High enhancement of sensitivity and reproducibility in label-free SARS-CoV-2 detection with graphene field-effect transistor sensors through precise surface biofunctionalization control.

The COVID-19 pandemic has taught us valuable lessons, especially the urgent need for a widespread, rapid and sensitive diagnostic tool. To this, the integration of bidimensional nanomaterials, particularly graphene, into point-of-care biomedical devices is a groundbreaking strategy able to potentially revolutionize the diagnostic landscape. Despite advancements in the fabrication of these biosensors, the relationship between their surface biofunctionalization and sensing performance remains unclear. Here, we demonstrate that the combination of careful sensor fabrication and its precise surface biofunctionalization is crucial for exalting the sensing performances of 2D biosensors. Specifically, we have biofunctionalized Graphene Field-Effect Transistor (GFET) sensors surface through different biochemical reactions to promote either random/heterogeneous or oriented/homogeneous immobilization of the Anti-SARS-CoV-2 spike protein antibody. Each strategy was thoroughly characterized by in-silico simulations, physicochemical and biochemical techniques and electrical characterization. Subsequently, both biosensors were tested in the label-free direct titration of SARS-CoV-2 virus in simulated clinical samples, avoiding sample preprocessing and within short timeframes. Remarkably, the oriented GFET biosensor exhibited significantly enhanced reproducibility and responsiveness, surpassing the detection sensitivity of conventional non-oriented GFET by more than twofold. This breakthrough not only involves direct implications for COVID-19 surveillance and next pandemic preparedness but also clarify an unexplored mechanistic dimension of biosensor research utilizing 2D-nanomaterials.

Improved antitumor activity through a tyramidyl maslinic acid derivative. Design and validation as drug-loaded electrospun polymeric nanofibers.

Among the most harmful tumors detected in the human body, such as breast, colon, brain or pancreas, breast (BC) and colorectal cancer (CRC) are the first and third most frequent cancer worldwide, respectively. The current existing chemotherapeutic treatments present serious side effects due to their intravenous administration can induce cytotoxicity in healthy cells. Thus, new treatment methods based on drug-loaded polymeric nanofibers (NFs) have gained significant potential for their use in localized cancer chemotherapy. Here, a deep in vitro comparative analysis between maslinic acid (MA) and a tyramine-maslinic acid (TMA) derivative is initially performed. This analysis includes a proliferation, and a cell cycle assay, and a genotoxicity, antiangiogenic and apoptosis study. Then, the TMA derivative has been incorporated into electrospun polymeric NFs obtaining an implantable dressing material with antitumor activity. Two types of patches containing TMA-loaded polymeric NFs of poly(caprolactone) (PCL), and a mixture of polylactic acid/poly(4-vinylpyridine) (PLA/PVP) were fabricated by the electrospinning technique. The characterization of the drug-loaded NFs showed an encapsulation capacity of 0.027 mg TMA/mg PCL and 0.024 mg TMA/mg PLA/PVP. Then, the cytotoxic activity of both polymeric systems was tested in CRC (T84), BC (MCF-7) and a no tumor (L929) cell lines exposed to TMA-loaded NFs and blank NFs for 48 h. Moreover, cell cycle assay, genotoxicity, angiogenesis and apoptosis tests were carried out to study the mechanism of action of TMA. Blank NFs showed no-toxicity in all cell lines tested and both drug-loaded NFs significantly reduced cell proliferation (relative proliferation of ≈44 % and ≈25 % respectively). Therefore, TMA was less genotoxic than maslinic acid (MA), and reduced VEGFA expression in MCF-7 cells (1.32 and 2.12-fold for MA and TMA respectively). These results showed that TMA-loaded NFs could constitute a promising biocompatible and biodegradable nanoplatform for the local treatment of solid tumors such as CRC or BC.

Circulating Tumor DNA as a Cancer Biomarker: An Overview of Biological Features and Factors That may Impact on ctDNA Analysis.

Cancer cells release nucleic acids, freely or associated with other structures such as vesicles into body fluids, including blood. Among these nucleic acids, circulating tumor DNA (ctDNA) has emerged as a minimally invasive biomarker for tumor molecular profiling. However, certain biological characteristics of ctDNA are still unknown. Here, we provide an overview of the current knowledge about ctDNA biological features, including size and structure as well as the mechanisms of ctDNA shedding and clearance, and the physio-pathological factors that determine ctDNA levels. A better understanding of ctDNA biology is essential for the development of new methods that enable the analysis of ctDNA.

ALK-Fusion Transcripts Can Be Detected in Extracellular Vesicles (EVs) from Nonsmall Cell Lung Cancer Cell Lines and Patient Plasma: Toward EV-Based Noninvasive Testing.

