Single-cell transcriptomics reveals cell atlas and identifies cycling tumor cells responsible for recurrence in ameloblastoma

Ameloblastoma is a benign tumor characterized by locally invasive phenotypes,leading to facial bone destruction and a high recurrence rate.However,the mechanisms governing tumor initiation and recurrence are poorly understood.Here,we uncovered cellular landscapes and mechanisms that underlie tumor recurrence in ameloblastoma at single-cell resolution.Our results revealed that ameloblastoma exhibits five tumor subpopulations varying with respect to immune response(IR),bone remodeling(BR),tooth development(TD),epithelial development(ED),and cell cycle(CC)signatures.Of note,we found that CC ameloblastoma cells were endowed with stemness and contributed to tumor recurrence,which was dominated by the EZH2-mediated program.Targeting EZH2 effectively eliminated CC ameloblastoma cells and inhibited tumor growth in ameloblastoma patient-derived organoids.These data described the tumor subpopulation and clarified the identity,function,and regulatory mechanism of CC ameloblastoma cells,providing a potential therapeutic target for ameloblastoma.

Graft-versus-host disease after liver transplantation:A comprehensive literature review

AIM： To determine the factors affecting mortality in pa- tients who developed graft-versus-host disease （GvHD） after liver transplantation （LT）. METHODS： We performed a review of studies of GvHD following LT published in the English literature and ac- cessed the PubMed, Medline, EBSCO, EMBASE, and Google Scholar databases. Using relevant search phras- es, 88 articles were identified. Of these, 62 articles con- raining most of the study parameters were considered eligible for the study. Risk factors were first examined using a univariate Kaplan-Meier model, and variables with a significant association （P 〈 0.05） were then sub- jected to multivariate analyses using a Cox proportional- hazards model. RESULTS： The 61 articles reported 87 patients, 58 male and 29 female, mean age, 40.4 ± 15.5 years （range： 8 mo to 74 years）, who met the inclusion criteria for the present study. Deaths occurred in 59 （67.8%） patients, whereas 28 （32.2%） survived after a mean follow-up period of 280.8 ± 316.2 d （range： 27-2285 d）. Among the most frequent symptoms were rash （94.2%）, fever （66.6%）, diarrhea （54%）, and pancytopenia （54%）. The average time period between LT and first symptom on- set was 60.6 ± 190.1 d （range： 2-1865 d）. The Kaplan- Meier analysis revealed that pancytopenia （42.8% vs 59.3%, P = 0.03）, diarrhea （39.2% vs 61.0%, P = 0.04）, age difference between the recipient and the donor （14.6 ± 3.1 years vs 22.6 ± 2.7 years, P 〈 0.0001）, and time From first symptom occurrence to diagnosis or treatment （13.3 ± 2.6 mo vs 15.0 ± 2.3 mo, P 〈 0.0001） were significant factors affecting mortality, whereas age, sex, presence of rash and fever, use of immunosuppressive agents, acute rejection before GvHD, etiological causes, time of onset, and donor type were not associated with mortality risk. The Cox proportional-hazards model, de- termined that an age difference between the recipient and donor was an independent risk Factor （P = 0.03; hazard ratio, 7.395, 95% confidence interval, 1.2-46.7）. CONCLUSION： This study showed that an age differ- ence between the recipient and donor is an independent risk factor for mortality in patients who develop GvHD after LT.

Outcomes and mechanisms of ischemic preconditioning in liver transplantation

BACKGROUND: Liver transplantation is so far the most effective therapeutic modality for end-stage liver diseases, but ischemia/reperfusion (I/R) injury represents a critical barrier to liver transplantation. Primary graft dysfunction and small-for-size syndrome are closely associated with I/R injury. Ischemic preconditioning (IPC) is defined as a brief period of liver ischemia followed by reperfusion, and has demonstrated protections against a prolonged I/R injury and improved the capacity of regeneration. The article aimed to review IPC literatures for the understanding of the effects of IPC on I/R injury involving in the procurement of donor liver and protective mechanisms. DATA SOURCES: A literature search of MEDLINE and Web of Science databases using 'liver transplantation', 'liver regeneration', 'hepatectomy', 'ischemia/reperfusion' and 'ischemic preconditioning' was performed, and then a large amount of related data was collected. RESULTS: The literature search provided a huge amount of evidence for the protective effects of IPC on I/R injury in liver transplantation, including reduction of blood loss in hepatectomy, intraoperative hemodynamic stability and its significant role in liver regeneration. The mechanism involves in balancing inflammatory cytokines, enhancing energy status and mitigating microcirculatory disturbance. CONCLUSION: IPC plays an essential role in hepatectomy before and after harvest of living donor liver and implantation of liver graft.

