AIM:To determine the concentration of ARV-471(a representative PROTAC drug)in mice plasma by establishing an HPLC-MS/MS method and to apply this method in pharmacokinetic studies in mice.METHODS:Verapamil was taken as the inter-nal standard,the mice plasma samples were extract-ed by methanol.After centrifugation,the superna-tant was analyzed by liquid chromatography-mass spectroscopy(LC-MS/MS).Chromatographic col-umn:ACQUITY UPLC HSS T3(1.8 μm,2.1 mm × 50 mm);The mobile phase consists of acetonitrile(containing 0.1％formic acid)and 2 mmol/L ammoni-um formate aqueous solution(containing 0.1％for-mic acid)for gradient elution;flow-rate of 0.6 mL/min;injection volume:2 μL.The electrospray ioniza-tion(ESI)is employed,operating in positive ion scan-ning mode with multiple reaction monitoring(MRM)detection.The specificity,standard curve and quanti-fication limit,precision and accuracy,recovery rate and matrix effect,stability and dilution reliability of this method were examined.Furthermore,the plas-ma concentration and pharmacokinetic parameters of ARV-471 in mice after intravenous injection of 5 mg/kg and oral gavage of 30 mg/kg were deter-mined and calculated.RESULTS:The results dem-onstrate that ARV-471 exhibits a good linear rela-tionship within the concentration range of 2.0 to 2 000.0 ng/mL.The intra-and inter-accuracy were between 80.0％and 120.0％,with the intra-and in-ter-precision less than 15％.The results of method-ological study of specificity,matrix effect,stability conformed to the requirements of the guideline.Pharmacokinetic parameters reveal that after oral administration of 30 mg/kg ARV-471 to male and fe-male mice,Cmax were(2 947.19±454.77)and(2 682.02±342.23)ng·mL-1;AUC0-twere(23 357.37±3 488.00)and(20 161.23±1 871.32)ng·h·mL-1;T1/2were(3.11±0.18)and(2.93±0.62)h.Tmax were(1.83±0.41)and(2.00±0.00)h for male and fe-male mice,respectively.After intravenous adminis-tration of 5 mg/kg ARV-471 to male and female mice,the AUC0-t values were found to be(18 219.07±2 059.41)and(17 238.01±2 380.55)ng·h·mL-1;T1/2 values were(2.76±0.23)and(2.73±0.20)h;The absolute bio-availability of ARV-471 were deter-mined to be(19.49±1.81)％and(21.37±3.19)％for male and female mice,respectively.CONCLU-SION:This study establishes a rapid and effective method for the pharmacokinetic research of ARV-471,laying the foundation for the pharmacokinetic studies of PROTAC drugs.

AIM:To determine the concentration of ARV-471(a representative PROTAC drug)in mice plasma by establishing an HPLC-MS/MS method and to apply this method in pharmacokinetic studies in mice.METHODS:Verapamil was taken as the inter-nal standard,the mice plasma samples were extract-ed by methanol.After centrifugation,the superna-tant was analyzed by liquid chromatography-mass spectroscopy(LC-MS/MS).Chromatographic col-umn:ACQUITY UPLC HSS T3(1.8 μm,2.1 mm × 50 mm);The mobile phase consists of acetonitrile(containing 0.1％formic acid)and 2 mmol/L ammoni-um formate aqueous solution(containing 0.1％for-mic acid)for gradient elution;flow-rate of 0.6 mL/min;injection volume:2 μL.The electrospray ioniza-tion(ESI)is employed,operating in positive ion scan-ning mode with multiple reaction monitoring(MRM)detection.The specificity,standard curve and quanti-fication limit,precision and accuracy,recovery rate and matrix effect,stability and dilution reliability of this method were examined.Furthermore,the plas-ma concentration and pharmacokinetic parameters of ARV-471 in mice after intravenous injection of 5 mg/kg and oral gavage of 30 mg/kg were deter-mined and calculated.RESULTS:The results dem-onstrate that ARV-471 exhibits a good linear rela-tionship within the concentration range of 2.0 to 2 000.0 ng/mL.The intra-and inter-accuracy were between 80.0％and 120.0％,with the intra-and in-ter-precision less than 15％.The results of method-ological study of specificity,matrix effect,stability conformed to the requirements of the guideline.Pharmacokinetic parameters reveal that after oral administration of 30 mg/kg ARV-471 to male and fe-male mice,Cmax were(2 947.19±454.77)and(2 682.02±342.23)ng·mL-1;AUC0-twere(23 357.37±3 488.00)and(20 161.23±1 871.32)ng·h·mL-1;T1/2were(3.11±0.18)and(2.93±0.62)h.Tmax were(1.83±0.41)and(2.00±0.00)h for male and fe-male mice,respectively.After intravenous adminis-tration of 5 mg/kg ARV-471 to male and female mice,the AUC0-t values were found to be(18 219.07±2 059.41)and(17 238.01±2 380.55)ng·h·mL-1;T1/2 values were(2.76±0.23)and(2.73±0.20)h;The absolute bio-availability of ARV-471 were deter-mined to be(19.49±1.81)％and(21.37±3.19)％for male and female mice,respectively.CONCLU-SION:This study establishes a rapid and effective method for the pharmacokinetic research of ARV-471,laying the foundation for the pharmacokinetic studies of PROTAC drugs.

