1.Eucommia ulmoides promotes alveolar bone formation in ovariectomized rats
Lin ZHENG ; Wenjun JIN ; Shanshan LUO ; Rui HUANG ; Jie WANG ; Yuting CHENG ; Zheqing AN ; Yue XIONG ; Zipeng GONG ; Jian LIAO
Chinese Journal of Tissue Engineering Research 2025;29(6):1159-1167
BACKGROUND:Eucommia ulmoides has a certain osteogenic effect,which can promote the proliferation and differentiation of osteoblasts.However,it is unclear whether Eucommia ulmoides has effects on alveolar bone formation and Wnt/β-Catenin signaling pathway. OBJECTIVE:To investigate the mechanism by which Eucommia ulmoides promotes alveolar bone formation in ovariectomized rats based on the Wnt/β-Catenin signaling pathway. METHODS:Sixty female Sprague-Dawley rats were selected and randomly divided into five groups:blank control group,sham-operation group,model group,low-dose group Eucommia ulmoides group,and high-dose Eucommia ulmoides group,with twelve rats in each group.Osteoporosis animal models were constructed by bilateral oophorectomy in the model group and the low-dose and high-dose Eucommia ulmoides groups.The sham-operation group underwent the same method to remove adipose tissue of equal mass around the bilateral ovaries.Three months after surgery,the low-and high-dose Eucommia ulmoides groups were given 2.1 g/kg/d and 4.2 g/kg/d Eucommia ulmoides by gavage,respectively.The sham-operation group and model group were given the same amount of physiological saline by gavage.After 12 weeks of drug intervention,the changes in alveolar bone mass of rats in each group were observed through Micro-CT;hematoxylin-eosin staining was used to observe the pathological structural changes of alveolar bone in rats;enzyme linked immunosorbent assay was used to detect the expression levels of alkaline phosphatase and osteocalcin in the serum of rats;western blot was used to detect the expression levels of β-Catenin and Frizzled9 receptor proteins in the alveolar bone of rats;and real-time fluorescence quantitative PCR was used to detect the expression of osteocalcin,Runt-related transcription factor 2(Runx2),alkaline phosphatase,β-catenin,and frizzled9 mRNAs in alveolar bone tissues of rats. RESULTS AND CONCLUSION:Compared with the blank control group,bone volume fraction,trabecular number,trabecular thickness,and bone mineral density were reduced in the model group(P<0.05),and trabecular separation was elevated(P<0.05).Pathological observation showed that the arrangement of trabeculae was disordered and irregular,the trabeculae were thinned or broken,and the marrow cavity was enlarged in the model group,with a significant reduction in bone volume;the level of alkaline phosphatase in the serum was increased(P<0.05),and the level of osteocalcin was decreased(P<0.05);mRNA expression of alkaline phosphatase,osteocalcin,Runx2,β-catenin,and frizzled9 were decreased(P<0.05);protein expression of β-Catenin and Frizzled9 was decreased(P<0.05).Compared with the model group,the low-and high-dose Eucommia ulmoides groups showed an increase in bone volume fraction,trabecular number,trabecular thickness,and bone mineral density(P<0.05)and a decrease in trabecular separation(P<0.05).In the low-and high-dose Eucommia ulmoides groups,bone trabeculae were slightly aligned and thickened,with a significant increase in bone mass.Compared with the model group,the serum level of alkaline phosphatase was reduced(P<0.05)and the serum level of osteocalcin was elevated(P<0.05)in the low-and high-dose Eucommia ulmoides groups.Compared with the model group,the mRNA expression of alkaline phosphatase,osteocalcin,Runx2,β-catenin,and frizzled9 were increased in the low-and high-dose Eucommia ulmoides groups(P<0.05).Compared with the model group,the protein expression of Frizzled9 was increased in the low-dose Eucommia ulmoides group(P<0.05),while the protein expression of β-Catenin and Frizzled9 was increased in the high-dose Eucommia ulmoides group(P<0.05).Compared with the low-dose Eucommia ulmoides group,the high-dose Eucommia ulmoides group had a more significant improvement in the above indexes.To conclude,Eucommia ulmoides can effectively promote the alveolar bone formation,and its mechanism of action might be related to the activation of the Wnt/β-catenin signaling pathway.
