Objective To prepare the recombinant Echinococcus granulosus Polo-like kinase 2 (rEgPLK2) protein and evaluate its immunoprotective efficacy against cystic echinococcosis, so as to provide insights into research and development of novel vaccines against echinococcosis. Methods The Polo-like kinase (PLK) protein sequences were retrieved from 12 species in the NCBI protein database, including E. granulosus and E. multilocularis. Multiple sequence alignment was performed using the Clustal Omega program, and structural visualization and homology analysis were conducted using the ESPript 3.2 program. The recombinant plasmid pET-30a-EgPLK2 was transformed into BL21(DE3) competent cells. Protein expression was induced with isopropyl-β-D-thiogalactoside (IPTG), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to characterize the expression and molecular weight of the rEgPLK2 protein. The purified rEgPLK2 protein was thoroughly emulsified with Freund’s complete adjuvant at a 1 : 1 volume ratio. Two New Zealand white rabbits were immunized with multipoint subcutaneous injection on the back at a dose of 300 μg per rabbit for primary immunization. For booster immunizations, the protein was emulsified with Freund’s incomplete adjuvant at a 1 : 1 volume ratio and administered on days 14, 28, and 42 after the primary immunization at a dose of 150 μg per rabbit. Serum was sampled from the rabbit ear vein on day 7 after the final immunization to yield anti-rEgPLK2 polyclonal antibodies. Antibody titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and antibody specificity was verified by Western blotting. The tissue localization of the EgPLK2 protein was detected in E. granulosus protoscoleces and adult worms using immunofluorescence assay (IFA). Eighteen 6- to 8-week-old female SPF-grade BALB/c mice were randomly divided into three groups, including the blank control group, rEgPLK2-ISA immunization group, and PBS-ISA adjuvant control group, of 6 mice each group. Mice in the rEgPLK2-ISA immunization group and PBSISA group received three primary immunizations via intramuscular injection, and animals in the rEgPLK2-ISA immunization group was inoculated with immunogens prepared by emulsifying rEgPLK2 protein with ISA 201 adjuvant at a 1 : 1 volume ratio (6 μg per mouse), while mice in the PBS-ISA adjuvant control group received an equal volume of PBS emulsified with ISA adjuvant at a 1 : 1 volume ratio. A fourth booster immunization was administered via intraperitoneal injection. Mice in the rEgPLK2-ISA immunization group received a booster immunization with 8 μg of rEgPLK2 protein per mouse, and animals in the PBS-ISA group received an equal volume of PBS, with immunizations given at 2-week intervals. Mice in the blank control group were given no treatment, and housed under standard conditions. Tail vein blood was collected from all mice 7 days after the final immunization, and levels of specific anti-rEgPLK2 IgG antibody and its subclasses (IgG1, IgG2a, IgG2b, IgG3) were measured by indirect ELISA. E. granulosus infection was modelled in mice through injection with 1 000 E. granulosus protoscoleces via intrahepatic portal vein in the rEgPLK2-ISA immunization group and PBS-ISA adjuvant control group 2 weeks after the last immunization. All mice were sacrificed and dissected. The number of cysts was counted in mouse livers, and the cyst reduction rate was calculated. Liver tissues were processed for paraffin sectioning and stained with hematoxylin and eosin (HE), and histopathological changes were examined under a light microscope. Results Sequence analysis revealed that EgPLK2 shared a high amino acid sequence homology with E. multilocularis PLK2 (EmPLK2) and contained the typical domains of the Polo-like kinase family, including the serine/threonine protein kinase catalytic domain (STKc) and Polo-box. The IPTG-induced rEgPLK2 protein was mainly expressed in the form of inclusion bodies, and the purified rEgPLK2 protein showed a relative molecular mass of approximately 70 kDa. The prepared rabbit anti-rEgPLK2 polyclonal antibody had a titer of 1 : 256 000, and