ObjectiveTo study the effect and mechanism of Xiangsha Liu Junzitang combined with phlegm-removing and detoxifying traditional Chinese medicine on immune escape in Lewis lung cancer mice. MethodA total of 60 specific-pathogen-free （SPF）-grade C57BL/6J male mice were injected subcutaneously with 0.2 mL of Lewis cell suspension （containing 2×10⁶ cells·mL^-1） in the right mid-axillary line. After 7 days， the mice that had been successfully modeled were randomly divided into six groups： the model group， the cisplatin group， the Xiangsha Liu Junzitang low-， medium-， and high-dose groups， and the combined group， with 10 mice in each group. The Xiangsha Liu Junzitang low-， medium- and high-dose groups were gavaged with 17.88， 35.75， 71.50 g·kg^-1 Xiangsha Liu Junzitang solution once a day， respectively， and the dosage of cisplatin intraperitoneally injected into the mice was converted to 5 mg·kg^-1 twice a week， and the tumour volumes of each group were measured every two days. The intervention lasted for 14 consecutive days. At the end of treatment， the tumour mass of mice in each group was weighed and the tumour inhibition rate was calculated. The morphological characteristics of tumours in each group were observed by hematoxylin-eosin （HE） staining. Fluorescent quantitative real-time polymerase chain reaction （Real-time PCR） assay was used to detect messenger ribonucleic acid （mRNA） contents of the natural killer group 2 member D （NKG2D） receptor， ribonucleic acid export-1 （RAE-1）， and γ interferon （IFN-γ） in the tumour tissues of each group. NKG2D， RAE-1， and IFN-γ mRNA in tumour tissues of each group. Immunohistochemistry （IHC） and Western blot were applied to detect the expressions of RAE-1， NKG2D， and IFN-γ in tumour tissues of each group， and Western blot was used to detect the expressions of interleukin-6 （IL-6）， Janus kinase 2 （JAK2）， p-JAK2， signal transducer and activator of transcription 3 （STAT3）， and p-STAT3 in tumour tissues of each group， as well as the protein levels of NKG2D， and RAE-1 in spleen tissues of each group. ResultCompared with that in the model group， the tumour mass decreased in all dose groups of Xiangsha Liu Junzitang， with no statistically significant difference. The tumour volume was reduced （P<0.05， P <0.01）. The pathological morphology was improved. The mRNA contents of NKG2D， RAE-1 and IFN-γ were increased in the medium-dose group （P<0.05， P<0.01）， and the protein expressions of NKG2D， RAE-1， and IFN-γ in tumour tissues were elevated （P<0.05， P<0.01）， and p-JAK2 and p-STAT3 protein expressions were decreased （P<0.05， P<0.01）. In spleen tissues， the protein expressions of NKG2D and RAE-1 in all dose groups of Xiangsha Liu Junzitang were significantly elevated （P<0.01）. Compared with those in the cisplatin group， NKG2D， RAE-1 and IFN-γ mRNA contents were elevated in the middle-dose group of Xiangsha Liu Junzitang， and the difference was not statistically significant. IHC showed that the protein expressions of NKG2D and IFN-γ in the combined group were significantly elevated （P<0.01）， and Western blot results showed that the protein expressions of RAE-1， NKG2D and IFN-γ were elevated （P<0.05， P<0.01）. p-JAK2 and p-STAT3 protein expressions were decreased in the combined group （P<0.05， P<0.01）. NKG2D and RAE-1 protein expressions were significantly increased in spleen tissues of the medium-dose groups and the combined group （P<0.01）. ConclusionXiangsha Liu Junzitang combined with phlegm-removing and detoxifying traditional Chinese medicine can inhibit the growth of tumours in Lewis lung cancer mice by up-regulating the expressions of RAE-1/NKG2D， promoting the activation of NK cells， and inhibiting immune escape， the mechanism of which may be related to down-regulation of the JAK2/STAT3 pathway.