2.Role and mechanism of platelet-derived growth factor BB in repair of growth plate injury
Hongcheng PENG ; Guoxuan PENG ; Anyi LEI ; Yuan LIN ; Hong SUN ; Xu NING ; Xianwen SHANG ; Jin DENG ; Mingzhi HUANG
Chinese Journal of Tissue Engineering Research 2025;29(7):1497-1503
BACKGROUND:In the initial stage of growth plate injury inflammation,platelet-derived growth factor BB promotes the repair of growth plate injury by promoting mesenchymal progenitor cell infiltration,chondrogenesis,osteogenic response,and regulating bone remodeling. OBJECTIVE:To elucidate the action mechanism of platelet-derived growth factor BB after growth plate injury. METHODS:PubMed,VIP,WanFang,and CNKI databases were used as the literature sources.The search terms were"growth plate injury,bone bridge,platelet-derived growth factor BB,repair"in English and Chinese.Finally,66 articles were screened for this review. RESULTS AND CONCLUSION:Growth plate injury experienced early inflammation,vascular reconstruction,fibroossification,structural remodeling and other pathological processes,accompanied by the crosstalk of chondrocytes,vascular endothelial cells,stem cells,osteoblasts,osteoclasts and other cells.Platelet-derived growth factor BB,as an important factor in the early inflammatory response of injury,regulates the injury repair process by mediating a variety of cellular inflammatory responses.Targeting the inflammatory stimulation mediated by platelet-derived growth factor BB may delay the bone bridge formation process by improving the functional activities of osteoclasts,osteoblasts,and chondrocytes,so as to achieve the injury repair of growth plate.Platelet-derived growth factor BB plays an important role in angiogenesis and bone repair tissue formation at the injured site of growth plate and intrachondral bone lengthening function of uninjured growth plate.Inhibition of the coupling effect between angiogenesis initiated by platelet-derived growth factor BB and intrachondral bone formation may achieve the repair of growth plate injury.
3.A Study on the In Vitro Antibacterial Effects of Colistin or Tigecycline Combined with Three Antimicrobial Agents Against Carbapenem-resistant Acinetobacter Baumannii
Weichen HUANG ; Kuo LI ; Lijing GUO ; Xinyi JIN ; Chaogui TANG ; Ning LIN ; Jingjing HUANG
Medical Journal of Peking Union Medical College Hospital 2025;16(5):1131-1137
To analyze the Non-duplicate CRAB strains isolated from clinical specimens between June and December 2023 at Huai'an First People's Hospital Affiliated to Nanjing Medical University were retrospectively collected. The minimum inhibitory concentrations of commonly used antimicrobial agents against these strains were determined using the broth microdilution method. A total of 48 CRAB strains were included. COL-based combination regimens all exhibited favorable synergistic or additive effects against CRAB. Among them, the COL+IPM group showed the highest synergistic rate (83.3%), while the COL+AMK and COL+CSL groups primarily demonstrated additive effects (additive rates of 66.7% and 70.8%, respectively). TGC-based combination regimens mainly exhibited additive or indifferent effects, with the TGC+CSL group showing the highest additive rate (75.0%) and the TGC+AMK group showing the highest indifferent rate (70.8%). No antagonistic effects were observed in any of the six combination regimens. Further analysis revealed that the antibacterial effect of the COL+IPM group against CRAB was significantly different from that of the other combination regimens ( The combination of COL and IPM demonstrates the optimal synergistic antibacterial effect against CRAB, with the highest synergistic rate, and can be used as an empirical combination regimen for the clinical treatment of CRAB infections.
