamic light scattering scanning and UV spectrophotometry. MTT assay was used to examine the inhibitory effect of WLTSL on mouse breast cancer cells (4T1) .@*RESULTS@#Warangalone showed stronger anti-proliferation effects than soy isoflavones and genistein in the 3 human breast cancer cell lines and significantly inhibited colony formation by MDA-MB-231 cells. Treatment with warangalone significantly inhibited migration of the breast cancer cells and down-regulated the cellular expressions of MMP2 and MMP9. The prepared TSL and WLTSL presented with a homogeneous, irregular spherical morphology, with a mean particle size of 56.23±0.61 nm, a polymer dispersity index of 0.241±0.014, a Zeta potential of -40.40±0.46 mV, and an encapsulation efficiency was 87.68±2.41%. WLTSL showed a good stability at 4 ℃ and 37 ℃ and a stronger inhibitory effect than warangalone in 4T1 cells.@*CONCLUSIONS@#Warangalone inhibits the proliferation, migration and invasion of breast cancer cells, and the prepared WLTSL possesses good physical properties and strong anti-breast cancer activity.

Study on process and rheological parameters of Kucha gynaecological gel / 中草药

Objective To investigate the preparation process of Kucha gynecological gel and prepare a temperature-sensitive in situ gel capable of rapid phase change in vagina. Methods Poloxamer P188 and Poloxamer P407 were used as gel matrix, Lactic acid and sodium lactate were used as the acid buffer solution. The central composite design was performed to optimize the prescription and technology, and the gelation temperature (GT), gelation time, and the effects of different acid buffer solutions on GT, gel stability, and related rheological properties were determined. Results Within a certain concentration range, the gelation temperature was gradually increased with the increase of poloxamer 188 concentration, and the gelation temperature was gradually decreased with the increase of poloxamer 407 concentration, and the optimized prescription by central composite design was poloxamer P188 with a concentration of 3.51% and poloxamer P407 with a concentration of 17.16%. The addition of different buffers has a deviation of 5% of the gelation temperature of the gel in vitro, but the variation in the vaginal fluid is large. The gelation temperatures of the citric acid-sodium citrate buffer and the lactic acid-sodium lactate buffer solution in the vaginal fluid were 68.5 ℃ and 35.8 ℃, respectively, and the rheological data showed that the gel retained better in vivo. Conclusion The Kucha gynecological gel preparation corresponded with the model employed by central composite design, and the screened process can prepare situ gels with good retention and stable properties.

Intranasal tetrandrine temperature-sensitive gel for treatment of post-traumatic stress disorder / 药学学报

The traditional systemic treatment of post-traumatic stress disorder (PTSD) requires a long time period for an effect and has obvious side effects. In this study, tetrandrine temperature-sensitive gel (TTG) was prepared for treatment of PTSD in mice by nasal administration. TTG was prepared with poloxamer as matrix, the gelation temperature was suitable (<32 ℃) and the gelation time was short (1.32 min). Rheology experiments demonstrated that TTG has temperature sensitivity. In vivo imaging system of small animals proved that TTG nasal cavity retention time was so long. The cilia toxic test of toad showed that the formulation was safe. Animal experiments were approved by the Ethics Committee of Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations. The mice were randomly assigned into healthy group, model group and TTG group. The PTSD model of mice was established by single prolonged stress (SPS) and foot-shock method to generate anxiety and fear behavior. On the day 0 of TTG administration, SPS model mice were evaluated by the elevated plus maze (EPM). Percentages of open arm entries number (OE), latency of open arm entries (OL) and the residence time of open arm entries (OT) all indicated that the SPS model was successfully established. On the 7th day of TTG administration, TTG increased the OE and OT, decreased the OL of SPS mice. The feard behavior of mice in the foot-shock model was tested using conditioned fear box, 7 days of TTG treatment can reduce the freezing time of the mice obviously. The pathological changes of hippocampus, prefrontal cortex and amygdala were observed by H&E histological sections and c-fos immunohistochemical expression. The main influenced areas of PTSD were revealed to be the CA1 of hippocampus, prefrontal cortex and amygdala. All of the above indicated that TTG is a convenient, safe and effective drug for PTSD treatment, and will provide a new choice for clinical management of PTSD.

