Objective:To obtain a prevalent respiratory syncytial virus (RSV) clinical isolate in Beijing and analyze the genotype and biological characteristics of the strain.Methods:A nasopharyngeal secretion specimen was collected from a child with RSV infection in Beijing in 2023 and used for viral isolation. Viral nucleic acid was amplified using qRT-PCR. The isolated virus was identified by transmission electron microscopy, indirect immunofluorescence assay, and plaque formation assay. A phylogenetic analysis was conducted based on the whole-genome sequencing results. Virus titers were determined, and replication characteristics were analyzed. The efficacy of the isolated strain for in vitro screening of antiviral drugs was validated. Results:A clinical RSV isolate, named hRSV/C-Tan/BJ 202301, was successfully isolated, which could form syncytia in Hep-2 cells. Spherical, filamentous, and irregular virus particles were observed by electron microscopy. Immunofluorescence detection showed green fluorescence in Hep-2 cells, and plaque assay showed round plaques, which were similar to the Long strain in morphology. Genomic sequence analysis showed that it belonged to ON1 genotype. It exhibited similar cell growth kinetics characteristics with the Long strain and could be used for antiviral drug screening in vitro. Conclusions:In this study, one RSV strain is successfully isolated and identified. The biological characteristics and the phylogenetic relationship of this strain reflect the characteristics of the circulating strains in Beijing, which provides experimental material for RSV vaccine development and antiviral drug screening in China.

Objective:To establish and optimize a novel method, focus forming assay (FFA), for quantitation of influenza A virus (FluA) and compare its application performance with traditional plague forming assay (PFA).Methods:The foci chromogenic effects of three peroxidase substrates in immunostaining were compared. The PFA and FFA methods were used to explore FluA incubation times and plaque morphology on 12-well plates, and to determine optimal incubation times and virus adsorption volumes for different FluA subtypes on 96-well plates. The correlation between FFA and PFA was evaluated, and the optimized FFA was applied to the in vitro antiviral efficacy analysis of Favipiravir and neutralization test against different subtypes of FluA. Results:TRUEBLUE substrate was identified as the optimal substrate for foci visualization. Compared with the PFA, the FFA showed improved sensitivity and reduced detection time in FluA titration, and good correlation was shown between the two methods′ results. By replacing the 96-well plate with the 12-well plate for FFA titration of different subtypes of FluA, the detection time was shortened, and the amount of serum samples used could be further reduced by optimizing the virus adsorption volume. The half-maximal effective concentration of favipiravir against influenza viruses assessed by the FFA and PFA methods showed no significant difference, and was consistent with the results obtained from quantitative PCR. Additionally, the focus reduction neutralization test and hemagglutination inhibition assays demonstrated strong correlation in determining antibody titers against FluA in serum neutralization assays.Conclusions:The improved FFA method developed here provides a more efficient experimental tool for FluA titration, antiviral drug screening and broad-spectrum vaccine evaluation.

Objective:To obtain a prevalent respiratory syncytial virus (RSV) clinical isolate in Beijing and analyze the genotype and biological characteristics of the strain.Methods:A nasopharyngeal secretion specimen was collected from a child with RSV infection in Beijing in 2023 and used for viral isolation. Viral nucleic acid was amplified using qRT-PCR. The isolated virus was identified by transmission electron microscopy, indirect immunofluorescence assay, and plaque formation assay. A phylogenetic analysis was conducted based on the whole-genome sequencing results. Virus titers were determined, and replication characteristics were analyzed. The efficacy of the isolated strain for in vitro screening of antiviral drugs was validated. Results:A clinical RSV isolate, named hRSV/C-Tan/BJ 202301, was successfully isolated, which could form syncytia in Hep-2 cells. Spherical, filamentous, and irregular virus particles were observed by electron microscopy. Immunofluorescence detection showed green fluorescence in Hep-2 cells, and plaque assay showed round plaques, which were similar to the Long strain in morphology. Genomic sequence analysis showed that it belonged to ON1 genotype. It exhibited similar cell growth kinetics characteristics with the Long strain and could be used for antiviral drug screening in vitro. Conclusions:In this study, one RSV strain is successfully isolated and identified. The biological characteristics and the phylogenetic relationship of this strain reflect the characteristics of the circulating strains in Beijing, which provides experimental material for RSV vaccine development and antiviral drug screening in China.

