[Mechanisms by which Mettl3 regulates pericyte-myofibroblast transdifferentiation through PI3K/AKT signaling pathway].

Objective: To investigate the role and underlying mechanisms of methyltransferase (Mettl) 3 in the process of angiotensin Ⅱ (Ang Ⅱ)-induced pericyte-to-myofibroblast transdifferentiation and renal fibrosis. Methods: C57BL/6J mice were used, in cell experiments, mouse renal pericytes were isolated and cultured using magnetic bead sorting. These pericytes were then induced to transdifferentiate into myofibroblasts with 1×106 mmol/L Ang Ⅱ, which was the Ang Ⅱ group, while pericytes cultured in normal conditions served as the control group. Successful transdifferentiation was verified by immunofluorescence staining, Western blotting, and real-time reverse transcription PCR (RT-qPCR) for α-smooth muscle actin (α-SMA). The levels of m6A modifications and related enzymes (Mettl3, Mettl14), Wilms tumor 1-associated protein (WTAP), fat mass and obesity protein (FTO), ALKBH5, YTHDF1, YTHDF2, YTHDC1, YTHDC2, YTHDC3 were assessed by Dot blot, RT-qPCR and Western blot. Mettl3 expression was inhibited in cells using lentivirus-mediated Mettl3-shRNA transfection, creating sh-Mettl3 and Ang Ⅱ+sh-Mettl3 groups, while lentivirus empty vector transfection served as the negative control (Ang Ⅱ+sh-NC group). The impact of Ang Ⅱ on pericyte transdifferentiation was observed, and the expression of downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway proteins, including PI3K, AKT, phosphorylated AKT at serine 473 (p-AKT (S473)), and phosphorylated AKT at threonine 308 (p-AKT (T308)), were examined. PI3K gene transcription was inhibited by co-culturing cells with actinomycin D, and the half-life of PI3K mRNA was calculated by measuring residual PI3K mRNA expression over different co-culture time. The reversibility of Mettl3 inhibition on Ang Ⅱ-induced pericyte-to-myofibroblast transdifferentiation was assessed by adding the AKT activator SC79 to the Ang Ⅱ+sh-Mettl3 group. In animal experiments, mice were divided into these groups: sham group (administered 0.9% sterile saline), Ang Ⅱ group (infused with Ang Ⅱ solution), sh-Mettl3 group (injected with Mettl3 shRNA lentivirus solution), Ang Ⅱ+sh-Mettl3 group (infused with Ang Ⅱ solution and injected with Mettl3 shRNA lentivirus solution), and Ang Ⅱ+sh-Mettl3+SC79 group (administered Ang Ⅱ solution and Mettl3 shRNA lentivirus, with an additional injection of SC79). Each group consisted of six subject mice. Blood pressure was measured using the tail-cuff method before and after surgery, and serum creatinine, urea, and urinary albumin levels were determined 4 weeks post-surgery. Kidney tissues were collected at 28 days and stained using hematoxylin-eosin (HE) and Masson's trichrome to assess the extent of renal fibrosis. Results: Primary renal pericytes were successfully obtained by magnetic bead sorting, and intervened with 1×106 mmol/L Ang Ⅱ for 48 hours to induce pericyte-to-myofibroblast transdifferentiation. Dot blot results indicated higher m6A modification levels in the Ang Ⅱ group compared to the control group (P<0.05). RT-qPCR and Western blot results showed upregulation of Mettl3 mRNA and protein levels in the Ang Ⅱ group compared to the control group (both P<0.05). In the Ang Ⅱ+sh-Mettl3 group, Mettl3 protein expression was lower than that in the Ang Ⅱ group, with reduced expression levels of α-SMA, vimentin, desmin, fibroblast agonist protein (FAPa) and type Ⅰ collagen (all P<0.05). Compared to the control group, PI3K mRNA expression level was elevated in the Ang Ⅱ group, along with increased p-AKT (S473) and p-AKT (T308) expressions. In the Ang Ⅱ+sh-Mettl3 group, PI3K mRNA expression and p-AKT (S473) and p-AKT (T308) levels were decreased (all P<0.05). The half-life of PI3K mRNA was shorter in the Ang Ⅱ+sh-Mettl3 group than that in the Ang Ⅱ+sh-NC group (2.34 h vs. 3.42 h). The ameliorative effect of Mettl3 inhibition on Ang Ⅱ-induced pericyte-to-myofibroblast transdifferentiation was reversible by SC79. Animal experiments showed higher blood pressure, serum creatinine, urea, and 24-hour urinary protein levels, and a larger fibrosis area in the Ang Ⅱ group compared to the sham group (all P<0.05). The fibrosis area was smaller in the Ang Ⅱ+sh-Mettl3 group than that in the Ang Ⅱ group (P<0.05), but increased again upon addition of SC79. Conclusion: Mettl3-mediated RNA m6A epigenetic regulation is involved in Ang Ⅱ-induced pericyte-to-myofibroblast transdifferentiation and renal fibrosis, potentially by affecting PI3K stability and regulating the PI3K/AKT signaling pathway.

