Objective To construct Raji-Luc lymphoma cells with CD19 knockout using CRISPR/Cas9 technology and preliminarily validate their immune escape ability.Methods PB-CRISPR-CD19 small guide RNA(sgRNA)plasmids was constructed,the optimal sgRNA sequence was screened,and Raji-Luc cells with pCAG-PBase,PB-CD19 sgRNA,and PB-CRISPR-Cas9 were co-transfected.Stable knockout monoclonal cell lines were screened by flow sorting and limit dilution method and the knockout effect was verified through gene sequence testing.The expression of luciferase on the surface of the cell line was detected by microplate reader,CD19 CAR-T and CD38 CAR-T previously constructed in the laboratory were used as effector cells,and the immune escape ability of Raji-Luc CD19 KO cell line was verified by universal luciferase chemiluminescence method.Results The transfection efficiency of Raji-Luc CD19 KO cells prepared by electro transfection was high,and the knockout efficiency of the two monoclonal cells was more than 99%.There was no significant difference in luciferase expression compared to the original Raji-Luc cells,and CD19 CAR-T cells could not be activated to the kill them.Conclusion Successfully constructed Raji-Luc CD19 KO lymphoma cell line.

Objective To explore EEG characteristics and the therapeutic effect in children with electrical status epilepticus during slow sleep(ESES).Methods The eligible ESES cases in our center from 2014 to 2020 were included.The age at diagnosis of ESES,the duration of ESES,spike wave index(SWI)during wakefulness and the distribution of spike wave during the period of ESES,age at seizure onset,the clinical syndromes and the outcomes after treatment were analyzed.The ESES cases were divided into 4 groups according to the distribution of spike wave:focal ESES,unilateral ESES,bilateral asymmetric ESES,multiple foci ESES.The SWI during the awake stage were divided into 3 groups based on the different rates:≤20%,21%～49%,≥50%.The therapeutic outcomes were classified into three groups:satisfactory response,seizure control and ineffective.Results 50 cases were included,with 32 males and 18 females.The average onset age of ESES was 6 years and 7 months,and the average duration of ESES was 28 months.A significant correlation between the distribution of ESES and the thera-peutic effects was found,bilateral asymmetric ESES had a good therapeutic effects,while multiple foci ESES showed a poor therapeutic effects.The duration of ESES was significantly correlated with therapeutic effects,and the efficacy was worse when the duration was longer than 1 year.A significant relationship between the SWI during wakefulness of ESES and the therapeutic effects was detected,the patient with SWI≤20%during wakefulness had a good therapeutic effect.There was a negative correlation between the onset age of ESES and the duration of ESES and SWI index during wakefulness.There was a positive correlation between the duration of ESES and SWI index during wakefulness.Conclusion Our results suggest that onset age,distribution,duration and SWI during wake-fulness of ESES were correlated with therapeutic outcomes,The patient with SWI≤20%during wakefulness had a good therapeutic effect and have unfavorable outcomes with ESES last more than 1 year.The earlier onset of ESES,the longer duration of ESES and higher SWI during wakefulness will be showed..

Objective:To study the current status of longitudinal extrauterine growth restriction (EUGR) in extremely preterm infants (EPIs) and to develop a prediction model based on clinical data from multiple NICUs.Methods:From January 2017 to December 2018, EPIs admitted to 32 NICUs in North China were retrospectively studied. Their general conditions, nutritional support, complications during hospitalization and weight changes were reviewed. Weight loss between birth and discharge > 1SD was defined as longitudinal EUGR. The EPIs were assigned into longitudinal EUGR group and non-EUGR group and their nutritional support and weight changes were compared. The EPIs were randomly assigned into the training dataset and the validation dataset with a ratio of 7∶3. Univariate Cox regression analysis and multiple regression analysis were used in the training dataset to select the independent predictive factors. The best-fitting Nomogram model predicting longitudinal EUGR was established based on Akaike Information Criterion. The model was evaluated for discrimination efficacy, calibration and clinical decision curve analysis.Results:A total of 436 EPIs were included in this study, with a mean gestational age of (26.9±0.9) weeks and a birth weight of (989±171) g. The incidence of longitudinal EUGR was 82.3%(359/436). Seven variables (birth weight Z-score, weight loss, weight growth velocity, the proportion of breast milk ≥75% within 3 d before discharge, invasive mechanical ventilation ≥7 d, maternal antenatal corticosteroids use and bronchopulmonary dysplasia) were selected to establish the prediction model. The area under the receiver operating characteristic curve of the training dataset and the validation dataset were 0.870 (95% CI 0.820-0.920) and 0.879 (95% CI 0.815-0.942), suggesting good discrimination efficacy. The calibration curve indicated a good fit of the model ( P>0.05). The decision curve analysis showed positive net benefits at all thresholds. Conclusions:Currently, EPIs have a high incidence of longitudinal EUGR. The prediction model is helpful for early identification and intervention for EPIs with higher risks of longitudinal EUGR. It is necessary to expand the sample size and conduct prospective studies to optimize and validate the prediction model in the future.

