1.Single-cell RNA sequencing and the pathogenesis of intervertebral disc degeneration
Haotian CHENG ; Xiaofeng ZHAO ; Xiangdong LU ; Yibo ZHAO ; Zhifeng FAN ; Detai QI ; Xiaonan WANG ; Runtian ZHOU ; Xinjie JIN ; Bin ZHAO
Chinese Journal of Tissue Engineering Research 2024;28(1):93-99
BACKGROUND:Intervertebral disc degeneration is clinically considered to be the main cause of low back pain,but due to the unclear pathogenesis of intervertebral disc degeneration,there is still a lack of effective means to delay the progression of the disease.Single-cell RNA sequencing technology can amplify and sequence mRNA at the single-cell level,reveal the gene expression intensity of a single cell,discover different cell subsets in tissues according to the heterogeneity of cells,study the pathogenesis of intervertebral disc degeneration at the molecular level,and provide a new theoretical basis for its early diagnosis and treatment. OBJECTIVE:To introduce the basic principles of single-cell RNA sequencing technology and review the research progress of single-cell RNA sequencing technology in intervertebral disc degeneration in recent years. METHODS:A computer was used to search PubMed,Web of Science,CNKI and WanFang databases for the literature published from 2012 to 2022.Key words were"single-cell RNA sequencing,intervertebral disc degeneration,sequencing Technology"in Chinese and English.Duplicate,poor-quality and irrelevant articles were excluded;a total of 70 articles were eventually included. RESULTS AND CONCLUSION:(1)We identified new cell subsets such as homeostatic chondrocytes,hypertrophy chondrocyte-like nucleus pulposus cells and fibrous nucleus pulposus cells,identified the marker genes and transcription factors of these cell subsets,and described the functions,differentiation paths and cell fate of these cell subsets during the development and progression of intervertebral disc degeneration,and proposed the concept of progenitor nucleus pulposus cells.A cell subpopulation with progenitor nucleus pulposus cells properties was identified and its effectiveness in treating intervertebral disc degeneration was verified in mice.(2)Fibro chondrocyte-like annulus fibrosus cells and annulus fibrosus stem cells with both cartilage and fiber properties were identified,and a new type of composite hydrogel was prepared by combining fibrous cartilage inducers silk fibroin and hyaluronic acid in vitro.Experiments in mice demonstrated that this hydrogel could repair both annulus fibrosus tissue and cartilage matrix,and was remarkably effective in the treatment of intervertebral disc degeneration.(3)Regulatory chondrocytes were found in endplate cartilage.Two distinct fates in the progression of intervertebral disc degeneration were analyzed and the differential genes in the two fates were identified.Intercellular communication analysis indicated that regulatory chondrocytes interact with endothelial cells to promote angiogenesis.(4)Immune cells such as macrophages,T cells,myeloid progenitor cells and neutrophils were identified in the degenerated intervertebral disc tissues,demonstrating the existence of immune response during intervertebral disc degeneration.It was found that apolipoprotein induced the polarization of macrophages M1 and M2 subtypes,and this polarization process affected the activity of progenitor nucleus pulposus cells by amplifying the inflammatory response through the MIF signaling pathway.
2.Disease burden of acute viral hepatitis in Guangdong Province, 1990-2019
Jiamin QIU ; Fangfang ZENG ; Chen CHENG ; Huiyan WEN ; Shiqi HUANG ; Dan LIU ; Jinlei QI ; Peng YIN ; Maigeng ZHOU ; Ying XU ; Zhiping LIU ; Qingsong MEI ; Heng XIAO ; Zheng XIANG ; Xiaofeng LIANG
Chinese Journal of Epidemiology 2024;45(3):365-372
Objective:To examine the burden and trends of acute viral hepatitis in Guangdong Province from 1990 to 2019, and provide reference evidences for hepatitis prevention and control in the province.Methods:Data on acute viral hepatitis (hepatitis A, B, C, and E) in Guangdong from 1990 to 2019 were extracted from the Global Burden of Disease Study 2019 database. The incidence, prevalence, mortality, and disability-adjusted life years (DALY) data were analyzed by age and gender, and the estimated annual percentage change (EAPC) was calculated to describe the changing trends in disease burden.Results:From 1999 to 2019, the standardized incidence, prevalence, mortality, and DALY of acute viral hepatitis in Guangdong were higher than the national averages. In 2019, 51.43% (2 245 087/4 365 221) of acute viral hepatitis cases in Guangdong Province were mainly attributed to hepatitis B, and 77.18% (106/138) of deaths were due to acute hepatitis B. In different age groups, except for acute hepatitis B, which was more common in adults, the incidence rates of other types of viral hepatitis such as hepatitis A, B, and E showed an overall decreasing trend with age. The mortality rates of different types of acute viral hepatitis, except for the <5 age group, increased with age. The overall incidence and mortality rates of acute viral hepatitis were higher in men than in women.Conclusions:The overall burden of acute viral hepatitis in Guangdong declined in 2019, but remained higher than the national level. Further efforts are needed to strengthen hepatitis prevention and screening in different population in Guangdong Province, especially in children and the elderly.