BACKGROUND: ALK rearrangements are present in 5% of nonsmall cell lung cancer (NSCLC) tumors and identify patients who can benefit from ALK inhibitors. ALK fusions testing using liquid biopsies, although challenging, can expand the therapeutic options for ALK-positive NSCLC patients considerably. RNA inside extracellular vesicles (EVs) is protected from RNases and other environmental factors, constituting a promising source for noninvasive fusion transcript detection. METHODS: EVs from H3122 and H2228 cell lines, harboring EML4-ALK variant 1 (E13; A20) and variant 3 (E6a/b; A20), respectively, were successfully isolated by sequential centrifugation of cell culture supernatants. EVs were also isolated from plasma samples of 16 ALK-positive NSCLC patients collected before treatment initiation. RESULTS: Purified EVs from cell cultures were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. Western blot and confocal microscopy confirmed the expression of EV-specific markers as well as the expression of EML4-ALK-fusion proteins in EV fractions from H3122 and H2228 cell lines. In addition, RNA from EV fractions derived from cell culture was analyzed by digital PCR (dPCR) and ALK-fusion transcripts were clearly detected. Similarly, plasma-derived EVs were characterized by NTA, flow cytometry, and the ExoView platform, the last showing that EV-specific markers captured EV populations containing ALK-fusion protein. Finally, ALK fusions were identified in 50% (8/16) of plasma EV-enriched fractions by dPCR, confirming the presence of fusion transcripts in EV fractions. CONCLUSIONS: ALK-fusion transcripts can be detected in EV-enriched fractions. These results set the stage for the development of EV-based noninvasive ALK testing.

Clinical Utility of Liquid Biopsy-Based Actionable Mutations Detected via ddPCR.

Cancer is one of the leading causes of death worldwide and remains a major public health challenge. The introduction of more sensitive and powerful technologies has permitted the appearance of new tumor-specific molecular aberrations with a significant cancer management improvement. Therefore, molecular pathology profiling has become fundamental not only to guide tumor diagnosis and prognosis but also to assist with therapeutic decisions in daily practice. Although tumor biopsies continue to be mandatory in cancer diagnosis and classification, several studies have demonstrated that liquid biopsies could be used as a potential tool for the detection of cancer-specific biomarkers. One of the main advantages is that circulating free DNA (cfDNA) provides information about intra-tumoral heterogeneity, reflecting dynamic changes in tumor burden. This minimally invasive tool has become an accurate and reliable instrument for monitoring cancer genetics. However, implementing liquid biopsies across the clinical practice is still ongoing. The main challenge is to detect genomic alterations at low allele fractions. Droplet digital PCR (ddPCR) is a powerful approach that can overcome this issue due to its high sensitivity and specificity. Here we explore the real-world clinical utility of the liquid biopsy ddPCR assays in the most diagnosed cancer subtypes.

A comprehensive database for integrated analysis of omics data in autoimmune diseases.

BACKGROUND: Autoimmune diseases are heterogeneous pathologies with difficult diagnosis and few therapeutic options. In the last decade, several omics studies have provided significant insights into the molecular mechanisms of these diseases. Nevertheless, data from different cohorts and pathologies are stored independently in public repositories and a unified resource is imperative to assist researchers in this field. RESULTS: Here, we present Autoimmune Diseases Explorer ( https://adex.genyo.es ), a database that integrates 82 curated transcriptomics and methylation studies covering 5609 samples for some of the most common autoimmune diseases. The database provides, in an easy-to-use environment, advanced data analysis and statistical methods for exploring omics datasets, including meta-analysis, differential expression or pathway analysis. CONCLUSIONS: This is the first omics database focused on autoimmune diseases. This resource incorporates homogeneously processed data to facilitate integrative analyses among studies.

NGS-based liquid biopsy profiling identifies mechanisms of resistance to ALK inhibitors: a step toward personalized NSCLC treatment.

Despite impressive and durable responses, nonsmall cell lung cancer (NSCLC) patients treated with anaplastic lymphoma kinase (ALK) inhibitors (ALK-Is) ultimately progress due to development of resistance. Here, we have evaluated the clinical utility of circulating tumor DNA (ctDNA) profiling by next-generation sequencing (NGS) upon disease progression. We collected 26 plasma and two cerebrospinal fluid samples from 24 advanced ALK-positive NSCLC patients at disease progression to an ALK-I. These samples were analyzed by NGS and digital PCR. A tool to retrieve variants at the ALK locus was developed (VALK tool). We identified at least one resistance mutation in the ALK locus in ten (38.5%) plasma samples; the G1269A and G1202R mutations were the most prevalent among patients progressing to first- and second-generation ALK-Is, respectively. Overall, 61 somatic mutations were detected in 14 genes: TP53, ALK, PIK3CA, SMAD4, MAP2K1 (MEK1), FGFR2, FGFR3, BRAF, EGFR, IDH2, MYC, MET, CCND3, and CCND1. Specifically, a deletion in exon 19 in EGFR, a non-V600 BRAF mutation (G466V), and the F129L mutation in MAP2K1 were identified in four patients who showed no objective survival benefit from ALK-Is. Potential ALK-I-resistance mutations were also found in PIK3CA and IDH2. Finally, a c-MYC gain, along with a loss of CCND1 and FGFR3, was detected in a patient progressing on a first-line treatment with crizotinib. We conclude that NGS analysis of liquid biopsies upon disease progression identified different putative ALK-I-resistance mutations in most cases and could be a valuable approach for therapy decision making.