Objective: To investigate the effects and mechanisms of Xuebijing injection (XBJ) on Chimeric antigen receptor-T (CAR-T) cell function and its therapeutic potential against CAR-T therapy-associated cytokine storms (CRS). Methods: Anti-CD19 CAR-T cells were established based on FMC63 antibodies. Different doses of XBJ (1 and 10 mg/mL) were added to the culture system. Untreated anti-CD19 CAR-T cells served as negative controls. After 48-h co-culture, the effects of XBJ on CAR-T cell function were assessed. Carboxyfluorescein diacetate succinimidyl ester staining was used to assess the effect of XBJ on CAR-T cell proliferation. Flow cytometry, luciferase reporter gene assays, and real time cellular analysis were employed to evaluate the effects of XBJ on CAR-T cell cytotoxicity in vitro. RNA-sequencing was performed to analyze the effects of XBJ on CAR-T cell gene expression. Network pharmacology predicted potential XBJ therapeutic targets for CRS, which were verified in a THP-1 macrophage inflammation model. Results: XBJ enhanced both the proliferation and tumor killing capacities of CAR-T cells. Transcriptome analysis showed that XBJ treatment affects multiple genes and pathways in CAR-T cells, with differential gene enrichment in multiple cell proliferation and growth factor pathways. Potential targets for CRS control by XBJ were predicted using network pharmacology, and the inhibitory effect of XBJ on the expression of relevant genes was verified using a macrophage model. Conclusion The results of this study indicate that XBJ can enhance the killing effect of CAR-T cells on tumor cells and that the mechanism is related to the regulation of T cell proliferation and activation. Moreover, XBJ inhibited excessive inflammation associated with CAR-T therapy. However, the current findings remain to be further validated through in vivo experiments.

BACKGROUND: Antenatal corticosteroids (ACS) can significantly improve the outcomes of preterm infants. This study aimed to describe the ACS use rates among preterm infants admitted to Chinese neonatal intensive care units (NICU) and to explore perinatal factors associated with ACS use, using the largest contemporary cohort of very preterm infants in China. METHODS: This cross-sectional study enrolled all infants born at 24 +0 to 31 +6 weeks and admitted to 57 NICUs of the Chinese Neonatal Network from January 1st, 2019 to December 30th, 2019. The ACS administration was defined as at least one dose of dexamethasone and betamethasone given before delivery. Multiple logistic regressions were applied to determine the association between perinatal factors and ACS usage. RESULTS: A total of 7828 infants were enrolled, among which 6103 (78.0%) infants received ACS. ACS use rates increased with increasing gestational age (GA), from 177/259 (68.3%) at 24 to 25 weeks' gestation to 3120/3960 (78.8%) at 30 to 31 weeks' gestation. Among infants exposed to ACS, 2999 of 6103 (49.1%) infants received a single complete course, and 33.4% (2039/6103) infants received a partial course. ACS use rates varied from 30.2% to 100% among different hospitals. Multivariate regression showed that increasing GA, born in hospital (inborn), increasing maternal age, maternal hypertension and premature rupture of membranes were associated with higher likelihood to receive ACS. CONCLUSIONS The use rate of ACS remained low for infants at 24 to 31 weeks' gestation admitted to Chinese NICUs, with fewer infants receiving a complete course. The use rates varied significantly among different hospitals. Efforts are urgently needed to propose improvement measures and thus improve the usage of ACS.
Humans ; Infant, Newborn ; Infant ; Pregnancy ; Female ; Gestational Age ; Infant, Premature ; Intensive Care Units, Neonatal ; Cross-Sectional Studies ; Adrenal Cortex Hormones/therapeutic use*

Humans ; Consensus ; Pancreatic Neoplasms/therapy* ; Neuroendocrine Tumors/therapy*

