Generation of induced secretome from adipose-derived stem cells specialized for disease-specific treatment:An experimental mouse model

BACKGROUND Recently,the exclusive use of mesenchymal stem cell(MSC)-secreted molecules,named as the secretome,have been evaluated for overcoming the limitations of cell-based therapy while maintaining its advantages.AIM To improve cell-free therapy by adding disease-specificity through stimulation of MSCs using disease-causing materials.METHODS We collected the secretory materials(named as inducers)released from AML12 hepatocytes that had been pretreated with thioacetamide(TAA)and generated the TAA-induced secretome(TAA-isecretome)after stimulating adipose-derived stem cells with the inducers.The TAA-isecretome was intravenously administered to mice with TAA-induced hepatic failure and those with partial hepatectomy.RESULTS TAA-isecretome infusion showed higher therapeutic potential in terms of(1)restoring disorganized hepatic tissue to normal tissue;(2)inhibiting proinflammatory cytokines(interleukin-6 and tumor necrosis factor-α);and(3)reducing abnormally elevated liver enzymes(aspartate aminotransferase and alanine aminotransferase)compared to the naïve secretome infusion in mice with TAA-induced hepatic failure.However,the TAA-isecretome showed inferior therapeutic potential for restoring hepatic function in partially hepatectomized mice.Proteomic analysis of TAA-isecretome identified that antioxidant processes were the most predominant enriched biological networks of the proteins exclusively identified in the TAA-isecretome.In addition,peroxiredoxin-1,a potent antioxidant protein,was found to be one of representative components of TAA-isecretome and played a central role in the protection of TAA-induced hepatic injury.CONCLUSION Appropriate stimulation of adipose-derived stem cells with TAA led to the production of a secretome enriched with proteins,especially peroxiredoxin-1,with higher antioxidant activity.Our results suggest that appropriate stimulation of MSCs with pathogenic agents can lead to the production of a secretome specialized for protecting against the pathogen.This approach is expected to open a new way of developing various specific therapeutics based on the high plasticity and responsiveness of MSCs.

Estimating prognosis of gastric neuroendocrine neoplasms using machine learning:A step towards precision medicine

Survival rates following radical surgery for gastric neuroendocrine neoplasms(g-NENs)are low,with high recurrence rates.This fact impacts patient prognosis and complicates postoperative management.Traditional prognostic models,including the Cox proportional hazards(CoxPH)model,have shown limited predictive power for postoperative survival in gastrointestinal neuroectodermal tumor patients.Machine learning methods offer a unique opportunity to analyze complex relationships within datasets,providing tools and methodologies to assess large volumes of high-dimensional,multimodal data generated by biological sciences.These methods show promise in predicting outcomes across various medical disciplines.In the context of g-NENs,utilizing machine learning to predict survival outcomes holds potential for personalized postoperative management strategies.This editorial reviews a study exploring the advantages and effectiveness of the random survival forest(RSF)model,using the lymph node ratio(LNR),in predicting disease-specific survival(DSS)in postoperative g-NEN patients stratified into low-risk and high-risk groups.The findings demonstrate that the RSF model,incorporating LNR,outperformed the CoxPH model in predicting DSS and constitutes an important step towards precision medicine.

Combining lymph node ratio to develop prognostic models for postoperative gastric neuroendocrine neoplasm patients