Effects of gender-affirming hormone therapy on insulin resistance and body composition in transgender individuals: A systematic review

BACKGROUND Transgender individuals receiving masculinising or feminising gender-affirming hormone therapy with testosterone or estradiol respectively,are at increased risk of adverse cardiovascular outcomes,including myocardial infarction and stroke.This may be related to the effects of testosterone or estradiol therapy on body composition,fat distribution,and insulin resistance but the effect of genderaffirming hormone therapy on these cardiovascular risk factors has not been extensively examined.AIM To evaluate the impact of gender-affirming hormone therapy on body composition and insulin resistance in transgender individuals,to guide clinicians in minimising cardiovascular risk.METHODS We performed a review of the literature based on PRISMA guidelines.MEDLINE,Embase and PsycINFO databases were searched for studies examining body composition,insulin resistance or body fat distribution in transgender individuals aged over 18 years on established gender-affirming hormone therapy.Studies were selected for full-text analysis if they investigated transgender individuals on any type of gender-affirming hormone therapy and reported effects on lean mass,fat mass or insulin resistance.RESULTS The search strategy identified 221 studies.After exclusion of studies that did not meet inclusion criteria,26 were included(2 cross-sectional,21 prospectiveuncontrolled and 3 prospective-controlled).Evidence in transgender men suggests that testosterone therapy increases lean mass,decreases fat mass and has no impact on insulin resistance.Evidence in transgender women suggests that feminising hormone therapy(estradiol,with or without anti-androgen agents)decreases lean mass,increases fat mass,and may worsen insulin resistance.Changes to body composition were consistent across almost all studies:Transgender men on testosterone gained lean mass and lost fat mass,and transgender women on oestrogen experienced the reverse.No study directly contradicted these trends,though several small studies of short duration reported no changes.Results for insulin resistance are less consistent and uncertain.There is a paucity of prospective controlled research,and existing prospective evidence is limited by small sample sizes,short follow up periods,and young cohorts of participants.CONCLUSION Further research is required to further characterise the impact of genderaffirming hormone therapy on body composition and insulin resistance in the medium-long term.Until further evidence is available,clinicians should aim to minimise risk by monitoring cardiovascular risk markers regularly in their patients and encouraging healthy lifestyle modifications.

Development of the Scientific,Transparent and Applicable Rankings (STAR) tool for clinical practice guidelines

Background:This study aimed to develop a comprehensive instrument for evaluating and ranking clinical practice guidelines,named Scientific,Transparent and Applicable Rankings tool(STAR),and test its reliability,validity,and usability.Methods:This study set up a multidisciplinary working group including guideline methodologists,statisticians,journal editors,clinicians,and other experts.Scoping review,Delphi methods,and hierarchical analysis were used to develop the STAR tool.We evaluated the instrument’s intrinsic and interrater reliability,content and criterion validity,and usability.Results:STAR contained 39 items grouped into 11 domains.The mean intrinsic reliability of the domains,indicated by Cronbach’sαcoefficient,was 0.588(95%confidence interval[CI]:0.414,0.762).Interrater reliability as assessed with Cohen’s kappa coefficient was 0.774(95%CI:0.740,0.807)for methodological evaluators and 0.618(95%CI:0.587,0.648)for clinical evaluators.The overall content validity index was 0.905.Pearson’s r correlation for criterion validity was 0.885(95%CI:0.804,0.932).The mean usability score of the items was 4.6 and the median time spent to evaluate each guideline was 20 min.Conclusion:The instrument performed well in terms of reliability,validity,and efficiency,and can be used for comprehensively evaluating and ranking guidelines.