Preparation and preliminary evaluation of chitosan composite KGF-2 mutant thermal sensitive dressing / 军事医学

Objective To develop chitosan composite keratinocyte growth factor-2 mutant(KGF-2M)temperature-sen-sitive dressing and evaluate its physicochemical properties and dynamic release rule were used.Methods Chitosan, chi-tosan quaternary ammonium salt,β-glycerophosphate and other adjuvant materials to configure different formulations which were compounded with KGF-2M in order to develop temperature-sensitive dressing.Gelling time, temperature,the release rate of KGF-2M and other indicators were measured to analyze the physical and chemical properties of the temperature -sen-sitive dressing.Results Chitosan-KGF-2M composite dressing with temperature-sensitive properties was obtained by opti-mizing the formulation components of chitosan and related adjuvant materials.When the liquid dressing was above 35℃,it could be converted from liquid to solid gelatin within 10 minutes.The compound KGF-2M released from the gel was more than 98%at 4 h,and its bioactivity remained stable.Conclusion The thermo-sensitive gel has the characteristics of good conformability,moisturizing(moisture),isolation,wound healing,and a controlled release effect,which has great potential in wartime for wound repair.

Preparation and characterization of composite delivery system of paclitaxel-loaded temperature sensitive liposome and siRNA-loaded gold nanostar / 中国药科大学学报

We prepared gold nanostar (GNS) through seed growth method firstly,then formation of COX-2 siRNA(siCOX-2) and GNS composite modified with polyethylene glyco (PEG),2-amino-2-deoxy-D-glucos (DG) and 9-D-arginin (9R) was prepared.Mterwords,paclitaxel temperature sensitive liposome (PTX-TSL) was prepared by film dispersion method.Finally,siCOX-2 delivery systerm (PTX-TSL-(siCOX-2(9R/DG-GNS)))was obtained by hydrosulfuryl ligand reaction between siCOX-2 (9R/DG-GNS) and PTX-TSL The successful build of siCOX-2 (9R/DG-GNS) was vetified by nuclear magnetic resonance (NMR),sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE),and ultraviolet spectrophotometry and agarose gel electrophoresis method.Particle size of PTX-TSL-(siCOX-2(9R/DG-GNS)) was (292 ± 14) nm and Zeta potential was about -(2.59 ± 0.12) mV,which were measured by Zetasizer Nano ZS90.The morphology of PTX-TSL-(siCOX-2 (9R/DG-GNS)) measured by transmissionelectronmicroscopy showed homogeneous star structure with phospholipid bilayer on the surface,and it showed good thermal conversion efficiency under radiation of 808 nm laser.Differential scanning calorimetry test showed that PTX-TSL phase transition temperature is about 42.6 ℃.The drug loading content(using dialysis method) and encapsulation efficiency of PTX-TSL were about 7.5％ and 95.4％,at the same time,the release process experiment of PTX-TSL showed that it had a good temperature sensitive release performance.It is hopeful that this siCOX-2 system can be used for reducing drug resistance of PTX and improving the treatment effect of PTX through the synergistic antitumor drug resistance effect of siCOX-2.

Purification and enzyme activity assay of filamentous temperature-sensitive protein Z in Streptococcus suis serotype 2 / 中国人兽共患病学报

We conducted purification of filamentous temperature-sensitive protein Z of Streptococcus suis serotype 2 (S.suis 2) and measured its GTPase activity.The ftsz gene in the genome of the Chinese 05ZYH33 strain of S.suis 2 was successfully amplified using PCR,and then the ftsz gene was cloned into prokaryotic expression plasmid pET28a,and the recombinant plasmid pET28a-ftsz was transformed into E.coli BL21.After induction by IPTG,the isolated FtsZ protein was analyzed with SDS-PAGE.Then the recombinant protein was purified by Ni2+-NTA affinity chromatography.The rabbit serum was harvested after immunization with recombinant FtsZ protein,and was analyzed by indirect ELISA and Western blotting.The GTPase activity of FtsZ was measured with the malachite green method.Results showed that successfully constructed recombinant plasmid pET28a-ftsz and the recombinant protein with high purity was obtained;Western blot result indicated that FtsZ could react with the His-tag antibody and the rabbit serum;the polyclonal antibody titer of the rabbit serum reached 1 ∶ 13 107 200;FtsZ have GTPase activity.We successfully prepared S.suis 2 recombinant protein FtsZ having GTPase activity and high titer antiserum would be useful for the further study of S.suis 2 cell division mechanism.