Long-term hematopoietic transfer of the anti-cancer and lifespan-extending capabilities of a genetically engineered blood system by transplantation of bone marrow mononuclear cells.

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1K74R/K74R or Klf1(K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1(K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1(K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1(K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging.

[Cancer survival analysis in Tianjin, 2010 to 2016].

Objective: Survival analysis of cancers' incidence data in Tianjin from 2010 to 2016 was conducted to provide the basis for formulating and evaluating regional health policies on cancer prevention and treatment. Methods: Registration data in Tianjin were used between January 1, 2010 to December 31, 2016 and patients were followed-up till 31 December, 2021. Life-table method was used to calculate the observed survival rate and Edered Ⅱ was used to calculate the relative survival rate. The data were stratified by year, gender, age group and cancer sites. Difference in survival curves between group was analyzed by Kaplan-Meier method and Log rank test. Joinpoint regression model was used to analyze the trend change. Results: The 5-year relative survival rates of cancer were 41.92% to 53.65% from 2010 to 2016 for residents in Tianjin, with an increasing trend (t=4.81, P=0.005), and the average was 48.56%. The survival rate of females was higher than that of males (57.71%vs. 39.20%), and the survival rate of urban residents was higher than that of rural residents (49.38% vs. 47.24%). The 5-year relative survival rates were 63.14%, 78.39%, 58.25% and 32.67% in 0-14, 15-44, 45-64 and 65 and above age groups, respectively. The median relative survival times of all cancer were 2.34 to 6.00 years from 2010 to 2016 in Tianjin, with an increasing trend (t=3.86, P=0.012). The average of median relative survival times was 4.11 years. The median survival time of females was longer than that of males (11.99 years vs. 2.03 years), and the time of urban residents were longer than that of rural residents (4.60 years vs. 3.43 years). The median relative survival time were 12.07, 11.92 and 1.34 years in 15-44, 45-64 and 65 and above age groups, respectively. Conclusions: The cumulative survival rate of cancer increased significantly from 2010 to 2016 in Tianjin, indicating that the prevention and treatment effect of cancer is obvious. The focus should be on male, rural areas, higher age group, and targeted prevention and treatment measures should be taken to lung, esophagus, liver, gallbladder and pancreatic cancer.

[Liver cancer treatment with mitochondrial homeostasis].

Systemic treatment, including molecular targeted therapy, immunotherapy, and chemotherapy, is an important means of achieving long-term survival in patients with intermediate-and advanced-stage liver cancer. However, some patients are insensitive to treatment and even develop drug resistance. Mitochondria are the center of cellular energy metabolism and, at the same time, are the priority targets for systemic therapy. Mitochondrial homeostasis plays an important role in the treatment of liver cancer. The relationship between the two advances is elucidated so as to provide better ideas for the clinical treatment of liver cancer.

Targeting glutamine metabolism exhibits anti-tumor effects in thyroid cancer.

BACKGROUND: Effective treatment for patients with advanced thyroid cancer is lacking. Metabolism reprogramming is required for cancer to undergo oncogenic transformation and rapid tumorigenic growth. Glutamine is frequently used by cancer cells for active bioenergetic and biosynthetic needs. This study aims to investigate whether targeting glutamine metabolism is a promising therapeutic strategy for thyroid cancer. METHODS: The expression of glutaminase (GLS) and glutamate dehydrogenase (GDH) in thyroid cancer tissues was evaluated by immunohistochemistry, and glutamine metabolism-related genes were assessed using real time-qPCR and western blotting. The effects of glutamine metabolism inhibitor 6-diazo-5-oxo-l-norleucine (DON) on thyroid cancer cells were determined by CCK-8, clone formation assay, Edu incorporation assay, flow cytometry, and Transwell assay. The mechanistic study was performed by real time-qPCR, western blotting, Seahorse assay, and gas chromatography-mass spectrometer assay. The effect of DON prodrug (JHU-083) on thyroid cancer in vivo was assessed using xenograft tumor models in BALB/c nude mice. RESULTS: GLS and GDH were over-expressed in thyroid cancer tissues, and GLS expression was positively associated with lymph-node metastasis and TNM stage. The growth of thyroid cancer cells was significantly inhibited when cultured in glutamine-free medium. Targeting glutamine metabolism with DON inhibited the proliferation of thyroid cancer cells. DON treatment did not promote apoptosis, but increased the proportion of cells in the S phase, accompanied by the decreased expression of cyclin-dependent kinase 2 and cyclin A. DON treatment also significantly inhibited the migration and invasion of thyroid cancer cells by reducing the expression of N-cadherin, Vimentin, matrix metalloproteinase-2, and matrix metalloproteinase-9. Non-essential amino acids, including proline, alanine, aspartate, asparagine, and glycine, were reduced in thyroid cancer cells treated with DON, which could explain the decrease of proteins involved in migration, invasion, and cell cycle. The efficacy and safety of DON prodrug (JHU-083) for thyroid cancer treatment were verified in a mouse model. In addition to suppressing the proliferation and metastasis potential of thyroid cancer in vivo, enhanced innate immune response was also observed in JHU-083-treated xenograft tumors as a result of decreased expression of cluster of differentiation 47 and programmed cell death ligand 1. CONCLUSIONS: Thyroid cancer exhibited enhanced glutamine metabolism, as evidenced by the glutamine dependence of thyroid cancer cells and high expression of multiple glutamine metabolism-related genes. Targeting glutamine metabolism with DON prodrug could be a promising therapeutic option for advanced thyroid cancer.

