Physical frailty, genetic predisposition, and the risks of severe non-alcoholic fatty liver disease and cirrhosis: a cohort study.

BACKGROUND: Frailty, defined as a phenotype of decreased physiological reserves and diminished ability to respond to stressors, has been linked to the development of chronic diseases. Epidemiological evidence connecting frailty to non-alcoholic fatty liver disease (NAFLD) and cirrhosis risks remain sparse. We aimed to assess the longitudinal associations of frailty with the risks of severe NAFLD and cirrhosis in middle-aged to older adults and further explore the modification role of genetic risk on these associations. METHODS: This study included a total of 398 386 participants from the UK Biobank. Incident cases of severe NAFLD and cirrhosis were ascertained through linked hospital records and death registries. Frailty status was assessed by a modified version of the frailty phenotype, encompassing five key components: weight loss, tiredness, physical activity, gait speed, and grip strength. Participants were classified as pre-frailty if they met one or two of these criteria, and as frailty if they met three or more. Genetic predisposition to NAFLD and cirrhosis was estimated by genetic risk score (GRS) and further categorized into high, intermediate, and low genetic risk levels according to tertiles of GRSs. Cox proportional hazards regression model was employed to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) for their associations. RESULTS: The mean (standard deviation) age of the study population was 56.6 (8.03) years. 214 408 (53.8%) of the participants was female; 14 924 (3.75%) of participants met the criteria for frailty, 170 498 (42.8%) for pre-frailty, and 212 964 (53.5%) for non-frailty. Over a median follow-up of 12.0 years, we documented 4439 incident severe NAFLD and 3323 incident cirrhosis cases, respectively. Compared with non-frailty, both pre-frailty (HR: 1.50; 95% CI: 1.40-1.60) and frailty (HR: 1.98; 95% CI: 1.77-2.21) were associated with increased risk of NAFLD. Similar associations were observed for cirrhosis, the corresponding HRs (95% CIs) for non-frailty, pre-frailty, and frailty were 1.00 (reference), 1.29 (1.20, 1.38), and 1.90 (1.66, 2.18). Such associations were consistent across all genetic risk levels, with no observed interactions between frailty and GRSs (all P for interactions ≥0.10). Compared with participants with frailty and a low level of genetic risk, the greatest risk increasement in developing severe NAFLD (HR: 3.36; 95% CI: 2.83-3.99) and cirrhosis (HR: 2.81; 95% CI: 2.29-3.44) was both observed in those with frailty and a high level of genetic risk. CONCLUSIONS: Our findings indicate that frailty is a significant predictor of severe NAFLD and cirrhosis, irrespective of genetic predisposition.

Intensity-Specific Physical Activity Measured by Accelerometer, Genetic Susceptibility, and the Risk of Kidney Stone Disease: Results From the UK Biobank.