Objective:To evaluate the diagnostic efficacy and practicality of the Jaundice color card (JCard) as a screening tool for neonatal jaundice.Methods:Following the standards for reporting of diagnostic accuracy studies (STARD) statement, a multicenter prospective study was conducted in 9 hospitals in China from October 2019 to September 2021. A total of 845 newborns who were admitted to the hospital or outpatient department for liver function testing due to their own diseases. The inclusion criteria were a gestational age of ≥35 weeks, a birth weight of ≥2 000 g, and an age of ≤28 days. The neonate′s parents used the JCard to measure jaundice at the neonate′s cheek. Within 2 hours of the JCard measurement, transcutaneous bilirubin (TcB) was measured with a JH20-1B device and total serum bilirubin (TSB) was detected. The Pearson′s correlation analysis, Bland-Altman plots and the receiver operating characteristic (ROC) curve were used for statistic analysis.Results:Out of the 854 newborns, 445 were male and 409 were female; 46 were born at 35-36 weeks of gestational age and 808 were born at ≥37 weeks of gestational age. Additionally, 432 cases were aged 0-3 days, 236 cases were aged 4-7 days, and 186 cases were aged 8-28 days. The TSB level was (227.4±89.6) μmol/L, with a range of 23.7-717.0 μmol/L. The JCard level was (221.4±77.0) μmol/L and the TcB level was (252.5±76.0) μmol/L. Both the JCard and TcB values showed good correlation ( r=0.77 and 0.80, respectively) and agreements (96.0% (820/854) and 95.2% (813/854) of samples fell within the 95% limits of agreement, respectively) with TSB. The JCard value of 12 had a sensitivity of 0.93 and specificity of 0.75 for identifying a TSB ≥205.2?μmol/L, and a sensitivity of 1.00 and specificity of 0.35 for identifying a TSB ≥342.0?μmol/L. The TcB value of 205.2?μmol/L had a sensitivity of 0.97 and specificity of 0.60 for identifying TSB levels of 205.2 μmol/L, and a sensitivity of 1.00 and specificity of 0.26 for identifying TSB levels of 342.0 μmol/L. The areas under the ROC curve (AUC) of JCard for identifying TSB levels of 153.9, 205.2, 256.5, and 342.0 μmol/L were 0.96, 0.92, 0.83, and 0.83, respectively. The AUC of TcB were 0.94, 0.91, 0.86, and 0.87, respectively. There were both no significant differences between the AUC of JCard and TcB in identifying TSB levels of 153.9 and 205.2 μmol/L (both P>0.05). However, the AUC of JCard were both lower than those of TcB in identifying TSB levels of 256.5 and 342.0 μmol/L (both P<0.05). Conclusions:JCard can be used to classify different levels of bilirubin, but its diagnostic efficacy decreases with increasing bilirubin levels. When TSB level are ≤205.2 μmol/L, its diagnostic efficacy is equivalent to that of the JH20-1B. To prevent the misdiagnosis of severe jaundice, it is recommended that parents use a low JCard score, such as 12, to identify severe hyperbilirubinemia (TSB ≥342.0 μmol/L).

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.
Humans ; Apolipoprotein E4/genetics* ; Cytosine ; Mutation ; Blastocyst ; Heterozygote ; Gene Editing ; CRISPR-Cas Systems

CRISPR-Associated Protein 9 ; Gene Editing ; CRISPR-Cas Systems/genetics*

Objective To quantify the setup errors for the different anatomical sites of patients who received intensity-modulated radiotherapy (IMRT) with linear accelerator on-board kilovolt fan beam CT(kV-FBCT) as non-isocenter IGRT and megavolt cone beam CT (MV-CBCT) as isocenter IGRT. Methods A retrospective analysis was performedon 70 patients who underwent radiotherapy, kV-FBCT, and/or MV-CBCT scans after each routine setup prior to IMRT. The average displacement (M), systematic error (Σ), and random error (б) at different treatment sites in the left-right, anterior-posterior, and cranial-caudal directions were calculated according to the individual displacements. The formula 2.5Σ+0.7б was used to estimate the PTV margin in respective direction. For each single patient, the root mean square in three directions was used as 3D displacement. Results A total of 1130 displacements were recorded in the 70 patients. The PTV margin was estimated to be 1.9-3.1 mm in head and neck cancer, 2.8-5.1 mm in thoracic cancer, 4.6-5.1 mm in breast cancer, 3.0-5.5 mm in upper abdominal cancer, and 3.5-6.8 mm in pelvic tumor. For the 3D mean displacements, the head and neck, thoracic, breast, upper abdominal, and pelvic cancer were 2.4±1.0, 4.0±1.6, 4.1±2.0, 4.6±2.1, and 4.6±2.1 mm, respectively. The average 3D displacement obtained by kV-FBCT and MV-CBCT were 4.1 and 3.4 mm, respectively (P=0.212). Conclusion The quantitative setup-error data can be obtained using linear accelerator on-board FBCT, and the non-isocenter IGRT induced set-up error cannot be negligible.

