Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Objective:To evaluate the healthcare accessibility and quality of diagnosis and treatment services of Internet hospitals in China.Methods:One hundred and eighty Internet hospitals in 60 cities were seleted based on the sampling of development levels in the eastern, central and western regions of China. From April to May 2023, standardized patients methodology was applied to evaluate the accessibility(including the number of Internet hospitals, functional settings, online doctor status, the doctor′s attending rate and consultation fees) and diagnosis and treatment service quality(including the diagnosis and treatment services quality, response speed and patient′s evaluation) of Internet hospitals.Results:The average opening rate of Internet hospitals in China was 52.9% (560/1 058), the average online rate of doctors was 64.2% (1 099/1 713), the average doctor′s attending rate was 33.6% (112/333), the average consultation fee was 4.85 yuan, the average score of consultation was 1.92 out of 9, the average score of diagnosis and treatment was 1.12 out of 4, the average score of the response speed was 1.70 out of 3, and patient satisfaction was 2.73 out of 3.Conclusions:The Internet hospital accessibility in China is unevenly developed, and the overall quality of diagnosis and treatment is low. It is recommended to accurately position and optimize the function of Internet hospital, establish the incentive mechanism for online consultation doctors, construct and improve the regulatory system of Internet hospital diagnosis and treatment, so as to improve the accessibility and quality of diagnosis and treatment of Internet hospitals.

Objective:Central nervous system leukemia(CNSL)is one of the main causes of recurrence and death in patients with acute leukemia.This study aims to dynamically monitor minimal residual disease(MRD)in cerebrospinal fluid and bone marrow of patients with different types of acute leukemia by flow cytometry(FCM),and to compare the timeliness and consistency of MRD detection between the 2 methods to further explore the application value of monitoring MRD in cerebrospinal fluid. Methods:A total of 199 patients with acute leukemia admitted to the Guangdong Provincial people's Hospital between October 2018 and January 2022 were retrospectively analyzed,and multiparametric FCM method was adopted to summarize and analyze MRD in cerebrospinal fluid of patients with different types of leukemia and MRD in cerebrospinal fluid and bone marrow specimens of the same patients,and its role in assessing the prognostic value of patients was discussed. Results:Among the 199 acute leukemia cases,a total of 31 cases(15.58%)were positive MRD in the cerebrospinal fluid,of which 18 cases(58%)were detected earlier than the corresponding bone marrow specimens.Among the 19 patients with acute T lymphoblastic leukemia,134 patients with acute B lymphoblastic leukemia,and 46 patients with acute myeloid leukemia counted,there were 4,18,and 9 patients with positive MRD in the cerebrospinal fluid.The Kappa value of the concordance test between the results of cerebrospinal fluid MRD and bone marrow MRD in different types of acute leukemia was only 0.156,demonstrating a low concordance between them. Conclusion:Dynamic monitoring of cerebrospinal fluid MRD by FCM can be used as a monitoring index for central nervous system leukemia,and monitoring cerebrospinal fluid can detect MRD earlier compared with bone marrow,which complements each other as a sensitive index for evaluating prognosis with significant guidance in clinic.