Objective:To establish and optimize a novel method, focus forming assay (FFA), for quantitation of influenza A virus (FluA) and compare its application performance with traditional plague forming assay (PFA).Methods:The foci chromogenic effects of three peroxidase substrates in immunostaining were compared. The PFA and FFA methods were used to explore FluA incubation times and plaque morphology on 12-well plates, and to determine optimal incubation times and virus adsorption volumes for different FluA subtypes on 96-well plates. The correlation between FFA and PFA was evaluated, and the optimized FFA was applied to the in vitro antiviral efficacy analysis of Favipiravir and neutralization test against different subtypes of FluA. Results:TRUEBLUE substrate was identified as the optimal substrate for foci visualization. Compared with the PFA, the FFA showed improved sensitivity and reduced detection time in FluA titration, and good correlation was shown between the two methods′ results. By replacing the 96-well plate with the 12-well plate for FFA titration of different subtypes of FluA, the detection time was shortened, and the amount of serum samples used could be further reduced by optimizing the virus adsorption volume. The half-maximal effective concentration of favipiravir against influenza viruses assessed by the FFA and PFA methods showed no significant difference, and was consistent with the results obtained from quantitative PCR. Additionally, the focus reduction neutralization test and hemagglutination inhibition assays demonstrated strong correlation in determining antibody titers against FluA in serum neutralization assays.Conclusions:The improved FFA method developed here provides a more efficient experimental tool for FluA titration, antiviral drug screening and broad-spectrum vaccine evaluation.

Objective:To explore the application effect of the attention and interpretation therapy based on Swanson′s caring theory in adolescents with epilepsy, aiming to reduce negative emotions and improve self-management levels in children.Methods:This study was a quasi-experimental research. A convenience sampling method was used to select 78 adolescents with epilepsy who were admitted to the Department of Neurology, the Affiliated Children′s Hospital of Xiangya School of Medicine, Central South University (Hunan Children′s Hospital) from March 1 to August 31, 2023. The participants were assigned into two groups, the control group ( n=39) and the intervention group ( n=39), using a random number table. The control group received standard anti-epileptic treatment and routine nursing care, while the intervention group received the same treatment and care with the addition of a caring team providing attention and interpretation therapy based on Swanson′s caring theory. Assessments were made at three time points: before the intervention, 4 weeks after the intervention, and 12 weeks after the intervention. The Depression-Anxiety-Stress Scale (DASS-21), Epilepsy Self-Management Scale (ESMS), and Pediaric Quality of Life Inventory Measurement Methods (PedsQL TM4.0) were used to assess the participants′ scores. Results:In the control group, 23 were male and 16 were female, with aged of (13.49 ± 1.43) years. In the intervention group, 24 were male and 15 were female, with aged of (13.51 ± 1.45) years. After 4 weeks of intervention, the scores of depression, anxiety, and stress on the DASS-21 for the intervention group were (9.32 ± 2.04), (6.79 ± 2.44), and (13.63 ± 2.67), respectively, which were lower than those of the control group: (10.43 ± 2.27), (8.43 ± 2.75) and (16.05 ± 3.32); the scores of ESMS and PedsQL TM4.0 in the intervention group were (66.66 ± 10.87) and (73.63 ± 9.85), respectively, which were higher than those of the control group: (60.70 ± 10.24) and (67.27 ± 12.33), the differences were statistically significant ( t values were -2.47 to 3.49, all P<0.05). After 12 weeks of intervention, the scores on the DASS-21 for the intervention group were (5.47 ± 1.66), (3.37 ± 1.68) and (8.42 ± 2.24) for depression, anxiety and stress, respectively, which were lower than those of the control group: (8.03 ± 1.98), (6.06 ± 2.64) and (11.28 ± 3.21) ;the scores for ESMS and PedsQL TM4.0 in the intervention group were (74.66 ± 9.15) and (85.24 ± 7.00), respectively, which were higher than those of the control group: (65.36 ± 10.86) and (75.67 ± 11.39), the differences were statistically significant ( t values were -4.33 to 6.03, all P<0.05). Additionally, there were statistically significant differences in the DASS-21 depression, anxiety, and stress scores, ESMS, and PedsQL TM4.0 scores across the different time points ( F values ranged from 4.65 to 1 134.03, all P<0.05). Conclusions:Attention and interpretation therapy based on Swanson′s caring theory can effectively reduce negative emotions in adolescents with epilepsy, improve their self-management skills, and further enhance their quality of life.