BACKGROUND Lymph node ratio(LNR)was demonstrated to play a crucial role in the prognosis of many tumors.However,research concerning the prognostic value of LNR in postoperative gastric neuroendocrine neoplasm(NEN)patients was limited.AIM To explore the prognostic value of LNR in postoperative gastric NEN patients and to combine LNR to develop prognostic models.METHODS A total of 286 patients from the Surveillance,Epidemiology,and End Results database were divided into the training set and validation set at a ratio of 8:2.92 patients from the First Affiliated Hospital of Soochow University in China were designated as a test set.Cox regression analysis was used to explore the relationship between LNR and disease-specific survival(DSS)of gastric NEN patients.Random survival forest(RSF)algorithm and Cox proportional hazards(CoxPH)analysis were applied to develop models to predict DSS respectively,and compared with the 8th edition American Joint Committee on Cancer(AJCC)tumornode-metastasis(TNM)staging.RESULTS Multivariate analyses indicated that LNR was an independent prognostic factor for postoperative gastric NEN patients and a higher LNR was accompanied by a higher risk of death.The RSF model exhibited the best performance in predicting DSS,with the C-index in the test set being 0.769[95%confidence interval(CI):0.691-0.846]outperforming the CoxPH model(0.744,95%CI:0.665-0.822)and the 8th edition AJCC TNM staging(0.723,95%CI:0.613-0.833).The calibration curves and decision curve analysis(DCA)demonstrated the RSF model had good calibration and clinical benefits.Furthermore,the RSF model could perform risk stratification and individual prognosis prediction effectively.CONCLUSION A higher LNR indicated a lower DSS in postoperative gastric NEN patients.The RSF model outperformed the CoxPH model and the 8th edition AJCC TNM staging in the test set,showing potential in clinical practice.

Disease-specific protein corona formed in pathological intestine enhances the oral absorption of nanoparticles

Protein corona(PC)has been identified to impede the transportation of intravenously injected nanoparticles(NPs)from blood circulation to their targeted sites.However,how intestinal PC(IPC)affects the delivery of orally administered NPs are still needed to be elucidated.Here,we found that IPC exerted“positive effect”or“negative effect”depending on different pathological conditions in the gastrointestinal tract.We prepared polystyrene nanoparticles(PS)adsorbed with different IPC derived from the intestinal tract of healthy,diabetic,and colitis rats(H-IPC@PS,D-IPC@PS,C-IPC@PS).Proteomics analysis revealed that,compared with healthy IPC,the two disease-specific IPC consisted of a higher proportion of proteins that were closely correlated with transepithelial transport across the intestine.Consequently,both D-IPC@PS and C-IPC@PS mainly exploited the recycling endosome and ER-Golgi mediated secretory routes for intracellular trafficking,which increased the transcytosis from the epithelium.Together,disease-specific IPC endowed NPs with higher intestinal absorption.D-IPC@PS posed“positive effect”on intestinal absorption into blood circulation for diabetic therapy.Conversely,CIPC@PS had“negative effect”on colitis treatment because of unfavorable absorption in the intestine before arriving colon.These results imply that different or even opposite strategies to modulate the disease-specific IPC need to be adopted for oral nanomedicine in the treatment of variable diseases.

蛋白质糖基化修饰:新一代肝病靶标的分子基础

肝病糖生物学研究是新一代肝病分子诊断标志物及肝癌治疗分子靶点研究的热点领域。不同肝病过程中伴随着复杂的蛋白质N-糖基化和O-糖基化修饰,这些糖基化修饰的变化导致了一些蛋白质翻译后修饰的改变,因而有可能成为未来新的肝病诊断标志物。这些修饰聚糖改变的基础在于肝细胞内部糖基转移酶和糖苷酶活性的变化,因而,对糖基转移酶和糖苷酶活性调控也有可能成为新一代抗癌药物研发的靶点。针对血清蛋白N-糖组分析也已经成为肝癌、肝硬化诊断的新方法。这些进展预示着肝病糖生物学研究将迎来一个新的时代。

血清ConA非结合型GPDA检测及其临床应用

目的:建立血清甘氨酰脯氨酸二肽氨基肽酶(GPDA)糖链异质性的检测法。方法:利用伴刀豆素A(ConA)与等量血清孵育法测定血清中ConA非结合型GPDA。结果:正常人血清中ConA非结合型GPDA为12.1￣42.1U/L,原发性肝癌时明显升高。结论:本法简便,灵敏度高,有助于肝癌的早期诊断。

糖蛋白微观分布的毛细管电泳分离

糖蛋白广泛存在于动植物体中，在生物过程中有重要或关键的作用，并具有药用价值，不少糖蛋白已开始用生物技术进行大规模生产［１～４］，但是糖蛋白的生物功能研究及其生物技术产品质量控制因无有效的分析方法而受挫。糖蛋白中的糖链及其与多肽链的连接是由酶控制合成的...