3.Clinical Validation of a Prototype Smart Non-Invasive Pregnancy Glucose Monitor
Heqing LU ; Lirui CHENG ; Qiaoling DU ; Xiaofeng ZHANG ; Zhenzhen SUN ; Zhigang ZHU
Chinese Journal of Medical Instrumentation 2024;48(5):539-543
The aim of this study is to evaluate the effectiveness of a smart non-invasive blood glucose monitor prototype during pregnancy through clinical validation.The monitor utilizes near-infrared spectroscopy combined with AI big data analysis of photoelectric volumetric pulse wave data to achieve non-invasive monitoring of blood glucose in women during pregnancy.The research team developed a monitor that employs a sensing chip,effectively overcoming the problems of weak signals and individual differences in non-invasive blood glucose monitoring.The user experience is enhanced by visualizing the test results on the accompanying cell phone APP(application)of the smart non-invasive pregnancy blood glucose monitor.Clinical validation revealed that the non-invasive monitoring data for pregnant women aged 20~30 years significantly differed from those obtained via traditional blood glucose measurement methods,whereas no significant difference(P<0.05)was observed for pregnant women aged 31~42 years.The study concluded that further calibration of the monitor and an expansion of the sample size are necessary to enhance consistency with invasive glucose monitoring results.
4.Surgical treatment and prognosis analysis of hilar cholangiocarcinoma
Xiangcheng LI ; Changxian LI ; Hui ZHANG ; Feng CHENG ; Feng ZHANG ; Liyong PU ; Chuanyong ZHANG ; Ke WANG ; Lianbao KONG ; Xiaofeng QIAN ; Donghua LI ; Wenxiong LU ; Ping WANG ; Aihua YAO ; Jianfeng BAI ; Xiaofeng WU ; Ruixiang CHEN ; Xuehao WANG
Chinese Journal of Surgery 2024;62(4):290-301
Objective:To investigate the surgical treatment effect and prognostic factors of hilar cholangiocarcinoma.Methods:This is an ambispective cohort study. From August 2005 to December 2022,data of 510 patients who diagnosed with hilar cholangiocarcinoma and underwent surgical resection at the Hepatobiliary Center of the First Affiliated Hospital of Nanjing Medical University were retrospectively collected. In the cohort,there were 324 males and 186 females,with an age of ( M (IQR)) 63(13)years (range:25 to 85 years). The liver function at admission was Child-Pugh A (343 cases,67.3%) and Child-Pugh B (167 cases,32.7%). Three hundred and seventy-two(72.9%) patients had jaundice symptoms and the median total bilirubin was 126.3(197.6) μmol/L(range: 5.4 to 722.8 μmol/L) at admission. Two hundred and fourty-seven cases (48.4%) were treated with percutaneous transhepatic cholangial drainage or endoscopic nasobiliary drainage before operation. The median bilirubin level in the drainage group decreased from 186.4 μmol/L to 85.5 μmol/L before operation. Multivariate Logistic regression was used to identify the influencing factors for R0 resection,and Cox regression was used to construct multivariate prediction models for overall survival(OS) and disease-free survival(DFS). Results:Among 510 patients who underwent surgical resection,Bismuth-Corlett type Ⅲ-Ⅳ patients accounted for 71.8%,among which 86.1% (315/366) underwent hemi-hepatectomy,while 81.9% (118/144) underwent extrahepatic biliary duct resection alone in Bismuch-Corlett type Ⅰ-Ⅱ patients. The median OS time was 22.8 months, and the OS rates at 1-,3-,5-and 10-year were 72.2%,35.6%,24.8% and 11.0%,respectively. The median DFS time was 15.2 months,and the DFS rates was 66.0%,32.4%,20.9% and 11.0%,respectively. The R0 resection rate was 64.5% (329/510), and the OS rates of patients with R0 resection at 1-,3-,5-and 10-year were 82.5%, 48.6%, 34.4%, 15.2%,respectively. The morbidity of Clavien-Dindo grade Ⅲ-Ⅴ complications was 26.1%(133/510) and the 30-day mortality was 4.3% (22/510). Multivariate Logistic regression indicated that Bismuth-Corlett type Ⅰ-Ⅲ ( P=0.009), hemi-hepatectomy