4.Correlation of serum metabolites and clinical features in patients with peripheral T-cell lymphoma
Yishuo DUAN ; Jun RAO ; Jing XIA ; Naya MA ; Shijia LIN ; Fu LI ; Shuhan TANG ; Sha ZHOU ; Yunjing ZENG ; Xinlei LI ; Dezhi HUANG ; Qiong LI ; Bangdong LIU ; Xianlan ZHAO ; Jin WEI ; Xi ZHANG
Journal of Army Medical University 2024;46(4):352-358
Objective To explore the changes in serum energy metabolites in patients with peripheral T-cell lymphoma,and investigate serum biomarkers for monitoring peripheral T-cell lymphoma from the perspective of energy metabolism.Methods Multiple/selected reaction monitoring(MRM/SRM)was used to detect the energy-related metabolites in the sera of 16 patients with newly diagnosed peripheral T-cell lymphoma admitted in the Hematology Medical Center of the Second Affiliated Hospital of Army Medical University from November 2020 to December 2021,as well as 10 recruited healthy volunteers.The corresponding clinical data including medical history,laboratory results and image data were collected and retrospectively analyzed.Results Significant differences were seen in the contents and expression profiles of serum energy metabolism-related products between the patients and the healthy volunteers.The patients had significantly reduced serum contents of cyclic AMP,succinate,citrate and cis-aconitate(P<0.05),and elevated D-glucose 6-phosphate content(P<0.05).The serum contents of citrate and succinate were negatively correlated with the risk stratification(low-,moderate-and high-risk)and clinical stage of the disease(P<0.05).Meanwhile,there was a negative correlation between the contents of L-malic acid and citrate and the mid-term efficacy evaluation results,such as complete/partial response(CR/PR)or stable disease(SD)(P<0.05).For patients with extranodal NK/T cell lymphoma(n=10),there were also significant reductions in the contents of cyclic AMP,succinate,citrate,isocitrate and cis-aconitate in the sera of patients compared with healthy volunteers(P<0.05),and the contents of citrate and succinate were negatively correlated with the clinical stage(P<0.05)and were rather correlated with mid-term efficacy evaluation results(CR/PR or SD)(P<0.05).For patients with angioimmunoblastic T-cell lymphoma(n=6),the serum contents of cyclic AMP,citrate and succinate were significantly lower,while the content of D-glucose 6-phosphate was higher when compared with the healthy volunteers(P<0.05),and the content of succinate was negatively correlated with both clinical stage and risk grade of the patients(P<0.05).Conclusion There are 5 serum differential metabolites identified between patients with peripheral T-cell lymphoma and healthy controls,and succinate and citrate are expected to be serum biomarkers of peripheral T-cell lymphoma.
5.Determination of the Contents of Three Lignans in Dendrobium fimbriatum Hook
Ying-Hua HUANG ; Lin ZHANG ; Jin-Yan LI ; Zhi-Bin LI ; Zhi-Yun LIANG ; Li-E YANG ; Gang WEI ; Yue-Chun HUANG
Journal of Guangzhou University of Traditional Chinese Medicine 2024;41(1):207-212
Objective To establish the method for content determination of three lignans of Dendrobium Fimbriatum Hook..Methods The lignans in Dendrobium tasselii were identified by high-performance liquid chromatography/multi-stage mass spectrometry(HPLC-ESI/MSn)coupled with ultraviolet absorption spectrometry(UV)coupled with retention time localization of high-performance liquid chromatography(HPLC).The separation was carried out on a Kromasil 100-5 C18 column(4.6 mm×250 mm,5 μm)using a gradient elution of acetonitrile-0.1%formic acid solution as the mobile phase,the volume flow rate was 0.8 mL·min-1 and the column temperature was 35℃,and the mass spectrometry was performed using an ESI ion source with the data collected in the negative ion mode.The HPLC content was determined on the same column as that of MS analysis,with the mobile phase methanol + acetonitrile(V/V=1∶1)-0.01 mol/L ammonium acetate solution,gradient elution,flow rate of 0.8 mL·min-1,column temperature of 40℃,and detection wavelength of 215 nm.Results Syringaresinol di-O-glucoside and(-)-Syringaresinol 4-O-β-D-glucopyranoside and DL-Syringaresinol were identified from Dendrobium fimbriatum Hook.,and the results of content determination showed that the linear ranges of above three components were respectively 0.1701-3.4020,0.1020-2.0400,0.0403-0.8060 μg(r≥0.9995),the average recoveries were in the range of 97.71%-101.67%,and the relative standard deviations(RSDs)were all less than 3.0%.The contents of Syringaresinol di-O-glucoside and(-)-Syringaresinol 4-O-β-D-glucopyranoside and DL-Syringaresinol in the 10 batches of samples were 0.7779-1.3852,0.0734-0.1966,0.0295-0.1882 mg·g-1.Conclusion This research method can provide a reference basis for the quality evaluation method of Dendrobium fimbriatum Hook..