Objective　To observe the influences of circular RNA HECT domain E3 ubiquitin ligase 1 (Circ_HECTD1) on the proliferation and apoptosis of HT22 cells and its regulatory mechanism on the microRNA-135a-5p (miR-135a-5p)/tumor protein 53-induced nuclear protein 1 (TP53INP1) axis by in vitro oxygen-glucose deprivation/reoxygenation (OGD/R)-induced hippocampal neuron damage.Methods　HT22 cells were routinely cultured,and the cells were separated into Con group,OGD/R group,si-NC group,and si-Circ_HECTD1 group,miR-NC group,miR-135a-5p group,si-Circ_HECTD1+anti-miR-NC group,and si-Circ_HECTD1+anti-miR-135a-5p group.qRT-PCR method was used to detect the expression of Circ_HECTD1,miR-135a-5p and TP53INP1 mRNA;MTT was used to detect cell viability;flow cytometry was used to detect apoptosis;dual-luciferase reporter gene experiment was applied to verify the targeting relationship between Circ_HECTD1 and miR-135a-5p,miR-135a-5p and TP53INP1;Western blot was applied to measure the protein expressions of Bax,Bcl-2 and TP53INP1.Results　After OGD/R induction,the expressions of Circ_HECTD1 and TP53INP1 in HT22 cells were up-regulated,the expression of miR-135a-5p was down-regulated,the cell survival rate and the expression of Bcl-2 protein were remarkably decreased,the apoptosis rate and the expression of Bax protein were remarkably increased (all P<0.05).Silencing the expression of Circ_HECTD1 could remarkably up-regulate the expression of miR-135a-5p in OGD/R-induced HT22 cells,down-regulate the expression of TP53INP1,increase cell survival rate and Bax protein expression,decrease cell apoptosis rate and Bcl-2 protein expression (all P<0.05).There was a targeting relationship between Circ_HECTD1 and miR-135a-5p,between miR-135a-5p and TP53INP1.Overexpression of miR-135a-5p could remarkably down-regulate the expression of TP53INP1,increase cell survival rate and Bcl-2 protein expression,decrease cell apoptosis rate and Bax protein expression (all P<0.05).Inhibition of miR-135a-5p expression could partially reverse the protective effect of silencing Circ_HECTD1 on HT22 cell damage.Conclusion　Silencing Circ_HECTD1 can regulate the miR-135a-5p/TP53INP1 axis and promote cell survival,inhibit cell apoptosis,and protect against OGD/R-induced hippocampal neuron damage.

Cerebral edema and its caused elevated intracranial pressure are one of the main causes of death in patients with acute ischemic stroke. The main pathogeneses of brain edema include cytotoxic edema, ionic edema, and angiogenic edema. At present, the treatment strategies used to control brain edema and reduce intracranial pressure are mainly osmotic drugs and hemicraniectomy decompression, which are symptomatic treatments to reduce intracranial pressure. In recent years, it has been proposed to inhibit the ion channel in the formation of brain edema as a therapeutic target, which provides a new direction for the treatment of brain edema.

Objective:To explore more clinical characteristics and outcomes of children with severe pneumonia induced by adenovirus type 7(Adv-7).Methods:Seventy children with severe pneumonia induced by Adv-7 admitted to the pediatric intensive care unit of Guangzhou Women and Children′s Medical Center from January 2016 to October 2019 were enrolled.The clinical features, treatments and outcomes of these children were recorded.Results:(1)There were 43 males(61.4%)and 27 females(38.6%). Thirty(42.9%)patients were 0-12 months old, 28(40.0%)patients were 13-36 months old, and 12(17.1%)patients were over 36 months old.(2)The average onset time of severe pneumonia induced by Adv-7 was(11.87±7.10)days before being admitted to PICU.The average sequential organ failure assessment score was 6.80±3.13.The average Murray lung injury score was 2.49±1.15.The average P/F value was(150.57±86.25)mmHg(1 mmHg=0.133 kPa). Sixty-four cases(91.4%)were involved in two or more area of lung on X-rays.All cases were diagnosed with sepsis.(3)Laboratory examination: white blood cell count was(7.6±5.5) ×10 9/L, platelet count was(238.8±164.2)×10 9/L, and C-reactive protein was(39.4±37.2)mg/L.(4)Treatments: intravenous gamma globulin was used in 65 cases(92.9%). Intravenous glucocorticoid was used in 45 cases(64.3%). Fiberoptic bronchoscopy was performed in 43 cases(61.4%). Blood purification treatment was performed in 21 cases(30%). Sixty-three patients(90.0%)were treated with non-invasive or invasive ventilator, high frequency ventilator-assisted ventilation was used in 20 cases(28.6%). Surfactant was used in six cases(8.6%). Extracorporeal membrane oxygenation was used in 19 cases(27.1%). (5)The average treatment time for ventilation was(13.10±11.58)days.The average time for temperature recover was(4.69±4.01)days in PICU.The average PICU stay time was(15.76±12.20)days.The average length of stay was(27.04±13.10)days.There were 16 patients died and the mortality was 22.9%. Conclusion:The children with severe pneumonia induced by Adv-7 were critical and had significant lung injuries.Suffering from active clinical treatment such as extracorporeal membrane oxygenation, some of these patients still had poor prognosis.