[Research progress on the mechanism and response strategies of molecular targeted drug resistance in liver cancer].

Molecular targeted drugs are one of the treatments for hepatocellular carcinoma (HCC), the primary factor influencing their therapeutic efficacy is drug resistance. Diminished drug intake, greater efflux, improved DNA damage repair capacity, aberrant signal pathways, hypoxia, epithelial-mesenchymal cell transition, and the cellular autophagy system are summarized herein as aspects of the drug resistance mechanism. Simultaneously, effective strategies for addressing drug resistance are elaborated, providing ideas for better clinical treatment of HCC.

[Association of category of dietary intake and physical activity with the risk of mortality in patients with type 2 diabetes mellitus: a prospective cohort study].

Objective: To investigate the association between dietary intake and physical activity category and their combined effects on all-cause and cause-specific mortality risk in patients with type 2 diabetes mellitus (T2DM). Methods: Between December 2013 and December 2021, a prospective cohort study was conducted on 19 863 T2DM patients in Changshu City, Qingjiangpu District (formerly Qinghe District), and Huai'an District, included in the national basic health service management. Information on deaths and underlying causes of death was obtained from the Jiangsu Provincial CDC and Prevention Death Surveillance System. Cox proportional hazards models were used to estimate the intensity of associations between dietary intake, physical activity, and their combined effects with all-cause and cause-specific mortality in patients with T2DM. Results: As of December 31, 2021, the research subjects had been followed up for 150 283 person-years, with a median follow-up time of 8.15 years. During the follow-up period, 3 293 people died, including 1 124 deaths from cardiovascular disease (CVD) and 875 deaths from cancer. Cox regression analysis showed that compared with the population of 0-1 recommended food group, those having more than five recommended food groups had a 19% lower risk of all-cause mortality [hazard ratio (HR)=0.81, 95%CI: 0.70-0.94] and a 33% lower risk of all-cause mortality (HR=0.67, 95%CI: 0.52-0.87). Compared with the T2DM population in the physical activity Q1 group, the risk of all-cause mortality, CVD mortality, and cancer mortality among the physical activity Q4 group reduced by 50% (HR=0.50, 95%CI: 0.45-0.56), 50% (HR=0.50, 95%CI: 0.41-0.61), and 27% (HR=0.73, 95%CI: 0.60-0.88), respectively. The combined effect showed that compared with the population in the intake of food categories 0-2 and low physical activity groups, the risk of all-cause, CVD mortality, and cancer mortality in the intake of food categories 4-9 and high physical activity groups reduced by 55% (HR=0.45, 95%CI: 0.38-0.53), 56% (HR=0.44, 95%CI: 0.32-0.59), and 40% (HR=0.60, 95%CI: 0.44-0.82), respectively. Conclusion: Type of dietary intake, physical activity, and their combined effects are associated with a reduced mortality risk in patients with T2DM.

[Expression and protective effect of chemerin in idiopathic pulmonary fibrosis].

Objective: To explore the expression and the role of chemerin in idiopathic pulmonary fibrosis (IPF). Methods: Quantitative PCR and Western blotting were used to determine the mRNA and protein levels of chemerin in lung tissues from IPF patients and the controls. Clinical serum level of chemerin was analyzed by enzyme-linked immunosorbent assay. The mouse lung fibroblasts isolated and cultured in vitro were divided into the control, TGF-ß, TGF-ß+chemerin and chemerin groups. Immunofluorescence staining was used to observe the expression of α-smooth muscle actin (α-SMA). C57BL/6 mice were randomly divided into the control, bleomycin, bleomycin+chemerin, and chemerin groups. Masson and immunohistochemical staining were performed to evaluate the severity of pulmonary fibrosis. Expression of epithelial to mesenchymal transition (EMT) markers was detected by quantitative PCR and immunohistochemical staining in the in vitro and in vivo models of pulmonary fibrosis, respectively. Results: Compared with the control group, the expression of chemerin was downregulated in both the lung tissue and the serum of IPF patients. Immunofluorescence showed that treatment of fibroblasts with TGF-ß alone resulted in a robust expression of α-SMA, whereas treatment with TGF-ß and chemerin together exhibited the similar expression levels of α-SMA as the control group. Masson staining indicated that the bleomycin-induced pulmonary fibrosis model was constructed successfully, while treatment of chemerin partially alleviated the damage of lung tissue. Immunohistochemical staining showed that the expression of chemerin in the lung tissue was significantly decreased in the bleomycin group. Quantitative PCR and immunohistochemistry showed that chemerin attenuated EMT induced by TGF-ß and bleomycin both in vitro and in vivo. Conclusions: The expression of chemerin was reduced in patients with IPF. Chemerin may play a protective role in the development of IPF by regulating EMT, providing a new idea for the clinical treatment of IPF.