RATIONALE & OBJECTIVE: Kidney stone disease (KSD), a significant health care problem within both developed and developing countries, has been associated with genetic risk factors. An association between physical activity and KSD risk also has been hypothesized, but studies have yielded inconsistent findings. This study investigated the association between the intensity of physical activity and the incidence of KSD accounting for genetic risk. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: A total of 80,473 participants from the UK Biobank Study. EXPOSURE: Physical activity levels, including total physical activity (TPA), moderate-to-vigorous intensity physical activity (MVPA), and light-intensity physical activity (LPA), were measured using accelerometers and quantified using a machine learning model. A polygenic risk score (PRS) for KSD was also constructed. OUTCOME: Individuals with KSD were identified using the International Classification of Diseases, Tenth Revision (ICD-10), and procedure codes for KSD surgery. ANALYTICAL APPROACH: A Fine and Gray survival model was used to estimate the associations of incident KSD with TPA, MVPA, LPA, and PRS (as categorical variables). Restricted cubic splines were used to examine potential nonlinear associations within the fully adjusted models. RESULTS: During an average follow-up of 6.19 years, 421 participants developed KSD. Participants in the highest quartiles of TPA, MVPA, and LPA had lower adjusted rates of KSD compared with those in the lowest quartiles: HR, 0.50 (95% CI, 0.44-0.56), 0.57 (95% CI, 0.51-0.64), and 0.66 (95% CI, 0.59-0.74), respectively. TPA, MVPA, and LPA were associated with a lower risk of KSD in participants with low and high genetic predisposition for KSD. LIMITATIONS: Selection bias as participants who provided accelerometry data may have been more adherent to health care. CONCLUSIONS: Physical activity was negatively associated with the risk of KSD, regardless of the genetic risk. Future large studies are warranted to confirm and explain the mechanisms underlying these associations. PLAIN-LANGUAGE SUMMARY: The association between the intensity of physical activity (PA) and the incidence of kidney stone disease (KSD) after accounting for genetic risk is unclear. We conducted a comprehensive prospective cohort study utilizing participants from the UK Biobank to assess the intensity of PA using accelerometers. Our study findings indicated that greater total PA, moderate-to-vigorous-intensity PA, and light-intensity PA were each associated with a lower risk of KSD irrespective of an individual's genetic risk. Our study informs the understanding of risk factors for KSD.

The sirtuin family in health and disease.

Sirtuins (SIRTs) are nicotine adenine dinucleotide(+)-dependent histone deacetylases regulating critical signaling pathways in prokaryotes and eukaryotes, and are involved in numerous biological processes. Currently, seven mammalian homologs of yeast Sir2 named SIRT1 to SIRT7 have been identified. Increasing evidence has suggested the vital roles of seven members of the SIRT family in health and disease conditions. Notably, this protein family plays a variety of important roles in cellular biology such as inflammation, metabolism, oxidative stress, and apoptosis, etc., thus, it is considered a potential therapeutic target for different kinds of pathologies including cancer, cardiovascular disease, respiratory disease, and other conditions. Moreover, identification of SIRT modulators and exploring the functions of these different modulators have prompted increased efforts to discover new small molecules, which can modify SIRT activity. Furthermore, several randomized controlled trials have indicated that different interventions might affect the expression of SIRT protein in human samples, and supplementation of SIRT modulators might have diverse impact on physiological function in different participants. In this review, we introduce the history and structure of the SIRT protein family, discuss the molecular mechanisms and biological functions of seven members of the SIRT protein family, elaborate on the regulatory roles of SIRTs in human disease, summarize SIRT inhibitors and activators, and review related clinical studies.

A Follow-Up Study of Ovarian Cancer (OOPS): A Study Protocol.

The ovarian cancer (OC) follow-up study (OOPS) is an on-going hospital-based large prospective longitudinal cohort study aimed to explore the relationship between pre/post-diagnostic biological, clinical, environmental, and lifestyle factors with focus on the diet and OC prognosis (including drug resistance, relapse, and mortality). Patients recruited during the baseline survey were between 18 and 79 years old, with histologically confirmed OC diagnosis. Their follow-up and medical treatment were conducted at the gynecological oncology ward at Shengjing Hospital of China Medical University, Shenyang, China after 2015. A total of 703 OC patients made up the final OOPS study population. The follow-up stage was conducted in both passive and active modes. In the passive mode, the follow-up was performed by linkage to the Liaoning Providence Center for Disease Control and Prevention every 6 months to obtain health outcome results. The status of lifestyle factors was re-estimated using the same measurements as those in the baseline survey. OC participants in the OOPS study completed a questionnaire and anthropometric examinations. In addition, biological specimens were collected during the baseline survey, which included blood, urine, and stool samples that were stored for further use. This article is intended to serve as an introduction to this project and to provide details for investigators who may be carry out related analysis.

Longitudinal Associations Between Household Solid Fuel Use and Handgrip Strength in Middle-Aged and Older Chinese Individuals: The China Health and Retirement Longitudinal Study.