Objective To analyze the individualized CTV-to-PTV margin dose and positioning errors in radiotherapy for esophageal cancer for improving the treatment accuracy while meeting dose requirements.Methods Fifty-four esophageal cancer patients admitted to Sun Yat-sen University Cancer Center at Huangpu District from June 2021 to June 2022 were enrolled.All of the patients underwent CBCT scans in each fraction,and a total of 1283 CBCT images were collected.The image registration between CBCT image before radiotherapy and planning CT image was carried out to obtain errors in vertical(VRT),longitudinal(LNG),lateral(LAT),Roll,Pitch,and YAW directions.The mean values of six-dimensional positioning errors in the first 5 fractions were calculated,and the results were compared with the total fractional errors using the single sample t-test method for determining the differences.The CTV-to-PTV margin was calculated with the formula(margin=2.5∑+0.7δ),and the calculated margins were divided into 5 groups:Group A(5 mm expansion in all directions),Group B(7.9 mm expansion in LAT direction,and 5 mm expansion in other directions),Group C(11.03 mm expansion in LNG direction,and 5 mm expansion in the other directions),Group D(6.36 mm expansion in VRT direction,and 5 mm expansion in the other directions),and Group E(7.9 mm expansion in LAT direction,11.03 mm expansion in LNG direction,and 6.36 mm expansion in VRT direction).Simulation planning was conducted for 10 patients.Results The proportions of differences between the mean values of six-dimensional errors in the first 5 fractions and the total fractional errors in 54 patients were analyzed.There was no significant difference in 192 out of the 324 directions in 54 patients,accounting for 59.26%(P>0.05).Among them,the LAT,LNG,VRT,Pitch,Roll and YAW directions accounted for 64.81%,57.41%,51.85%,64.81%,57.41%and 59.26%of the total cases.The calculated CTV-to-PTV margin was 7.90,11.03 and 6.36 mm in LAT,LNG and VRT directions.The statistical analysis showed that the differences in the coverage rates of organs-at-risk and target areas among the 5 groups of CTV-to-PTV margins were trivial(P>0.05).Conclusion Using the positioning errors in the first 5 fractions of radiotherapy for esophageal cancer to predict subsequent positioning errors is feasible.The reasonable individualized margin in radiotherapy for esophageal cancer can reduce the inter-fractional off-target rate without increasing the dose delivered to organs-at-risk.The study provides a reference for the target volume margin of esophageal cancer and an important basis for precision treatment.

Objective:To standardize the naming of organ at risk (OAR) and target area during cervical cancer radiotherapy based on AAPM TG-263.Methods:After self-programming of Matlab software to implement the reading and resolution of radiotherapy structure files, the naming of each substructure was automatically output, recorded and restored. After naming all substructures, the structure names were classified by keywords. According to TG-263, a standard naming conversion table of OAR and target area was developed, and the classified structure names were standardized through procedures. Finally, the standardized named radiotherapy structure files were output and imported into the treatment planning system (TPS).Results:The radiation structure of 144 patients with cervical cancer was successfully transformed and displayed correctly in TPS. Before the transformation, the naming of OAR and target area lacked of uniform norms and standards, and the naming of the same structure significantly differed. After the transformation, 43 naming methods of OAR and 74 naming methods of the target area were unified into 20 and 8 naming methods, which were more convenient for staff understanding and communication.Conclusion:The standardization of cervical cancer radiotherapy structure naming can reduce the inconsistency of naming and provide reference for the standardized naming of pelvic tumors.