Objective:To explore the application effect of the attention and interpretation therapy based on Swanson′s caring theory in adolescents with epilepsy, aiming to reduce negative emotions and improve self-management levels in children.Methods:This study was a quasi-experimental research. A convenience sampling method was used to select 78 adolescents with epilepsy who were admitted to the Department of Neurology, the Affiliated Children′s Hospital of Xiangya School of Medicine, Central South University (Hunan Children′s Hospital) from March 1 to August 31, 2023. The participants were assigned into two groups, the control group ( n=39) and the intervention group ( n=39), using a random number table. The control group received standard anti-epileptic treatment and routine nursing care, while the intervention group received the same treatment and care with the addition of a caring team providing attention and interpretation therapy based on Swanson′s caring theory. Assessments were made at three time points: before the intervention, 4 weeks after the intervention, and 12 weeks after the intervention. The Depression-Anxiety-Stress Scale (DASS-21), Epilepsy Self-Management Scale (ESMS), and Pediaric Quality of Life Inventory Measurement Methods (PedsQL TM4.0) were used to assess the participants′ scores. Results:In the control group, 23 were male and 16 were female, with aged of (13.49 ± 1.43) years. In the intervention group, 24 were male and 15 were female, with aged of (13.51 ± 1.45) years. After 4 weeks of intervention, the scores of depression, anxiety, and stress on the DASS-21 for the intervention group were (9.32 ± 2.04), (6.79 ± 2.44), and (13.63 ± 2.67), respectively, which were lower than those of the control group: (10.43 ± 2.27), (8.43 ± 2.75) and (16.05 ± 3.32); the scores of ESMS and PedsQL TM4.0 in the intervention group were (66.66 ± 10.87) and (73.63 ± 9.85), respectively, which were higher than those of the control group: (60.70 ± 10.24) and (67.27 ± 12.33), the differences were statistically significant ( t values were -2.47 to 3.49, all P<0.05). After 12 weeks of intervention, the scores on the DASS-21 for the intervention group were (5.47 ± 1.66), (3.37 ± 1.68) and (8.42 ± 2.24) for depression, anxiety and stress, respectively, which were lower than those of the control group: (8.03 ± 1.98), (6.06 ± 2.64) and (11.28 ± 3.21) ;the scores for ESMS and PedsQL TM4.0 in the intervention group were (74.66 ± 9.15) and (85.24 ± 7.00), respectively, which were higher than those of the control group: (65.36 ± 10.86) and (75.67 ± 11.39), the differences were statistically significant ( t values were -4.33 to 6.03, all P<0.05). Additionally, there were statistically significant differences in the DASS-21 depression, anxiety, and stress scores, ESMS, and PedsQL TM4.0 scores across the different time points ( F values ranged from 4.65 to 1 134.03, all P<0.05). Conclusions:Attention and interpretation therapy based on Swanson′s caring theory can effectively reduce negative emotions in adolescents with epilepsy, improve their self-management skills, and further enhance their quality of life.