Western blotting assay showed that this anti-body specifically recognized the rEgPLK2 protein with a relative molecular mass of approximately 70 kDa. Immunofluorescence assay showed that the EgPLK2 protein was localized in the excretory bladder and rostellum of E. granulosus protoscoleces, as well as the tegument, suckers, and inter-proglottid junctions of adult worms. Immunoprotective assay showed that the serum levels of specific anti-rEgPLK2 IgG, IgG1, IgG2a, and IgG2b antibodies were 2.92 ± 0.49, 0.33 ± 0.10, 0.31 (0.36), and 3.12 (1.73) in mice in the rEgPLK2-ISA immunization group, which were all significantly higher than those in the PBS-ISA adjuvant control group (0.14 ± 0.04, 0.07 ± 0.01, 0.12 ± 0.04, and 0.11 ± 0.04, respectively) (t = 19.28 and 8.46, Z = 3.75 and 4.15; all P values < 0.001); however, there was no significant difference in the serum anti-IgG3 antibody level between the rEgPLK2-ISA immunization group and the PBS-ISA adjuvant control group [0.07 (0.01) vs. 0.073 (0.07); Z = 0.69, P > 0.05)]. In the mouse model of E. granulosus infections, the area of hepatic lesions was reduced and the inflammatory infiltration was alleviated in the rEgPLK2-ISA immunization group than in the PBS-ISA adjuvant control group, and the number of hepatic cysts was higher in the PBS-ISA adjuvant control group than in the rEgPLK2-ISA immunization group [8.00 (2.00) vs. 1.00 (0.75); Z = −2.93, P < 0.01], with a cyst reduction rate of 80.40%. Indirect ELISA assay measured higher serum levels of specific anti-rEgPLK2 IgG (3.28 ± 0.48 vs. 0.11 ± 0.04; t = 15.86, P < 0.01), IgG1 (0.29 ± 0.02 vs. 0.09 ± 0.01; t = 15.67, P < 0.01), IgG2a [3.71 (1.09) vs. 0.08 (0.03); Z = 2.88, P < 0.01], and IgG2b antibodies [3.34 (1.01) vs. 0.08 (0.03); Z = 2.88, P < 0.01] in the rEgPLK2-ISA immunization group than in the PBS-ISA adjuvant control group, and there was no significant difference in the serum level of the specific anti-rEgPLK2 IgG3 antibody between the rEgPLK2-ISA immunization group and the PBS-ISA adjuvant control group (0.07 ± 0.01 vs. 0.07 ± 0.01; t = 1.29, P > 0.05). Conclusions The prokaryotic expression system has been successfully constructed for the EgPLK2 gene and the anti-rEgPLK2 polyclonal antibody has been obtained. The rEgPLK2 protein exhibits a high immunogenicity, and is effective to protect against E. granulosus infection, and inhibits cyst development, which is a promising candidate vaccine target against cystic echinococcosis.

Objective To explore the diagnostic value of ultrasonography in ischiofemoral impinge-ment syndrome(IFI).Methods Fifty-six patients who underwent hip MRI with confirmed IFI diagnosis and completed ultrasonography examinations were enrolled as the IFI group,including 44 females and 12 males.Twenty healthy volunteers were concurrently recruited as the control group,consisting of 10 females and 10 males.The control group underwent ultrasonography examinations of bilateral hip joints,whiletheischialfemoralspace(IFS)andquadratusfemoristhickness(QFT)of both groups were measured and recorded.Then measurements were compared within(by laterality and gender)and between the two groups using independent-samples t-tests.Moreover,receiver operating characteristic adults,males exhibited significantly higher IFS and QFT values than females(P<0.05).Within the IFI group,males with affected hips had significantly higher IFS than females(P<0.05),while no sig-nificant differences were observed in QFT between different genders(P>0.05).Moreover,affected hips in the IFI group showed significantly narrower IFS and thicker QFT compared to both contralateral hips and the control group(P<0.001).In addition,the diagnostic cut-off values of IFS and QFT for ultrasound diagnosis of IFI were 22.93 mm and 16.48 mm,respectively.At these thresholds,the ar-eas under the curve(AUC)were 0.997 and 0.977,with sensitivities of 97.8%and 91.8%,and speci-ficities of 98.4%and 97.8%,respectively.Conclusion Ultrasound can serve as a reliable diagnostic technique for IFI,where narrowing of the IFS and thickening of the QFT should raise suspicion of this condition.