[The distribution of blood pressure and associated factors of the elderly with type 2 diabetes in Jiangsu Province].

Objective: To investigate the distribution of blood pressure and analyze the associated factors of blood pressure of the elderly with type 2 diabetes in Jiangsu Province. Methods: The elderly over 60 years old participants with type 2 diabetes in the communities of Huai'an City and Changshu City, Jiangsu Province were selected in this study. They were divided into two groups: taking antihypertensive drugs and not taking antihypertensive drugs. The demographic characteristics, such as age and sex, and relevant factors were collected by questionnaire. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by physical examination. The percentile of SBP and DBP in each age group of men and women were described. The kernel density estimation curve was used to show the blood pressure distribution. The trend of blood pressure with age was fitted by locally weighted regression. The logistic regression model was used to analyze relevant factors of blood pressure. Results: A total of 12 949 participants were included in this study, including 7 775 patients in the antihypertensive drug group and 5 174 patients in the group without antihypertensive drugs. The SBP of participants was concentrated at 140-160 mmHg, and their DBP was concentrated at 75-85 mmHg. There were significant differences in the distribution of blood pressure among the subgroups of body mass index (BMI) and rural areas whether taking antihypertensive drugs and not. For participants aged under 80 years old, the SBP showed an increasing trend with age and the DBP showed a decreasing trend with age. Age, BMI ≥24 kg/m2, fasting blood glucose ≥7.0 mmol/L, living in rural areas and no smoking were influencing factors of the elevated SBP; BMI ≥24 kg/m2, male, living in rural areas, no smoking, drinking alcohol and not receiving drug hypoglycemic treatment were influencing factors of the elevated DBP. Conclusion: The SBP of older diabetic adults in Jiangsu Province is at a high level, and the distribution of blood pressure is significantly different between men and women in taking antihypertensive drugs group. The SBP presents a rising trend and the DBP is decreasing at the age of 60-80 years. The blood pressure level of this population are mainly affected by age, BMI, urban and rural areas, smoking.

[Liver transplantation for the treatment of acute liver failure in 3 cases with NBAS gene deficiency and literature review].

Objective: To investigate the clinical efficacy of liver transplantation in the treatment of acute liver in children with NBAS gene deficiency disease and their outcome. Methods: This retrospective study enrolled children with NBAS gene deficiency who were admitted to the Children's Hospital of Fudan University for liver transplantation from January 2013 to June 2022. The clinical data were collected and analyzed. Medical literature published before June 2022 was searched with the keywords of "NBAS" "neuroblastoma amplified sequence recurrent" "acute liver failure" "SOPH syndrome" "short stature with optic nerve atrophy" "Pelger-Huët anomaly" in PubMed, China National Knowledge Infrastructure and Wanfang database. Results: Liver transplantation was performed in 3 patients (2 males and 1 female) with NBAS deficiency. All patients presented with fever-triggered recurrent acute liver failure. The genetic detection found compound heterozygous NBAS gene pathogenic variants in them. The total episodes of acute liver failure before liver transplantation were 11, 2, and 4 respectively, and the age at liver transplantation was 3.5, 2.3, and 2.0 years respectively. During liver transplantation, patient 1 was in the convalescent phase of acute liver failure, patient 2 was in the acute phase, presenting with hepatic encephalopathy (grade V) and respiratory failure, and patient 3 was considered to be in the acute phase. After liver transplantation, patient 1 recovered normal liver function within 1 month and had no liver transplantation-related complications. Patient 2 had secondary epilepsy, intellectual disability, movement disorder, and transiently elevated transaminases. Patient 3 died of severe infection within 1 month. There was no literature in Chinese, 6 in English, 8 NBAS-deficient patients who were treated with liver transplantation. Total 11 patients presented with fever-triggered recurrent acute liver failure. Their age at liver transplantation ranged from 0.9 to 5.0 years. Postoperative complications occurred in 3 patients. Until the last visit, they were followed up for 0.7 to 14.0 years. Total 2 patients died and the 9 surviving patients did not develop acute liver failure. Conclusions: Liver transplantation is effective for the treatment of acute liver failure associated with NBAS gene disease. However, postoperative complications of liver transplantation may occur. The timing of liver transplantation still needs further research.

[Pancreatic ß-cell dedifferentiation detected by flow cytometry].

