1.Analysis of serum differential proteomics in patients with acute cervical spondylotic radiculopathy
Xianzhong BU ; Baoxian BU ; Wei XU ; Chi ZHANG ; Yisheng ZHANG ; Yuanming ZHONG ; Zhifei LI ; Fubo TANG ; Wei MAI ; Jinyan ZHOU
Chinese Journal of Tissue Engineering Research 2024;28(4):535-541
BACKGROUND:The specific molecular mechanism of the transformation from normal healthy people to acute cervical spondylotic radiculopathy has not been clear,which needs to be further studied. OBJECTIVE:To investigate the differential expression of serum proteomics between normal healthy people and patients with acute cervical spondylotic radiculopathy,and to find and identify potential specific serum markers between them. METHODS:The serum samples of eight patients with acute cervical spondylotic radiculopathy and eight normal healthy people were collected,and the proteomic screening and analysis were performed by tandem mass tag combined with liquid chromatography-tandem mass spectrometry technology,in order to explore and identify serum proteins differentially expressed in patients with acute cervical spondylotic radiculopathy. RESULTS AND CONCLUSION:A total of 183 significantly differential proteins were screened by tandem mass tag technology,and 11 significantly differential proteins were identified(P<0.05).Compared with normal healthy people,three differential proteins were significantly up-regulated,including human leukocyte antigen-A,secretoglobin family 1a member 1,and protein 4-hydroxyphenylpyruvate dioxygenase,and seven differential proteins were significantly down-regulated,such as immunoglobulin heavy constant gamma 3,skin factor,and myosin light chain 3,in patients with acute cervical spondylotic radiculopathy.Gene ontology enrichment analysis showed that these differential proteins participated in antigen binding,immunoglobulin receptor binding and other molecular functions.Protein-protein interaction analysis showed that among the common differential proteins between normal healthy people and patients with acute cervical spondylotic radiculopathy,HLA-A,HPD,PSMA3,DMKN,SCGB1A1,and MYL3 were located at the nodes of the functional network,and were closely related to the systems of body immunity,cellular inflammatory response,energy metabolism,and mechanical pressure.The significantly differential proteins HLA-A,HPD and MYL3 were verified by western blot,and the results were consistent with those of proteomics.To conclude,tandem mass tag combined with liquid chromatography-tandem mass spectrometry technology can be used to find the differentially expressed proteins in serum between normal healthy people and patients with acute cervical spondylotic radiculopathy.It is preliminarily believed that HLA-A,HPD and MYL3 may be specific serum markers of acute cervical spondylotic radiculopathy,providing a new direction for further research on its pathogenesis.
2.Finite element analysis of the correlation between tilt angle of titanium cage and postoperative subsidence of titanium cage after anterior subtotal cervical corpectomy,decompression and fusion
Zhifei LI ; Yin YANG ; Hualong CHEN ; Qinqiu LIANG ; Yuanming ZHONG ; Yisheng ZHANG
Chinese Journal of Tissue Engineering Research 2024;28(9):1313-1319
BACKGROUND:Anterior subtotal corpectomy,decompression and fusion is a conventional method to treat cervical degenerative diseases.A titanium cage is an important implant to maintain the stability of the cervical spine after subtotal corpectomy.In recent years,many patients have complications such as titanium cage sinking,which are highly controversial. OBJECTIVE:To investigate the internal biomechanical relationship between the tilt angle of the titanium cage and postoperative titanium cage subsidence after anterior subtotal cervical corpectomy,decompression and fusion. METHODS:A three-dimensional finite element model of the C4-C6 segment was established by CT images of a normal human cervical spine,in which the anterior subtotal resection,decompression and fusion of the C5 vertebral body were simulated,and titanium cages with different tilt angles(-6° to-1° negative angle,that is,the front edge of titanium cage is shorter than the rear edge of titanium cage;1° to 6° positive angle,that is,the front edge of titanium cage is longer than the rear edge of titanium cage)were placed.After setting the boundary conditions,preloads of 50,100 and 150 N were applied respectively on the C4 vertebral body.The stress value of each contact point between the titanium cage and C4 lower-end plate and C6 lower-end plate(seven stress contact points on the contact surface of titanium mesh)was recorded and statistical analysis was conducted. RESULTS AND CONCLUSION:(1)The tilt angles of the titanium cage of the positive angle group and negative angle group under 50,100 and 150 N stress respectively were found by Mann Whitn test,with P<0.05,which was statistically significant.The dispersion coefficients of the positive angle group were smaller than those of the negative angle group under 50,100 and 150 N stress conditions.(2)Under 50,100 and 150 N stress conditions,the Wilcoxon sign rank test in the positive angle group of titanium cage tilt angle found that when the angle was set to 1° to 5°,the difference was not statistically significant(P>0.05).However,when the tilt angle of the titanium cage was set to 6°,the difference was statistically significant(P<0.05).(3)Under 50,100 and 150 N stress conditions,the Wilcoxon sign rank test in the negative angle group of titanium cage tilt angle found that when the tilt angle was set to-1° to-6°,the difference was not statistically significant(P>0.05).(4)It is concluded that in the sagittal position,the titanium cage with a positive tilt angle is more stable than with a negative tilt angle,which is more suitable for clinical use.The tilt angle of the titanium cage is relatively stable in the range of 1° to 5°.When the tilt angle is 6°,the stability starts to decline,which is easy to cause complications of titanium cage sinking after surgery.It is more suitable to select the titanium cage with a tilt angle of 1° to 5° according to the clinical situation during surgery to improve the efficacy.