Objective:To summarize the clinical characteristics, diagnosis and treatment of caspase recruitment domain-containing protein 9 (CARD9) gene deficiency associated invasive candidiasis, and report a novel mutation in CARD9 gene.Methods:The clinical characteristics, laboratory tests, treatment and the outcome of follow-up in a boy with invasive candidiasis were described. The boy′s main clinical manifestations were central nervous system infection and retroperitoneal mass. Whole-exome sequencing was performed and Sanger sequencing was verified to identify the CARD9 gene mutations in the patient and his parents. A literature search for “CARD9”and “invasive candidiasis”was conducted in PubMed, Wanfang and CNKI databases from their establishment to May 2020.Results:A 10-year-old boy suffered onset symptom of chronic diarrhea, which lasted for two months. The symptom was followed by progressive neurological symptoms such as headache, vomiting, seizures and disorder of consciousness. His unusual medical history was absent. Candida albicans were cultured several times in cerebrospinal fluid and blood, and yeast-like fungi were found in the stool high power field of vision. Cerebral magnetic resonance imaging indicated obstructive hydrocephalus and abdominal CT scan showed retroperitoneal mass and thickening of the intestinal wall. The whole-exome sequencing analyses of blood samples from the boy and his parents were performed. The results showed that there was a homozygous mutation of c.952-12_956delinsAG in the CARD9 gene, which was an unreported pathogenic mutation. This was confirmed by Sanger sequencing. There was no significant relief from intravenous combined antifungal medications. After lateral ventricular drainage surgery and injection of amphotericin B into the lateral ventricle, improvement of clinical symptoms and cerebral spinal fluid abnormalities was observed after nine weeks, and the retroperitoneal mass shrank. At follow-up after four-month oral combined antifungal medications, the child had no complaint except fatigue. However, cerebral spinal fluid analysis showed increased protein level and decreased glucose. Persistent hydrocephalus and periventricular white matter abnormal signals were revealed on the brain magnetic resonance imaging and the smaller retroperitoneal mass than before on the abdominal CT scan. In addition to this case, totally 21 cases with CARD9 gene deficiency associated invasive candidiasis have been reported worldwide, most of which featured central nervous system infections.Conclusions:CARD9 gene deficiency is an autosomal recessive primary immunodeficiency that confers human susceptibility to fungal disease. The associated invasive candidiasis often affects the central nervous system and makes the patient severely ill. Adequate systemic antifungal therapies should be given, and patients with hydrocephalus need surgical treatment. A novel mutation is reported that expands the variant diversity of CARD9 gene. For patients with unexplained invasive candidiasis, including those without a history of previous recurrent infection, genetic testing is recommended for primary immunodeficiency including CARD9 gene deficiency.

Objective To evaluate the role of a color jaundice card (6 colors) as a possible screening tool for detecting neonatal hyperbilirubinemia.Methods During February 1,2016 and May 31,2017,neonates were enrolled in the study,with gestational age ≥35 weeks,birth weight ≥2 000 g,postnatal age 3-28 days,who were the outpatients or inpatients of the 9th People's Hospital of Wuxi Affiliated to Soochow University and the People's Hospital of Anyang.In a well-lighted room,the card measurements were performed at the infants' forehead,the cheek and the sternum.The skin was pressed with a finger for 2 seconds and left quickly,and then the card was used to compare with the exposed yellow skin.Within 2 hours after jaundice card measurement,blood was obtained by venipuncture and total serum bilirubin (TSB) levels were measured.The sensitivity,specificity,positive predictive value (PPV),negative predictive value (NPV),positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were calculated at each measurement sites.Results One hundred and thirty-two neonates were enrolled,of whom 68 cases (51.5％) were male and 64 cases(48.5％) were female and 18 cases (13.6％) were preterm and 114 cases (86.4％) were term neonates.Among all neonates,TSB was ＜5.00 mg/dL(1 mg/dL =17.1 μmol/L) in 21 cases (15.9％),5.00-9.99 mg/dL in 26 cases (19.7％),10.00-14.99 mg/dL in 34 cases (25.8％),15.00-19.99 mg/dL in 37 cases (28.0％) and ≥ 20.00 mg/dL in 14 cases (10.6％).The card had the highest cap ability to recognize jaundice at the cheek,slightly lower at the sternum and the worst in the forehead.The cut-off of ≥ 12 on the six-color card at the cheek had a sensitivity of 95.95％,specificity of 74.14％,PPV of 82.56％,NPV of 93.48％,PLR of 3.710 and NLR of 0.055 for identifying neonates with TSB ≥ 12 mg/dL,with sensitivity being 98.08％,specificity 57.50％,PPV 60.00％,NPV 97.87％,PLR 2.308 and NLR 0.033 for TSB≥ 15 mg/dL.The identification rate was as follows:sensitivity of 100.00％,specificity of 46.00％,PPV of 37.21％,NPV of 100.00％ and PLR of 1.852 for predicting TSB ≥ 17 mg/dL.In addition,in the forehead,cheeks and sternum,the sensitivity of the cut-off of ≥ 12 on the card was 100.00％ for identifying neonates with TSB≥20 mg/dL.In the cheeks and the sternum,the cut-off of ≥ 15 on the card was with a sensitivity of 100.00％ for predicting TSB ≥ 20 mg/dL.Conclusion The six-color jaundice card is a potential screening tool for neonatal hyperbilirubinemia,and the cheek is the best measurement site.