Objective: To establish a method for detecting pancreatic ß-cell dedifferentiation using flow cytometry. Methods: Experimental study. Min6 (mouse ß cell line), αTC1-6 (mouse α cell line), HepG2 (human hepatocellular carcinoma cells) and mouse F9 cells (mouse teratocarcinoma cell) were cultured with conventional medium. Min6 cells were treated with interleukin-1ß (IL-1ß) in combined with tumor necrosis factor α (TNFα), or palmitic acid (PA) overnight and stained with anti-chromogranin A (ChgA), anti-insulin (Ins), anti-glucagon (Gcg), anti-SRY-box transcription factor 9 (Sox9) and anti-octamer binding transcription factor 4 (Oct4) antibodies, respectively. Flow cytometry was applied to detect the pression of ChgA, Ins, Gcg, Sox9, and Oct4 in the cells, respectively. Unpaired Student t test was used for statistical analysis. Results: Flow cytometry analyses showed that Ins and ChgA were highly expressed in Min6 cells, Gcg was highly expressed in αTC1-6, Sox9 was highly expressed in HepG2, and Oct4 was highly expressed in F9 cells, respectively (around 90%). Treatment of Min6 cells with IL-1ß+TNFα significantly decreased Ins positive staining cells (92.775%±1.702% vs. 97.125%±0.246%, P=0.045), while increased Sox9 positive staining cells (41.675%±0.390% vs. 25.875%±3.348%, P=0.003). No significant changes in ChgA and Oct4 expression could be viewed (both P>0.05). PA treatment elevated the number of Gcg positive staining cells (54.500%±3.597% vs. 41.160%±3.007%, P=0.022). The levels of mRNA expression by qPCR of the above proteins were in consistent with the levels of protein expression by flow cytometry in Min6 cells. Conclusion: Flow cytometry can be used to detect proteins expressed in dedifferentiated models of ß cells, which provides a new method for identify dedifferentiation of pancreatic ß cells.

[Analysis of risk factors and prognosis of cytomegalovirus infection post umbilical cord blood stem cell transplantation in children with primary immunodeficiency diseases].

Objective: To investigate the risk factors and outcomes of cytomegalovirus (CMV) infection post umbilical cord blood stem cell transplantation (UCBT) in children with primary immunodeficiency diseases (PID). Methods: Clinical data of 143 PID children who received UCBT in the Children's Hospital of Fudan University from January 2015 to June 2020 were collected retrospectively. CMV-DNA in the plasma was surveilled once or twice a week within 100 days post-UCBT. According to the CMV-DNA test results, children were divided into the CMV-infected group and the CMV-uninfected group. The incidence and risk factors of CMV infection were analyzed. At 1-month post-UCBT, the absolute lymphocyte count, ratio of lymphocyte subsets and immunoglobulin levels were compared between those whose CMV infection developed 1-month later post-UCBT and those not. Mann-Whitney U test and chi-squared test were used for comparision between groups. Kaplan-Meier survival analysis was used to analyze the impact of CMV infection on survival. Results: Among 143 patients, there were 113 males and 30 females, with a age of 14 (8, 27) months at UCBT. Chronic granulomatosis disease (n=49), very-early-onset inflammatory bowel disease (n=43) and severe combined immunodefiency (n=29) were the three main kinds of PID. The rate of CMV infection was 21.7% (31/143), and the time of infection occurring was 44 (31, 49) days post-UCBT. The incidence of recurrent CMV infection was 4.2% (6/143) and refractory CMV infection was 4.9% (7/143).There was no significant difference in the first time CMV-DNA copy and peak CMV-DNA copy during treatment between the recurrent CMV infection group and the non-recurrent CMV infection group (32.8 (18.3, 63.1)×106 vs. 22.5 (13.2, 31.9)×106 copies/L, Z=-0.95, P=0.340;35.2 (20.2, 54.6)×106 vs. 28.4 (24.1, 53.5)×106copies/L, Z=-0.10, P=0.920), so were those between the refractory CMV infection group and non-refractory CMV infection group (21.8 (13.1, 32.2)×106 vs. 25.9 (14.2, 12.2)×106copies/L, Z=-1.04, P=0.299; 47.7 (27.9, 77.6)×106 vs. 27.7 (19.7,51.8)×106copies/L, Z=-1.49, P=0.137). The CMV-infected group accepted more reduced-intensity conditioning (RIC) regimen than the CMV-uninfected group (45.2% (14/31) vs. 25.0% (28/112), χ2=4.76, P<0.05). The rate of CMV-seropositive recipients and Ⅱ-Ⅳ acute graft versus host diseases (aGVHD) are significantly higher in the CMV-infected group than the CMV-uninfected group (100% (31/31) vs. 78.6% (88/112), 64.5% (20/31) vs. 26.8% (30/112), χ2=7.98,15.20, both P<0.05). The follow-up time was 31.6 (13.2, 45.9) months, CMV infection had no effect on overall survival (OS) rate (χ2=0.02, P=0.843). There was significant difference in the survival rate among three groups of refractory CMV infection, non-refractory CMV infection and the CMV-uninfected (4/7 vs.95.8% (23/24) vs. 86.6% (97/112), χ2=5.91, P=0.037), while there was no significant difference in the survival rate among three groups of recurrent CMV infection, non-recurrent CMV infection and the CMV-uninfected (5/6 vs. 88.0% (22/25) vs. 86.6% (97/112), χ2=0.43, P=0.896). Children who developed CMV infection after 30 days post-UCBT had lower absolute count and rate of CD4+ T cells and immunoglobulin G (IgG) level than those in the CMV-uninfected group (124.1 (81.5, 167.6) ×106 vs. 175.5 (108.3, 257.2) ×106/L, 0.240 (0.164, 0.404) vs. 0.376 (0.222, 0.469), 9.3 (6.2, 14.7) vs. 13.6 (10.7, 16.4) g/L, Z=-2.48, -2.12,-2.47, all P<0.05), but have higher rate of CD8+T cells than those in CMV-uninfected group (0.418 (0.281, 0.624) vs. 0.249 (0.154, 0.434), Z=-2.56, P=0.010). Conclusions: RIC regimen, grade Ⅱ-Ⅳ aGVHD and CMV-seropositive recipients are the main risk factors associated with CMV infection in PID patients post-UCBT. Survival rate of children with refractory CMV infection after UCBT is reduced. Immune reconstitution in children after UCBT should be regularly monitored, and frequency of CMV-DNA monitoring should be increased for children with delayed immune reconstitution.