and extended resection ( P=0.001),T1 and T2 patients without vascular invasion (T2 vs. T1: OR=1.43 (0.61-3.35), P=0.413;T3 vs. T1: OR=2.57 (1.03-6.41), P=0.010;T4 vs. T1, OR=3.77 (1.37-10.38), P<0.01) were more likely to obtain R0 resection. Preoperative bilirubin,Child-Pugh grade,tumor size,surgical margin,T stage,N stage,nerve infiltration and Edmondson grade were independent prognostic factors for OS and DFS of hilar cholangiocarcinoma patients without distant metastasis. Conclusions:Radical surgical resection is necessary to prolong the long-term survival of hilar cholangiocarcinoma patients. Hemi-hepatectomy and extended resection,regional lymph node dissection and combined vascular resection if necessary,can improve R0 resection rate.
5.Effect of anesthesia mode during endovascular treatment on neurological functional outcomes in patients with acute posterior circulation stroke
Xiaofeng ZHU ; Zheyu ZHANG ; Wansi ZHONG ; Yaode HE ; Zhongyu LUO ; Ningyuan ZHANG ; Chaochan CHENG ; Jianhong YANG ; Min LOU
Journal of Zhejiang University. Medical sciences 2024;53(2):151-159
Objective:To compare the effect of anesthesia mode on the neurological functional outcomes in patients undergoing endovascular treatment for acute posterior circulation ischemic stroke.Methods:Clinical data of 656 patients undergoing intravascular therapy for acute posterior circulation ischemic stroke registered in online Acute Stroke Patients for Stroke Management Quality Evaluation Database from January 2017 to December 2022 were retrospectively analyzed.The data included 163 cases with conscious sedation and 493 cases with general anesthesia during the procedure.After propensity score matching,428 patients were included in the analysis,including 155 cases in the conscious sedation group and 273 cases in the general anesthesia group.The differences of operation mode,etiology type,vascular recanalization,hemorrhagic transformation at 24 h,modified Rankin Scale(mRS)score at 3 months and mortality within 3 months were compared between the two groups.Binary logistic regression was used to explore the effect of different anesthesia mode on neurological functional outcomes.Results:There was a significant difference in operation mode between the two groups(P<0.01),while there were no significant differences in etiology type,vascular recanalization,hemorrhagic transformation at 24 h,mRS score at 3 months or mortality within 3 months(all P>0.05).Binary logistic regression analysis revealed that anesthesia modes were not significantly associated with functional outcomes of patients(OR=1.151,95%CI:0.751-1.765,P>0.05).Conclusion:Anesthesia mode(conscious sedation or general anesthesia)will not affect the neurological functional outcomes in patients with acute posterior circulation ischemic stroke undergoing endovascular treatment.
6.Research progress of non-coding RNA in methamphetamine-induced addiction and neurotoxicity
Shuwei ZHANG ; Hao CHENG ; Haowei WANG ; Lin MIAO ; Yi LI ; Lina GUAN ; Xiaofeng ZENG
Chinese Journal of Comparative Medicine 2024;34(2):114-121
Methamphetamine(METH)is highly addictive and neurotoxic,which causes cognitive and memory dysfunction in abusers.The harm of METH lies not only in its own toxicity,but also in the high physical and mental dependence of drug addicts,often causing mental disorders and violent behavior,bringing great safety risks to society.Non-coding RNA(ncRNA)does not encode proteins and is an important factor in regulating gene expression at the post-transcriptional level.Studies have shown that ncRNA plays an important regulatory role in methamphetamine-induced addiction and neurotoxicity,but the specific mechanism is unclear.This article reviews the current research progress of ncRNA in regulating METH-induced addiction and neurotoxicity to provide a reference for ncRNA as a forensic identification index and potential therapeutic target for METH abusers.