肺癌与肺良性疾病特异血清免疫炎性蛋白质N-糖基化修饰差异

目的探索肺癌与肺良性疾病特异血清免疫炎性蛋白质N-糖基化修饰差异。方法应用非变性聚丙烯酰胺凝胶电泳(native-PAGE)从肺腺癌和慢性肺炎患者血清中分离得到一组疾病特异的血清免疫炎性蛋白质复合物(IIRPCs),经胶内酶解、石墨相氮化碳富集分离得到完整糖肽。应用高效液相色谱-串联质谱(HPLC-MS/MS)技术,结合GPSeeker数据库检索软件进行糖肽鉴定。结果共鉴定了来自12种蛋白质的89种糖肽,其中21种仅与肺良性疾病相关,38种仅与肺癌相关。89种糖肽中包括63种糖型,其中10种为肺良性疾病特有糖型,21种为肺癌特有糖型。两种疾病的特有糖型在其结构和连接位点上均存在差异。结论发现了两种疾病血清特异免疫炎性蛋白质N-糖基化修饰的差异,为糖基化修饰在慢性疾病的诊断、预后评估和分子机制等方面的研究提供了新视角。

质谱法定量测定高密度脂蛋白结合蛋白的糖基化水平

采用质谱法对4种高密度脂蛋白(HDL)的结合蛋白重组人载脂蛋白血清淀粉样蛋白A(SAA)、α1-抗胰蛋白酶(A1AT)、α2-人体血清糖蛋白(A2HSG)和A载脂蛋白C3(Apo C3)从蛋白质含量(蛋白的绝对定量)、位点特异性糖基化(糖肽的相对定量)及聚糖位点占有率等方面进行了研究.利用四极杆-飞行时间质谱仪(Q-TOF)测量糖蛋白标样酶解产物的二级质谱碎片离子,用Byonic软件发现了新的糖基化位点信息,即增加了原位点处聚糖糖型的种类.对于A2HSG,新增了N-糖基化156位点上的4种糖型, N -糖基化176位点上的6种糖型, O-糖基化319位点的4种O-聚糖和 O-糖基化346位点上的1种糖型.对于Apo C3,只有O-糖基化 94一个位点,在此位点上新增了9种糖型.同时,调整了用于定量蛋白的多肽,使得定量更加准确.采用三重四极杆串级质谱仪(UPLC-ESI-QQQ)研究了4种结合蛋白中多肽和糖肽的多反应监测(MRM)行为,并重新计算了每种聚糖的位点占有率,优化了现有的定量方法.

E-cadherin expression pattern in primary colorectal carcinomas and their metastases reflects disease outcome

AIM： To investigate the changes that occur in E-cadherin expression during the process of metastasis in colorectal cancer.METHODS： E-cadherin expression was detected by immunohistochemistry and two indices of expression were calculated which reflected the level of expression and the locations （membrane and cytoplasm）. Univariate and multivariate survival analyses were used to assess the value of these two E-cadherin indices as predictors of both disease-free （DFS） and disease-specific （DSS） survival. RESULTS： E-cadherin membrane index （MI）, but not cytoplasmic index （CI）, was significantly higher in primary tumors than their metastases （P = 0.0001）. Furthermore, both primary tumor MI and CI were higher among the patients who developed subsequent metastasis （P = 0.022 and P = 0.007, respectively）. Interestingly, both indices were higher in liver metastase compared to other anatomic sites （MI, P = 0.034 and CI, P = 0.022）. The CI of the primary tumors was a significant predictor of DFS （P = 0.042, univariate analysis）, with a strong inverse correlation between CI and DFS （P = 0.006, multivariate analysis）. Finally, the MI of primary tumor proved to be a significant independent predictor of DSS, with higher indices being associated with a more favorable outcome （P = 0.016）. CONCLUSION： Examination of E-cadherin expression and distribution in colorectal tumors can be extremely valuable in predicting disease recurrence. The observation that aberrant cytoplasmic expression of E-cadherin can predict disease recurrence is obviously of great importance for both patients and clinicians, and significantly affects decisions concerning the therapy and management of the patients.