[The prevention and therapy of corneal ulcers in patients with large-area thermal burns].

Objective: To evaluate the characteristics of ocular injury in patients with severe extensive thermal burns, and to explore the effective methods to prevent and treat corneal ulcers related to severe burns. Methods: A retrospective case series study. Between 2010 and 2019, Sixteen severe thermal burn patients with burn sizes>70% of the total body surface area involving the ocular region were admitted to the Burns and Plastic Surgery Division of Chinese PLA General Hospital, and consult with Ophthalmology Division. There were deep second-degree to fourth-degree burns in the eyelids. In the eleven surviving patients, 22 eyes presented ectropion. Eyelid full-thickness skin grafting (EFTSG) combined with or without tarsorrhaphy was performed in 20 eyes due to severe corneal exposure. Two eyes received partial blepharorrhaphy because of mild ectropion. The ocular manifestations and treatment outcomes were reviewed and assessed. Results: The majority of the patients were youth, and the average age was (36.8±10.4) years. The burn area was 84.0%±9.1% of the body surface area. Corneal ulcers secondary to lagophthalmos occurred at (35.1±15.6) days after burning in 75% (24/32) of eyes. Perforation was found in 18.8% (6/32) of eyes. Among the 22 operated eyes, the corneal ulcer was repaired in all 9 eyes receiving EFTSG with tarsorrhaphy, whereas ectropion recurred in 8 of 11 eyes only receiving EFTSG, and 4 eyes underwent further surgery due to corneal epithelial defects. Conclusions: In patients with severe large-area thermal burns, corneal ulcers are common complications. Prevention of corneal exposure is vital because the treatment of corneal ulceration is difficult due to eyelid deformity, inflammation and the absence of donor skin. Timely full-thickness skin grafting and blepharorrhaphy are effective approaches to preventing exposure keratopathy. To severe ulcers occur, conjunctival flap or Tenon's capsule covering combined with eyelid EFTSG and tarsorrhaphy is useful to rescue visual function.

[Whole-transcriptome sequencing analysis of placental differential miRNA expression profile in Down syndrome].

OBJECTIVE: To identify new biomarkers and molecular pathogenesis of Down syndrome (DS) by analyzing differentially expressed miRNAs in the placentas and their biological pathways. METHODS: Whole transcriptome sequencing was used to identify the differentially expressed miRNAs in DS (n=3) and normal placental samples (n=3) diagnosed by prenatal diagnosis. The target genes were predicted using miRWalk, Targetscan and miRDB, and GO and KEGG pathway analyses were performed for gene enrichment studies. RESULTS: We identified a total of 82 differentially expressed miRNAs in the placental tissues of DS, including 29 up-regulated miRNAs (fold change ≥2, P < 0.05) and 15 down-regulated miRNAs (fold change ≥2, P < 0.05), among which 10 miRNAs with relatively high expression abundance were selected for further analysis, including 4 up-regulated and 6 down-regulated miRNAs. These selected miRNAs shared the common target genes BTBD3 and AUTS2, both of which were associated with neurodevelopment. GO analysis showed that the target genes of the selected miRNAs were mainly enriched in protein binding, hydrolytic enzymes, metal ion binding protein combining, transferase activity, nucleotide, cytoplasmic constituents, nucleus composition, transcriptional regulation, RNA metabolism regulation, DNA-dependent RNA polymerase Ⅱ promoter transcriptional regulation, eye development, and sensory organ development. KEGG enrichment analysis showed that the target genes of these differentially expressed miRNAs were involved in the signaling pathways including tumor-related signaling pathway, PI3K-Akt signaling pathway, Ras signaling pathway, Rap1 signaling pathway, cytoskeletal regulatory signaling pathway, purine metabolization-related signaling pathway and P53 signaling pathway. CONCLUSION: The differentially expressed miRNAs may play important roles in placental damage and pregnancy pathology in DS and provide clues for the prevention and treatment of mental retardation-related diseases.