Background: Household solid fuel have been associated with changes of handgrip strength (HGS). However, no study has explored the longitudinal associations between household solid fuel use and HGS. Thus, the aim of our cohort study was to investigate the longitudinal associations between household fuel use and HGS. Methods: The study was based on the China Health and Retirement Longitudinal Study. A handheld dynamometer was used to measure HGS. Household fuel use statuses were collected using questionnaires. Analyses of covariance were performed to examine the associations between household fuel use and HGS. Results: The study included 9,382 participants during a 4-year follow-up. The participants who used solid fuel for cooking had more decreases of HGS than those who used clean fuel (P < 0.0001). The least square means (95% CIs) of changes of HGS for participants who used solid fuel and those who used clean fuel for cooking were -1.67 (-2.15, -1.19) and-2.27 (-2.75, -1.79), respectively. The association between fuel use for heating and HGS was non-significant (P = 0.63). The interaction terms of sex to cooking fuel (P = 0.04) and smoking to cooking fuel (P < 0.001) were significant; men and participants who had ever smoked had higher decreases in HGS. Conclusion: Using household solid fuel for cooking but not heating was associated with more decreases in HGS. Proper ventilation and clean fuel should be promoted for public health.

Pre-diagnosis meat intake and cooking method and ovarian cancer survival: results from the Ovarian Cancer Follow-Up Study (OOPS).

Objectives: The relationships between pre-diagnosis meat intake and ovarian cancer (OC) survival were limited and controversial. To date, no study has taken account of cooking methods. Thus, we aimed to firstly clarify these associations based on the Ovarian Cancer Follow-Up Study. Methods: This prospective cohort study, including 853 OC patients between 2015 and 2020, was conducted to examine the aforementioned associations. All women completed a food frequency questionnaire. Deaths were ascertained up to March 31, 2021 via medical records and active follow-up. We used the Cox proportional hazards model to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Results: During the median follow-up of 37.17 months, 130 women died. Pre-diagnosis fish and seafood intake was associated with better survival (HRT3 vs. T1 = 0.46, 95% CI = 0.26-0.82, p trend <0.05), whereas processed red meat (HR = 1.54, 95% CI = 1.04-2.26) and a high frequency of fried fish intake (HR = 1.49, 95% CI = 1.03-2.16) were associated with worse survival than consuming none. After considering the interaction of cooking methods, we found that compared with the lowest tertile of fish and seafood intake and almost no fried fish cooking, women with the highest tertile of intake and almost no fried fish cooking had better survival (HR = 0.35, 95% CI = 0.13-0.92). Additionally, compared with the lowest tertile of fish and seafood intake and almost no baked fish cooking, women with the lowest tertile of intake and consuming baked fish had worse survival (HR = 3.75, 95% CI = 1.53-9.15). Conclusions: Pre-diagnosis fish and seafood intake was associated with better OC survival, whereas processed red meat intake was associated with worse survival. Cooking methods, especially for fried or baked fish, may play interaction effects with fish intake on OC survival.

Cruciferous vegetable consumption and multiple health outcomes: an umbrella review of 41 systematic reviews and meta-analyses of 303 observational studies.

