In-depth study of the components of polymyxins is the key to controlling the quality of this class of antibiotics.Similarities and variations of components present significant analytical challenges.A two-dimensional(2D)liquid chromatography-mass spectrometry(LC-MS)method was established for screening and comprehensive profiling of compositions of the antibiotic colistimethate sodium(CMS).A high concentration of phosphate buffer mobile phase was used in the first-dimensional LC system to get the components well separated.For efficient and high-accuracy screening of CMS,a targeted method based on a self-constructed high resolution(HR)mass spectrum database of CMS components was established.The database was built based on the commercial MassHunter Personal Compound Database and Library(PCDL)software and its accuracy of the compound matching result was verified with six known components before being applied to genuine sample screening.On this basis,the unknown peaks in the CMS chromatograms were deduced and assigned.The molecular formula,group composition,and origins of a total of 99 compounds,of which the combined area percentage accounted for more than 95％of CMS components,were deduced by this 2D-LC-MS method combined with the MassHunter PCDL.This profiling method was highly efficient and could distinguish hundreds of components within 3 h,providing reliable results for quality control of this kind of complex drugs.

Neuropathic pain, a debilitating condition caused by dysfunction of the somatosensory nervous system, remains difficult to treat due to limited understanding of its molecular mechanisms. Bioinformatics analysis identified cerebellin 2 (CBLN2) as highly enriched in human and murine proprioceptive and nociceptive neurons. We found that CBLN2 expression is persistently upregulated in dorsal root ganglia (DRG) following spinal nerve ligation (SNL) in mice. In addition, transcription factor SOX11 binds to 12 cis-regulatory elements within the Cbln2 promoter to enhance its transcription. SNL also induced SOX11 upregulation, with SOX11 and CBLN2 co-localized in nociceptive neurons. The siRNA-mediated knockdown of Sox11 or Cbln2 attenuated SNL-induced mechanical allodynia and thermal hyperalgesia. High-throughput sequencing of DRG following intrathecal injection of CBLN2 revealed widespread gene expression changes, including upregulation of numerous NF-κB downstream targets. Consistently, CBLN2 activated NF-κB signaling, and inhibition with pyrrolidine dithiocarbamate reduced CBLN2-induced pain hypersensitivity, proinflammatory cytokines and chemokines production, and neuronal hyperexcitability. Together, these findings identified the SOX11/CBLN2/NF-κB axis as a critical mediator of neuropathic pain and a promising target for therapeutic intervention.
Animals ; Neuralgia/metabolism* ; Ganglia, Spinal/metabolism* ; Up-Regulation ; Mice ; NF-kappa B/metabolism* ; SOXC Transcription Factors/genetics* ; Male ; Neuroinflammatory Diseases/metabolism* ; Mice, Inbred C57BL ; Nerve Tissue Proteins/genetics* ; Hyperalgesia/metabolism* ; Signal Transduction ; Spinal Nerves

In-depth study of the components of polymyxins is the key to controlling the quality of this class of antibiotics. Similarities and variations of components present significant analytical challenges. A two-dimensional (2D) liquid chromatography-mass spectrometr (LC-MS) method was established for screening and comprehensive profiling of compositions of the antibiotic colistimethate sodium (CMS). A high concentration of phosphate buffer mobile phase was used in the first-dimensional LC system to get the components well separated. For efficient and high-accuracy screening of CMS, a targeted method based on a self-constructed high resolution (HR) mass spectrum database of CMS components was established. The database was built based on the commercial MassHunter Personal Compound Database and Library (PCDL) software and its accuracy of the compound matching result was verified with six known components before being applied to genuine sample screening. On this basis, the unknown peaks in the CMS chromatograms were deduced and assigned. The molecular formula, group composition, and origins of a total of 99 compounds, of which the combined area percentage accounted for more than 95% of CMS components, were deduced by this 2D-LC-MS method combined with the MassHunter PCDL. This profiling method was highly efficient and could distinguish hundreds of components within 3 h, providing reliable results for quality control of this kind of complex drugs.