Objective:To analyze the effects of continuous nursing intervention on patients with intestinal stoma using WeChat, China′s most popular messaging App.Methods:A total of 60 patients undergoing permanent intestinal stoma surgery, who were admitted to Dalian Third People′s Hospital from January 2018 to December 2019, were selected as the research objects. They were divided into the control group and the observation group with 30 cases each using random number table method. The control group adopted the traditional continuous nursing model while the observation group received both traditional continuous nursing model and continuous nursing interventions via WeChat. By using the Exercise of Self-Care Agency Scale (ESCA), City of Hope-Quality of Life-Ostomy Questionnaire Chinese Version (C-COH), and Ostomy Adjustment Inventory (OAI), the differences in self-care capability, quality of life, stoma adaptability, incidence of intestinal stoma complications, and patients′ satisfaction with the new nursing model in the two groups were compared before the intervention, 1 month and 3 months after the intervention, respectively.Results:Before intervention, there was no significant difference between the two groups in ESCA total score, C-COH score and OAI score ( P>0.05). After 1 month and 3 months of intervention, the observation group had the total ESCA scores of (91.50 ± 2.86) and (104.87 ± 3.45) points, which were higher than control group′s (81.37 ± 2.98) and (92.80 ± 2.91) points, respectively, and the differences between the two groups were statistically significant ( t=13.13, 14.38, both P<0.05); the observation group had the C-COH scores of (5.00 ± 1.13) and (6.37 ± 1.22) points, which were higher than the control group′s (3.90 ± 1.14) and (4.67 ± 1.04) points, respectively, and the differences between the two groups were statistically significant ( t=3.71, 5.69, both P<0.05); the observation group had the OAI scores of (48.13 ± 8.55) and (60.07 ± 7.43) points, which were higher than the control group′s (39.57 ± 7.43) and (43.47 ± 7.39) points, respectively, and the difference between the two groups was statistically significant ( t=4.07, 8.53, both P<0.05); after 3-month intervention, the observation group had a 16.7 % (5/30) incidence of stoma complications, which was lower than the control group′s 46.7% (14/30), and the difference between the two groups was statistically significant ( χ2=6.24, P<0.05); after 3-month intervention, the observation group had a score of (95.90 ± 1.66) points for the patient′s satisfaction with continuous care, which was higher than the control group′s (89.80 ± 2.44) points, and the difference between the two groups was statistically significant (t value was 11.13, P<0.05). Conclusions:Delivering the WeChat continuous nursing interventions for patients with intestinal stoma has improved the patients′ self-care capability, enhanced stoma adaptation level and quality of life, reduced the incidence of intestinal stoma complications, and improved patients′ satisfaction. Thereby, continuous nursing on WeChat is worthy of clinical application.

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

Objective Establishment of orthotopic transplantation tumor model of human choriocarcinoma in nude mice. Methods Human choriocarcinoma cell line JAR was cultured and the single cell suspension was subcutaneously injected into five 8-week-old BALB / c nude mice to establish subcutaneous xenograft model. After subcutaneous tumor was formed in nude mice, the tumor tissue was taken under aseptic conditions and cut into 1 mm