[Advancement in endovascular therapy of aortoiliac occlusive disease].

Aortoiliac occlusive disease (AIOD) refers to the stenosis and occlusion of the distal abdominal aorta and(or) bifurcation of the aortoiliac artery,which is mainly caused by atherosclerosis,leading to pelvic and lower limb ischemia.Open surgery has always been the main treatment for complex AIOD.However,in recent years,with the development of endovascular surgery technologies and medical instruments,its treatment concept has been greatly changed.More and more clinical evidence has proved that the long-term efficacy of endovascular therapy is not inferior to that of traditional open surgery,so minimally invasive endovascular therapy has become the preferred treatment for AIOD.

[Safety evaluation of hyperthermic intraperitoneal chemotherapy in patients with local advanced gastric cancer after radical resection for prevention of peritoneal metastasis].

Objective: Patients with advanced gastric cancer have a poor prognosis and a possibility of peritoneal metastasis even if receiving gastrectomy. Hyperthermic intraperitoneal chemotherapy (HIPEC) can effectively kill free cancer cells or small lesions in the abdominal cavity. At present, preventive HIPEC still lacks safety evaluation in patients with locally advanced gastric cancer. This study aims to explore the safety of radical resection combined with HIPEC in patients with locally advanced gastric cancer. Methods: A descriptive case series study was carried out. Clinicopathological data of 130 patients with locally advanced gastric cancer who underwent radical resection + HIPEC at the Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from January 2020 to February 2021 were retrospectively analyzed. Inclusion criteria: (1) locally advanced gastric adenocarcinoma confirmed by postoperative pathology; (2) no distant metastasis was found before surgery; (3) radical resection; (4) at least one HIPEC treatment was performed. Exclusion criteria: (1) incomplete clinicopathological data; (2) tumor metastasis was found during operation; (3) concomitant with other tumors. HIPEC method: all the patients received the first HIPEC immediately after D2 radical resection, and returned to the ward after waking up from anesthesia; the second and the third HIPEC were carried out according to the patient's postoperative recovery and tolerance; interval between two HIPEC treatments was 48 h. Observation indicators: (1) basic information, including gender, age, body mass index, etc.; (2) treatment status; (3) perioperative adverse events: based on the standard of common adverse events published by the US Department of Health and Public Health (CTCAE 5.0), the adverse events of grade 2 and above during the treatment period were recorded, including hypoalbuminemia, bone marrow cell reduction, wound complications, abdominal infection, lung infection, gastroparesis, anemia, postoperative bleeding, anastomotic leakage, intestinal obstruction, pleural effusion, abdominal distension, impaired liver function, and finally a senior professional title chief physician reviewed the above adverse events and made a safety evaluation of the patient; (4) association between times of HIPEC treatment and adverse events in perioperative period; (5) analysis of risk factors for adverse events in perioperative period. Results: Among the 130 patients, 79 were males and 51 were females with a median age of 59 (54, 66) years and an average body mass index of (23.9±7.4) kg/m(2). The tumor size was (5.4±3.0) cm and 100 patients (76.9%) had nerve invasion. All the 130 patients received radical resection + HIPEC and 125 (96.2%) patients underwent laparoscopic surgery. The mean operative time was (345.6±52.3) min and intraoperative blood loss was (82.0±36.5) ml. One HIPEC treatment was performed in 54 patients (41.5%), 2 HIPEC treatments were in 57 (43.8%), and 3 HIPEC treatments were in 19 (14.6%). The average postoperative hospital stay was (13.1±7.5) d. A total of 57 patients (43.8%) had 71 cases of postoperative complications of different degrees. Among them, the incidence of hypoalbuminemia was 22.3% (29/130), and the grade 2 and above anemia was 15.4% (20/130), lung infection was 3.8% (5/130), bone marrow cell suppression was 3.7% (4/130), abdominal cavity infection was 2.3% (3/130), and liver damage was 2.3% (3/130), wound complications was 1.5% (2/130), abdominal distension was 1.5% (2/130), anastomotic leakage was 0.8% (1/130), gastroparesis was 0.8% (1/130) and intestinal obstruction was 0.8% (1/130), etc. These adverse events were all improved by conservative treatments. There were no statistically significant differences in the incidence of adverse events during the perioperative period among patients undergoing 1, 2, and 3 times of HIPEC treatments (all P>0.05). Univariate and multivariate logistic analyses showed that age > 60 years (OR: 2.346, 95%CI: 1.069-5.150, P=0.034) and neurological invasion (OR: 2.992, 95%CI: 1.050-8.523, P=0.040) were independent risk factors for adverse events in locally advanced gastric cancer patients undergoing radical resection+HIPEC (both P<0.05). Conclusions: Radical surgery + HIPEC does not significantly increase the incidence of perioperative complications in patients with advanced gastric cancer. The age >60 years and nerve invasion are independent risk factors for adverse events in these patients.

[Establishment of a cytokine release syndrome associated with chimeric antigen receptor T cell treatment in SCID/Beige mice model].