3.Finite element analysis of characteristics of spinal cord compression in patients with early cervical spondylotic myelopathy under dynamic position
Chengwei LI ; Yisheng ZHANG ; Zhifei LI ; Yuanming ZHONG ; Jiwen MENG ; Qinqiu LIANG ; Hualong CHEN
Chinese Journal of Tissue Engineering Research 2024;33(33):5257-5264
BACKGROUND:Cervical spondylotic myelopathy is a progressive disease leading to dysfunction in the middle-aged and elderly,and early diagnosis is difficult.In recent years,some clinical scholars have found that dynamic magnetic resonance imaging technology can detect spinal cord compression in a dynamic position earlier,but its specific biomechanical mechanism needs to be clarified. OBJECTIVE:To investigate the biomechanical compression characteristics of early cervical spondylotic myelopathy in hyperextension and flexion position,and to verify the effectiveness of dynamic magnetic resonance imaging in the diagnosis of early cervical spondylotic myelopathy. METHODS:A retrospective analysis was made on the patients who underwent cervical dynamic magnetic resonance imaging in the Department of Orthopedics of First Affiliated Hospital of Guangxi University of Chinese Medicine from January to June 2022.16 subjects were selected and divided into two groups.The pathological group included 8 patients with early cervical spondylotic myelopathy with hypertrophy of ligamentum flavum as the main sign,with 5 male patients and 3 female patients.The normal group included 8 normal degenerative people,with 4 male patients and 4 female patients.All patients were photographed with cervical CT plain scan,magnetic resonance imaging plain scan,and dynamic magnetic resonance imaging plain scan.This study was divided into the following three parts:(1)collect the dynamic magnetic resonance imaging image DCOM data of two groups of subjects,and collect the cervical vertebra CT and neutral magnetic resonance imaging image DCOM data to understand the bone and soft tissue of the two groups of subjects in the neutral position.(2)Based on the DCOM data of magnetic resonance imaging and CT plain scan,the three-dimensional finite element models of lower cervical vertebra(C3-7)of normal degenerative population and early cervical spondylotic myelopathy patients were established by reverse engineering software.The equivalent stress and equivalent elastic strain of the spinal cord and posterior dura were analyzed,and the distribution of stress and strain was observed.(3)After obtaining the stress and strain data,the data between groups were compared to analyze the mechanical characteristics of spinal cord compression caused by early cervical spondylotic myelopathy in a dynamic position and to verify the effectiveness of dynamic magnetic resonance imaging in the diagnosis of early cervical spondylotic myelopathy. RESULTS AND CONCLUSION:(1)When simulating the posterior extension,flexion and neutral position of the lower cervical vertebrae(C3-7)in the two groups,the values of stress and strain in the posterior part of the spinal cord were in the following order:extension>flexion>neutral(P<0.05).The strain values from large to small were as follows:extension>flexion>neutral(P<0.05).(2)Compared with the normal degenerative population model,the equivalent stress and strain of the spinal cord in the pathological group were higher than those in the normal group under two degrees of freedom of flexion and extension(P<0.05).The distribution area of stress and strain in the posterior part of the spinal cord was irregular.(3)In the neutral position,there was no significant difference in the strain value of the spinal cord between the two groups(P>0.05),and the strain distribution was uniform and regular.(4)It is indicated that in the cervical extension position,the dural sac and the posterior part of the spinal cord were compressed and deformed in the early cervical spondylotic myelopathy patients with the hypertrophy of ligamentum flavum as the main sign,and the degree of compression deformation of the spinal cord was significantly higher than that in the anterior flexion position and neutral position.In the neutral position,there were no obvious signs of spinal cord deformation in patients with early cervical spondylotic myelopathy.This study verified the role of dynamic magnetic resonance imaging in the diagnosis of early cervical spondylotic myelopathy from the point of view of biomechanics.