Recent Progress and Future Direction for the Application of Multiomics Data in Clinical Liver Transplantation

Omics data address key issues in liver transplantation(LT)as the most effective therapeutic means for end-stage liver disease.The purpose of this study was to review the current application and future direction for omics in LT.We reviewed the use of multiomics to elucidate the pathogenesis leading to LT and prognostication.Future directions with respect to the use of omics in LT are also described based on perspectives of surgeons with experience in omics.Significant molecules were identified and summarized based on omics,with a focus on post-transplant liver fibrosis,early allograft dysfunction,tumor recurrence,and graft failure.We emphasized the importance omics for clinicians who perform LTs and prioritized the directions that should be established.We also outlined the ideal workflow for omics in LT.In step with advances in technology,the quality of omics data can be guaranteed using an improved algorithm at a lower price.Concerns should be addressed on the translational value of omics for better therapeutic effects in patients undergoing LT.

Role of 1, 25-dihydroxyvitamin D3 in preventing acute rejec tion of allograft following rat orthotopic liver transplantation

Background We investigated the role of 1, 25-dihydro xyvitamin D3 (1, 25-(OH) 2D 3) in preventing allograft from acute rejection following orthotopic liver transplantation. Methods A rat orthotopic liver transplantation model was used i n this study. SD-Wistar rats served as a high responder strain combination. Recipients were subjected to administration of 1, 25-(OH) 2 D 3 at dosages ranging from 0.25 μg·kg -1 ·d -1 to 2.5 μg·kg -1 ·d -1 . Survival a fter transplantation as well as pathological rejection grades and IFN-γ mRNA, IL-10 mRNA transcription intragraft on day 7, and day 30 post-transplantation were observed. Results After recipients were treated with 1, 25(OH) 2 D 3 at dosages of 0.5 μg·kg -1 ·d -1 or 1.0 μ g ·kg -1 ·d -1 , survivals of recipients were prolonged. Ninety-five percent confidence intervals of survival were 46-87 days and 69-102 days (both P=0.0005 vs control group), respectively. On day seven post-transplantation, relative levels of IFN-γ mRNA transcription were 0.59±0.12 and 0.49±0.16, which was higher t han the control group (P=0.005, P=0.003, respectively). Relative leve ls of IL-10 mRNA transcription were 0.83±0.09 and 0.76±0.09, which was lower than the con trol group (P=0.002, P=0.003, respectively). At a dosage of 0.5 μg·kg -1 ·d -1 , the median of pathological rejection gra de on day seven and on day thirty post-transplantation were 1.5 and 2.0 in comparison with the CsA-treated g roup (P=0.178, P=0.171, respectively). At a dosage of 0.5 μg·kg -1 ·d -1 , the median of pathological rejection grade on day seven and day thirty post-transplantation were 1.5 and 1.5 in comparison with CsA-treated group (P=0.350, P=0.69 3, respectively).Conclusion After each recipient was treated with 1,25-(OH) 2 D 3 at a dosage of (0.5-1.0) μg·kg -1 ·d -1 , transcription of cytokine intragraft was accommodated effectively and deviated to Th2 type, resulting in alleviation of acute rejection. 1, 25-(OH) 2 D 3 can prolong survival of recipient after orthotopic liver transplantation.

Autoantibodies against beta cells to predict early insulin requirements in pediatric patients with clinically diagnosed type 2 diabetes

BACKGROUND Autoimmunity has emerged as a probable disease modifier in patients with clinically diagnosed type 2 diabetes mellitus(T2DM),that is,patients who have insulin resistance,obesity,and other cardiovascular risk factors,suggesting that the presence of glutamic acid decarboxylase(anti-GAD65),islet antigen 2(anti-IA2),and zinc transporter 8(anti-Zn8T)antibodies could have deleterious effects on beta cell function,causing failure and earlier requirement for insulin treatment.AIM To evaluate anti-GAD65,anti-IA2 and anti-Zn8T as predictors of early insulin requirement in adolescents with a clinical diagnosis of T2DM.METHODS This was a case–control study in patients with clinically diagnosed with T2DM(68 cases and 64 controls with and without early insulin dependence respectively),male and female,aged 12–18 years.Somatometry,blood pressure,glucose,insulin,C-peptide,glycated hemoglobin A1c,and lipid profiles were assessed.ELISA was used to measure anti-GAD65,anti-IA2,and anti-Zn8T antibodies.Descriptive statistics,Pearson'sχ2 test,Student's t test,and logistic regression was performed.P<0.05 was considered statistically significant.RESULTS There were 132 patients(53.8%female),with a mean age was 15.9±1.3 years,and there was a disease evolution time of 4.49±0.88 years.The presence of anti-GAD65,anti-IA2,and anti-Zn8T positivity was found in 29.5%,18.2%,and 15.9%,respectively.Dividing the groups by early or no insulin dependence showed that the group with insulin had a higher frequency of antibody positivity:anti-GAD65 odds ratio(OR):2.42(1.112–5.303,P=0.026);anti-IA2:OR:1.55(0.859–2.818,P=0.105);and anti-Zn8T:OR:7.32(2.039–26.279,P=0.002).CONCLUSION Anti-GAD65 positivity was high in our study.Anti-GAD65 and anti-Zn8T positivity showed a significantly depleted beta cell reserve phenotype,leading to an increased risk of early insulin dependence.