Objective To investigate the expression of semaphorin 3A (Sema3A) and its receptor neuropilin-1 (NRP-1) in gastric cancer and its correlation with microvessel density (MVD), and then to explore the effect of recombinant human Sema3A on angiogenesis of gastric cancer and the associated mechanisms. Methods Forty cases of gastric cancer tissues and its corresponding adjacent normal tissues were used to detecte the expression of Sema3A, NRP-1 and MVD in tissues by immunohistochemistry method . The expression level of Sema3A in serum of gastric cancer patient group and normal control group were measured by Enzyme-linked immuno-sorbent assay (ELISA). Western blotting was used to detect the expression of Sema3A and NRP-1 in five gastric cancer cell lines (MGC-803,HGC-27,MKN-28,SGC-7901,MKN-45) and human gastric mucosal epithelial cell (GES-1). Transwell chamber was used to construct non-contact in vitro co-culture system, in which the effects of different concentrations of recombinant human Sema3A on angiogenesis in gastric cancer were analyzed by tube formation assay preliminarily. The expression levels of vascular endothelial growth factor receptor 2 (VEGFR2) and NRP-1 in co-culture system were detected by Western blotting. Results The expression levels of Sema3A in gastric cancer tissues, cell lines and patient serum were significantly lower than that in the control group(P<0. 05), while the expression of NRP-1 in gastric cancer tissues and MKN-28 cells was significantly increased, and both of them were associated with TNM staging of gastric cancer (P < 0. 05) . In vitro co-culture system, The tube forming abilities of human umbilical vein endothelial cell (HUVEC) were decreased in recombinant human Sema3A treated group, and this phenomenon was concentration dependent. The expression of VEGFR2 protein was down-regulated by recombinant human Sema3A. Conclusion The expression of Sema3A was decreased in gastric cancer tissues, cell lines and patient serum, and negatively correlated with microvessel density. The recombinant human Sema3A could inhibit the angiogenesis of gastric cancer in vitro, which may be related to down-regulation of VEGFR2 protein expression.

Objective To achieve the purpose of promoting movement function of the injury nerve by using the joint therapy of NT- 3- HUMSCs and SOCS3 gene silencing on SD rats'spinal cord injury. Methods (1) We used adherence method in vitro human umbilical cord-derived mesenchymal cells (HUMSC) during separation, purification and identification. (2) Then constructed NT-3 gene eukaryotic expression vector, which was transfected into its HUMSC, and constructed NT-3- HUMSC cell survival in vitro assay conditions and NT-3 expression. (3) We selected specific targets for SOCS3 screening and for sequence homology analysis. A negative control group was established. siRNA was designed and synthesized in vitro detection. (4) SD rats with spinal cord injury model were divided into two categories: (1) sham group with 10 rats; (2) T12 whole spinal cord injury model with 40 rats. The 40 rats were randomly divided into four groups with 10 rats in each group (saline treatment group,siRNA +NT-3-HUMSCs treatment group,NT-3-HUMSCs treatment group and siRNA treated group) . Motor function of the rats were evaluated respectively in 1, 2 and 3 months after the modeling was established successfully.Results(1) siRNA + NT-3-HUMSCs treatment group's BBB scores was significantly higher than NT-3-HUMSCs, SOCS3-siRNA and physiological saline groups ( P＜0.05) . (2) The grid climbing experiments showed that the neural functional recovery performed better in siRNA+the NT- 3- HUMSCs treatment group compared to the NT - 3 - HUMSCs, SOCS3 - siRNA and physiological saline groups (P＜0.05) . Conclusion The NT- 3- HUMSCs joint SOCS3 gene silencing in the treatment of SD rat spinal cord injury can improve the motor function of SD rat spinal cord injury.