Establishment of a Stable PrP^(Sc) Panel from Brain Tissues of Experimental Hamsters with Scrapie Strain 263K

Objective To establish a stable PrP^Sc panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals＇ prion diseases. Methods Thirty brain tissue samples from hamsters intracerebrally infected with scrapie strain 263K and another 30 samples from normal hamsters were selected to prepare 10%, 1%, and 0.5% brain homogenates, which were aliquoted into stocks. PrP^Sc in each brain homogenate was determined by proteinase K digestions followed by Western blot assay and partially by immunohistochemistry. Stability and glycoforms of PrP^Sc were repeatedly detected by prp^SC-specific Western blots in half a year and 3 years later. Results PrPsc signals were observed in all 10% brain homogenates of infected hamsters. Twenty out of 30 stocks and 19 out of 30 stocks were PrPsc positive in 1% and 0.5% brain homogenatesof infected hamsters, respectively. TWenty-seven out of 30 stocks presented three positive bands in 10% brain homogenates, whereas none of 1% and 0.5% homogenates contained 3 bands. The detection of prpSc-specific signals stored in half a year and 3 years later demonstrated that the ratio of PrPsc positive samples and glycoforms was almost unchanged. All normal hamsters＇ brain homogenates were PrP^Sc negative. Conclusion A PrP^Sc panel of prion disease can be established, which displays reliably stable prp^Sc-specific signals and glycoforms.

Nuclear β-catenin expression as a prognostic factor in advanced colorectal carcinoma

AIM： To investigate the changing pattern of β-catenin expression and its prognostic value in advanced colorectal cancer （CRC）. METHODS： Archival tumor samples were analyzed for β-catenin using immunohistochemistry （IHC） in 95 patients with advanced CRC. RESULTS： Membranous β-catenin expression was found in the normal colorectal epithelium. Almost 100% of CRC cases showed membranous and cytoplasmic expression, and 55 （58%） cases showed nuclear expression. In univariate （Kaplan-Meier） survival analysis, only the nuclear index （NI） was a significant predictor of disease free survival （DFS） （P = 0.023; n = 35）, with a NI above the median associated with longer DFS （34.2 too） than those with a NI below the median （15.5 too） （P = 0.045, ANOVA）. The other indices were not significant predictors of DFS, and none of the three tested indices （for membranous, cytoplasmic, or nuclear expression） predicted diseasespecific survival （DSS）. However, when dichotomized as positive or negative nuclear expression, the former was a significant predictor of more favorable DFS （P = 0.041） and DSS （P = 0.046）. CONCLUSION： Nuclear β-catenin expression provides additional information in predicting patient outcome in advanced CRC.

A novel nomogram individually predicting disease-specific survival after D2 gastrectomy for advanced gastric cancer

Background:Few studies have shown nomograms that may predict disease-specific survival(DSS)probability after curative D2 gastrectomy for advanced gastric cancer(AGC),particularly among Chinese patients.This study sought to develop an elaborative nomogram that predicts long-term DSS for AGC in Chinese patients.Methods:A retrospective study was conducted on 6753 AGC patients undergoing D2 gastrectomy between January 1,2000 and December 31,2012 from three large medical hospitals in China.We assigned patients from Sun Yat-sen University Cancer Center to the training set,and patients from the First Affiliated Hospital of China Medical University and Tianjin Medical University Cancer Hospital to two separate external validation sets.A multivariate survival analysis was performed using Cox proportional hazards regression model in a training set,and a nomogram was constructed.Harrell’s C-index was used to evaluate discrimination and calibration plots were used to validate similarities between survival probabilities predicted by the nomogram model and actual survival rates in two validation sets.Results:The multivariate Cox regression model identified age,tumor size,location,Lauren classification,lymphatic/venous invasion,depth of invasion,and metastatic lymph node ratio as covariates associated with survival.In the training set,the nomogram exhibited superior discrimination power compared with the 8th American Joint Com-mittee on Cancer TNM classification(Harrell’s C-index,0.82 vs.0.74;P<0.001).In two validation sets,the nomogram’s discrimination power was also excellent relative to TNM classification(C-index,0.83 vs.0.75 and 0.81 vs.0.74,respec-tively;P<0.001 for both).After calibration,the nomogram produced survival predictions that corresponded closely with actual survival rate.Conclusions:The established nomogram was able to predict 3-,5-,and 10-year DSS probabilities for AGC patients.Validation revealed that this nomogram exhibited excellent discrimination and calibration capacity,suggesting its clinical utility.