Spiritual Forests—A Nature Conservation Model of Ethnic Minority Communities in Central Viet Nam

This paper aims to analyze and evaluate a model of forest conservation and management of ethnic minority (or indigenous) people in central Vietnam, often referred to as the spiritual forest. These forests, called sacred forest or ghost forests by the ethnic minority people in Thua Thien Hue province, have existed for a long time among forest residents. However, they have recently declined, both in quality and quantity, due to various factors, including changes in society, economy, environment, and perception, among other reasons. Based on research conducted in A Luoi district, Thua Thien Hue province with household interviews, group discussions, and field surveys, we find that spiritual forest retains religious and human significance. They are also often among the last remaining natural forests left due to deforestation by human activities. The research results indicate challenges that conservation of spiritual forest may face, while giving recommendations derived from communities for sustainable forest development and conservation in the region.

Research on stability of laminated composite plate under nonlinear aerodynamic load

In this paper,we propose an finite element approach based on classical plate theory to investigate the dynamic stability of a layered composite plate subject to nonlinear aerodynamic load.This study considers the influence of temperature,nonlinear geometry,and nonlinear aerodynamic load on composite plate structures simultaneously.Specifically,the present work conduct comparison the results of the critical pressure value between the nonlinear aerodynamic load and the linear aerodynamic load,thereby pointing out some necessary cases which must consider the nonlinearity of aerodynamic load for calculating the aerospace structures.We determine the critical pressure value and vibrational amplitude response of the plate by means of calculation.The outcomes of our calculations can be useful in designing and repairing body shells and wings of aircraft equipment.

智能创造生活 制造改变社会——访2019国际自主智能机器人大赛发起人王志华教授

《微纳电子与智能制造》杂志社有幸采访到2019国际自主智能机器人大赛主要发起人——清华大学王志华教授,请他为我们介绍一下2019国际自主智能机器人大赛,相信经过王老师的详细讲解,一定能让大家更加了解本届大赛,更加深入地认识自主智能机器人。

Designing and generating a mouse model:frequently asked questions

Genetically engineered mouse(GEM)models are commonly used in biomedical research.Generating GEMs involve complex set of experimental procedures requiring sophisticated equipment and highly skilled technical staff.Because of these reasons,most research institutes set up centralized core facilities where custom GEMs are created for research groups.Researchers,on the other hand,when they begin thinking about generating GEMs for their research,several questions arise in their minds.For example,what type of model(s)would be best useful for my research,how do I design them,what are the latest technologies and tools available for developing my model(s),and finally how to breed GEMs in my research.As there are several considerations and options in mouse designs,and as it is an expensive and time-consuming endeavor,careful planning upfront can ensure the highest chance of success.In this article,we provide brief answers to several frequently asked questions that arise when researchers begin thinking about generating mouse model(s)for their work.

Temperature dependence of spin pumping in YIG/NiO(x)/W multilayer

We report the temperature dependence of the spin pumping effect for Y_(3)Fe_(5)O_(12)(YIG,0.9μm)/NiO(tNiO)/W(6 nm)(tNiO=0 nm,1 nm,2 nm,and 10 nm)heterostructures.All samples exhibit a strong temperature-dependent inverse spin Hall effect(ISHE)signal I_(c)and sensitivity to the NiO layer thickness.We observe a dramatic decrease of I_(c)with inserting thin NiO layer between YIG and W layers indicating that the inserting of NiO layer significantly suppresses the spin transport from YIG to W.In contrast to the noticeable enhancement in YIG/NiO(tNiO≈1-2 nm)/Pt,the suppression of spin transport may be closely related to the specific interface-dependent spin scattering,spin memory loss,and spin conductance at the NiO/W interface.Besides,the I_(c)of YIG/Ni O/W exhibits a maximum near the TNof the AF NiO layer because the spins are transported dominantly by incoherent thermal magnons.