Background: Epidemiological studies evaluating the associations between the consumption of cruciferous vegetables (CV) and diverse health outcomes have generated inconsistent findings. Therefore, we carried out an umbrella review to systematically summarize existing evidence on this topic. Methods: This study had been registered at PROSPERO (no. CRD42021262011). Relevant systematic reviews and meta-analyses of observational studies were identified by searching PubMed, Web of science, and Embase databases from inception up to March 15, 2021. Observational studies investigating the association between CV intake and multiple health outcomes in humans were eligible for inclusion. The validated AMSTAR (A Measurement Tool to Assess Systematic Reviews) instrument was utilized for assessing the methodological quality of the included systematic reviews. For each meta-analysis, we assessed the summary effect size by using fixed and random effects models, 95% prediction intervals, heterogeneity, evidence of small-study effects, and excess significance bias. Results: Our umbrella review included 41 meta-analyses of 303 individual studies involving 13 394 722 participants. Twenty-four health outcomes including cancers (n = 23), cardiovascular disease (n = 12), mortality (n = 5), and metabolic diseases (n = 1) were evaluated. The summary random effects estimates were significant at P < 0.05 in 24 meta-analyses - all of which reported decreased risks of health outcomes. All were of moderate methodological quality in our study. Of the 41 meta-analyses, we observed suggestive evidence for beneficial associations between gastric cancer, lung cancer, endometrial cancer, and all-cause mortality. Moreover, 16 associations were supported by weak evidence, including breast cancer, lung cancer, renal cell carcinoma, bladder cancer, prostate cancer, ovarian cancer, endometrial cancer, colon cancer, colorectal adenoma, colorectal neoplasm, non-Hodgkin lymphoma, and total cancer. Conclusions: It revealed that CV intake might be associated with beneficial effects on several health-related outcomes (gastric cancer, lung cancer, endometrial cancer, and all-cause mortality). Other associations could be genuine, but substantial uncertainty remains. Additional studies are needed to evaluate the relationship between the consumption of CV and various health outcomes as well as robust randomized controlled trials in the future.

Long-term PM2.5 exposure and various health outcomes: An umbrella review of systematic reviews and meta-analyses of observational studies.

Adverse effects from exposure to particulate matter <2.5 µm in diameter (PM2.5) on health-related outcomes have been found in a range of experimental and epidemiological studies. This study aimed to assess the significance, validity, and reliability of the relationship between long-term PM2.5 exposure and various health outcomes. The Embase, PubMed, Web of Science, CNKI, WANFANG, VIP, and SinoMed databases and reference lists of the retrieved review articles were searched to obtain meta-analysis and systematic reviews focusing on PM2.5-related outcomes as of August 31, 2021. Random-/fixed-effects models were applied to estimate summary effect size and 95% confidence intervals (CIs). The quality of included meta-analyses was evaluated based on the AMSTAR 2 tool. Small-study effect and excess significance bias studies were conducted to further assess the associations. Registered PROSPERO number: CRD42020200606. This included 24 articles involving 71 associations between PM2.5 exposure and the health outcomes. The evidence for the positive association of 10 µg/m3 increments of long-term exposure to PM2.5 and stroke incidence in Europe was convincing (effect size = 1.07, 95% CI: 1.05-1.10). There was evidence that was highly suggestive of a positive association between 10 µg/m3 increments of long-term exposure to PM2.5 and the following health-related outcomes: mortality of lung cancer (effect size = 1.11, 95% CI: 1.08-1.13) and Alzheimer's disease (effect size = 4.79, 95% CI: 2.79-8.21). There was highly suggestive evidence that chronic obstructive pulmonary disease risk is positively associated with higher long-term PM2.5 exposure versus lower long-term PM2.5 exposure (effect size = 2.32, 95% CI: 1.88-2.86). In conclusion, the positive association of long-term exposure to PM2.5 and stroke incidence in Europe was convincing. Given the validity of numerous associations of long-term exposure to PM2.5 and health-related outcomes is subject to biases, more robust evidence is urgently needed.

Associations of Nutritional, Lifestyle, and Metabolic Factors With Non-alcoholic Fatty Liver Disease: An Umbrella Review With More Than 380,000 Participants.