6.Effect of sarcopenia on balance function in patients with Parkinson's disease
Yaxi ZHANG ; Caidan LIN ; Haimin JIN ; Xiangdong HUANG
China Modern Doctor 2024;62(2):29-32
Objective To investigate the effect of sarcopenia on balance function in patients with Parkinson's disease(PD).Methods Sixty patients with PD diagnosed by our hospital from January 2021 to June 2021 were selected as the study objects.All patients were divided into myopenia group and control group according to the diagnostic criteria established by the Asian Working Group for Sarcopenia(AWGS)in 2014.The UPDRS Ⅲ,Berg balance scale,timed-up-go test and activity balance confidence scale were compared between the two groups.Results The grip strength,walking speed and RASM of the myopenia group were lower than those of the control group(P<0.05);Compared with the control group,the number of falls,UPDRS Ⅲ,Berg score and TUG duration in the myopenia group were significantly increased(P<0.05).The total score of ABC,ABC-2,ABC-3,ABC-5,ABC-6,ABC-7,ABC-8,ABC-9,ABC-11,ABC-14,ABC-15,ABC-16 in myopenia group were poor,and the differences were statistically significant(P<0.05).Further analyzing the differences between sex genders,we found that female sarcopenia had lower scores on ABC-2,ABC-3,ABC-8,ABC-9,ABC-11,ABC-13,ABC-14,ABC-15,ABC-16,and total score indexes,with statistically significant differences.Conclusion Myopenia significantly affects the balance function of PD and increases the risk of falls.
7.Clinical trial of roxadustat in the treatment of maintenance hemodialysis patients with renal anemia
Jin-Bao WANG ; Lin HUANG ; Wei-Dong ZHANG
The Chinese Journal of Clinical Pharmacology 2024;40(2):180-184
Objective To investigate the clinical efficacy and microinflammatory response of roxadustat and recombinant human erythropoietin(rHuEPO)in the treatment of renal anemia(RA)in maintenance hemodialysis(MHD).Methods MHD RA patients with uremia were analyzed retrospectively.The control group was given subcutaneous injection of 120 U·kg-1 rHuEPO and iron sucrose injection 100 mg.After 2 h of hemodialysis,rHuEPO was slowly dropped into the intravenous end of the dialyser for more than 30 min,once a week.The treatment group was additionally treated with roxadustat capsule,starting at 100 mg,3 times a week for 3 months.The clinical efficacy and serum creatinine(Scr),hemoglobin(Hb),hematocrit(Hct),urea nitrogen(BUN),serum creatinine(SCR),ferritin(SF),transferrin saturation(TSAT),transferrin(TRF),centriocyte absolute value/lymphocyte absolute value(NLR)and absolute value of neutrophils/absolute value of lymphocytes(NLR),absolute value of platelets/absolute value of lymphocytes(PLR),and high-sensitivity C-reactive protein(hs-CRP)levels,as well as the occurrence of adverse drug reactions were compared.Results Thirty-two cases were included in the treatment group and twenty-eight cases in the control group.After treatment,the total effective rate of the treatment group and the control group were 93.75%(30 cases/32 cases)and 75.00%(21 cases/28 cases),respectively,and the difference was statistically significant(P<0.05).Hb in treatment group and control group were(113.64±12.58)and(104.39±11.67)g·L-1;Hct were(31.51±4.33)%and(28.72±5.47)%;TSAT were(21.11±1.26)%and(19.57±1.29)%,respectively;SF were(161.25±7.91)and(210.68±9.02)ng·mL-1;TRF were(13.06±1.29)and(16.57±1.45)g·L-1,respectively;BUN were(21.90±4.41)and(24.37±3.51)mmol·L-1;Scr were(862.56±97.81)and(980.30±99.67)μmnol·L-1;NLR were(1.30±0.29)%and(2.01±0.41)%,respectively;PLR levels were(86.57±16.55)%and(104.82±26.31)%,and hs-CRP levels were(4.82±0.36)and(6.57±0.55)mg·L-1,respectively.Compared with control group,the above indexes except BUN and Scr in treatment group were statistically significant(all P<0.05).The adverse drug reactions of the treatment group were mainly digestive tract reaction and liver function injury,while the adverse drug reactions of the control group were mainly elevated blood potassium,digestive tract reaction and liver function injury.The incidence of total adverse drug reactions in treatment group and control group were 12.50%and 21.43%,respectively,with no statistical significance(P>0.05).Conclusion roxadustat can effectively improve the efficacy of MHD RA patients with uremia,relieve anemia and reduce microinflammatory response.