Objective:To investigate the effect of total alkaloid of harmaline (TAH) on inducing cellular autophagy and degradating of neurotoxic proteins Tau and α-synuclein (α-Syn).Methods:(1) The in vitro cultured PC12 cells were divided into blank control group, and 1, 2.5, 5, 10, 20 and 50 μg/mL TAH groups, respectively; and they were treated with 0, 1, 2.5, 5, 10, 20 and 50 μg/mL TAH for 24 h; cell morphology and number were observed, and cell survival rate was determined by MTT assay. (2) PC12 cells were divided into blank control group, rapamycin group, and 1, 2.5, 5, 10 and 20 μg/mL TAH groups; these cells were treated with same amount of solvent, 50 nmol/L autophagy activator rapamycin, and 1, 2.5, 5, 10 and 20 μg/mL TAH for 4 h, respectively, and the number of autophagosomes was detected by immunofluorescent staining. (3) PC12 cells were divided into blank control group, rapamycin group, and 10 μg/mL TAH group; these cells were treated with same amount of solvent, 50 nmol/L rapamycin, and 10 μg/mL TAH for 4 h; the protein expression levels of p62 and microtubule-associated protein 1 light chain 3 II (LC3-II) was detected by Western blotting. (4) PC12 cells were divided into blank control group, chloroquine group, TAH group, and TAH+chloroquine group; these PC12 cells were treated with 50 nmol/L autophagy inhibitor chloroquine, 10 μg/mL TAH, and 10 μg/mL TAH+50 nmol/L chloroquine for 4 h, respectively; the LC3-II protein expression was detected by Western blotting. (5) PC12 cells were divided into TAH group and blank control group; 10 μg/mL TAH and same amount of solvent were given to each group for 4 h, and then, the phosphorylated mammalian target of rapamycin (p-mTOR) and phosphorylated 70-KD ribosomal protein S6 kinase (p-P70S6K) protein expression levels were detected by Western blotting. (6) Tet on HEK293 cells with Tau-green fluorescent protein (GFP) overexpression were divided into blank control group, TAH group, doxycycline group, doxycycline+TAH group, doxycycline+TAH+3-MA group, and doxycycline+TAH+chloroquine group. Cells in the later 4 groups were treated with 200 ng/mL Tet system inducer doxycycline for 24 h; cells in the blank control group were treated with same amount of solvent, those in the TAH group were treated with 10 μg/mL TAH, and cells in the latter 3 groups were treated with 10 μg/mL TAH, 10 μg/mL TAH+5 mmol/L 3-MA, and 10 μg/mL TAH+50 nmol/L chloroquine, respectively, for 24 h; the changes of green fluorescence intensity of these cells were observed under laser confocal microscope. The Tau-GFP and LC3-II protein expression levels were detected by Western blotting. (7) HEK293 cells with stable α-Syn expression were divided into blank control group, chloroquine group, TAH group and TAH+chloroquine group; these cells were treated with same amount of solvent, 50 nmol/L chloroquine, 10 μg/mL TAH and 10 μg/mL TAH+50 nmol/L chloroquine for 24 h, respectively; the α-Syn and LC3-II protein expression levels were detected by Western blotting. Results:(1) As compared with that in the blank control group, the cell survival rate in 20 and 50 μg/mL TAH groups was significantly lower, and that in the 50 μg/mL TAH group was statistically lower than that in 20 μg/mL TAH group ( P<0.05). (2) As compared with that in the blank control group, the number of autophagosomes in rapamycin group, and 10 and 20 μg/mL TAH groups was significantly increased, and that in 10 μg/mL TAH group was statistically higher than that in 20 μg/mL TAH group ( P<0.05); 10 μg/mL TAH group was selected for subsequent experiments. (3) As compared with the blank control group, the rapamycin group and TAH group had significantly decreased P62 protein expression and significantly increased LC3-II protein expression ( P<0.05). (4) As compared with that in the blank control group, the LC3-II protein expression in the chloroquine group, TAH group and TAH+chloroquine group was significantly increased, and LC3-II protein expression in TAH+chloroquine group was statistically higher than that in chloroquine group ( P<0.05). (5) The p-mTOR and p-p70S6K expression levels in the TAH group were significantly decreased as compared with those in the blank control group ( P<0.05). (6) The Tau-GFP protein expression in doxycycline group was significantly increased as compared with that in the blank control group ( P<0.05); that in doxycycline+TAH group was significantly decreased as compared with that in the doxycycline group ( P<0.05); that in the doxycycline+TAH+3-MA group and doxycycline+TAH+chloroquine group was statistically increased as compared with that in doxycycline+TAH group ( P<0.05). The LC3-II protein expression in the TAH group was significantly increased as compared with that in the control group, that in the doxycycline+TAH group was significantly increased as compared with that in the doxycycline group, that in the doxycycline+TAH+3-MA group was significantly decreased as compared with that in the doxycycline+TAH group, and that in doxycycline+TAH+ chloroquine group was significantly increased as compared with that in the doxycycline+TAH group ( P<0.05). Conclusion:TAH may activate autophagy by inhibiting the mTOR/p70S6K signaling pathway, which in turn promotes the degradation of neurotoxic proteins Tau and α-Syn.

Objective To evaluate and compare the effectiveness of propofol-remifentanil closed-loop and open-loop anesthesia in gynecological laparoscopic operation under bispectral index (BIS) monitoring.Methods Forty female patients undergoing elective gynecological laparoscopic operation were recruited and randomly divided into closed-loop (group Ⅰ) and open-loop (group Ⅱ) groups.During anesthesia maintenance,the closed-loop group was administered with a BIS-feedback system to regulate the target effect-site concentration;whereas,the open-loop group was administered the target effect-site concentration according BIS value manually.The variation of non-invasive mean arterial pressure (MAP),heart rate (HR),SpO2,BIS,extubation time,consumption of propofol and remifentanil,Ramsay index,and subjective comfort grade were recorded at the selected time points.Results The extubation time in group Ⅰ was shorter than in group Ⅱ.The total dosage of propofol administered in group Ⅰ was less than that in group Ⅱ,but there was no significant difference in the total consumption of remifentanil.HR in group Ⅰ was steadier than in group Ⅱ.However,the MAP and Ramsay index were similar in both the groups.The subjective comfort grade in group Ⅰ was higher than in group Ⅱ.Conclusion The use of propofol-remifentanil closed-loop system by BIS-feedback anesthesia is safer,more controllable,with higher degree of satisfaction and sparing side-effects,we therefore recommend it during gynecological laparoscopic operations.