Membrane bound vesicles, including microvesicles and exosomes, are secreted by both normal and cancerous cells into the extracellular space and in blood circulation. These circulating extracellular ...vesicles (cirEVs) and exosomes in particular are recognized as a potential source of disease biomarkers. However, to exploit the use of circulatory exosomes as a biomarker, a rapid, high-throughput and reproducible method is required for their isolation and molecular analysis. We have developed a simple, low cost microfluidic-based platform to isolate cirEVs enriched in exosomes directly from blood serum allowing simultaneous capture and quantification of exosomes in a single device. To capture specific exosomes, we employed "ExoChip", a microfluidic device fabricated in polydimethylsiloxane (PDMS) and functionalized with antibodies against CD63, an antigen commonly overexpressed in exosomes. Subsequent staining with a fluorescent carbocyanine dye (DiO) that specifically labels the exosomes, we quantitated exosomes using a standard plate-reader. Ten independent ExoChip experiments performed using serum obtained from five pancreatic cancer patients and five healthy individuals revealed a statistically significant increase (2.34 ± 0.31 fold, p < 0.001) in exosomes captured in cancer patients when compared to healthy individuals. Exosomal origins of ExoChip immobilized vesicles were further confirmed using immuno-electron-microscopy and Western blotting. In addition, we demonstrate the ability of ExoChip to recover exosomes with intact RNA enabling profiling of exosomal-microRNAs through openarray analysis, which has potential applications in biomarker discovery. Based on our findings, ExoChip is a well suited platform to be used as an exosome-based diagnostic and research tool for molecular screening of human cancers.
The textile industry has emerged as a major pollution source owing to a rise in carbon footprint, the spike in greenhouse gas emission, and increasing landfill waste. Sustainable fashion has become a ...new style statement and industries are shifting their orientation towards environment-friendly manufacturing. The theory of planned behavior (TPB) model was employed with environmental concern, personal moral norms, and perceived consumer effectiveness to better predict the eco-friendly apparel purchase intention of educated Indian youths. Variance-based partial least square-structural equation modeling (PLS-SEM) was applied to evaluate the hypothesized model. Findings indicated that perceived behavioral control has a strong significant positive influence on purchase intention followed by personal moral norms, attitude, and perceived consumer effectiveness. Environmental concern was found to have an indirect effect on purchase intention through three primary TPB variables and personal moral norms. Multi-group analysis (MGA) was performed to examine the moderating effect of perceived consumer effectiveness on an attitude-intention relationship. The highly perceived consumer effectiveness group was shown to have a more consistent attitude-purchase intention relationship as compared to the low-perceived consumer effectiveness group. The study promulgates insights to professionals and policymakers to formulate sustainable marketing strategies and policies to cope with the indigenous market conditions.
Pancreatic cancer (PC) is lethal malignancy with very high mortality rate. Absence of sensitive and specific marker(s) is one of the major factors for poor prognosis of PC patients. In pilot studies ...using small set of patients, secreted acute phase proteins neutrophil gelatinase associated lipocalin (NGAL) and TGF-β family member macrophage inhibitory cytokine-1 (MIC-1) are proposed as most potential biomarkers specifically elevated in the blood of PC patients. However, their performance as diagnostic markers for PC, particularly in pre-treatment patients, remains unknown. In order to evaluate the diagnostic efficacy of NGAL and MIC-1, their levels were measured in plasma samples from patients with pre-treatment PC patients (n = 91) and compared it with those in healthy control (HC) individuals (n = 24) and patients with chronic pancreatitis (CP, n = 23). The diagnostic performance of these two proteins was further compared with that of CA19-9, a tumor marker commonly used to follow PC progression. The levels of all three biomarkers were significantly higher in PC compared to HCs. The mean (± standard deviation, SD) plasma NGAL, CA19-9 and MIC-1 levels in PC patients was 111.1 ng/mL (2.2), 219.2 U/mL (7.8) and 4.5 ng/mL (4.1), respectively. In comparing resectable PC to healthy patients, all three biomarkers were found to have comparable sensitivities (between 64%-81%) but CA19-9 and NGAL had a higher specificity (92% and 88%, respectively). For distinguishing resectable PC from CP patients, CA19-9 and MIC-1 were most specific (74% and 78% respectively). CA19-9 at an optimal cut-off of 54.1 U/ml is highly specific in differentiating resectable (stage 1/2) pancreatic cancer patients from controls in comparison to its clinical cut-off (37.1 U/ml). Notably, the addition of MIC-1 to CA19-9 significantly improved the ability to distinguish resectable PC cases from CP (p = 0.029). Overall, MIC-1 in combination with CA19-9 improved the diagnostic accuracy of differentiating PC from CP and HCs.