4.Accurate diagnosis of neurography and nerve root sealing in treating multi-segment lumbar spinal stenosis with lumbar instability using Endo-P/TLIF
Yisheng ZHANG ; Yaru SUN ; Fubo TANG ; Zhifei LI ; Yi MO ; Yuanming ZHONG
The Journal of Practical Medicine 2023;39(21):2827-2833
Objective To explore the clinical value of neurography and nerve root sealing in treatment of multilevel lumbar spinal stenosis with lumbar instability using Endo-P/TLIF.Methods A total of 60 patients with multi-segment lumbar spinal stenosis and lumbar instability hospitalized in our hospital were included in this study From January 1,2022 to June 21,2022.All patients underwent nerve root closure angiography before surgery to confirm the responsible segments,and then the responsible segments were treated with Endo-P/TLIF.The patients were followed up for 6 months.The basic information on the age,gender,course of disease,surgical time,intraop-erative bleeding,hospitalization time,and off-bed ambulation time was collected.Then the data on VAS score,ODI score,JOA score,lumbar lordosis angle,intervertebral height,dural cross-sectional area,sacral inclination angle,pelvic projection angle,and pelvic inclination angle before,right after,3 months and 6 months after the operation were calculated.The number of responsible segments indicated by MRI and confirmed by nerve root closure angiography and the number of the single segment,double segments,3 segments,and above finally decompressed were statisti-cally analyzed.Results All patients went through the surgery safely.During the 6-month follow-up,one patient did not return to the hospital for consultation on time,and one patient was out of contact.Finally,the follow-up data of 58 patients were completely collected for statistical analysis.Fifty-five cases were remarkably improved,2 better,and 1 moderately,6 months after the operation,with a total effectiveness rate of 100%.The number of unilateral and bilateral single responsible segments confirmed by nerve root angiography and sealing was significantly larger than by MRI(P<0.05),but the number of unilateral and unilateral double,or multiple responsible segments was signifi-cantly smaller(P<0.05).There were statistically significant differences in terms of postoperative VAS score,ODI score,JOA score,VAS score,ODI score,JOA score,lumbar lordosis angle,intervertebral height,dural cross-sectional area,sacral inclination angle,pelvic inclination angle as compared to the preoperative data(P<0.05).The pelvic projection angle was insignificantly improved as compared to the preoperative condition(P ? 0.05).Conclusion The accurate diagnosis with selective neurography and nerve root sealing improves the confirmation of responsible nerve segments before operation.Base on the accurate diagnosis,multi-segment lumbar spinal canal stenosis with lumbar instability can be effectively treated with Endo-P/TLIF,the responsible segment decompressed,trauma and bleeding reduced,hospital stay shortened,spinal physiological curvature well recovered,and clinical efficacy improved.Therefore,the method is worthy of extensive application in clinical practice.