Poly(lactic-co-glycolide)(PLGA)is a key excipient in long-acting sustained-release preparations,and its degradation properties directly affect the drug release behavior.In this study,PLGA microspheres were prepared by microfluidic techniques,and the morphology changes of the microspheres were observed by scanning electron microscopy(SEM).In alkaline environment,due to the accelerated hydrolysis of ester bonds,the surface of the microspheres was rapidly dissolved and eroded,and the degradation rate was significantly higher than that in acidic environment.High temperature accelerated the degradation of PLGA microspheres.Under neutral and alkaline conditions,the microspheres showed aggregation and adhesion.Under acidic conditions,the microspheres gradually decomposed into irregular fragments.The high ionic strength further promoted the surface corrosion of the microspheres,especially under extreme pH conditions.Simultaneously,PLGA microspheres encapsulating coumarin were prepared to simulate the microsphere formulation.The release rate of coumarin after degradation of the microspheres under different conditions was observed by measuring the absorbance with ultraviolet-visible spectrophotometry.The results were consistent with those of the blank microspheres.This study revealed that the degradation of PLGA microspheres was significantly pH-dependent,temperature sensitive and ion strength responsive.These findings not only helped to understand and optimize the long-term stability and controlled release performance of drug-carrying microspheres,but also provided a theoretical basis for further improvement of PLGA-based drug carrier design.

Objective To explore the intervention effect and mechanism of Zhiwei Fuwei pills on precancerous lesions of gastric cancer(PLGC)model rats based on hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF)signaling pathway.Methods PLGC model rats were established by the combination of N-methyl-N'-nitro-N-nitrosoguanidine and ammonia drinking,hunger and satiation disorder,ethanol gavage and ranitidine feeding,and were randomly divided into model group,control group and experimental-H,-M,-L groups,with 8 rats in each group.Another 8 healthy rats were selected as blank group.Experimental-H,-M,-L groups were given 1.67,0.84 and 0.42 g·kg-1 Zhiwei Fuwei pills solution by intragastric administration,respectively;control group was given 2.00 × 10-3g·kg-1 folic acid solution by intragastric administration;blank group and model group were given 0.9％NaCl by intragastric administration,once a day for 4 weeks.The structural changes of gastric microvessels were detected by transmission electron microscopy,and the expression levels of HIF-1α,VEGF,vascular endothelial growth factor receptor-2(VEGFR-2)and Angiopoietin-2(Ang-2)were detected by western blot.Results Compared with the model group,the abnormality of gastric microvascular structure in experimental-H,-M,-L groups was improved to some extent,and the effect of high dose experimental group was the most obvious.The relative expression levels of HIF-1 α protein in experimental-H,-M groups and control group,model group and blank group were 0.43±0.03,0.66±0.04,0.77±0.01,0.80±0.02 and 0.37±0.02;the relative expression levels of VEGF protein were 0.97±0.06,1.21±0.06,1.51±0.07,1.54±0.05 and 0.88±0.02;the relative expression levels of VEGFR-2 protein were 1.03±0.06,1.18±0.04,1.37±0.05,1.45±0.02 and 0.70±0.02;the relative expression levels of Ang-2 protein were 0.51±0.03,0.61±0.02,0.71±0.01,0.78±0.03 and 0.34±0.01,respectively.Compared with the model group,the above indexes in the experimental-H,-M groups were statistically significant(all P＜0.01).Conclusion Zhiwei Fuwei pills may inhibit the abnormal activation of HIF-1α/VEGF signaling pathway,improve hypoxia microenvironment,reduce angiogenesis,and then effectively interfere with the progression of PLGC.