7.Sampling inspection results of disinfection products in Shaanxi Province in 2020 - 2023
Yongbing CHENG ; Baobao CHEN ; Xiaoling ZHANG ; Jianfeng XUE ; Xiaofeng DONG ; Zhaowei MENG
Journal of Public Health and Preventive Medicine 2024;35(6):93-96
Objectives To understand the quality status of disinfection products in the market in Shaanxi Province, and to provide a basis for taking targeted management measures. Methods From 2020 to 2023, in accordance with the national disinfection product management regulations and standards, the physicochemical and microbial tests of disinfection products in Shaanxi Province were carried out through supervised sampling, and the test results were analyzed according to the active ingredients, illegal addition, stability, and bactericidal or bacteriostatic performance. Results The overall qualification rate of the active ingredient content of disinfectants sampled in Shaanxi Province was 90.48%, with the lowest qualification rate in 2023 (84.38%). The overall qualification rate of quantitative sterilization test of disinfectants was 90.12%, showing a decreasing trend year by year (P=0.272). The overall qualification rate of anti-bacterial products was 88.59%, and the bactericidal test results of 20 antibacterial products were all qualified. The overall qualification rate of bacteriostatic performance testing for bacteriostatic products was 86.17%, with the pass rate of bacteriostatic product germicidal efficacy testing of different components in descending order being quaternary ammonium salt/silver ion > guanidine > others > lysozyme, and there was statistically significant difference (P=0.004). The detection rate of illegal additives in antimicrobial products was 7.07%, with the main detection indicators being miconazole nitrate, clobetasol propionate, and dexamethasone acetate. Conclusion The qualified rate of disinfection products in Shaanxi Province is relatively high, but there is a downward trend. It is necessary to continue to strengthen the daily supervision and product testing of disinfection product manufacturers, and promote the continuous improvement of disinfection product quality.
8.Epidemiological and clinical characteristics of respiratory syncytial virus infection among people aged 60 and above in Beijing City
Xiaofeng WEI ; Maozhong LI ; Yiting WANG ; Qi HUANG ; Cheng GONG ; Luodan SUO ; Fang HUANG
Chinese Journal of Preventive Medicine 2024;58(7):952-958
Objective:To investigate the epidemiological and clinical characteristics of RSV among patients aged ≥60 years in Beijing from 2015 to 2023.Methods:Based on the respiratory pathogen surveillance system, samples of upper respiratory tract infections (URTI), non-severe community-acquired pneumonia (nsCAP) and severe community-acquired pneumonia (sCAP) among people aged ≥60 years were collected from 28 sentinel hospitals in 16 districts of Beijing from January 2015 to December 2023. Swab samples were collected from URTI within one week, and lower respiratory tract samples from nsCAP and sCAP were collected. Demographic and epidemiological data were also collected. Various respiratory pathogens including RSV were detected.Results:From January 2015 to December 2023, a total of 20 349 cases of acute respiratory infections aged ≥60 years were included, with the RSV-positive rate of 1.54% (313/20 349, 95% CI: 1.39%-1.68%). Among them, the total RSV-positive rates of older people during the pre-pandemic, pandemic, and post-pandemic periods of COVID-19 were 1.59% (207/13 006, 95% CI: 1.38%-1.81%), 0.82% (38/4 650, 95% CI: 0.56%-1.08%) and 2.53% (68/2 693, 95% CI: 1.93%-3.12%), respectively. The difference in RSV-positive rate was statistically significant ( P<0.001). Based on the sampling time of cases, the RSV epidemic season for older people in Beijing was from October to March of the following year, with a peak period in December or January of the following year. In the post COVID-19 pandemic, there were very few RSV-positive cases detected in the elderly from April to June 2023, with only one positive case detected in May and one in June. The RSV-positive rate of older people increased significantly from October to December, reaching 11.75% (51/383) in December. Among 263 RSV-positive cases in the elderly, RSV-A, RSV-B and unclassified type accounted for 43.35% (114/263), 29.28% (77/263) and 27.38% (72/263), respectively. Since 2020, there has been a subtype conversion, with RSV-B being the main focus. Among 197 elderly cases that have complete clinical data, the main symptoms were cough (86.8%, 171/197), sputum (80.2%, 158/197) and fever (73.60%, 145/197). About 24.87% (49/197) of elderly cases experienced complications. The hospitalization mortality rate was 4.57% (9/197), and the hospitalization rate was 78.68% (155/197). The ICU occupancy rate was 1.99% (36/197). The mechanical ventilation usage rate was 13.32% (33/197), and the length of hospital stay [ M ( Q1, Q3)] was 12 (9, 16) days. Conclusion:In Beijing, the RSV infection rate is relatively low during the COVID-19 pandemic, and the prevalence of COVID-19 is relatively high. In 2023, there was no out-of-season outbreak of RSV infection among the elderly. Elderly RSV infection cases have multiple complications, severe diseases, and poor prognosis.