Preparation and Quality Analysis of Osthole Microcapsules-Temperature-sensitive Gel / 中国药学杂志

OBJECTIVE: To optimize the preparation process of osthole microcapsules-temperature-sensitive gel and set up its quality standard. METHODS: Using gelling temperature as the indicator, P407, P188 and the concentration of propylene glycol were investigated by single factor test, and orthogonal experiment was conducted to optimize the preparation process of osthole microcapsules-temperature-sensitive gel. Osthole content was determined by HPLC. The quality standard of osthole microcapsules-temperature-sensitive gel was established. RESULTS: The optimal formulation of osthole microcapsules-temperature-sensitive gel was as followsP407-P188-propylene glycol=18%∶1%∶15%. Osthole contentin the osthole microcapsules-temperature-sensitive gel should not be less than 31.77 μg·mL-1, and the gelling temperature should be 36-37℃. CONCLUSION: Osthole microcapsules-temperature-sensitive gel prepared in this study has reasonable composition, simple preparation process, and stable quality standards, indicating a hopeful application prospect.

Formula Screening and in vitro Drug Release of Tegafur Thermo-sensitive Gel for Intratumor Injection / 中国药师

Objective:To screen the best formula of tegafur temperature-sensitive gel for intratumor injection and investigate the in vitro drug release behavior. Methods:The drug dose was determined by cytotoxicity experiment. The thermo-sensitive gel was prepared with PLGA-PEG-PLGA and HPMC as the matrix. With the in vitro release as the index, the effects of PLGA-PEG-PLGA and HPMC at different concentrations on gel were investigated. The gelation temperature, viscosity and pH were detected. Results:The best formula was as follows:25% PLGA-PEG-PLGA, 1% HPMC, and tegafur dose of 1 mg·ml-1 . The average gelation temperature was 36. 7℃, the average viscosity was 7550 mPa·s, and the average pH was 7. 2. Conclusion:Tegafur thermo-sensitive gel for intratumor in-jection shows temperature sensitivity and obvious sustained-release property, which provides experimental basis for the further clinical research.

Research status on Ts-SV40-mediated temperature sensitive cells / 天津医药

The in vitro passage number and proliferation of non-immortalized cells are limited, which restrictions cell therapy or in vitro studies. Cells transfected with temperature sensitive simian virus 40 large T antigen (ts-SV40LT) gene could show the greatest proliferation. The cells can be amplified with compensating the lack of limited number of cells under the permissive temperature. Non-permissive temperature can be used in studying the cell therapy or its other physiological characteristics. This research field involves peritoneal stromal cells, satellite cells of urinary tract, oral epithelial cells, adre?nal medullary cells, bone marrow-derived endothelial cells, retinal progenitor cells, mesenchymal stem cells, hematopoietic stem cells, mast cells, podocytes and Kupffer cells. In this study, the current research on Ts-SV40-mediated temperature-sensitive cells was reviewed.

Optimization of the prescription of Calcein-loaded novel temperature-sensitive liposomes / 中国药学杂志

OBJECTIVE: To optimize the prescription of the novel temperature-sensitive liposomes(NTSL). METHODS: Calcein-loaded temperature-sensitive liposomes with different phospholipid composition (DPPC-MSPC-DSPE-PEG200=86:10:4 and N (20% HSPC + 80% DPPC) -MSPC-DSPE-PEG200=92:4:4) were prepared by the thin film -hydration method. Based on the fluorescence self-quenching off characteristics of calcein, the release behavior of the above liposomes was compared when exposed to 37°C (normal temperature) as well as their respective phase transition temperature (Tm). RESULTS: Compared with the traditional temperature-sensitive liposomes (TTSL), the NTSL showed more favorable temperature-sensitive release performance and more stable encapsulation ability to calcein, even with the amount of lysophospholipids was reduced by 60 percent. CONCLUSION: The application of NTSL helps to enhance the effect of temperature-sensitive release.

Synthesis and Characterization of biodegradable Poly (Vinyl caprolactam) grafted on to sodium alginate and its microgels for controlled release studies of an anticancer drug.