8.National bloodstream infection bacterial resistance surveillance report (2022) : Gram-negative bacteria
Zhiying LIU ; Yunbo CHEN ; Jinru JI ; Chaoqun YING ; Qing YANG ; Haishen KONG ; Haifeng MAO ; Hui DING ; Pengpeng TIAN ; Jiangqin SONG ; Yongyun LIU ; Jiliang WANG ; Yan JIN ; Yuanyuan DAI ; Yizheng ZHOU ; Yan GENG ; Fenghong CHEN ; Lu WANG ; Yanyan LI ; Dan LIU ; Peng ZHANG ; Junmin CAO ; Xiaoyan LI ; Dijing SONG ; Xinhua QIANG ; Yanhong LI ; Qiuying ZHANG ; Guolin LIAO ; Ying HUANG ; Baohua ZHANG ; Liang GUO ; Aiyun LI ; Haiquan KANG ; Donghong HUANG ; Sijin MAN ; Zhuo LI ; Youdong YIN ; Kunpeng LIANG ; Haixin DONG ; Donghua LIU ; Hongyun XU ; Yinqiao DONG ; Rong XU ; Lin ZHENG ; Shuyan HU ; Jian LI ; Qiang LIU ; Liang LUAN ; Jilu SHEN ; Lixia ZHANG ; Bo QUAN ; Xiaoping YAN ; Xiaoyan QI ; Dengyan QIAO ; Weiping LIU ; Xiusan XIA ; Ling MENG ; Jinhua LIANG ; Ping SHEN ; Yonghong XIAO
Chinese Journal of Clinical Infectious Diseases 2024;17(1):42-57
Objective:To report the results of national surveillance on the distribution and antimicrobial resistance profile of clinical Gram-negative bacteria isolates from bloodstream infections in China in 2022.Methods:The clinical isolates of Gram-negative bacteria from blood cultures in member hospitals of national bloodstream infection Bacterial Resistant Investigation Collaborative System(BRICS)were collected during January 2022 to December 2022. Antibiotic susceptibility tests were conducted by agar dilution or broth dilution methods recommended by Clinical and Laboratory Standards Institute(CLSI). WHONET 5.6 and SPSS 25.0 software were used to analyze the data.Results:During the study period,9 035 strains of Gram-negative bacteria were collected from 51 hospitals,of which 7 895(87.4%)were Enterobacteriaceae and 1 140(12.6%)were non-fermenting bacteria. The top 5 bacterial species were Escherichia coli( n=4 510,49.9%), Klebsiella pneumoniae( n=2 340,25.9%), Pseudomonas aeruginosa( n=534,5.9%), Acinetobacter baumannii complex( n=405,4.5%)and Enterobacter cloacae( n=327,3.6%). The ESBLs-producing rates in Escherichia coli, Klebsiella pneumoniae and Proteus spp. were 47.1%(2 095/4 452),21.0%(427/2 033)and 41.1%(58/141),respectively. The prevalence of carbapenem-resistant Escherichia coli(CREC)and carbapenem-resistant Klebsiella pneumoniae(CRKP)were 1.3%(58/4 510)and 13.1%(307/2 340);62.1%(36/58)and 9.8%(30/307)of CREC and CRKP were resistant to ceftazidime/avibactam combination,respectively. The prevalence of carbapenem-resistant Acinetobacter baumannii(CRAB)complex was 59.5%(241/405),while less than 5% of Acinetobacter baumannii complex was resistant to tigecycline and polymyxin B. The prevalence of carbapenem-resistant Pseudomonas aeruginosa(CRPA)was 18.4%(98/534). There were differences in the composition ratio of Gram-negative bacteria in bloodstream infections and the prevalence of main Gram-negative bacteria resistance among different regions,with statistically significant differences in the prevalence of CRKP and CRPA( χ2=20.489 and 20.252, P<0.001). The prevalence of CREC,CRKP,CRPA,CRAB,ESBLs-producing Escherichia coli and Klebsiella pneumoniae were higher in provinicial hospitals than those in municipal hospitals( χ2=11.953,81.183,10.404,5.915,12.415 and 6.459, P<0.01 or <0.05),while the prevalence of CRPA was higher in economically developed regions(per capita GDP ≥ 92 059 Yuan)than that in economically less-developed regions(per capita GDP <92 059 Yuan)( χ2=6.240, P=0.012). Conclusions:The proportion of Gram-negative bacteria in bloodstream infections shows an increasing trend,and Escherichia coli is ranked in the top,while the trend of CRKP decreases continuously with time. Decreasing trends are noted in ESBLs-producing Escherichia coli and Klebsiella pneumoniae. Low prevalence of carbapenem resistance in Escherichia coli and high prevalence in CRAB complex have been observed. The composition ratio and antibacterial spectrum of bloodstream infections in different regions of China are slightly different,and the proportion of main drug resistant bacteria in provincial hospitals is higher than those in municipal hospitals.
