In previous studies we described a decreased alpha-l-fucosidase activity in colorectal tumors, appearing as a prognostic factor of tumoral recurrence. The aim of this work was to extend the knowledge about tissue alpha-l-fucosidase in colorectal cancer by quantifying the expression of its encoding gene FUCA1 in tumors and healthy mucosa. FUCA1 mRNA levels were measured by RT-qPCR in paired tumor and normal mucosa tissues from 31 patients. For the accuracy of the RT-qPCR results, five candidate reference genes were validated in those samples. In addition, activity and expression of alpha-l-fucosidase in selected matched tumor and healthy mucosa samples were analyzed. According to geNorm and NormFinder algorithms, RPLP0 and HPRT1 were the best reference genes in colorectal tissues. These genes were used for normalization of FUCA1 expression levels. A significant decrease of more than 60% in normalized FUCA1 expression was detected in tumors compared to normal mucosa (p = 0.002). Moreover, a gradual decrease in FUCA1 expression was observed with progression of disease from earlier to advanced stages. These findings were confirmed by Western blot analysis of alpha-l-fucosidase expression. Our results demonstrated diminished FUCA1 mRNA levels in tumors, suggesting that expression of tissue alpha-l-fucosidase could be regulated at transcriptional level in colorectal cancer.

No abstract

Background:Novel non-invasive biomarkers for the precise diagnosis of malignancy in pleural effusion (PE) are needed. The aim of this study was to determine the diagnostic accuracy of calprotectin for predicting malignancy in patients with exudative PE.Methods:Calprotectin concentration was measured in 156 individuals diagnosed with exudative PE (67 malignant and 89 benign). Calprotectin accuracy for discriminating between malignant and benign PE was evaluated using receiver operating characteristic (ROC) curves. Univariate and multivariate logistic regression were performed to test the association between calprotectin levels and malignant PE.Results:Calprotectin levels were significantly lower in malignant pleural fluid (257.2 ng ml−1, range: 90.7–736.4) than benign effusions (2627.1 ng ml−1, range: 21–9530.1). The area under the curve was 0.963. A cutoff point of ⩽736.4 ng ml−1 rendered a sensitivity of 100%, with a specificity of 83.15%, which could prove useful to delimit those patients with negative cytology tests that should be referred for more invasive diagnostic procedures. Logistic regression demonstrated a strong association between calprotectin and malignancy (adjusted OR 663.14).Conclusion:Calprotectin predicts malignancy in pleural fluid with high accuracy and could be a good complement to cytological methods.

Taking advantage of eight established cell lines from colorectal cancer patients at different stages of the disease and the fact that all of them could form spheres, cell surface biomarkers of cancer stem cells and epithelial-mesenchymal transition were tested. The aim was to investigate cancer stem cells and metastatic stem cells in order to provide functional characterization of circulating tumor cells and promote the development of new anti-metastatic therapies. Our model showed an important heterogeneity in EpCAM, CD133, CD44, LGR5, CD26 and E-cadherin expression. We showed the presence of a subset of E-cadherin + (some cells being E-cadherin high ) expressing CD26 + (or CD26 high ) together with the well-known CSC markers LGR5 and EpCAM high , sometimes in the absence of CD44 or CD133. The already described CD26 + /E-cadherin low or negative and CD26 + /EpCAM − /CD133 − subsets were also present. Cell division drastically affected the expression of all markers, in particular E-cadherin, so new-born cells resembled mesenchymal cells in surface staining. CD26 and/or dipeptidyl peptidase 4 inhibitors have already shown anti-metastatic effects in pre-clinical models, and the existence of these CD26 + subsets may help further research against cancer metastasis.