AIM:To investigate the inhibitory ef-fect and mechanism of hesperetin(HST)on human gefitinib-resistant NCI-H1975 lung adenocarcinoma cells.METHODS:CCK-8 assay was used to detect the effects of HST on the proliferation of NCI-H1975 cells;Annexin V-FITC/PI double staining was used to detect HST-induced apoptosis of NCI-H1975 cells;flow cytometry was used to observe the effects of HST and HST+acetylcysteine(NAC)combined on the levels of reactive oxygen species(ROS)in NCI-H1975 cells;Western blot was used to detect the expression of Bcl-2,Bax,Cleaved Cas-pase-3,p-eIF2α,eIF2α and CHOP proteins in NCI-H1975 cells by HST,NAC+HST and Salubrinal+HST.The antitumor effect of HST in vivo was stud-ied by constructing a xenograft model in nude mice;HE staining was used to observe the effect of HST on the histopathological morphology of heart,liver,kidney and xenograft in tumor-bearing mice;immunohistochemistry was used to detect the ef-fect of HST on p-eIF2α protein in xenograft tissues.RESULTS:Compared with the control group,HST at 37.5 μmol/L for 24 h significantly inhibited the via-bility of NCI-H1975 cells(P＜0.05),and NAC attenu-ated the inhibitory effect of HST;concentrations greater than 150 μmol/L increased intracellular ROS levels(P＜0.05),induced apoptosis(P＜0.05),and increased Caspase3 activity(P＜0.01),and com-pared with HST 300 μmol/L group,ROS levels,apoptosis rate,and Caspase3 activity were signifi-cantly decreased in NAC+HST 300 μmol/L group(P＜0.01);HST up-regulated Bax,Cleaved Caspase-3,CHOP,and p-elF2α expression and down-regulated Bcl-2 expression in a concentration-dependent manner(P＜0.01),and compared with HST 300μmol/L group,Bax and Cleaved Caspase-3 expres-sion was decreased and Bcl-2 expression was in-creased in Sal+HST 300 μmol/L group;p-eIF2αand CHOP expression were significantly down-regu-lated in the NAC+HST 300 μmol/L and Sal+HST 300 μmol/L groups(P＜0.01).In vivo,experiments showed that HST could significantly inhibit the growth of transplanted tumors and up-regulate p-eIF2α protein expression(P＜0.05),and had no sig-nificant adverse effects on the growth status,body weight and important organs(heart,liver and kid-ney)of nude mice.CONCLUSION:HST inhibits the proliferation of gefitinib-resistant NCI-H1975 lung adenocarcinoma cells in vitro and in vivo,and the mechanism may be related to HST mediating ER stress-induced apoptosis of NCI-H1975 cells through ROS.