5.The value of left atrial structural and functional parameters combined with cardiac biomarkers in predicting left atrial spontaneous echo contrast in patients with non‐valvular atrial fibrillation
Ying TAN ; Ji WU ; Decai ZENG ; Ting ZHANG ; Guoqiang ZHONG ; Hongyuan XU ; Yisheng ZHENG
Chinese Journal of Ultrasonography 2019;28(3):230-234
Objective To investigate the relationships between the structure ,function of left atrial as well as cardiac biomarkers and left atrial spontaneous echo contrast ( SEC ) in patients with non‐valvular atrial fibrillation ( NVAF ) ,and evaluate its predictive value for left atrial SEC . Methods T hirty‐four control subjects and 80 patients with NVAF were included ,patients with NVAF were divided into positive group ( 30 cases) and negative group ( 50 cases) according to w hether SEC was presented on transesophageal echocardiography ( T EE ) . Clinical and laboratory data were collected ,including medical history ,cardiac troponin I ( cT nI) ,and pro‐brain natriuretic peptide ( pro‐BNP ) ,etc . T hen CHA2 DS2‐VASc scores were calculated .The left atrial appendage width ( W LAA ) ,left atrial appendage depth ( DLAA ) ,left atrial appendage blood flow velocity ( V LAA ) ,left atrial volume index ( LAVI) and other parameters were measured by T EE and transthoracic echocardiography ( T T E) ,and left atrial ejection fraction ( LAEF ) was calculated . T he clinical and T EE parameters were compared between the positive and negative group ,T T E parameters and cardiac biomarkers levels were compared among the three groups ,and ROC curve was used to evaluate the diagnostic efficiency . Results ①Compared with those in control group ,the value of LAVI and pro‐BNP were significantly increased and LAEF was significantly decreased in negative group ( all P < 0 .01 ) . ②Compared with those in negative group ,CHA 2 DS2‐VASc scores ,WLAA ,DLAA ,LAVI and pro‐BNP levels in positive group showed a significant increase ( all P <0 .01) ,LAEF and VLAA showed a significant reduction ( all P <0 .01) . ③cT nI between negative group and control group did not show statisticant difference ( P>0 .05) ,but presented a significant increase in positive group compared with those in control group and negative group (all P <0 .01).T he AUC ,sensitivity and specificity of CHA 2 DS2‐VASc scores for SEC were 0 .71 (95% CI :0 .60 ~ 0 .82 ) ,73 .3% and 62 .0%,respectively .When left atrial structural and functional parameters (LAVI and LAEF ) combined with cardiac biomarkers (cT nI and pro‐BNP ) ,the AUC , sensitivity and specificity for SEC were 0 .90 (95% CI :0 .84 ~ 0 .97 ) ,74 .0% and 100%,respectively ,both enhanced w hen compared with CHA 2 DS2‐VASc scores alone ( P < 0 .01 ).Conclusions Left atrial enlargement ,decreased function and elevated levels of cardiac biomarkers are associated with left atrial SEC in patients with NVAF .CHA2 DS2‐VASc scores can be used for perdicting left atrial SEC ,left atrial structural and functional parameters (LAVI and LAEF) combined with cardiac biomarkers (cT nI and pro‐BNP) provide significant increments in prediction of left atrial SEC .
6.Association between serotonin transporter promoter gene polymorphism and clinicopathological factors and effect of the polymorphism on the prognosis of colorectal cancer patients.
Minrui LUO ; Chuyuan HONG ; Yaguang ZHOU ; Yanrui LIANG ; Zhihua LIANG ; Zhen ZHONG ; Yisheng WEI
Chinese Journal of Gastrointestinal Surgery 2018;21(7):814-818
OBJECTIVETo examine the association between the genotype (LL, LS and SS) of serotonin transporter promoter gene polymorphism(5-HTTLPR) and clinicopathological factors, and to investigate the effect of 5-HTTLPR on the prognosis of colorectal cancer patients.
METHODSData of peripheral blood samples of 161 colorectal cancer patients at the Second Affiliated Hospital of Guangzhou Medical University from October 2009 to January 2014 were collected retrospectively. The genotyping of 5-HTTLPR was determined by PCR and agarose gel electrophoresis. Coincidence Chi-square test was used to examine the 5-HTTLPR genotype with Hardy-Weinberg law. Chi-square test and Cox multifactor model were used to analyze the association of 5-HTTLPR genotype with clinicopathology and prognosis. All the patients were informed and agreed to participate in the study. This study was approved by the Hospital Ethics Committee (2015056).