Revalidation of a prognostic score model based on complete blood count for nasopharyngeal carcinoma through a prospective study

Objective： In our previous work, we incorporated complete blood count （CBC） into TNM stage to develop a new prognostic score model, which was validated to improve prediction efficiency of TNM stage for nasopharyngeal carcinoma （NPC）. The purpose of this study was to revalidate the accuracy of the model, and its superiority to TNM stage, through data from a prospective study.Methods： CBC of 249 eligible patients from the 863 Program No. 2006AA02Z4B4 was evaluated. Prognostic index （PI） of each patient was calculated according to the score model. Then they were divided by the PI into three categories： the low-, intermediate-and high-risk patients. The 5-year disease-specific survival （DSS） of the three categories was compared by a log-rank test. The model and TNM stage （Tth edition） were compared on efficiency for predicting the 5-year DSS, through comparison of the area under curve （AUC） of their receiver-operating characteristic curves.Results： The 5-year DSS of the low-, intermediate- and high-risk patients were 96.0%, 79.1% and 62.2%, respectively. The low- and intermediate-risk patients had better DSS than the high-risk patients （P〈0.001 and P〈0.005, respectively）. And there was a trend of better DSS in the low-risk patients, compared with the intermediate-risk patients （P=0.049）. The AUC of the model was larger than that of TNM stage （0.726 vs. 0.661, P：0.023）. Conclusions： A CBC-based prognostic score model was revalidated to be accurate and superior to TNM stage on predicting 5-year DSS of NPC.

Ehealth monitoring in irritable bowel syndrome patients treated with low fermentable oligo-, di-, mono-saccharides and polyols diet

In the present study we report on changes in irritable bowel syndrome-severity scoring system (IBS-SSS) and irritable bowel syndrome-quality of life (IBS-QoL) in 19 IBS patients, aged 18 to 74 years (F/M: 14/5), during 12 wk registering their symptoms on the web-application (www.ibs.constant-care.dk). During a control period of the first 6-wk patients were asked to register their IBS-SSS and IBS-QoL on the web-application weekly without receiving any intervention. Thereafter, low fermentable oligo-, di-, mono-saccharides and polyols (FODMAP) diet (LFD) was introduced for the next 6 wk while continuing the registration. Though a small sample size a significant improvement in disease activity (IBS-SSS) was observed during both the control period, median: 278 (range: 122-377), P = 0.02, and subsequently during the LFD period, median: 151 (range: 29-334), P &#x0003c; 0.01. The IBS-QoL solely changed significantly during the LFD period, median: 67 (37-120), P &#x0003c; 0.01. The significant reduction in disease activity during the control period shows a positive effect of the web-application on IBS symptoms when presented as a &#x0201c;traffic light&#x0201d;. However adding the diet reduced IBS-SSS to &#x0003c; 150, inactive to mild symptoms. In the future results from larger scale trials are awaited.

A systematic review: How to choose appropriate health-related quality of life (HRQOL) measures in routine general practice?

In more recent times, health-related quality of life (HRQOL) measurements have formed an important part of as- sessing the quality of routine care in general practice. For a measure to have clinical usefulness it must not only be valid, appro- priate, reliable, responsive, and capable of being interpreted, but it must also be simple, fast to complete, easy to score, and provide useful clinical data. The Two-step method of choosing appropriate measures is introduced. Then through comparison of generic instruments with disease-specific instruments, we can conclude that sometimes a combination of generic and disease-specific HRQOL measures may be more appropriate for monitoring changes in a patient’s health status due to an intervention.