Background & Aims: Nonalcoholic fatty liver disease (NAFLD) is the most common liver injury. We performed this umbrella review of meta-analyses to summarize the evidence on the associations of nutritional, lifestyle, and metabolic factors with NAFLD. Methods: We searched the PubMed, Embase, and Web of Science databases from inception until July 2, 2020, to identify meta-analyses of observational studies which explored the associations of nutritional, lifestyle, and metabolic factors with NAFLD. Evidence levels were assessed using summary effect sizes, 95% prediction intervals, between-study heterogeneity, evidence of small-study effects, and evidence of excess significance bias for each meta-analysis. (No. of PROSPERO, CRD42020200124). Results: Twenty two risk or protective factors from 10 published meta-analyses were included and studied. Three risk factors (sugar-sweetened beverage consumption, serum fetuin-A, and waist circumference) with highly suggestive levels of evidence and three risk factors (soft drink consumption, former smoking, and body mass index) with suggestive levels of evidence were identified. Only two protective factors (physical activity and serum vitamin D level [among adults in Western countries]) with suggestive levels of evidence were identified. Furthermore, other six risk factors and two protective factors with weak levels of evidence were identified. Conclusions: We found varying levels of evidence of associations of nutritional, lifestyle, and metabolic factors and NAFLD. The results suggest that nutritional and lifestyle management should be considered as a major primary preventive strategy for NAFLD. Moreover, considering the low quality of included meta-analyses and limited area of research topics, future high-quality original studies and meta-analyses should be performed to study these associations.

Umbrella Review on Associations Between Single Nucleotide Polymorphisms and Lung Cancer Risk.

The aim is to comprehensively and accurately assess potential relationships between single nucleotide polymorphisms (SNP) and lung cancer (LC) risk by summarizing the evidence in systematic reviews and meta-analyses. This umbrella review was registered with the PROSPERO international prospective register of systematic reviews under registration number CRD42020204685. The PubMed, Web of Science, and Embase databases were searched to identify eligible systematic reviews and meta-analyses from inception to August 14, 2020. The evaluation of cumulative evidence was conducted for associations with nominally statistical significance based on the Venice criteria and false positive report probability (FPRP). This umbrella review finally included 120 articles of a total of 190 SNP. The median number of studies and sample size included in the meta-analyses were five (range, 3-52) and 4 389 (range, 354-256 490), respectively. A total of 85 SNP (in 218 genetic models) were nominally statistically associated with LC risk. Based on the Venice criteria and FPRP, 13 SNP (in 22 genetic models), 47 SNP (in 99 genetic models), and 55 SNP (in 94 genetic models) had strong, moderate, and weak cumulative evidence of associations with LC risk, respectively. In conclusion, this umbrella review indicated that only 13 SNP (of 11 genes and one miRNA) were strongly correlated to LC risk. These findings can serve as a general and helpful reference for further genetic studies.

Dietary Carbohydrate and Diverse Health Outcomes: Umbrella Review of 30 Systematic Reviews and Meta-Analyses of 281 Observational Studies.

Background and Aims: The associations between dietary carbohydrate and diverse health outcomes remain controversial and confusing. To summarize the existing evidence of the association between dietary carbohydrate intake and diverse health outcomes and to evaluate the credibility of these sources of evidence. We performed this umbrella review of evidence from meta-analyses of observational studies. Methods: PubMed, Embase, Web of Science databases, and manual screening of references up to July 2020 were searched. Systematic reviews with meta-analyses of observational studies in humans investigating the association between dietary carbohydrate intake and multiple health outcomes were identified. We assessed the evidence levels by using summary effect sizes, 95% prediction intervals, between-study heterogeneity, evidence of small-study effects, and evidence of excess significance bias for each meta-analysis. Results: We included 43 meta-analyses of observational research studies with 23 health outcomes, including cancer (n = 26), mortality (n = 4), metabolic diseases (n = 4), digestive system outcomes (n = 3), and other outcomes [coronary heart disease (n = 2), stroke (n = 1), Parkinson's disease (n = 1), and bone fracture (n = 2)]. This umbrella review summarized 281 individual studies with 13,164,365 participants. Highly suggestive evidence of an association between dietary carbohydrate intake and metabolic syndrome was observed with adjusted summary odds ratio of 1.25 [95% confidence interval (CI) 1.15-1.37]. The suggestive evidences were observed in associations of carbohydrate consumption with esophageal adenocarcinoma (0.57, 95% CI = 0.42-0.78) and all-cause mortality (adjusted summary hazard ratio 1.19, 95% CI = 1.09-1.30). Conclusions: Despite the fact that numerous systematic reviews and meta-analyses have explored the relationship between carbohydrate intake and diverse health outcomes, there is no convincing evidence of a clear role of carbohydrate intake. However, there is highly suggestive evidence suggested carbohydrate intake is associated with high risk of metabolic syndrome, suggestive evidence found its association with increased risk of all-cause mortality and decreased risk of esophageal adenocarcinoma. Systematic Review Registration: CRD42020197424.