Objective To investigate the effect of quercetin on paraptosis induced by low-concentration hydrogen peroxide(H2O2)in human lens epithelial cells(LECs).Methods The human lens epithelial cell line SRA01/04 was cul-tured in vitro and randomly divided into the following groups:control group,H2O2 group(50 μmol·L-1 H2O2),and H2O2+Quercetin group(50 μmol·L-1 H2O2 and 20 μmol·L-1 quercetin).The area and number of endoplasmic reticulum vacuoles in the cells were observed using transmission electron microscopy and super-resolution laser confocal microscopy.Intracellular reactive oxygen species(ROS)levels were detected using the DCFH-DA fluorescent probe.Western blot was performed to detect the expression levels of the following proteins:insulin-like growth factor 1 receptor(IGF1R),phos-phorylated IGF1R(p-IGF1R),ALG-2-interacting protein X(ALIX),glucose-regulated protein 78(GRP78),extracellular signal-regulated kinase(ERK),phosphorylated ERK(p-ERK),P38,and phosphorylated P38(p-P38).Additionally,len-ses from 8-week-old Sprague-Dawley rats were subjected to ex vivo culture and divided into the normal group,the H2O2 treatment group(50 μmol·L-1 H2O2),and the combination treatment group(50 μmol·L-1 H2O2 and 20 μmol·L-1 quer-cetin).The degree of lens opacity was observed under a stereomicroscope,and the area of lens opacity was quantified.The ultrastructure of the endoplasmic reticulum in the rat LECs was observed by transmission electron microscopy.Western blot was used to detect the expression levels of the aforementioned proteins in the rat LECs.Results Compared with the control group,the H2O2 group exhibited significant increases in the area and number of endoplasmic reticulum vacuoles,ROS levels,and the relative protein expression levels of p-IGF1R,p-ERK,p-P38,and GRP78,while the relative expression of ALIX protein was decreased(all P＜0.001).Compared with the H2 O2 group,the H2O2+quercetin group showed signifi-cant reductions in the area and number of endoplasmic reticulum vacuoles,ROS levels,and the relative expression levels of p-IGF1R,p-ERK,p-P38,and GRP78,while the relative expression of ALIX protein was increased(all P＜0.01).In the ex vivo cultured rat lens model,compared with the normal group,the H2O2 group displayed a significant increase in lens opac-ity area,expanded endoplasmic reticulum area in LECs,elevated relative expression levels of p-IGF1R,p-ERK,p-P38,and GRP78 proteins,and decreased ALIX expression(all P＜0.000 1).In contrast,the combination treatment group showed significantly reduced lens opacity area,decreased endoplasmic reticulum area in LECs,lower relative expression of p-IGF1R,p-ERK,p-P38,and GRP78,and increased ALIX expression compared to the H2O2 group(all P＜0.01).Conclu-sion Quercetin inhibits activation of the IGF1R/MAPK signaling pathway and alleviates endoplasmic reticulum stress,thereby effectively attenuating paraptosis in lens epithelial cells.These findings provide a novel strategy for the prevention and treatment of early age-related cataract.