9.Simultaneous content determination of six constituents in Gaultheria leucocarpa var.yunnanensis by UPLC-MS/MS
Lin JIN ; Zu-Ying ZHOU ; Jia-Hua ZHOU ; Ming-Yan CHI ; Lin ZHENG ; Yong HUANG ; Wen HUANG
Chinese Traditional Patent Medicine 2024;46(1):22-28
AIM To establish an UPLC-MS/MS method for simultaneous content determination of protocatechuic acid,epicatechin,chlorogenic acid,quercitrin,gaultherin and gaultheroside A in Gaultheria leucocarpa var.yunnanensis.METHODS The analysis was performed on a 40℃thermostatic Waters BEH C18 column(100 mm×2.1 mm,1.7 μm),with the mobile phase comprising of water(containing 0.1%formic acid)-acetonitrile(containing 0.1%formic acid)flowing at 0.3 mL/min in a gradient elution manner,and electron spray inoization source was adopted in positive and negative ion scanning with multiple reaction monitoring(MRM)mode.Hierarchical cluster analysis(HCA)and principal component analysis(PCA)was used to screen important components that affect the quality of medicinal materials.RESULTS Six constituents showed good linear relationships within their own ranges(R2≥0.998 2),whose average recoveries were 98.76%-101.88%with the RSDs of 1.0%-2.5%.The constituents of G.leucocarpa in the roots and aerial parts were quite different.Gaultherin,epicatechin and protocatechuic acid may be the quality mark constituents of G.leucocarpa.CONCLUSION This accurate and efficient method can be used for the quality control of G.leucocarpa.
10.Aesthetic Investigation of the Pretarsal Shows and Morphology of Double Eyelids in Chinese Populations: A Questionnaire-based Survey
Yarong CHI ; Zhujun LI ; Lin JIN ; Nanze YU ; Jiuzuo HUANG ; Xiao LONG
Medical Journal of Peking Union Medical College Hospital 2024;15(6):1334-1341
To explore the ideal pretarsal shows and morphological characteristics of double eyelids in Chinese populations through a morphometric analysis. This study employed a convenience sampling method to survey Chinese adults who did not reside abroad for an extended period (with cumulative overseas stay of over one year). From November 2023 to May 2024, electronic questionnaires were distributed to collect attractiveness ratings of nine different images (including one image featuring single eyelid and eight images with varying pretarsal shows and morphologies of double eyelids). Stratified comparisons were conducted based on gender, age, occupation, etc., to determine the ideal pretarsal show and morphology of double eyelids. Quantitative analysis was performed on the aesthetic features of the nine images depicting double eyelids to validate and elucidate the survey findings. A total of 493 questionnaires were filled out, and 397 valid questionnaires were included for data analysis after quality control. As for the whole cohort, the images feathering 2 mm double eyelid were deemed to be the most attractive, with the in-fold type scoring (3.72±0.97) points and the out-fold type scoring (3.65±1.04) points. The next was 1 mm, followed by 3 mm, single eyelid, and finally, 4 mm. As for morphology, in-fold type achieved higher scores than out-fold type in images with the same pretarsal show. However, statistically significant differences were only found in ratings for the images with 3 mm and 4 mm double eyelids (all This study validates that the pretarsal show of 2 mm and the presence of either an in-fold or out-fold morphology are considered optimal for double eyelids among Chinese individuals. These findings hold significant implications for the planning of double eyelid surgeries, assessment of surgical outcomes, and evaluation of other periocular procedures associated with double eyelid surgeries.

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