AIM:To investigate the inhibitory ef-fect and mechanism of hesperetin(HST)on human gefitinib-resistant NCI-H1975 lung adenocarcinoma cells.METHODS:CCK-8 assay was used to detect the effects of HST on the proliferation of NCI-H1975 cells;Annexin V-FITC/PI double staining was used to detect HST-induced apoptosis of NCI-H1975 cells;flow cytometry was used to observe the effects of HST and HST+acetylcysteine(NAC)combined on the levels of reactive oxygen species(ROS)in NCI-H1975 cells;Western blot was used to detect the expression of Bcl-2,Bax,Cleaved Cas-pase-3,p-eIF2α,eIF2α and CHOP proteins in NCI-H1975 cells by HST,NAC+HST and Salubrinal+HST.The antitumor effect of HST in vivo was stud-ied by constructing a xenograft model in nude mice;HE staining was used to observe the effect of HST on the histopathological morphology of heart,liver,kidney and xenograft in tumor-bearing mice;immunohistochemistry was used to detect the ef-fect of HST on p-eIF2α protein in xenograft tissues.RESULTS:Compared with the control group,HST at 37.5 μmol/L for 24 h significantly inhibited the via-bility of NCI-H1975 cells(P＜0.05),and NAC attenu-ated the inhibitory effect of HST;concentrations greater than 150 μmol/L increased intracellular ROS levels(P＜0.05),induced apoptosis(P＜0.05),and increased Caspase3 activity(P＜0.01),and com-pared with HST 300 μmol/L group,ROS levels,apoptosis rate,and Caspase3 activity were signifi-cantly decreased in NAC+HST 300 μmol/L group(P＜0.01);HST up-regulated Bax,Cleaved Caspase-3,CHOP,and p-elF2α expression and down-regulated Bcl-2 expression in a concentration-dependent manner(P＜0.01),and compared with HST 300μmol/L group,Bax and Cleaved Caspase-3 expres-sion was decreased and Bcl-2 expression was in-creased in Sal+HST 300 μmol/L group;p-eIF2αand CHOP expression were significantly down-regu-lated in the NAC+HST 300 μmol/L and Sal+HST 300 μmol/L groups(P＜0.01).In vivo,experiments showed that HST could significantly inhibit the growth of transplanted tumors and up-regulate p-eIF2α protein expression(P＜0.05),and had no sig-nificant adverse effects on the growth status,body weight and important organs(heart,liver and kid-ney)of nude mice.CONCLUSION:HST inhibits the proliferation of gefitinib-resistant NCI-H1975 lung adenocarcinoma cells in vitro and in vivo,and the mechanism may be related to HST mediating ER stress-induced apoptosis of NCI-H1975 cells through ROS.

Objective:To investigate the effect and mechanism of NOD-like receptor family pyrin domain containing 12 (NLRP12) knockdown on inflammatory factor levels and retinal injury in retinal ganglion cells (RGCs) of rats with high intraocular pressure.Methods:Seventy SPF adult male SD rats were selected and randomized into control group, high intraocular pressure (IOP) group, high IOP+ small interfering RNA negative control (siNC) group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ recombinant rat caspase-1 (rrcaspase-1) group, with 14 rats in each group.Rats in the control group were only treated with conjunctival incision in the right eye, and ocular hypertension model was established in the other four groups with external scleral vein cauterization.High IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group were injected with siNC, siNLRP12 and siNLRP12+ rrcaspase-1 reagent via the tail vein, respectively.The IOP of the right eye was measured at 1 day, 1, 2 and 3 weeks after the operation.Three weeks after the operation, the retinal structure was observed by hematoxylin-eosin staining, and the number of RGCs in each group was counted.RGCs were divided into control group, rrcaspase-1 group, siNC+ rrcaspase-1 group, siNLRP12+ rrcaspase-1 group.The cells in rrcaspase-1 group, siNC+ rrcaspase-1 group and siNLRP12+ rrcaspase-1 group were treated with rrcaspase-1, siNC+ rrcaspase-1 and siNLRP12+ rrcaspase-1 reagent for 24 hours, respectively.No treatment was given to the control group.The expression levels of NLRP12, caspase-1 and cleaved-caspase-1 proteins in RGCs and retinal tissue were detected by Western blot.The concentrations of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in rat serum or cell culture supernatant were detected by enzyme-linked immunosorbent assay.The study protocol was approved by the Animal Ethics Committee of the First People's Hospital of Chenzhou (No.2020086).Results:Compared with control group, the IOP was higher in high IOP group at 1, 2 and 3 weeks after cauterization, and the differences were statistically significant (all at P<0.05). The retinal tissue was clear with the RGCs in a single layer arrangement in the control group.In the high IOP