Objective To investigate the effect of quercetin on paraptosis induced by low-concentration hydrogen peroxide(H2O2)in human lens epithelial cells(LECs).Methods The human lens epithelial cell line SRA01/04 was cul-tured in vitro and randomly divided into the following groups:control group,H2O2 group(50 μmol·L-1 H2O2),and H2O2+Quercetin group(50 μmol·L-1 H2O2 and 20 μmol·L-1 quercetin).The area and number of endoplasmic reticulum vacuoles in the cells were observed using transmission electron microscopy and super-resolution laser confocal microscopy.Intracellular reactive oxygen species(ROS)levels were detected using the DCFH-DA fluorescent probe.Western blot was performed to detect the expression levels of the following proteins:insulin-like growth factor 1 receptor(IGF1R),phos-phorylated IGF1R(p-IGF1R),ALG-2-interacting protein X(ALIX),glucose-regulated protein 78(GRP78),extracellular signal-regulated kinase(ERK),phosphorylated ERK(p-ERK),P38,and phosphorylated P38(p-P38).Additionally,len-ses from 8-week-old Sprague-Dawley rats were subjected to ex vivo culture and divided into the normal group,the H2O2 treatment group(50 μmol·L-1 H2O2),and the combination treatment group(50 μmol·L-1 H2O2 and 20 μmol·L-1 quer-cetin).The degree of lens opacity was observed under a stereomicroscope,and the area of lens opacity was quantified.The ultrastructure of the endoplasmic reticulum in the rat LECs was observed by transmission electron microscopy.Western blot was used to detect the expression levels of the aforementioned proteins in the rat LECs.Results Compared with the control group,the H2O2 group exhibited significant increases in the area and number of endoplasmic reticulum vacuoles,ROS levels,and the relative protein expression levels of p-IGF1R,p-ERK,p-P38,and GRP78,while the relative expression of ALIX protein was decreased(all P＜0.001).Compared with the H2 O2 group,the H2O2+quercetin group showed signifi-cant reductions in the area and number of endoplasmic reticulum vacuoles,ROS levels,and the relative expression levels of p-IGF1R,p-ERK,p-P38,and GRP78,while the relative expression of ALIX protein was increased(all P＜0.01).In the ex vivo cultured rat lens model,compared with the normal group,the H2O2 group displayed a significant increase in lens opac-ity area,expanded endoplasmic reticulum area in LECs,elevated relative expression levels of p-IGF1R,p-ERK,p-P38,and GRP78 proteins,and decreased ALIX expression(all P＜0.000 1).In contrast,the combination treatment group showed significantly reduced lens opacity area,decreased endoplasmic reticulum area in LECs,lower relative expression of p-IGF1R,p-ERK,p-P38,and GRP78,and increased ALIX expression compared to the H2O2 group(all P＜0.01).Conclu-sion Quercetin inhibits activation of the IGF1R/MAPK signaling pathway and alleviates endoplasmic reticulum stress,thereby effectively attenuating paraptosis in lens epithelial cells.These findings provide a novel strategy for the prevention and treatment of early age-related cataract.

Objective To analyze the relationship between serum levels of Clara cell protein 16(Cc16),E-selectin(CD62E),the severity of neonatal respiratory distress syndrome(NRDS)and lung ultrasound scores.Methods A total of 200 neonates with NRDS were selected in this study.The severity of the disease was assessed using oxygenation index(P/F).The neonates were divided into three groups:the mild group(46 cases),the moderate group(65 cases)and the severe group(89 cases).The serum levels of Cc16 and CD62E were compared between the three groups.In addition,based on the lung ultrasound scores,neonates were divided into the low-score group(score＜12 points,n=127),the medium-score group(score 12-24 points,n=51)and the high-score group(score>24 points,n=22).The receiver operating characteristic(ROC)curve was used to evaluate the predictive efficacy of the combined detection of serum Cc16,CD62E and lung ultrasound score for severe NRDS.Results Compared with the mild group,the serum levels of Cc16 and CD62E,and lung ultrasound scores were higher in the moderate group and the severe group(P＜0.05).Compared with the low lung ultrasound score group,the serum levels of Cc16 and CD62E were higher in the moderate lung ultrasound score group and the high lung ultrasound score groups(P＜0.05).Cc16 and CD62E were positively correlated with the severity of NRDS(rs=0.679 and 0.680,P＜0.01),and they were also positively correlated with lung ultrasound scores(r=0.692 and 0.685,P＜0.01).ROC analysis showed that the combined detection of Cc16,CD62E and lung ultrasound scores had a relatively high diagnostic value for severe NRDS(P＜0.05).Conclusion The serum levels of Cc16 and CD62E in NRDS neonates are positively correlated with disease severity and lung ultrasound scores.