RESULTSOf 161 colorectal cancer patients, 89 were male and 72 were female; the median age was 64 (25-85) years; 86 (53.5%) cases were colon cancer and 75 (46.5%) were rectal cancer. Genotype was LL in 12 cases, LS in 59 cases and SS in 90 cases, which complied with the law of Hardy-Weinberg genetic balance (χ²=0.288, P=0.592). Univariate analysis showed that 5-HTTLPR gene polymorphism was only associated with lymph node metastasis [lymph node metastasis rate: LL and LS genotype 21.1% (15/71);SS genotype 40.0% (36/90), χ²= 6.532, P=0.011]. The 3-year and 5-year overall survival rates of whole patients were 71% and 63% respectively. Multivariate analysis revealed that the SS genotype was an independent risk factor affecting the overall survival of colorectal cancer patients(HR=1.933, 95%CI:1.090-3.428, P=0.024).
CONCLUSIONAmong genotypes of 5-HTTLPR gene, colorectal cancer patients with SS genotype have higher risk of lymph node metastasis and poorer prognosis.
Adult ; Aged ; Aged, 80 and over ; Colorectal Neoplasms ; genetics ; pathology ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Prognosis ; Retrospective Studies ; Serotonin Plasma Membrane Transport Proteins ; genetics
7.Changes of Kir4.1 and TASK-1 expression in rat Müller cell induced by SCH442416 at an elevated hydrostatic pressure in vitro
Zijian YANG ; Ya CHENG ; Huiping YAO ; Ting SHEN ; Yanwei CHEN ; Yisheng ZHONG
Chinese Journal of Experimental Ophthalmology 2018;36(8):590-594
Objective To evaluate the effect of adenosine receptor antagonist SCH442416 on the expression of Kir2.1,Kir4.1 and TASK-1 in rat Müller cell at an elevated hydrostatic pressure in vitro.Methods Thirty SPF Sprague Dawley rats were purchased from Shanghai Slack Laboratory Animals Ltd.Cultured Müller cells were divided into normal control group (n =6),40 mmHg/24 hours (1 mmHg =0.133 kPa) group (n =6) and adenosine + SCH442416 intervention group (n =6).Müller cells were treated with 40 mmHg pressure for 24 hours in 40 mmHg/24 hours group,and Müller cells were treated with 40 mmHg pressure for 24 hours + 10 μ mol/L adenosine + 100 nmol/L SCH442416 in adenosine + SCH442416 intervention group.The real-time PCR,Western blot,whole-cell patch-clamp recordings and immunohistochemistry were used to detect Kir2.1,Kir4.1 and TASK-1 expression and Müller cells Kir currents.The experimental procedures were in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory,and follow the 3R principle.Results Western blot assay showed that,following 40 mmHg pressure cultured for 24 hours,the expression of Kir4.1 and TASK-1 protein in Müller cell were significantly decreased by 38.6% and 52.6% compared with the normal control group,with significant differences between the two groups (both at P =0.000);Kir2.1 protein expression decreased by 14.7%,with insignificant difference between the two groups (P =0.082).Kir4.1 and TASK protein expression in adenosine + SCH442416 intervention group was increased by 60.7% and 61.4% compared with the 40 mmHg/24 hours group,with significant differences between the two groups (both at P =0.000);Kir2.1 protein expression in adenosine + SCH442416 intervention group was increased by 8.8% compared with the 40 mmHg/24 hours group,with insignificant difference between them (P =0.354).Real-time PCR assay showed that,following 40 mmHg pressure cultured for 24 hours,Kir2.1,Kir4.1 and TASK-1 mRNA expression in Müller cells were significantly decreased compared with the normal control group,with significant differences between the two groups (P =0.047,0.001,0.000);Kir4.1 and TASK-1 mRNA expression in Müller cells in the adenosine + SCH442416 intervention group was significantly increased compared with the 40 mmHg/24 hours group,with significant differences between the two groups (P =0.038,0.030);however,there is no significant change in Kir2.1 mRNA expression (P =0.612).Conclusions SCH442416 upregulates the expression of Kir4.1 and TASK-1 mRNA and protein,but weakly affects the expression of Kir2.1.