9.Reference values of carotid intima-media thickness and arterial stiffness in Chinese adults based on ultrasound radio frequency signal: A nationwide, multicenter study
Changyang XING ; Xiujing XIE ; Yu WU ; Lei XU ; Xiangping GUAN ; Fan LI ; Xiaojun ZHAN ; Hengli YANG ; Jinsong LI ; Qi ZHOU ; Yuming MU ; Qing ZHOU ; Yunchuan DING ; Yingli WANG ; Xiangzhu WANG ; Yu ZHENG ; Xiaofeng SUN ; Hua LI ; Chaoxue ZHANG ; Cheng ZHAO ; Shaodong QIU ; Guozhen YAN ; Hong YANG ; Yinjuan MAO ; Weiwei ZHAN ; Chunyan MA ; Ying GU ; Wu CHEN ; Mingxing XIE ; Tianan JIANG ; Lijun YUAN
Chinese Medical Journal 2024;137(15):1802-1810
Background::Carotid intima-media thickness (IMT) and diameter, stiffness, and wave reflections, are independent and important clinical biomarkers and risk predictors for cardiovascular diseases. The purpose of the present study was to establish nationwide reference values of carotid properties for healthy Chinese adults and to explore potential clinical determinants.Methods::A total of 3053 healthy Han Chinese adults (1922 women) aged 18-79 years were enrolled at 28 collaborating tertiary centers throughout China between April 2021 and July 2022. The real-time tracking of common carotid artery walls was achieved by the radio frequency (RF) ultrasound system. The IMT, diameter, compliance coefficient, β stiffness, local pulse wave velocity (PWV), local systolic blood pressure, augmented pressure (AP), and augmentation index (AIx) were then automatically measured and reported. Data were stratified by age groups and sex. The relationships between age and carotid property parameters were analyzed by Jonckheere-Terpstra test and simple linear regressions. The major clinical determinants of carotid properties were identified by Pearson’s correlation, multiple linear regression, and analyses of covariance.Results::All the parameters of carotid properties demonstrated significantly age-related trajectories. Women showed thinner IMT, smaller carotid diameter, larger AP, and AIx than men. The β stiffness and PWV were significantly higher in men than women before forties, but the differences reversed after that. The increase rate of carotid IMT (5.5 μm/year in women and 5.8 μm/year in men) and diameter (0.03 mm/year in both men and women) were similar between men and women. For the stiffness and wave reflections, women showed significantly larger age-related variations than men as demonstrated by steeper regression slopes (all P for age by sex interaction <0.05). The blood pressures, body mass index (BMI), and triglyceride levels were identified as major clinical determinants of carotid properties with adjustment of age and sex. Conclusions::The age- and sex-specific reference values of carotid properties measured by RF ultrasound for healthy Chinese adults were established. The blood pressures, BMI, and triglyceride levels should be considered for clinical application of corresponding reference values.