In recent years, many studies have found that white matter hyperintensity (WMH) of presumed vascular origin is closely related to cognitive impairment, which reflects the negative effects of white matter macrostructure damage on cognition, while diffusion tensor imaging (DTI) can quantify white matter microstructure damage through different imaging parameters. A variety of DTI parameters are associated with cognitive impairment in WMH, and are of great value in early diagnosis, evaluation of progress risk and efficacy of cognitive impairment. On the basis of DTI, the relevant parameters of brain structural network obtained by joint the graph theory analysis method are also considered to be related to the cognitive impairment with WMH. This paper reviews the latest progress of DTI derived parameters, brain structural network parameters and their relations with WMH cognitive impairment.

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People’s Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories’ results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

BACKGROUND:Smal-needle-knife therapy for knee osteoarthritis has no uniform location, operation and mechanisms of action. Studies have proved that exercise therapy can enhance muscle strength, increase stability of the knee, improve joint range of motion, and effectively relieve pain.
OBJECTIVE:To observe the clinical effect of smal-needle-knife therapy combined with exercise therapy for treatment of knee osteoarthritisvia a randomized controled clinical trial.
METHODS:122 patients were randomly divided into treatment group (n=61; smal-needle-knife therapy combined with exercise therapy) and control group (n=61; low-frequency therapy combined with exercise therapy). Then, clinical efficacy in the two groups were assessed by statistical analysis of visual analog scale, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), sweling degree of the knee joint, quadriceps circumference, flexion and extension of the knee joint before and after treatment. Meanwhile, adverse reactions in patients were recorded for safety evaluation.
RESULTS AND CONCLUSION:(1) The visual analog scale and WOMAC scores in the two groups were both significantly improved at 2 weeks after treatment (P < 0.05). Moreover, these scores in the treatment group were significantly lower than those in the control group (P < 0.05). (2) At 12 weeks after treatment, the WOMAC score in the treatment group was better than that in the control group (P< 0.05), and the range of motion of the knee joint was also better in the treatment group than the control group (P< 0.05). (3) According to the full analysis set and per protocol set, the total efficiency rats in the treatment group were both superior to those in the control group (P< 0.001). (4) In the treatment group, there were four cases of surgery, four cases lost to folow-up, and two cases of mild adverse reactions; in the control group, there were six cases of surgery, three cases lost to folow-up, and no adverse reaction. Taken together, smal-needle-knife therapy and physiotherapy both have certain clinical effects on knee osteoarthritis. Smal-needle-knife therapy combined with exercise therapy is superior to physiotherapy combined with exercise therapy in the total efficiency. Folow-up results of 3 months have been confirmed, but long-term effects need further exploration.

Objective To study the clinical efficacy and safety of iguratimod in the treatment of active rheumatoid arthritis. Methods Ninety patients with rheumatoid arthritis were randomly divided into three groups, with 30 cases in each group. Group A: oral administration of iguratimod, 25 mg two times a day, and oral administration of methotrexate, 10 mg once a week. Group B:oral administration of iguratimod, 25 mg two times a ady. Group C: oral administration of methotrexate, 15 mg once a week. According to the American College of Rheumatology criteria for judging 20%, 50%and 70%(ACR20, ACR 50 and ACR 70) improvement of swollen and tender joint was judged according to the American College Of Rheumatology criteria, and the adverse reactions were observed. Results After the treatment in group A and group B ACR20, ACR50 and ACR70 were higher than those in group C [76.67%(23/30) and 60.00% (18/30) than 40.00% (12/30), 50.00% (15/30) and 33.33% (10/30) than 20.00% (6/30), 23.33%(7/30) and 13.33%(4/30) than 6.67%(2/30)], and in group A was higher than that in group B. The differences were statistically significant (P<0.05). The adverse reaction rate in group A and group B was significantly lower than that in group C:16.67%(5/30) and 13.33%(4/30) than 30.00%(9/30), and the difference was statistically significant (P<0.05); the adverse reaction occurred rate in group A and group B, had no significant difference (P>0.05). Conclusions Monotherapy with iguratimod in the treatment of active rheumatoid arthritis is superior to methotrexate, and has fewer side effects. The combined application of the two drugs is more effective, and can reduce the dose of methotrexate and reduce the incidence of side effects, which is worthy of clinical application.