Objective To analyze the relationship between serum levels of Clara cell protein 16(Cc16),E-selectin(CD62E),the severity of neonatal respiratory distress syndrome(NRDS)and lung ultrasound scores.Methods A total of 200 neonates with NRDS were selected in this study.The severity of the disease was assessed using oxygenation index(P/F).The neonates were divided into three groups:the mild group(46 cases),the moderate group(65 cases)and the severe group(89 cases).The serum levels of Cc16 and CD62E were compared between the three groups.In addition,based on the lung ultrasound scores,neonates were divided into the low-score group(score＜12 points,n=127),the medium-score group(score 12-24 points,n=51)and the high-score group(score>24 points,n=22).The receiver operating characteristic(ROC)curve was used to evaluate the predictive efficacy of the combined detection of serum Cc16,CD62E and lung ultrasound score for severe NRDS.Results Compared with the mild group,the serum levels of Cc16 and CD62E,and lung ultrasound scores were higher in the moderate group and the severe group(P＜0.05).Compared with the low lung ultrasound score group,the serum levels of Cc16 and CD62E were higher in the moderate lung ultrasound score group and the high lung ultrasound score groups(P＜0.05).Cc16 and CD62E were positively correlated with the severity of NRDS(rs=0.679 and 0.680,P＜0.01),and they were also positively correlated with lung ultrasound scores(r=0.692 and 0.685,P＜0.01).ROC analysis showed that the combined detection of Cc16,CD62E and lung ultrasound scores had a relatively high diagnostic value for severe NRDS(P＜0.05).Conclusion The serum levels of Cc16 and CD62E in NRDS neonates are positively correlated with disease severity and lung ultrasound scores.

OBJECTIVES: To analyze MYO1B expression in gastric cancer, its association with long-term prognosis and its role in regulating biological behaviors of gastric cancer cells. METHODS: We analyzed MYO1B expression in gastric cancer and its correlation with tumor grade, tumor stage, and patient survival using the Cancer Public Database. We also examined MYO1B expression with immunohistochemistry in gastric cancer and paired adjacent tissues from 105 patients receiving radical surgery and analyzed its correlation with cancer progression and postoperative 5-year survival of the patients. GO and KEGG enrichment analyses were used to explore the biological functions of MYO1B and the key pathways. In cultured gastric cancer cells, we examined the changes in cell proliferation, migration and invasion following MYO1B overexpression and knockdown. RESULTS: Data from the Cancer Public Database showed that MYO1B expression was significantly higher in gastric cancer tissues than in normal tissues with strong correlations with tumor grade, stage and patient prognosis (P<0.05). In the clinical tissue samples, MYO1B was significantly overexpressed in gastric cancer tissues in positive correlation with Ki67 expression (r=0.689, P<0.05) and the parameters indicative of gastric cancer progression (CEA ≥5 μg/L, CA19-9 ≥37 kU/L, G3-4, T3-4, and N2-3) (P<0.05). Kaplan-Meier analysis and multivariate Cox regression analysis suggested that high MYO1B expression was associated with decreased postoperative 5-year survival and was an independent risk factor (HR: 3.522, 95%CI: 1.783-6.985, P<0.05). MYO1B expression level was a strong predictor of postoperative survival (cut-off value: 3.11, AUC: 0.753, P<0.05). GO and KEGG analyses suggested that MYO1B may regulate cell migration and the mTOR signaling pathway. In cultured gastric cancer cells, MYO1B overexpression significantly enhanced cell proliferation, migration, and invasion and promoted the phosphorylation of Akt and mTOR. CONCLUSIONS High MYO1B expression promotes proliferation, migration and invasion of gastric cancer cells and is correlated with poor patient prognosis.