Adherence to Cancer Prevention Guidelines and Endometrial Cancer Risk: Evidence from a Systematic Review and Dose-Response Meta-analysis of Prospective Studies.

PURPOSE: The evidence of adherence to cancer prevention guidelines and endometrial cancer (EC) risk has been limited and controversial. This study summarizes and quantifies the relationship between adherence to cancer prevention guidelines and EC risk. MATERIALS AND METHODS: The online databases PubMed, Web of Science, and EMBASE were searched for relevant publications up to June 2, 2020. This study had been registered at PROSPERO. The registration number is CRD42020149966. Study quality evaluation was performed based on the Newcastle-Ottawa Scale. The I2 statistic was used to estimate heterogeneity among studies. Egger's and Begg's tests assessed potential publication bias. Summary hazard ratios (HRs) and 95% confidence intervals (CIs) for the relationship between adherence to cancer prevention guidelines score was assigned to participants by summarizing individual scores for each lifestyle-related factor. The scores ranged from least healthy (0) to most healthy (20) and the EC risk was calculated using a randomeffects model. RESULTS: Five prospective studies (four cohort studies and one case­cohort study) consisted of 4,470 EC cases, where 597,047 participants were included. Four studies had a low bias risk and one study had a high bias risk. Summary EC HR for the highest vs. lowest score of adherence to cancer prevention guidelines was 0.54 (95% CI, 0.40 to 0.73) and had a high heterogeneity (I2=86.1%). For the dose-response analysis, an increment of 1 significantly reduced the risk of EC by 6%. No significant publication bias was detected. CONCLUSION: This study suggested that adherence to cancer prevention guidelines was negatively related to EC risk.

Three-Dimensional Liver Image Segmentation Using Generative Adversarial Networks Based on Feature Restoration.

Medical imaging provides a powerful tool for medical diagnosis. In the process of computer-aided diagnosis and treatment of liver cancer based on medical imaging, accurate segmentation of liver region from abdominal CT images is an important step. However, due to defects of liver tissue and limitations of CT imaging procession, the gray level of liver region in CT image is heterogeneous, and the boundary between the liver and those of adjacent tissues and organs is blurred, which makes the liver segmentation an extremely difficult task. In this study, aiming at solving the problem of low segmentation accuracy of the original 3D U-Net network, an improved network based on the three-dimensional (3D) U-Net, is proposed. Moreover, in order to solve the problem of insufficient training data caused by the difficulty of acquiring labeled 3D data, an improved 3D U-Net network is embedded into the framework of generative adversarial networks (GAN), which establishes a semi-supervised 3D liver segmentation optimization algorithm. Finally, considering the problem of poor quality of 3D abdominal fake images generated by utilizing random noise as input, deep convolutional neural networks (DCNN) based on feature restoration method is designed to generate more realistic fake images. By testing the proposed algorithm on the LiTS-2017 and KiTS19 dataset, experimental results show that the proposed semi-supervised 3D liver segmentation method can greatly improve the segmentation performance of liver, with a Dice score of 0.9424 outperforming other methods.

Multiplex Immuno-Liquid Chromatography-Mass Spectrometry-Parallel Reaction Monitoring (LC-MS-PRM) Quantitation of CD8A, CD4, LAG3, PD1, PD-L1, and PD-L2 in Frozen Human Tissues.