BackgroundThe need for novel biomarkers that could aid in non-small cell lung cancer (NSCLC) detection, together with the relevance of Matrix Metalloproteases (MMPs) -1, -2, -7, -9 and -10 in lung tumorigenesis, prompted us to assess the diagnostic usefulness of these MMPs and the Tissue Inhibitor of Metalloproteinase (TIMP) -1 in NSCLC patients.MethodsMarkers were evaluated in an initial study cohort (19 NSCLC cases and 19 healthy controls). Those that better performed were analyzed in a larger sample including patients with benign lung diseases. Serum MMPs and TIMP-1 were determined by multiplexed immunoassays. Logistic regression was employed for multivariate analysis of biomarker combinations.ResultsMMPs and TIMP-1 were elevated in the serum of NSCLC patients compared to healthy controls. MMP-1, -7 and -9 performed at best and were further evaluated in the sample including benign pathologies, corroborating the superiority of MMP-9 in NSCLC discrimination, also at early-stage NSCLC. The optimal diagnostic value was obtained with the model including MMP-9, gender, age and smoking history, that demonstrated an AUC of 0.787, 85.54% sensitivity and 64.89% specificity.ConclusionOur results suggest that MMP-9 is a potential biomarker for NSCLC diagnosis and its combined measurement with other biomarkers could improve NSCLC detection.Electronic supplementary materialThe online version of this article (10.1186/s12885-017-3842-z) contains supplementary material, which is available to authorized users.

Avian reovirus sigmaA is a double-stranded RNA (dsRNA)-binding protein that has been shown to stabilize viral core particles and to protect the virus against the antiviral action of interferon. To continue with the characterization of this viral protein, we have investigated its intracellular distribution in avian cells. Most sigmaA accumulates into cytoplasmic viral factories of infected cells, and yet a significant fraction was detected in the nucleolus. The protein also localizes in the nucleolus of transfected cells, suggesting that nucleolar targeting is not facilitated by the viral infection or by viral factors. Assays performed in both intact cells and digitonin-permeabilized cells demonstrate that sigmaA is able to enter the nucleus via a nucleoporin-dependent nondiffusional mechanism that does not require added cytosolic factors or energy input. These results indicate that sigmaA by itself is able to penetrate into the nucleus using a process that is mechanistically different from the classical nuclear localization signal/importin pathway. On the other hand, two sigmaA arginines that are necessary for dsRNA binding are also required for nucleolar localization, suggesting that dsRNA-binding and nucleolar targeting are intimately linked properties of the viral protein.Avian reoviruses are members of the Orthoreovirus genus, one of the 12 genera of the Reoviridae family. These agents, which are ubiquitous in commercial poultry, induce several disease conditions that lead to important economic losses in the poultry industry. Avian reoviruses are nonenveloped viruses that replicate in the cytoplasm of infected cells and that induce fusion of the host cells. They contain a genome of 10 linear double stranded-RNA (dsRNA) segments encased within two concentric protein shells. Avian reoviruses express at least 10 different structural proteins (lambdaA, -B, and -C; muA, -B, -BC, and -BN; and sigmaA, -B, and -C) and four nonstructural proteins (muNS, sigmaNS, p10, and p17) (for a recent review on avian reovirus, see reference 6 and references therein).Avian reovirus replication starts with the extracellular attachment of viral particles to the host cell, which is mediated by specific interactions between the outer-capsid protein sigmaC with still-unknown cell surface receptors (43). The virus penetrates by receptor-mediated endocytosis, and the acidification of virus-containing endosomes promotes virus uncoating (14, 37). Uncoated viral cores are then able to cross the endosomal membrane and reach the cytoplasm, where a core-associated RNA polymerase catalyzes the synthesis of all 10 viral mRNAs, which display a dual function: to program viral protein synthesis at the ribosomes and to serve as templates for the production of dsRNA minus strands. Minus-strand synthesis and virus morphogenesis occurs within globular cytoplasmic inclusions, termed viral factories, which are initially formed by the nonstructural protein muNS (60, 61). Core assembly occurs within the first 30 min after the synthesis of its protein comp...