Objective To explore the intervention effect and mechanism of Zhiwei Fuwei pills on precancerous lesions of gastric cancer(PLGC)model rats based on hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF)signaling pathway.Methods PLGC model rats were established by the combination of N-methyl-N'-nitro-N-nitrosoguanidine and ammonia drinking,hunger and satiation disorder,ethanol gavage and ranitidine feeding,and were randomly divided into model group,control group and experimental-H,-M,-L groups,with 8 rats in each group.Another 8 healthy rats were selected as blank group.Experimental-H,-M,-L groups were given 1.67,0.84 and 0.42 g·kg-1 Zhiwei Fuwei pills solution by intragastric administration,respectively;control group was given 2.00 × 10-3g·kg-1 folic acid solution by intragastric administration;blank group and model group were given 0.9％NaCl by intragastric administration,once a day for 4 weeks.The structural changes of gastric microvessels were detected by transmission electron microscopy,and the expression levels of HIF-1α,VEGF,vascular endothelial growth factor receptor-2(VEGFR-2)and Angiopoietin-2(Ang-2)were detected by western blot.Results Compared with the model group,the abnormality of gastric microvascular structure in experimental-H,-M,-L groups was improved to some extent,and the effect of high dose experimental group was the most obvious.The relative expression levels of HIF-1 α protein in experimental-H,-M groups and control group,model group and blank group were 0.43±0.03,0.66±0.04,0.77±0.01,0.80±0.02 and 0.37±0.02;the relative expression levels of VEGF protein were 0.97±0.06,1.21±0.06,1.51±0.07,1.54±0.05 and 0.88±0.02;the relative expression levels of VEGFR-2 protein were 1.03±0.06,1.18±0.04,1.37±0.05,1.45±0.02 and 0.70±0.02;the relative expression levels of Ang-2 protein were 0.51±0.03,0.61±0.02,0.71±0.01,0.78±0.03 and 0.34±0.01,respectively.Compared with the model group,the above indexes in the experimental-H,-M groups were statistically significant(all P＜0.01).Conclusion Zhiwei Fuwei pills may inhibit the abnormal activation of HIF-1α/VEGF signaling pathway,improve hypoxia microenvironment,reduce angiogenesis,and then effectively interfere with the progression of PLGC.

OBJECTIVE: To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection. METHODS: The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs. RESULTS: A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01). CONCLUSION DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.
Humans ; Autophagy ; Cell Death ; Cell Nucleus ; Human Umbilical Vein Endothelial Cells/virology* ; Dengue ; Proteome

BACKGROUND: Patients with atrial fibrillation (AF) and prior stroke history have a high risk of cardiovascular events despite anticoagulation therapy. It is unclear whether catheter ablation (CA) has further benefits in these patients. METHODS: AF patients with a previous history of stroke or systemic embolism (SE) from the prospective Chinese Atrial Fibrillation Registry study between August 2011 and December 2020 were included in the analysis. Patients were matched in a 1:1 ratio to CA or medical treatment (MT) based on propensity score. The primary outcome was a composite of all-cause death or ischemic stroke (IS)/SE. RESULTS: During a total of 4.1 ± 2.3 years of follow-up, the primary outcome occurred in 111 patients in the CA group (3.3 per 100 person-years) and in 229 patients in the MT group (5.7 per 100 person-years). The CA group had a lower risk of the primary outcome compared to the MT group [hazard ratio (HR) = 0.59, 95% CI: 0.47-0.74, P < 0.001]. There was a significant decreasing risk of all-cause mortality (HR = 0.43, 95% CI: 0.31-0.61, P < 0.001), IS/SE (HR = 0.73, 95% CI: 0.54-0.97, P = 0.033), cardiovascular mortality (HR = 0.32, 95% CI: 0.19-0.54, P < 0.001) and AF recurrence (HR = 0.33, 95% CI: 0.30-0.37, P < 0.001) in the CA group compared to that in the MT group. Sensitivity analysis generated consistent results when adjusting for time-dependent usage of anticoagulants. CONCLUSIONS In AF patients with a prior stroke history, CA was associated with a lower combined risk of all-cause death or IS/SE. Further clinical trials are warranted to confirm the benefits of CA in these patients.

How the brain perceives objects and classifies perceived objects is one of the important goals of visual cognitive neuroscience. Previous research has shown that when we see objects, the brain's ventral visual pathway recognizes and classifies them, leading to different ways of interacting with them. In this paper, we summarize the latest research progress of the ventral visual pathway related to the visual classification of objects. From the perspective of the neural representation of objects and its underlying mechanisms in the visual cortex, we summarize the current research status of the two important organizational dimensions of object animacy and real-world size, provide new insights, and point out the direction of further research.
Brain Mapping/methods* ; Magnetic Resonance Imaging ; Pattern Recognition, Visual ; Photic Stimulation ; Visual Cortex ; Visual Pathways