Humans ; Stomach Neoplasms/metabolism* ; Cell Proliferation ; Prognosis ; Cell Movement ; Myosin Type I/genetics* ; Neoplasm Invasiveness ; Cell Line, Tumor ; Female ; Male

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Objective To explore the diagnostic value of ultrasonography in ischiofemoral impinge-ment syndrome(IFI).Methods Fifty-six patients who underwent hip MRI with confirmed IFI diagnosis and completed ultrasonography examinations were enrolled as the IFI group,including 44 females and 12 males.Twenty healthy volunteers were concurrently recruited as the control group,consisting of 10 females and 10 males.The control group underwent ultrasonography examinations of bilateral hip joints,whiletheischialfemoralspace(IFS)andquadratusfemoristhickness(QFT)of both groups were measured and recorded.Then measurements were compared within(by laterality and gender)and between the two groups using independent-samples t-tests.Moreover,receiver operating characteristic adults,males exhibited significantly higher IFS and QFT values than females(P<0.05).Within the IFI group,males with affected hips had significantly higher IFS than females(P<0.05),while no sig-nificant differences were observed in QFT between different genders(P>0.05).Moreover,affected hips in the IFI group showed significantly narrower IFS and thicker QFT compared to both contralateral hips and the control group(P<0.001).In addition,the diagnostic cut-off values of IFS and QFT for ultrasound diagnosis of IFI were 22.93 mm and 16.48 mm,respectively.At these thresholds,the ar-eas under the curve(AUC)were 0.997 and 0.977,with sensitivities of 97.8%and 91.8%,and speci-ficities of 98.4%and 97.8%,respectively.Conclusion Ultrasound can serve as a reliable diagnostic technique for IFI,where narrowing of the IFS and thickening of the QFT should raise suspicion of this condition.

Objective To investigate the expression level of basic leucine zipper and W2 domain-containing protein 1(BZW1)in gastric cancer,its impact on patient prognosis and the underlying mechanisms.Methods TIMER,UALCAN and Kaplan-Meier Plotter databases were used for analyzing BZW1 expression level gastric cancer tissues and its correlation with tumor grade and stage and the patients'prognosis.We further analyzed BZW1 expressions,disease progression,and postoperative 5-year survival in 102 patients undergoing radical surgery for gastric cancer at our hospital between January,2014 and December,2016.Gastric cancer MGC803 cells were examined for changes in migration,invasion,and epithelial-mesenchymal transition(EMT)following lentivirus-mediated BZW1 overexpression or knockdown.Results The protein and mRNA expressions of BZW1 in gastric cancer tissues were 3.30 and 6.54 times of those in adjacent tissues,respectively(P<0.01).BZW1 expression in gastric cancer tissues were positively correlated with peripheral blood CEA and CA199 levels(P<0.01).A high BZW1 expression was an independent risk factor for 5-year survival of gastric cancer patients after radical surgery(P<0.05,HR=2.070,95%CI:1.021-4.196).At the cut-off value of 3.61,BZW1 expression had a sensitivity of 75.56%and a specificity of 71.93%for predicting postoperative 5-year mortality(P<0.01).In MGC803 cells,BZW1 overexpression obviously promoted cell migration and invasion(P<0.05),enhanced cellular expressions of N-cadherin and vimentin(P<0.05)and inhibited the expression of E-cadherin(P<0.05).Enrichment analysis suggested the involvement of BZW1 in the Wnt/β-catenin signaling pathway.Western blotting confirmed that BZW1 overexpression promoted while BZW1 knockdown inhibited the expressions of Wnt3a,β-catenin and C-myc in MGC803 cells(P<0.05).Conclusion BZW1 is highly expressed in gastric cancer tissues to affect the patient prognosis possibly by activation the Wnt/β-catenin signaling pathway to promote EMT of gastric cancer cells.