Discriminating between malignant pleural effusion (MPE) and benign pleural effusion (BPE) remains difficult. Thus, novel and efficient biomarkers are required for the diagnosis of pleural effusion (PE). The aim of this study was to validate calprotectin as a diagnostic biomarker of PE in clinical settings. A total of 425 patients were recruited, and the pleural fluid samples collected had BPE in 223 cases (53.7%) or MPE in 137 patients (33%). The samples were all analysed following the same previously validated clinical laboratory protocols and methodology. Calprotectin levels ranged from 772.48 to 3,163.8 ng/mL (median: 1,939 ng/mL) in MPE, and 3,216-24,000 ng/mL in BPE (median: 9,209 ng/mL; p < 0.01), with an area under the curve of 0.848 [95% CI: 0.810-0.886]. For a cutoff value of ≤ 6,233.2 ng/mL, we found 96% sensitivity and 60% specificity, with a negative and positive predictive value, and negative and positive likelihood ratios of 96%, 57%, 0.06, and 2.4, respectively. Multivariate analysis showed that low calprotectin levels was a better discriminator of PE than any other variable [OR 28.76 (p < 0.0001)]. Our results confirm that calprotectin is a new and useful diagnostic biomarker in patients with PE of uncertain aetiology which has potential applications in clinical practice because it may be a good complement to cytological methods. The diagnosis of pleural effusion (PE) is a clinical challenge because it can be produced by over 60 diseases 1,2. Nevertheless, in clinical practice the priority is to establish whether the PE is malignant or not. A diagnosis of malignant PE (MPE) implies the presence of advanced-stage tumours and is therefore associated with a poor prognosis 1,3 which requires urgent diagnosis. Thoracocentesis is the first and most simple procedure for the diagnosis of PE 2-4. Unfortunately, while its specificity for establishing malignancy is 100%, the diagnostic sensitivity of pleural fluid (PF) cytological analysis is low. Although the odds of establishing an MPE diagnosis by immunohistochemistry are improved by applying a panel of different antibodies, its diagnostic sensitivity is still only approximately 60% for metastatic PE and less than 30% for mesothelioma 5,6. When the cytology results are negative, more invasive methods such as a pleural biopsy or thoracoscopy are necessary 2,4,7. In this context, more groups are searching for PF biomarkers for malignancy with the aim of avoiding these invasive procedures 8-10. Recent meta-analyses have evaluated the ability of new biomarkers such

Zeolitic imidazolate framework-8 (ZIF-8) is a metal organic framework with exceptional intrinsic properties, high tunability, cost effectiveness, and producibility, which has boosted the research development of the field. ZIF-8-based materials have shown high capabilities for multiple purposes as catalysts, capacitors, electrodes, drug delivery systems, or adsorption/separation membranes. Herein, we report the synergistic combination of ZIF-8, plasmonic nanoparticles, and rationally designed protein adaptors and antibodies for fabricating novel surface-enhanced Raman scattering (SERS) tags with enhanced sensing capabilities. The SERS tags consist of Au@Ag core−shell nanorods individually encapsulated within a multifunctional ZIF-8 matrix encoded with Raman reporters. While the role of the plasmonic core is to enhance the Raman, the ZIF-8 traps the Raman active molecules and, more importantly, facilitates the active targeting of the SERS tag surface through the modular assembly with conventional (i.e., immunoglobulins) and recombinant antibodies (i.e., nanobodies) mediated by the specific interaction of Zn 2+ with polyhistidine-tagged protein G and SpyCatcher. Evidence of the capabilities of the Au@Ag@ZIF-8 nanotags for the SERS detection of EGFR and CD44 cell surface receptors in vitro illustrates the potential of these optical nanoprobes for imaging and multiplex biodetection. The reported modular assembly approach for the functionalization of ZIF-8 SERS nanotags with different classes of antibodies based on polyhistidinetagged peptides and protein−protein interactions can not only be applied to the ever-increasing number of reported MOFs structures but also can be further exploited as a universal means for the functionalization of other transition metal surfaces.