Pancreatic cancer (PC) is a lethal malignancy that lacks specific diagnostic markers. The present study explores the diagnostic potential of the most differentially overexpressed secretory mucin ...MUC5AC alone and in combination with CA19-9 using multi-center training and validation sets.
The expression of MUC5AC in benign pancreatic pathologies, PC precursor lesions, primary PC tissues and metastatic lesions was evaluated by immunohistochemistry. Circulating MUC5AC levels were measured using sandwich ELISA assay developed in-house, and CA19-9 was measured using radioimmunoassay. A combined training set (n=346) was used to evaluate the diagnostic (n=241) and predictive (n=105, total samples 201 from pre- and post-surgical and chemotherapy set) significance of MUC5AC. Results were further validated with a pre-defined cut-off value using independent sets from the Mayo Clinic (n=94) and the University of Pittsburgh Medical Center (n=321).
Tissue expression analyses indicated the de novo expression of MUC5AC in pancreatic intraepithelial precursor lesions 1A (PanIN1A); the expression was maintained through all stages of progression to invasive adenocarcinoma. The median circulating MUC5AC levels in patients with resectable early-stage PC (EPC) (stage 1/2; 67.2 ng/ml, IQR: 23.9-382.1) and unresectable late-stage PC (LPC) (stage 3/4; 389.7 ng/ml, IQR: 87.7-948.6) were significantly higher compared with (P-value ≤0.0001) benign controls (BC) (7.2 ng/ml, IQR: 0.4-26.5) and (P-value ≤0.0001) chronic pancreatitis (CP) controls (8.4 ng/ml, IQR: 1.5-19.2). In the diagnostic training set (n=241), MUC5AC efficiently differentiated EPC from healthy controls (HC) (83%/80% sensitive (SN)/specific (SP)), BC (67%/87% SN/SP), and CP (83%/77% SN/SP). Independent validation sets from the Mayo Clinic and UPMC confirmed the diagnostic potential of MUC5AC to differentiate EPC from BC (68%/73%; 65%/83%) and CP (68%/79%; 65%/72%). Furthermore, MUC5AC and CA19-9 combination significantly improved (p-value < 0.001) the diagnostic accuracy for differentiating resectable cases from controls.
MUC5AC is a valuable diagnostic biomarker, either alone or in combination with CA19-9, to differentiate PC from CP and benign controls.
In brain tissues from multiple sclerosis (MS) patients, clusters of activated HLA-DR-expressing microglia, also referred to as preactive lesions, are located throughout the normal-appearing white ...matter. The aim of this study was to gain more insight into the frequency, distribution and cellular architecture of preactive lesions using a large cohort of well-characterized MS brain samples.
Here, we document the frequency of preactive lesions and their association with distinct white matter lesions in a cohort of 21 MS patients. Immunohistochemistry was used to gain further insight into the cellular and molecular composition of preactive lesions.
Preactive lesions were observed in a majority of MS patients (67%) irrespective of disease duration, gender or subtype of disease. Microglial clusters were predominantly observed in the vicinity of active demyelinating lesions and are not associated with T cell infiltrates, axonal alterations, activated astrocytes or blood-brain barrier disruption. Microglia in preactive lesions consistently express interleukin-10 and TNF-α, but not interleukin-4, whereas matrix metalloproteases-2 and -9 are virtually absent in microglial nodules. Interestingly, key subunits of the free-radical-generating enzyme NADPH oxidase-2 were abundantly expressed in microglial clusters.
The high frequency of preactive lesions suggests that it is unlikely that most of them will progress into full-blown demyelinating lesions. Preactive lesions are not associated with blood-brain barrier disruption, suggesting that an intrinsic trigger of innate immune activation, rather than extrinsic factors crossing a damaged blood-brain barrier, induces the formation of clusters of activated microglia.
Pancreatic ductal adenocarcinoma (PDAC) is an incredibly deadly disease with a 5-year survival rate of 9%. The presence of pancreatic cystic lesions (PCLs) confers an increased likelihood of future ...pancreatic cancer in patients placing them in a high-risk category. Discerning concurrent malignancy and risk of future PCL progression to cancer must be carefully and accurately determined to improve survival outcomes and avoid unnecessary morbidity of pancreatic resection. Unfortunately, current image-based guidelines are inadequate to distinguish benign from malignant lesions. There continues to be a need for accurate molecular and imaging biomarker(s) capable of identifying malignant PCLs and predicting the malignant potential of PCLs to enable risk stratification and effective intervention management. This review provides an update on the current status of biomarkers from pancreatic cystic fluid, pancreatic juice, and seromic molecular analyses and discusses the potential of radiomics for differentiating PCLs harboring cancer from those that do not.