8.Effects of A2a adenosine receptor antagonist SCH442416 and ZM241385 on GS and GLAST expression in rat retina under chronic intraocular hypertension
Yong LI ; Yingjun MIN ; Lili LANG ; Yisheng ZHONG ; Runqi ZHANG
Recent Advances in Ophthalmology 2017;37(7):615-618
Objective To observe the effects of A2a adenosine receptor antagonist SCH442416 and ZM241385 on the expression of glutamine synthetase(GS) and L-Glutamate/L-Aspartate Transporter(GLAST) in rat retina under chronic ocular hypertension model.Methods Rat chronic ocular hypertension models were induced in the right eye of 12 male Sprague Dawley rats by blocking three episcleral veins,the left eye as control one.Intraocular pressure (IOP) was measured and compared at postoperative 1 week,2 weeks and 3 weeks.54 male chronic ocular hypertension rats were divided into 3 groups randomly,topically applying A2a adenosine receptor antagonist SCH442416,ZM241385 and carrier,respectively,three times a day for three weeks.At three weeks,mRNA and protein expression of GS and GLAST in rat retina were analyzed by RealTime-PCR and Western-blot.Results The average IOP of the modeling eyes at postoperative 1 week,2 weeks and 3 weeks were higher than that of the control eyes (all P < 0.05).The mRNA and protein expression of GS and GLAST in the retina of SCH442416 and ZM241385 groups increased significantly compared to the carrier group (all P < 0.05).However,the differences of mRNA and protein expression of GS and GLAST between SCH442416 and ZM241385 groups was not significant(all P > 0.05).Conclusion Rat chronic ocular hypertension model can be induced by blocking three episceral veins successfully and effectively.A2a adenosine receptor antagonist SCH442416 and ZM241385 increase the expression of GS and GLAST.There seems no difference between the effects of these two drugs.
9.Effect and Mechanism of Reactive Oxygen Species on Apoptosis of Human Hepatocellular Carcinoma Cells Induced by Fenretinide
Xiaoqin WU ; Hui YANG ; Yisheng WEI ; Aiqun LI ; Yun ZHONG ; Hang SU ; Yuanwei DING ; Manpeng LIN
Chinese Journal of Gastroenterology 2014;(7):399-403
Background:Fenretinide,which is capable of generating reactive oxygen species( ROS ),has emerged as a promising antineoplastic agent based on numerous in vitro and in vivo studies and clinical chemoprevention trials. Preliminary studies showed that fenretinide could induce apoptosis in human hepatocellular carcinoma( HCC)cells in vitro, however,the precise mechanism was not clarified. Aims:To elucidate the effect of ROS on apoptosis of human HCC cells induced by fenretinide and the underlying mechanism. Methods:Human HCC cell line Huh-7 was treated with antioxidant vitamin E,fenretinide or their combination,respectively. ROS in live cells was evaluated by confocal microscopy and flow cytometry;cell viability and apoptosis were assessed by CellTiter-Glo Luminescent Cell Viability Assay Kit and Caspase-Glo3/7 Assay Kit;expression and intracellular localization of nuclear receptor Nur77,as well as expression of stress-induced transcription factor GADD153 were measured by immunofluorescence staining and Western blotting,respectively. Results:Vitamin E pretreatment fully blocked the fenretinide-induced ROS production. In Huh-7 cells pretreated with vitamin E,cell apoptosis induced by fenretinide was significantly reduced(P<0. 05). Furthermore,effect of vitamin E pretreatment was noteworthy on reducing fenretinide-induced GADD153 expression, while no significant impact on fenretinide-induced Nur77 expression and translocation was observed. Conclusions:Elimination of ROS by vitamin E can abrogate the pro-apoptotic effect of fenretinide on Huh-7 cells,which indicates the participation of ROS in fenretinide-induced apoptosis of human HCC cells. Its mechanism might be associated with induction of GADD153 protein expression.
10.Practice and thinking of standardized training for ophthalmology residents
Xi SHEN ; Qin JIAO ; Yu CHENG ; Bing XIE ; Yisheng ZHONG
Chinese Journal of Medical Education Research 2013;(7):732-735
As one of the first resident standardized training bases, department of ophthalmology of Shanghai Ruijin Hospital participated in this reform process from 2010. Relevant rules and regulations (training management system , training scheme implementation system and evaluation system ) were strictly obeyed. When new problems emerged, under the guidance of department in charge, a series of regimens were formulated and improved gradually by Ophthalmology Professional Committee of Shanghai Resident Standardized Training Department. Based on reviewing and summarizing the work in the last 3 years, some thoughts and suggestions on the resident standardized training in future were put forward ,in-cluding how to better solve the“heavily used, lightly cultured” problem, the“disregarding medical ethics establishment”problem, the“disregarding assessment of teachers”problem and the“disregard-ing obtaining employment”problem.

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