土工格栅与碎石-土混合料的界面摩擦受力特性分析

土工格栅与土的界面摩擦受力特性直接影响加筋土结构的安全稳定、筋材拉力发挥以及结构极限破坏型式,是加筋土结构设计、变形及稳定分析最为关键的技术指标。为研究土工格栅与碎石-土混合料的界面摩擦受力特性,在15k Pa、45k Pa、75k Pa法向应力下进行土工栅与碎石-土混合料界面的室内拉拔试验,分析了土工格栅不同测段格栅的受力和应变规律以及不同纵向埋深处测点的位移。结果表明:土工格栅不同测段格栅的应变随着法向应力的增加而增大,随着格栅纵向埋深的增加而依次递减,且75k Pa法向应力下AB段格栅应变约为DE段格栅应变的3.2倍;预设测点的位移以及不同测段格栅的应变随拉拔力的增大而增加,但格栅应变及测点位移会随着格栅纵向埋深的增加而依次递减;土中格栅应变相对于裸露在空气中的格栅应变要小,且远小于格栅的断裂伸长率;土工格栅应变与拉拔力呈现良好的幂函数关系。

关于比较文学研究的九个命题和三条建议

从事比较文学研究的学者无法回避下列问题:怎样区分文学和非文学,或曰虚构和非虚构?文学生产、流通、接受过程中的文学文本如何不同于其他文本?文学是怎样被书写出来的?文学审美成规的运作机制是什么?从20世纪文学研究的发展史和源流看,有9个反映文学写作和阅读审美性问题的命题,3条建议可供文学研究者参考。

Organization：A new focus on mine safety improvement in a complex operational and business environment

The daily operations in the mining industry are still a significant source of risk with regard to occupational safety and health(OS & H). Various research studies and statistical data world-wide show that the number of serious injuries and fatalities still remains high despite substantial efforts the industry has put in recent years in decreasing those numbers. This paper argues that the next level of safety performance will have to consider a transition from coping solely with workplace dangers, to a more systemic model taking organizational risks in consideration. In this aspect, lessons learned from the nuclear industry may be useful, as organizational learning processes are believed to be more universal than the technologies in which they are used. With the notable exception of major accidents, organizational performance has not received all the attention it deserves. A key element for reaching the next level of performance is to include organizational factors in low level events analyses, and approach the management as a risk control system. These factors will then appear not only in the event analysis,but in supervision activities, audits, change management and the like. Many recent event analyses across various industries have shown that organizational factors play a key role in creating conditions for triggering major accidents(aviation, railway transportation, nuclear industry, oil exploitation, mining, etc.).In this paper, a perspective that may be used in supervisory activities, self-assessments and minor events investigations, is presented. When ingrained in an organizational culture, such perspective has the highest potential for continuous safety improvement.

Investigation on Reduction of Pressure Fluctuation for a Double-Suction Centrifugal Pump

Double-suction centrifugal pumps have been applied extensively in many areas,and the significance of pressure fluctuations inside these pumps with large power is becoming increasingly important.In this study,a double-suction centrifugal pump with a high-demand for vibration and noise was redesigned by increasing the flow uniformity at the impeller discharge,implemented by combinations of more than two parameters.First,increasing the number of the impeller blades was intended to enhance the bounding effect that the blades imposed on the fluid.Subsequently,increasing the radial gap between the impeller and volute was applied to reduce the rotor-stator interaction.Finally,the staggered arrangement was optimized to weaken the efficacy of the interference superposition.Based on numerical simulation,the steady and unsteady characteristics of the pump models were calculated.From the fluctuation analysis in the frequency domain,the dimensionless pressure fluctuation amplitude at the blade passing frequency and its harmonics,located on the monitoring points in the redesigned pumps(both with larger radial gap),are reduced a lot.Further,in the volute of the model with new impellers staggered at 12°,the average value for the dimensionless pressure fluctuation amplitude decreases to 6%of that in prototype pump.The dimensionless rootmean-square pressure contour on the mid-span of the impeller tends to be more uniform in the redesigned models(both with larger radial gap);similarly,the pressure contour on the mid-section of the volute presents good uniformity in these models,which in turn demonstrating a reduction in the pressure fluctuation intensity.The results reveal the mechanism of pressure fluctuation reduction in a double-suction centrifugal pump,and the results of this study could provide a reference for pressure fluctuation reduction and vibration performance reinforcement of doublesuction centrifugal pumps and other pumps.