The immune status of tumors critically influences their responsiveness to PD1 blockades and other immune-based therapies. Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) is a clinically validated predictive biomarker of response to checkpoint-inhibitor therapy in a limited number of clinical settings but is poorly predictive in most. With emerging evidence that multiple pathways and immune-checkpoint proteins may coordinately contribute to the adaptive immune resistance, the identification and quantitation of multiple immune markers in tumor tissue could help identify the controlling pathways in a given patient, guide the selection of optimal therapy, and monitor response to treatment. We developed and validated a sensitive and robust immuno-liquid chromatography-parallel reaction monitoring assay to simultaneously quantify the expression levels of six immune markers (CD8A, CD4, LAG3, PD1, PD-L1, and PD-L2) using as little as 1-2 mg of fresh frozen tissue. The lower limit of quantitation ranged from 0.07 ng/mg protein for PD1 to 1.0 ng/mg protein for CD4. The intrabatch accuracy was within -16.6% to 15.0% for all proteins at all concentrations, and the variation ranged from 0.8% to 14.7%, while interbatch accuracy was within -6.3% to 8.6%, and the variation ranged from 1.3% to 12.8%. The validated assay was then applied to quantify all six biomarkers in different tissues and was confirmed to have sufficient sensitivity (0.07-1.00 ng/mg protein) and reproducibility (variation ranged from 4.3 to 12.0%). In an analysis of 26 cervical tumors, CD8A and CD4 were detected in all tumors, followed by PD-L1 in 85%, LAG-3 in 65%, PD1 in 50%, and PD-L2 in 35%. The strongest correlations were observed between CD8A and CD4 ( r = 0.88) and CD8A and LAG-3 ( r = 0.86). PD1 was not significantly correlated with any of the other proteins tested. This method can be applied to survey the immune signatures across tumor types and tailored to incorporate additional markers as needed.

Analysis of glycoproteins for biomarker discovery.

Glycoproteins play an important role in cell signaling and cell-cell interaction. The alterations of glycoproteins are often relevant to progression of diseases, and these changed glycoproteins can be important biomarkers. The lectin-based glycoproteomic technology has extensively been used for high-throughput screening of potential glycoprotein biomarkers. Here we describe a multi-lectin affinity chromatography and label-free quantitative glycoproteomic approach for discovery of glycoprotein biomarkers relevant to differentiation of glioblastoma stem cells.

Immunohistochemical staining, laser capture microdissection, and filter-aided sample preparation-assisted proteomic analysis of target cell populations within tissue samples.

An important problem involves isolating subpopulations of cells defined by protein markers in clinical tissue samples for proteomic studies. We describe a method termed Immunohistochemical staining, laser capture microdissection (LCM) and filter-aided sample preparation (FASP)-Assisted Proteomic analysis of Target cell populations within tissue samples (ILFAPT). The principle of ILFAPT is that a target cell population expressing a protein of interest can be lit up by immunohistochemical staining and isolated from tissue sections using LCM for FASP and proteomic analysis. Using this method, we isolated a small population of CD90(+) stem-like cells from glioblastoma multiforme tissue sections and identified 674 high-confidence (false discovery rate < 0.01) proteins from 32 nL of CD90(+) cells by LC-MS/MS using an Orbitrap Elite mass spectrometer. We further quantified the relative abundance of proteins identified from equal volumes of LCM-captured CD90(+) and CD90(-) cells, where 109 differentially expressed proteins were identified. The major group of these differentially expressed proteins was relevant to cell adhesion and cellular movement. This ILFAPT method has demonstrated the ability to provide in-depth proteome analysis of a very small specific cell population within tissues. It can be broadly applied to the study of target cell populations within clinical specimens.

Characterization of membrane-associated glycoproteins using lectin affinity chromatography and mass spectrometry.

Membrane-associated glycoproteins play critical roles in many biological processes and are often the therapeutic targets for drug discovery. Lectin affinity chromatography is one of the most widely used approaches for enrichment of glycoproteins at the protein level. Here, we describe a strategy for the characterization of membrane glycoproteins including membrane protein extraction, lectin affinity chromatography, protein digestion, and analysis by LC-MS/MS.