10.Inhibitory effect of Lactobacillus reuteri on rotavirus replication in vivo and in vitro and its effect on expression of immune factors
Xiaofeng LI ; Meihui CHENG ; Yang LIU ; Changcheng LIU ; Xuejiao JIA ; Mengqi LIU ; Wei ZHAO
Journal of Jilin University(Medicine Edition) 2024;50(6):1597-1605
Objectives:To discuss the inhibitory effect of Lactobacillus reuteri on the replication of rotavirus(RV)strain SA11 in vivo and in vitro,and to clarify its effect on the expression of related immune factors.Methods:For in vitro experiments,Lactobacillus reuteri was cultured and identified,and the standard curve and growth curve were plotted to screen the optimal time and concentration for Lactobacillus reuteri cultivation.The cells were infected with Lactobacillus reuteri at the concentrations of 5×108,10×108,50×108,100×108,200×108,and 500×108 CFU·mL-1,and the surival rates of Caco-2 cells were detected by trypan blue staining method.Various concentrations of Lactobacillus reuteri were co-incubated with RV in vitro and applied to the Caco-2 cells.The cells were divided into negative control group(NC group),positive control group(PC group),and 107,108,109,and 1010 CFU·mL-1 Lactobacillus reuteri groups.Immunofluorescence focus method was used to detect the viral titers in the Caco-2 cells after treated with Lactobacillus reuteri and real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the copy numbers of RV VP6 gene in the Caco-2 cells after treated with various concentrations of Lactobacillus reuteri.In in vivo experiments,25 litters of SPF suckling mice were divided into control group,RV group(infected with SA11 strain),Ab-NC group(treated with antibiotic to deplete gut microbiota),Ab-RV group(depleting gut microbiota and then infected with SA11 strain),and Ab-Lac-RV group(depleting gut microbiota,treated with Lactobacillus reuteri,and then infected with SA11 strain).The fecal samples were collected on days 2,4,6,8,and 10 gavage,colon tissue sample were collected on day 4 of and RT-qPCR method was used to detect the copy numbers of RV VP6 gene in feces and the mRNA expression levels of interleukin(IL)-1β,IL-8,IL-10,interferon-γ(IFN-γ),and tumor necrosis factor-α(TNF-α)in colon tissue of the suckling mice in vartious groups.Results:The Lactobacillus reuteri grew well,with round,smooth,and milky white convex colonies and neat edges.After Gram staining,the bacteria appeared purple,irregular,and square-shaped rods.16SrDNA sequencing showed 99%sequence homology,indicating successful activation of Lactobacillus reuteri.The number of live Lactobacillus reuteri was linearly related to the absorbance(A)value,and the standard curve for regression analysis was Y=0.437 5X+0.000 6,R2=0.999 4.During the 0-2 h cultivation period,the bacteria were at the logarithmic growth phase with slow growth;from 2-14 h,the bacteria grew rapidly and stabilized at 14-16 h,reaching the growth rate peak at 16 h,after which they entered the decline phase.Infection with Lactobacillus reuteri at concentrations of 5×108,10×108,50×108,100×108,and 200×108 CFU·mL-1 resulted in the survival rates of Caco-2 cells were all>90%,so these concentrations were selected for the further experiments.Compared with PC group,the copy numbers of RV VP6 gene in the Caco-2 cells in 5×108,10×108,50×108,100×108,and 200×108 CFU·mL-1 Lactobacillus reuteri groups were significantly decreased(P<0.01).Compared with PC group,the viral titers in the Caco-2 cells in 107,108,109,and 1010 CFU·mL-1 Lactobacillus reuteri groups were significantly decreased(P<0.01).Compared with control group,the numbers of gut microbiota colonies in Ab-NC,Ab-RV,and Ab-Lac-RV groups were significantly decreased,indicating successful depletion of gut microbiota in the suckling mice.On days 2 and 4 after gavage,the RV VP6 gene copy number in the feces in Ab-RV group was significantly lower than that in RV group(P<0.05).On days 4,6,8,and 10 after gavage,the RV VP6 gene copy number in the feces in Ab-Lac-RV group was significantly lower than that in Ab-RV group(P<0.05).Compared with control group,the expression levels of IL-1β,IL-10,IFN-γ,and TNF-α mRNA in colon tissue in Ab-RV and Ab-Lac-RV groups were significantly increased(P<0.05 or P<0.01),while the expression level of IL-8 mRNA was significantly decreased(P<0.05),and the expression level of IL-10 mRNA in colon tissue in Ab-LAC-RV group was significantly increased(P<0.01).Conclusion:Lactobacillus reuteri may inhibit the RV replication by upregulating the expressions of IL-1β,IL-10,IFN-γ,and TNF-α mRNA and downregulating the expression of IL-8 mRNA.


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