Potentially Toxic Element Concentration in Fruits Collected from Markazi Province(Iran): A Probabilistic Health Risk Assessment

Objective This study was conducted to evaluate the concentration of potentially toxic elements(PTEs)such as arsenic(As),cadmium(Cd),mercury(Hg),and lead(Pb)in fruit samples collected from Markazi Province,Iran.A probabilistic health risk assessment due to ingestion of PTEs through the consumption of these fruits was also conducted.Methods The concentration of PTEs in 90 samples of five types of fruits(n=3)collected from six geographic regions in Markazi Province was measured.The potential health risk was evaluated using a Monte Carlo simulation model.Results A significant difference was observed in the concentration of PTEs between fruits as well as soil and water samples collected from different regions in Markazi Province.The order of PTE concentration in the soil and water samples was as follows:Pb>As>Hg>Cd.Furthermore,the highest level of transfer factor for Cd and Hg correlated with the grape.The estimated daily intake for adults and children was lower than the recommended tolerable daily intake.Conclusion The population in Markazi Province,Iran,is not at considerable noncarcinogenic or carcinogenic risk due to the ingestion of PTEs through the consumption of the examined fruits.

Development of a PARCS/Serpent model for neutronics analysis of the Dalat nuclear research reactor

Cross-sectional homogenization for full-core calculations of small and complex reactor configurations,such as research reactors,has been recently recognized as an interesting and challenging topic.This paper presents the development of a PARCS/Serpent model for the neutronics analysis of a research reactor type TRIGA Mark-II loaded with Russian VVR-M2 fuel(known as the Dalat Nuclear Research Reactor or DNRR).The full-scale DNRR model and a supercell model for a shim/safety rod and its surrounding fuel bundles with the Monte Carlo code Serpent 2 were proposed to generate homogenized fewgroup cross sections for full-core diffusion calculations with PARCS.The full-scale DNRR model with Serpent 2 was also utilized as a reference to verify the PARCS/Serpent calculations.Comparison of the effective neutron multiplication factors,radial and axial core power distributions,and control rod worths showed a generally good agreement between PARCS and Serpent 2.In addition,the discrepancies between the PARCS and Serpent 2 results are also discussed.Consequently,the results indicate the applicability of the PARCS/Serpent model for further steady state and transient analyses of the DNRR.

Virtual bronchoscopic navigation without fluoroscopy guidance for peripheral pulmonary lesions in inexperienced pulmonologist

Objective:Fluoroscopy guidance is generally required for endobronchial ultrasonography with guide sheath(EBUS-GS)in peripheral pulmonary lesions(PPLs).Virtual bronchoscopic navigation(VBN)can guide the bronchoscope by creating virtual images of the bronchial route to the lesion.The diagnostic yield and safety profiles of VBN without fluoroscopy for PPLs have not been evaluated in inexperienced pulmonologist performing EBUS-GS.Methods:Between January 2016 and June 2017,consecutive patients with PPLs referred for EBUS-GS at a single cancer center were enrolled.The diagnostic yield as well as safety profiles was retrospectively analyzed,and our preliminary experience was shared.Results:A total of 109 patients with 109 lesions were included,99(90.8%)lesions were visible on EBUS imaging.According to the procedure time needed to locate the lesion on EBUS,24.8%(27/109)were deemed technically difficult procedures;however,no significant relationships were identified between candidate parameters and technically difficult procedures.The overall diagnosis yield was 74.3%(81/109),and the diagnostic yield of malignancy was 83.7%(77/92).Lesions larger than 20 mm[odds ratio(OR),2.758;95%confidence interval(95%CI),1.077-7.062;P=0.034]and probe of within type(OR,3.174;95%CI,1.151-8.757,P=0.026)were independent factors leading to a better diagnostic yield in multivariate analysis.About 30 practice procedures were needed to achieve a stable diagnostic yield,and the proportion of technically difficult procedures decreased and stabilized after 70 practice procedures.Regarding complications,one patient(0.9%)had intraoperative hemorrhage(100 mL)which was managed under endoscopy.Conclusions:VBN without fluoroscopy guidance is still useful and safe for PPLs diagnosis,especially for malignant diseases when performed by pulmonologist without previous experience of EBUS-GS.VBN may simplify the process of lesion positioning and further multi-center randomized studies are warranted.