Biodegradable sodium alginate-g-poly (vinyl caprolactam) synthesized by graft copolymerization of N-vinyl caprolactam (VCL) on to sodium alginate (NaAlg) via free radical initiation mechanism using a redox initiation system. Grafting (%), efficiency (%), and conversion (%) were all found to depend on the content of potassium persulfate (KPS), VCL reaction temperature and time. The maximum % of grafting was ascertained to be 251 at the optimum conditions of 65 oC reaction temperature, 180 min of reaction time, 1.1098X10-3 mol of KPS and 7.1844X10-3 mol of VCL. Evidence of graft copolymerization was obtained by fouriertransform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). Further, graft copolymer was used for preparation of microgels (MGs) using Ca+2 as a crosslinking agent. SEM results showed that the MGs are spherical in structure with smooth surface. The effects of pH and temperature on the swelling behaviour of MGs were studied and ascertained that they were sensitive to both pH and temperature. 5-FU drug was successfully loaded in to these MGs and encapsulation efficiency was found 84%. The release of 5-FU was systematically investigated as a function of temperature, pH, amount of crosslinker and % of drug loading concentration. The results indicate that the responsive MGs have the potential to be used as an effective pH and temperature controlled delivery of 5-FU for more than 12 h.

Development and evaluation of protective capacity of Salmonella Enteritidis polyphosphate kinase-deleted and temperature-sensitive mutant / 대한수의학회지

This study was focusing on evaluating the protection of polyphosphate kinase (ppk) deleted and/or temperature-sensitive (ts) Salmonella Enteritidis (SE) as an attenuated vaccine in chickens. We constructed SEppk, SEts and SEppk::ts mutants and screened those mutants by growth capability in vitro, protection study in mice model and antibody response in chickens. Among the mutants, SEppk::ts-3 was selected because it showed higher growth capability, good protection against highly virulent SE in mice model, and good antibody response in chickens. SEppk::ts-3 also showed good protection against highly virulent SE isolate because it decreased colonization of virulent SE challenge strain in spleen, liver and cecum compared with the non-vaccinated control. The SEppk::ts-3 mutant showed cross-protection against S. Gallinarum (SG) challenge although the its cross-protection rate was a little lower than that of SG9R, a commercial vaccine against SG infection. To use for live attenuated vaccine in chickens, it should further be characterized.

Systhesis of magnetic thermosensitive hydrogel and its heat effect under alternating magnetic field / 中国药学杂志

OBJECTIVE: To synthesize magnetic thermosensitive hydrogel and study its heat effect under alternating magnetic field in vitro. METHODS: PLGA-PEG-PLGA triblock copolymer was synthesized by ring-opening polymerization of D, L-lactide and glycolide with PEG1500 in the presence of stannous iso caprylate. Magnetic thermosensitive hydrogel of different concentrations were prepared using different currents. The influences of the concentration of magnetic fluid and current of magnetic field on the heat effect were separately observed. RESULTS: The synthetized PLGA-PEG-PLGA triblock copolymer was excellently temperature sensitive. It retained the thermo-sensitivity of original hydrogel when the magnetic fluid was loaded. The 5, 10 and 20 min heating ability of magnetic fluid was positively linearly correlated with its concentration (r=0.9985, 0.9893 and 0.9711, respectively, n=3) and current of magnetic field (r=0.9948, 0.9977 and 0.9994, respectively, n=4). CONCLUSION: The magnetic thermosensitive hydrogel has excellent temperature sensitivity. When alternating magnetic field is applied, the temperature of the system can rise and reach above LCST of hydrogel. The temperature can be controlled by changing the concentration of magnetic fluid and the current of magnetic field. Copyright 2012 by the Chinese Pharmaceutical Association.

Construction and Genetic Analysis of Murine Hepatitis Virus Strain A59 Nsp16 Temperature Sensitive Mutant and the Revertant Virus / 中国病毒学·英文版

Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu"-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu"-ts18(cd) were indistinguishable from the reported ts mutant Wu"-ts 18. Then we cultured the ts mutant Wu"-ts 18(cd) at non-permissive temperature 39.5℃, which "forced" the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu"-ts 18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.

Screening of Cell Cycle-Related Genes of Pleurotus eryngii Using Yeast Mutant Strains

Temperature-sensitive yeast mutants were used to screen for cell cycle-related genes from Pleurotus eryngii genomic DNA. A mushroom genomic DNA library was established and each gene was screened for the ability to rescue seven Saccharomyces cerevisiae temperature-sensitive strains. Hundreds of yeast transformants were selected at restrictive temperatures over 30degrees C. Plasmids from the transformants that survived were isolated and transformed back into their host strains. The temperature sensitivity of the resulting transformants was tested from 30degrees C to 37degrees C. Ten DNA fragments from P. eryngii were able to rescue yeast temperature-sensitive strains, and their DNA sequences were determined.