Objective To observe the effects of live combined bifidobacterium,lactobacillus and enterococcus powder on immunoglobulin E (IgE) and interleukin-17 (IL-17) in atopic children with bronchiolitis.Methods Sixty cases of atopic children with bronchiolitis were randomly divided into the therapy group (30 cases) and the control group (30 cases).Twenty-five healthy children were enrolled as the healthy control group.Both the therapy group and the control group were given traditional therapy.The therapy group received live combined bifidobacterium,lactobacillus and enterococcus powder for 2 months.The change of IgE and IL-17 levels were observed during the acutestage,remission stage and after receiving live combined bifidobacterium,lactobacillus and enterococcus powder for 2months.Results (1) The levels of IgE and IL-17 of therapy group[(132.36 ±9.50) μg/L and (77.76 ±7.95)μg/L] during acute stage were markedly higher than those in the healthy control group [(52.80 ±4.92) μg/L and (46.92 ±4.79) μg/L] (all P ＜0.001).The levels of IgE and IL-17 of control group [(128.83 ± 8.06) μg/L and (76.61 ±6.18) μg/L] during remission stage were markedly higher than those in the healthy control group [(52.80 ±4.92) μg/L and (46.92 ± 4.79) μg/L] (all P ＜ 0.001).(2) The levels of IgE of therapy group (56.67 ± 9.20)μg/L after receiving live combined bifidobacterium,lactobacillus and enterococcus powder for 2 months were markedly lower than those in the control group (70.50 ± 11.38) μg/L (P ＜ 0.001).The levels of IL-17 of therapy group [(49.63 ± 6.35) μg/L] at the time after receiving live combined bifidobacterium,lactobacillus and enterococcus powder for 2 months were markedly lower than these in the control group (54.77 ± 6.33) μg/L (P =0.003).Conclusion Receiving live combined bifidobacterium,lactobacillus and enterococcus powder for two months can decrease the IgE and IL-17 levels in atopic children with bronchiolitis.

Objective To observe the effects of live trigeminal bifidobacterium, lactobacillus and enterococcus powder on the recurrence of wheezing, and the levels of peripheral blood eosinophil ( EOS) and serum transforming growth factor-beta 1( TGF-β1) in atopic children with bronchiolitis.Methods Sixty atopic children with bronchiolitis were randomly divided into the therapy group (30 cases) and the conventional treatment group (30 cases) and another 25 healthy children were recruited as the healthy control group. The conventional treatment group was given routine therapy, and the therapy group received live trigeminal bifidobacterium, lactobacil-lus and enterococcus, in addition to routine therapy for 2 months.The levels of EOS and TGF-β1 were detected at the acute stage and 2 months after receiving trigeminal bifidobacterium, lactobacillus and enterococcus.Results (1) The recurrent rate of wheezing after medication for the therapy group (0.67 ±0.13) was significantly lower than that for the conventional treatment group (1.27 ±0.17), with statistical significance (P<0.05).(2)The levels of EOS of the therapy group [(0.72 ±0.13) ×109/L] and the conventional treatment group [(0.70 ±0.13) ×109/L] at the acute stage were markedly higher than those of the healthy control group [(0.16 ± 0.09) ×109/L], also with statistical significance (P<0.05).The levels of TGF-β1 of the therapy group [(1.20 ±0.13) ng/L] and the conventional treatment group(1.22 ±0.11) at acute stage were all considerably lower than those of the control group [(1.45 ± 0.13) ng/L], with statistical significance (P<0.05).The level of EOS in the therapy group [(0.27 ±0.12) ×109/L] 2 months af-ter medication of oral live trigeminal bifidobacterium, lactobacillus and enterococcus powder was lower than that in the conventional treatment group [(0.36 ±0.14) ×109/L], also with statistical significance (P<0.05).The level of TGF-β1 of the therapy group [(1.41 ±0.09) ng/L] 2 months after medication was markedly higher than that of the conventional treatment group [(1.34 ±0.10) ng/L], also with statistical significance (P<0.05).Conclusion Oral medication of live trigeminal bifidobacterium, lactobacillus and enterococcus powder for 2 months could obviously reduce the recurrent rate of wheezing within 6 months after the onset of bronchiolitis and could also up-regulate the levels of EOS and TGF-β1 in atopic children with bronchiolitis.