Lack of CD44 variant 6 expression in rectal cancer invasive front associates with early recurrence

AIM: To investigate the prognostic value of CD44 variant 6 (CD44v6), a membranous adhesion molecule, in rectal cancer. METHODS: Altogether, 210 rectal cancer samples from 214 patients treated with short-course radiotherapy (RT, n = 90), long-course (chemo) RT (n = 53) or surgery alone (n = 71) were studied with immunohistochemistry for CD44v6. The extent and intensity of membranous and cytoplasmic CD44v6 staining, and the intratumoral membranous staining pattern, were analyzed.RESULTS: Membranous CD44v6 expression was seen in 84% and cytoplasmic expression in 81% of the cases. In 59% of the tumors with membranous CD44v6 expression, the staining pattern in the invasive front was determined as "front-positive" and in 41% as "front-negative". The latter pattern was associated with narrower circumferential margin (P = 0.01), infiltrative growth pattern (P < 0.001), and shorter disease-free survival in univariate survival analysis (P = 0.022) when compared to the "front-positive" tumors. CONCLUSION: The lack of membranous CD44v6 in the rectal cancer invasive front could be used as a method to identify patients at increased risk for recurrent disease.

原发性肝癌患者血清及癌组织GPDA糖链异质体的研究

目的 :研究原发性肝癌 (PHC)患者血清、肝癌组织甘氨酰脯氨酸二肽氨基肽酶 (GPDA)糖链异质体的变化和对 PHC的诊断价值。方法 :测定 2 6 5例 PHC、良性肝病患者及正常对照血清 GPDA总活性 (TGPDA) ,利用刀豆素 A(Con A)与等量血清孵育法将 GPDA分为刀豆素 A结合型与非结合型两种异质体。测定非结合型活性 ,计算结合型活性 (总活性减去非结合型活性 ) ;同方法测定 PHC患者肝组织中 GPDA总活性和非结合型活性 ,根据测定的蛋白浓度分别计算两者的比活性。结果 :PHC患者血清非结合型活性显著高于慢性肝炎、肝硬化和正常对照组 (P均 <0 .0 1)及急性肝炎组 (P<0 .0 5 ) ;各受检组血清结合型活性均显著高于与该组非结合型的活性 (P均 <0 .0 1) ;PHC患者血清结合型活性与正常人比无显著差异 (P>0 .0 1)。结论 :分别以 TGPDA及非结合型作为 PHC诊断的指标 ,两者特异性相近 ,而敏感性、准确率均高于 TGPDA。推测非结合型异常增高可能源于肿瘤组织的膜结合部分的过量合成 。

基于纳升电喷雾-轨道阱超高分辨质谱对完整单克隆抗体药物进行分子量测定及糖型鉴定的条件探究

整体分子量测定和糖型鉴定是抗体药物研发及生产中不可或缺的表征内容,而在完整蛋白层面进行测定可获得最为直接的测定结果。实现此类测定有赖于可提供较高分辨能力及质量精度的仪器;测定中涉及的相对复杂的参数设置可能受多种条件的干扰而产生误导性结果。该研究考察了利用纳升电喷雾离子源-轨道阱质谱仪以非变性质谱方式测定完整单克隆抗体药时,仪器分辨率、源内裂解、碰撞诱导碎裂、溶液组成等条件对单克隆抗体药物分子量及糖型测定结果的影响,以期为此类质谱分析提供有益的借鉴。

液质联用技术分析可溶性CD95-Fc融合蛋白N-糖组成

目的:分析鉴定可溶性CD95-Fc融合蛋白的N-糖组成。方法:采用Glyco Works Rapi Fluor-MS N-糖分析试剂盒酶解并标记、纯化N-糖基,糖基纯化产物经Waters BEH Glycan糖蛋白分析柱分离后,依次进入荧光检测器和飞行时间质谱仪检测。结果:结合荧光和质谱检测结果,鉴定出15种糖型:G0-GN,G0FGN,G0,G0F,Man5,G1F,G1F,G2F,G2F+SA,G2F+SA,G3F,G2F+2SA,G3F+SA,G3F+2SA和G3F+3SA;各糖型组分实测单同位素分子量与理论值差异均不足0.1Da;所占比例最高的糖型依次为G0F(30%),G1F(25%),G2F+SA(14%)和G2F(13%);不同批次相同糖基组分保留时间的RSD均低于0.2%,峰面积百分比的RSD均低于15%。结论:本研究成功鉴定出可溶性CD95-Fc融合蛋白的15种主要糖型,不同批次的糖型荧光图谱基本一致,可作为UPLC法对可溶性CD95-Fc融合蛋白N-糖定性定量的标准参考图谱。