Objective To investigate the effect of joint therapy by NT-3-HUMSCs and SOCS3 gene silencing in promoting the injury nerve regeneration repair after spinal cord injury in SD rats. Methods (1) Adherence method was used to culture human umbilical cord-derived mesenchymal cells (HUMSC) in vitro for separation, purification and identification. (2) We constructed NT-3 gene eukaryotic expression vector, and used gene transfection technology into its HUMSC, and tested the survival of NT-3-HUMSC cells and NT-3 expression in cells. (3) We screened specific targets of SOCS3, made sequence homology analysis, and set a negative control, designed and synthesized siRNA and detected the function. (4) SD rats model of spinal cordinjury were established and divided into: 1. sham group 10; 2.T12 whole spinal cord injury model 40, were randomly divided into four groups, respectively; saline treatment group 10; siRNA + NT-3-HUMSCs treatment group 10; NT-3-HUMSCs treatment group 10; siRNA treated group 10. After each group above modeling success, they received respectively the neural electrophysiological monitoring for 12 weeks survival. (5) We perfused SD rats for fixation and collect samples, and observed the local glial scar degradation situation and axon regeneration, meanwhile, used biotin glucan fluorescent (BDA) anterograde tracing. The injury transplant area-host junction spinal cord tissues were collected to observe the corticospinal tract regeneration under microscope. Results (1) In siRNA + NT-3-HUMSCs treatment group, the transection syringomyelia was significantly reduced as compared with normal saline group (P ＜ 0.05). (2) BDA anterograde tracing results showed that in the siRNA + NT-3-HUMSCs treatment group, neural axon grew significantly compared with the normal saline group. (3) Neural electrophysiological testing 12 weeks after injury: in the treatment group, the incubation period P40 was shorter as compared with control group; in siRNA + NT-3-HUMSCs treatment group, the incubation period was shorter obviously than normal saline, but the amplitude increased obviously (P ＜ 0.05). Conclusion NT-3-HUMSCs joint with SOCS3 gene silencing can promote the injury nerve regeneration repair in the treatment of SD rat spinal cord injury.

BACKGROUNDPyroptosis is the term for caspase-1-dependent cell death associated with pro-inflammatory cytokines. The role of alveolar macrophage (AM) pyroptosis in the pathogenesis of the acute lung injury and acute respiratory distress syndrome (ALI/ARDS) remains unclear.

METHODSC57BL/6 wild-type mice were assigned to sham, lipopolysaccharide (LPS) + vehicle, LPS + acetyl-tyrosyl-valyl- alanyl-aspartyl-chloromethylketone (Ac-YVAD-CMK) and LPS + Z-Asp-Glu-Val-Asp-fluoromethylketone groups. Mice were given intraperitoneal (IP) injections of LPS. Drugs were IP injected 1 h before LPS administration. Mice were sacrificed 16 h after LPS administration, and AMs were isolated. Western blot analysis for active caspase-1 and cleaved caspase-3, evaluation of lung injury and a cytokine release analysis were performed. AMs were treated with LPS and adenosine triphosphate (ATP); caspase-1-dependent cell death was evaluated using flow cytometry; the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) pyroptosomes were examined by immunofluorescence.

RESULTSThe expression of activated caspase-1 in AMs was enhanced following LPS challenge compared with the sham group. In the ex vivo study, the caspase-1/propidium iodide-positive cells, caspase-1 specks and ASC pyroptosomes were up-regulated in AMs following LPS/ATP stimulation. The specific caspase-1 inhibitor Ac-YVAD-CMK inhibited the activation of caspase-1 and pyroptotic cell death. Ac-YVAD-CMK also reduced the lung injury, pulmonary edema and total protein in bronchoalveolar lavage fluid (BALF). In addition, Ac-YVAD-CMK significantly inhibited interleukin-α2 (IL-1α2) release both in serum and BALF and reduced the levels of IL-18, tumor necrosis factor-α± (TNF-α±), High Mobility Group Box 1 (HMGB1) in BALF during LPS-induced ALI/ARDS.

CONCLUSIONSThis study reported AM pyroptosis during LPS-induced ALI/ARDS in mice and has demonstrated that Ac-YVAD-CMK can prevent AM-induced pyroptosis and lung injury. These preliminary findings may form the basis for further studies to evaluate this pathway as a target for prevention or reduction of ALI/ARDS.

Acute Lung Injury ; chemically induced ; prevention & control ; Amino Acid Chloromethyl Ketones ; pharmacology ; Animals ; Lipopolysaccharides ; toxicity ; Macrophages, Alveolar ; drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Oligopeptides ; pharmacology ; Pyroptosis ; drug effects

Glioma is the most common intracranial malignant tumor,with high incidence and recurrence rate,high fatality rate and the characteristics of low cure rate.Current treatment is given priority to with surgical treatment,auxiliary comprehensive therapy such as radiotherapy and chemotherapy.Hydrogen peroxide (H2O2) is a intermediate product of the cell oxygen metabolism which is a universal phenomenon in aerobic organisms.H2O2 is indispensable at various stages of tumor cell proliferation,infiltration and metastasis.Studies of the production of H2O2 and its function,the mechanism of apoptosis and the relationship between H2O2 and glioma cells can provide corresponding guidances for looking for a target gene for the treatment of gliomas.