Identification and confirmation of differentially expressed fucosylated glycoproteins in the serum of ovarian cancer patients using a lectin array and LC-MS/MS.

In order to discover potential glycoprotein biomarkers in ovarian cancer, we applied a lectin array and Exactag labeling based quantitative glycoproteomics approach. A lectin array strategy was used to detect overall lectin-specific glycosylation changes in serum proteins from patients with ovarian cancer and those with benign conditions. Lectins, which showed significant differential response for fucosylation, were used to extract glycoproteins that had been labeled using isobaric chemical tags. The glycoproteins were then identified and quantified by LC-MS/MS, and five glycoproteins were found to be differentially expressed in the serum of ovarian cancer patients compared to benign diseases. The differentially expressed glycoproteins were further confirmed by lectin-ELISA and ELISA assay. Corticosteroid-binding globulin (CBG), serum amyloid p component (SAP), complement factor B (CFAB), and histidine-rich glycoprotein (HRG) were identified as potential markers for differentiating ovarian cancer from benign diseases or healthy controls. A combination of CBG and HRG (AUC = 0.825) showed comparable performance to CA125 (AUC = 0.829) in differentiating early stage ovarian cancer from healthy controls. The combination of CBG, SAP, and CA125 showed improved performance for distinguishing stage III ovarian cancer from benign diseases compared to CA125 alone. The ability of CBG, SAP, HRG, and CFAB to differentiate the serum of ovarian cancer patients from that of controls was tested using an independent set of samples. Our findings suggest that glycoprotein modifications may be a means to identify novel diagnostic markers for detection of ovarian cancer.

Identification of glycoprotein markers for pancreatic cancer CD24+CD44+ stem-like cells using nano-LC-MS/MS and tissue microarray.

Pancreatic adenocarcinoma is characterized by late diagnosis due to lack of early symptoms, extensive metastasis, and high resistance to chemo/radiation therapy. Recently, a subpopulation of cells within pancreatic cancers, termed cancer stem cells (CSCs), has been characterized and postulated to be the drivers for pancreatic cancer and responsible for metastatic spread. Further studies on pancreatic CSCs are therefore of particular importance to identify novel diagnosis markers and therapeutic targets for this dismal disease. Herein, the malignant phenotype of pancreatic cancer stem-like CD24+CD44+ cells was isolated from a human pancreatic carcinoma cell line (PANC-1) and demonstrated 4-fold increased invasion ability compared to CD24-CD44+ cells. Using lectin microarray and nano LC-MS/MS, we identified a differentially expressed set of glycoproteins between these two subpopulations. Lectin microarray analysis revealed that fucose- and galactose-specific lectins, UEA-1 and DBA, respectively, exhibit distinctly strong binding to CD24+CD44+ cells. The glycoproteins extracted by multilectin affinity chromatography were consequently analyzed by LC-MS/MS. Seventeen differentially expressed glycoproteins were identified, including up-regulated Cytokeratin 8/CK8, Integrin ß1/CD29, ICAM1/CD54, and Ribophorin 2/RPN2 and down-regulated Aminopeptidase N/CD13. Immunohistochemical analysis of tissue microarrays showed that CD24 was significantly associated with late-stage pancreatic adenocarcinomas, and RPN2 was exclusively coexpressed with CD24 in a small population of CD24-positive cells. However, CD13 expression was dramatically decreased along with tumor progression, preferentially present on the apical membrane of ductal cells and vessels in early stage tumors. Our findings suggest that these glycoproteins may provide potential therapeutic targets and promising prognostic markers for pancreatic cancer.

B lymphocytes as effector cells in the immunotherapy of cancer.

Over the years, the role of B cells in the host immune response to malignancy has been overshadowed by our focus on T cells. Nevertheless, B cells play important roles as antigen-presenting cells and in the production of antibodies. Furthermore, B cells can function as effector cells that mediate tumor destruction on their own. This review will highlight the various functions of B cells that are involved in the host response to tumor.