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Herein, we report a novel sensor to detect trypsin using a purpose-designed fluorescein-labelled peptide with negatively charged carbon nanoparticles (CNPs) modified by acid oxidation. The fluorescence of the fluorescein-labelled peptide was quenched by CNPs. The sensor reacted with trypsin to cleave the peptide, resulting in the release of the dye moiety and a substantial increase in fluorescence intensity, which was dose-and time-dependent, and trypsin could be quantified accordingly. Correspondingly, the biosensor has led to the development of a convenient and efficient fluorescent method to measure trypsin activity, with a detection limit of 0.7μg/mL. The method allows rapid determination of trypsin activity in the normal and acute pancreatitis range, suitable for point-of-care testing. Furthermore, the applicability of the method has been demonstrated by detecting trypsin in spiked urine samples.
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OBJECTIVE: To explore the molecular mechanism of curcumin in inhibiting the nucleotide-binding oligomerization domain like receptor family pyrin domain-containing(NLRP3) inflammatory bodies induced by silica(SiO_2) in mouse alveolar macrophages(AM). METHODS: AMs were isolated from the bronchoalveolar lavage fluid of specific pathogen free C57 BL/6 mice and divided into 6 groups. Among them, the AM of the control group received no stimulation; the AM in the SiO_2 stimulation group was stimulated with SiO_2 suspension at the final mass concentration of 50 mg/L; the AM in nuclear factor(NF-κB)inhibition group was pretreated with 5-(4-fluorophenyl)-2-urea-thiophene-3-formamide with a final concentration of 200 nmoL/L for 1 hour, the AM in the low-, medium-and high-dose curcumin groups were pretreated with curcumin with the final concentrations of 20, 40 and 50 μmol/L for 1 hour, respectively, and then stimulated with SiO_(2 )suspension with a final concentration of 50 mg/L. Samples were collected after 6 hours of incubation. The mRNA expression of NLRP3 inflammasome related genes such as NLRP3, Caspase-1 and interleukin(IL)-1β was detected by real-time fluorescence quantitative polymerase chain reaction. The secretion level of maturation IL-1β(mIL-1β) and IL-18 in AM was detected by enzyme-linked immunosorbent assay. The protein expression and secretion level of cleaved Caspase-1, precursor-IL-1β(pro-IL-1β) and mIL-1β were analyzed by Western blotting. RESULTS: The mRNA relative expression of NLRP3, Caspase-1 and IL-1β, and the secretion levels of mIL-1β and IL-18, and the protein relative expression of Caspase-1, pro-IL-1β and mIL-1β, as well as the secretion levels of cleaved Caspase-1 and mIL-1β increased in the SiO_2 stimulated group compared with the control group(P<0.05). Except for the relative expression and the secretion level of cleaved Caspase-1, the other 8 indexes in the NF-κB inhibition group were lower than that in the SiO_2 stimulation group(P<0.05). Except for the relative expression of cleaved Caspase-1 and mIL-1β proteins in the low-dose curcumin group, the relative expression of all the above 10 indexes was lower in the three curcumin treated groups than that in the SiO_2 stimulation group(P<0.05). In addition, all the above indexes decreased with the increase of curcumin intervention dose(P<0.05). The mRNA relative expression of NLRP3 and IL-1β, and the protein relative expression of pro-IL-1β increased in the medium-dose curcumin group(P<0.05), the secretion levels of mIL-1β and IL-18, as well as the protein relative expression and secretion levels of cleaved Caspase-1 and mIL-1β decreased(P<0.05), compared with the NF-κB inhibition group. CONCLUSION: Curcumin can inhibit SiO_2-induced AM NLRP3 inflammasome activation in a dose-response relationship. This process may be related to the inhibition of NF-κB signaling pathway by curcumin and the down-regulating NLRP3 inflammasome-related genes at the transcriptional level. The important mechanism may be that curcumin directly blocks the activation, assembly, and downstream shearing of NLRP3 in inflammasomes.
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OBJECTIVE:To establish the method for simultaneous determination of 6 components in Lonicera japonica,and to study the content changes of them before and after before and after carbonized. METHODS:UPLC method was adopted. The deter-mination was performed on Agilent Eclipse Plus C18 RRHD column with mobile phase consisting of 0.1% phosphoric acid solu-tion-acetonitrile(gradient elution)at the flow rate of 0.2 mL /min. The detection wavelength was set at 350 nm,and column tem-perature was 25 ℃. The sample size was 1 μL. RESULTS:The linear ranges of chlorogenic acid,rutin,galuteolin,isochlorogenic acid A,isochlorogenic acid B and isochlorogenic acid C were 21.2-424 μg(r=0.9993),1.17-23 μg(r=0.9995),2.18-43 μg(r=0.9998),5.10-102 μg(r=0.9993),2.60-52 μg(r=0.9991),4.95-99 μg(r=0.9998),respectively. RSDs of precision,stability and repeatability tests were all lower than 2.0%. Recoveries were 97.11%-99.76%(RSD=1.20%,n=6),95.20%-99.90%(RSD=2.20%,n=6),95.71%-100.30%(RSD=2.20%,n=6),95.00%-96.98%(RSD=0.88%,n=6),96.47%-103.00%(RSD=2.40%, n=6),95.78%-103.80%(RSD=3.20%,n=6). Compared with before processing,the contents of rutin,isochlorogenic acid B and isochlorogenic acid C in L. japonica were increased along with processing,the contents of chlorogenic acid and isochlorogenic acid A were decreased significantly,while the content of galuteolin had no significant change. CONCLUSIONS:The method is sim-ple,precise,stable and repeatable,and can be used for simultaneous determination of 6 components in L. japonica. Those chemi-cal components have certain changes before and after carbonized.
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Objective To conduct the molecular epidemiologic analysis of Staphylococcus aureus (S.aureus) in the intensive care units(ICUs) and general wards and to compare their clinical characteristics.Methods Ninety-six clinically isolated strains of S.aureus(43 strains from the emergency intensive care unit(EICU) and neurosurgical intensive care unit(NICU) and 53 strains from the general wards) collected from Sepetember 2015 to April 2016 were performed the bacterial identification and antibiotic susceptibility test.The molecular typing was performed by adopting staphylococcal protein A (spa) typing method.Results Among 96 strains of S.aureus,the detection rate of methicillin-resistant S.aureus(MRSA) was 40.6%(39/96),which among 43 strains in ICU was 62.8%(27/43) and which among 53 strains in the general words was 22.6%(12/53).The resistance rates of strains from ICUs to gentamicin,levofloxacin,clindamycin,fosfomycin and minocycline were 23.3%,48.8%,46.5%,32.6% and 32.5% respectively,while which from the general wards were 7.5%,24.5%,18.9%,2.1% and 0% respectively.The Spa typing results showed that the main types of ICUs were t002,t091 and t311.The major epidemic strain was t002(n=16,37.2%) and mainly isolated from EICUs(12 strains),26 spa types were identified among the general wards trains,mainly were t189,t377,t571,t034,t091,t127.Conclusion The detection rate of MRSA in ICUs is higher than that in the general wards,these strains have high resistant rate to routine antibacterial drugs.t002 is the major epidemic strain.The general wards have more spa types with higher genetic diversity.