Objective: To establish a cytokine release syndrome (CRS) mouse model related to CAR-T cell therapy and provide a research model for the clinical phenomena. Methods: CAR-T cells targeting human CD19 molecule were constructed by molecular cloning and lentiviral transfection. Flow cytometry (FACS) was used to detect the transfection efficiency of CAR-T cells. The tumor-killing efficiency of CAR-T cells was detected by ELISA and flow cytometry. The CAR-T cells were injected into the tumor-bearing SCID/Beige mice through tail vein, and divided into phosphate buffered solution (PBS) group, low-burden group (1×10(5) Raji-Luc2 cells) and high-burden group (5×10(5) Raji-Luc2 cells). The tumor treatment effect was detected by animal in vivo imaging. Serum levels of cytokines including human IFN-γ, human IL-2, mouse IL-6, and mouse GM-CSF were measured by ELISA. The health status of the mice was evaluated by pathological examination. Results: The health scores of T cell group and T cell+ OKT-3 group were (1.15±0.08) and (2.90±0.15), respectively, after the injection of human T cell and T cell + OKT-3 antibody through tail vein, and the difference was statistically significant (P<0.001). The serum levels of human IL-2, human IFN-γ, human IL-15, mouse IL-6 and mouse GM-CSF in T cell+ OKT-3 group were (1 064.00±50.14), (1 285.00±193.90), (202.4±18.76), (1 478.00±289.20) and (350.70±42.27) pg/ml, respectively, higher than (22.67±6.36), (23.67±3.71), (44.33±14.45), (147.30±36.20), (138.00±22.74) pg/ml in T cell group (P<0.05). OKT-3 combined with human T cells caused a rapid increase in serum levels of human IL-2, human IFN-γ, mouse IL-6 and mouse GM-CSF, accompanied by an increase in body temperature and weight loss. CD19-targeting CAR-T cells were successfully constructed, and the positive rate of CAR-T cells was >30% detected by flow cytometry. ELISA results showed that in the presence of CD19 antigen, IL-2 and IFN-γ secreted by CAR-T19 cells co-incubated with Raji and Nalm were (561.00±37.07), (680.30±71.27), (369±25.71) and (523.00±26.31) pg/ml, respectively, higher than (55.00±20.53) and (64.00±7.55) pg/ml in the co-incubated with K562 group (P<0.001). Activated CAR-T19 cells were reinjected through the tail vein on the seventh day after tumor formation. Imaging experiments in mice showed that on the thirteenth day after tumor formation, the fluorescence intensities of tumors in the low-burden and high-burden groups were lower than on the seventh day of tumor inoculation, and the fluorescence intensity of tumors in the high-burden group decreased from 144.00±24.69 to 5.02±2.35 (P=0.005). The fluorescence intensity of low burden group decreased from 58.47±9.36 to 3.48±1.67 (P=0.004). The serum levels of T cell activation related cytokines IL-2, IL-15 and IFN-γ increased rapidly, and the secretion of monocyte related cytokines IL-16 and GM-CSF increased, accompanied by the typical characteristics of CRS such as increased body temperature and weight loss at 72 hours after injection of CAR-T19 cells. Conclusions: CAR-T cells targeting CD19 molecule are successfully constructed, and CRS phenomenon is verified in tumor-bearing mice by CAR-T cell re-infusion, providing an animal model for the mechanism of CAR-T treatment-related CRS and CRS prevention strategies.

[Drug-coated balloons versus bare metal stent for treatment of femoropopliteal lesions:36 month follow-up results of single center].

Objective: To examine the therapeutic effects of drug-coated balloon (DCB) and bare metal stent (BMS) on primary femoropopliteal disease (FPAD) in the real world. Methods: This was a retrospective analysis of single-center follow-up results at 12,24,and 36 months of patients with FPAD lesions that were treated with DCB and BMS at Department of Aortic and Vascular Surgery, Fu Wai Hospital.One-to-one propensity score matching(PSM) was performed to balance the covariance between DCB group (137 cases) and BMS group (100 cases). Freedom from clinically driven target lesion reintervention rate(fCD-TLR) was determined by Kaplan-Meier curve.Log-rank test was used to compare the rates of fCD-TLR between DCB and BMS groups at 12,24,36 months post-operation. Results: After PSM, there were both 71 patients in each group,aged (68.0±9.6) years(range: 46 to 90 years) and (68.8±7.3) years(range: 48 to 87 years),lesion lengths were (119.6±14.2)mm(range:40 to 380 mm) and (110.8±13.1)mm(range:40 to 400 mm). The median follow-up period were 24.3 months (range:5.8 to 55.1 months).There was no death,amputation or reintervention within the 30 days after operation.The rates of fCD-TLR for DCB group at 12,24 and 36 months were 97.2%,85.9%,69.1%, and 95.8%,83.1%,59.2% for BMS group.There was no statistical difference between the two groups by Log-rank test (P=0.551). Conclusion: DCB and BMS can both maintain favorable clinical effects in FPAD patients at 12,24,36 months post-operation.