group and the high IOP+ siNC group, the RGCs layer was loose and the inner plexiform layer was thin.The inner plexiform layer was thickened in high IOP+ siNLRP12 group compared with high IOP group, and the RGCs layer was loose in the high IOP+ siNLRP12 group and the high IOP+ siNLRP12+ rrcaspase-1 group.The number of RGCs in control group, high IOP group, high IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group was 119.31±23.25, 89.19±16.98, 88.87±13.92, 109.33±10.25 and 92.89±12.58, respectively, showing a statistically significant overall difference ( F=201.932, P<0.001). The number of RGCs was lower in the high IOP group, high IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group than the control group, higher in the high IOP+ siNLRP12 group than the high IOP+ siNC group, and lower in the high IOP+ siNLRP12+ rrcaspase-1 group than the high IOP+ siNLRP12 group, and the differences were statistically significant (all at P<0.05). The relative expressions of caspase-1 and cleaved-caspase-1 proteins and the concentrations of TNF-α and IL-1β in the retinal tissue were higher in high IOP group, high IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group than control group, higher in high IOP+ siNLRP12 group than high IOP+ siNC group, and higher in high IOP+ siNLRP12+ rrcaspase-1 group than high IOP+ siNLRP12 group (all at P<0.05). Relative expression levels of caspase-1 and cleaved-caspase-1 protein were increased in rrcaspase-1 group and siNC+ rrcaspase-1 group compared with control group, and relative expression levels of NLRP12, caspase-1 and cleaved-caspase-1 protein were decreased in siNLRP12+ rrcaspase-1 group compared with control group (all at P<0.05). The relative mass concentrations of TNF-α and IL-1β were increased in rrcaspase-1 group, siNC+ rrcaspase-1 group and siNLRP12+ rrcaspase-1 group compared with the control group (all at P<0.05). Relative expression levels of NLRP12, caspase-1 and cleaved-caspase-1 proteins and relative mass concentrations of TNF-α and IL-1β in siNLRP12+ rrcaspase-1 group were lower than those in siNC+ rrcaspase-1 group (all at P<0.05). Conclusions:Knockdown of NLRP12 can reduce the inflammatory response and retinal injury induced by high IOP by inhibiting the activation of caspase-1.

Objective:To explore the intervention effect of motivational interviews based on timing theory on self-efficacy, negative affect and coping styles of parents with infantile spasms children.Methods:Cluster sampling was used to select 82 parents of infantile spasms hospitalized in the Department of Neurology of a children’s hospital, a three-A hospital from January 2019 to October 2019. They were divided into control group and observation group with 41 cases each according to random number table. The control group received routine health education, and the observation group received five motivational interviews based on timing theory interventions on the basis of routine care. The effect of the intervention was evaluated by General Self-Efficacy Scale (GSES), Hospital Anxiety and Depression Scale (HADS), and the Chinese version of Coping Health Inventory for Parents (CHIP) before intervention, on the day of discharge, and 3 months after discharge.Results:Before the intervention, there was no significant difference in the scores of GSES, HADS and CHIP scales between the parents of the two groups ( P>0.05). After intervention, The GSES scores of the observation group on the day of discharge and 3 months after discharge were (19.63±0.87) and (22.58±1.28) points, which were significantly higher than (18.92±0.74) and (19.46±1.25) points of the control group. The difference between both groups was statistically significant ( t values were -3.865, -10.926, P<0.01). HADS-A/HADS-D scores of the observation group on the day of discharge and 3 months after discharge were (12.50±0.82), (10.50±0.87) and (9.78±0.80), (8.63±0.87) points, respectively. The HADS-A/HADS-D scores of the control group on the day of discharge and 3 months after discharge were (12.92±0.74), (11.72±0.99) and (10.23±0.78), (9.38±1.04) points, respectively. The difference was statistically significant ( t values were 2.412-5.764, P<0.05 or 0.01). The observation group scores on CHIP subscales on the day of discharge and 3 months after discharge are higher than the control group, the difference was statistically significant (t values were -7.93--2.490, P<0.05 or 0.01). Conclusions:Motivational interviews based on timing theory can enhance parents’ self-efficacy, improve their negative emotions and family coping styles, and thereby promote the recovery of children.