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ABSTRACT
Purpose
Recurrence of colon cancer, which affects nearly 50% of patients treated by conventional therapeutics, is thought to be due to re-emergence of chemotherapy-resistant cancer ...stem/stem-like cells (CSCs). Therefore, development of therapeutic strategies for targeted elimination of CSCs would be a novel strategy. The current study examines whether diflourinated-curcumin (CDF), a novel analog of the dietary ingredient of curcumin, in combination with 5-fluorouracil and oxaliplatin (5-FU + Ox), the mainstay of colon cancer chemotherapeutic, would be effective in eliminating colon CSCs.
Methods
Multiple methodologies that include real-time RT-PCR, Western blot, MTT assay, caspase-3 activity, colonosphere formation, Hoechst-33342 dye exclusion and NF-κB-ELISA were used.
Results
We observed that CDF together with 5-FU + Ox were more potent than curcumin in reducing CD44 and CD166 in chemo-resistant colon cancer cells, accompanied by inhibition of growth, induction of apoptosis and disintegration of colonospheres. These changes were associated with down-regulation of the membrane transporter ABCG2 and attenuation of EGFR, IGF-1R, and NF-κB signaling consistent with inactivation of β-catenin, COX-2, c-Myc and Bcl-xL and activation of the pro-apoptotic Bax.
Conclusions
Our results suggest that CDF together with the conventional chemotherapeutics could be an effective treatment strategy for preventing the emergence of chemo-resistant colon cancer cells by eliminating CSCs.
Background
Evaluation of pulmonary arterial pressure is crucial among cirrhotic patients, considering that moderate portopulmonary hypertension (POPH) is a contraindication for liver transplantation. ...Although right heart catheterization (RHC) is the most accurate method to diagnose POPH, it is invasive.
Objective
The aim of the study is to evaluate the performance of echocardiography in detecting POPH in liver transplant candidates.
Methods
A Literature search was performed, and pooled sensitivity, specificity, positive likelihood ratio (LR), negative LR, and area under the summary receiver operating curve (AUC) were calculated. Subgroup analyses were performed based on different cutoff values for echocardiography and diagnostic criteria of RHC.
Results
Sensitivity, specificity, positive LR, negative LR, and AUC of echocardiography for detection of POPH were 0.86 (0.74, 0.94), 0.87 (0.84, 0.90), 7.17 (3.59, 14.31), 0.22 (0.13, 0.38), and 0.807 while they were 0.82 (0.74, 0.89), 0.81 (0.78, 0.84), 117.75 (16.03, 865.08), 0.28 (0.16, 0.50), and 0.876for detection of moderate POPH, respectively. Performance of echocardiography was not significantly different in the subgroup analyses of stringency of POPH criteria and pulmonary arterial systolic pressure (ePASP) cutoffs.
Conclusions
Our meta‐analysis supports utilization of echocardiography for screening of POPH. However, RHC remains essential in highly suspicious cases. Echocardiographic data other than ePASP should be evaluated in future studies.
The objective of this study was to evaluate the cytotoxicity of (+)-cyanidan-3-ol (CD-3) in human hepatocellular carcinoma cell line (HepG2) and chemopreventive potential against hepatocellular ...carcinoma (HCC) in Balb/c mice. The HepG2 cell line was treated with CD-3 at various concentrations and the proliferation of the HepG2 cells was measure by 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO), Acridine orange/ethylene dibromide (AO/EB) staining, DNA fragmentation analysis and the apoptosis rate was detected by flow cytometry. The HCC tumor model was established in mice by injecting N-nitrosodiethylamine/carbon tetrachloride (NDEA/CCl4) and the effect of CD-3 on tumor growth in-vivo was studied. The levels of liver injury markers, tumor markers, and oxidative stress were measured. The expression levels of apoptosis-related genes in in-vitro and in vivo models were determined by RT-PCR and ELISA. The CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes under fluorescent microscopy and DNA fragmentation analysis. Annexin V/PI assay demonstrated that apoptosis increased with increase in the concentration of CD-3. The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-κB activities were significantly suppressed by CD-3. Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors. Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.
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