Potential Therapeutic Effects of the Neural Stem Cell-Targeting Antibody Nilo1 in Patient-Derived Glioblastoma Stem Cells.

Glioblastoma (GBM) is the most devastating and least treatable brain tumor with median survival <15 months and extremely high recurrence rates. Promising results of immune checkpoint blockade obtained from pre-clinical studies in mice did not translate to clinic, and new strategies are urgently needed, particularly those targeting GBM stem cells (GSCs) that are held responsible for drug resistance and tumor recurrence. Patient-derived GSC cultures are critical for finding effective brain tumor therapies. Here, we investigated the ability of the recently described monoclonal antibody Nilo1 to specifically recognize GSCs isolated from GBM surgical samples. We employed five patient-derived GSC cultures with different stemness marker expression and differentiation potential, able to recapitulate original tumors when xenotransplanted in vivo. To answer whether Nilo1 has any functional effects in patient-derived GSCs lines, we treated the cells with Nilo1 in vitro and analyzed cell proliferation, cell cycle, apoptosis, sphere formation, as well as the expression of stem vs. differentiation markers. All tested GSCs stained positively for Nilo1, and the ability of Nilo1 to recognize GSCs strongly relied on their stem-like phenotype. Our results showed that a subset of patient-derived GSCs were sensitive to Nilo1 treatment. In three GSC lines Nilo1 triggered differentiation accompanied by the induction of p21. Most strikingly, in one GSC line Nilo1 completely abrogated self-renewal and led to Bax-associated apoptosis. Our data suggest that Nilo1 targets a molecule functionally relevant for stemness maintenance and pinpoint Nilo1 as a novel antibody-based therapeutical strategy to be used either alone or in combination with cytotoxic drugs for GSC targeting. Further pre-clinical studies are needed to validate the effectiveness of GSC-specific Nilo1 targeting in vivo.

BRAF V600E Detection in Liquid Biopsies from Pediatric Central Nervous System Tumors.

Pediatric Central Nervous System (CNS) tumors are the most fatal cancer diseases in childhood. Due to their localization and infiltrative nature, some tumor resections or biopsies are not feasible. In those cases, the use of minimally invasive methods as diagnostic, molecular marker detection, prognostic or monitoring therapies are emerging. The analysis of liquid biopsies which contain genetic information from the tumor has been much more widely explored in adults than in children. We compare the detection of BRAF V600E targetable mutation by digital-PCR from cell-free-DNA and EV-derived DNA (ctDNA) in serum, plasma and cerebrospinal fluid (CSF) isolated from a cohort of 29 CNS pediatric patients. Here we demonstrate that ctDNA isolated from serum and plasma could be successfully analyzed to obtain tumor genetic information which could be used to guide critical treatment decisions.

mCSEA: detecting subtle differentially methylated regions.

MOTIVATION: The identification of differentially methylated regions (DMRs) among phenotypes is one of the main goals of epigenetic analysis. Although there are several methods developed to detect DMRs, most of them are focused on detecting relatively large differences in methylation levels and fail to detect moderate, but consistent, methylation changes that might be associated to complex disorders. RESULTS: We present mCSEA, an R package that implements a Gene Set Enrichment Analysis method to identify DMRs from Illumina450K and EPIC array data. It is especially useful for detecting subtle, but consistent, methylation differences in complex phenotypes. mCSEA also implements functions to integrate gene expression data and to detect genes with significant correlations among methylation and gene expression patterns. Using simulated datasets we show that mCSEA outperforms other tools in detecting DMRs. In addition, we applied mCSEA to a previously published dataset of sibling pairs discordant for intrauterine hyperglycemia exposure. We found several differentially methylated promoters in genes related to metabolic disorders like obesity and diabetes, demonstrating the potential of mCSEA to identify DMRs not detected by other methods. AVAILABILITY AND IMPLEMENTATION: mCSEA is freely available from the Bioconductor repository. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Multiple immunofluorescence assay identifies upregulation of Active ß-catenin in prostate cancer.

OBJECTIVES: To apply a systems pathology-based approach to the quantification of nuclear Active ß-catenin and human leukocyte antigen class I, and assess the biomarker involvement in a cohort of prostate tumor patients. RESULTS: The systems pathology approach applied allows a precise quantification of the marker expression in the different cell compartments as well as the determination of the areas that coexpress two markers. Our data shows that the accumulation of ß-catenin in the nuclear compartment is significantly decreased in the adjacent normal areas when compared to tumor of the same patients (p < 0.001). In conclusion, the application of this novel multiple immunofluorescence assay demonstrates that the upregulation of Active ß-catenin is a relatively common feature of prostate tumor development, and further supports the activation of the Wnt/ß-catenin pathway in prostate cancer progression.

ImaGEO: integrative gene expression meta-analysis from GEO database.

SUMMARY: The Gene Expression Omnibus (GEO) database provides an invaluable resource of publicly available gene expression data that can be integrated and analyzed to derive new hypothesis and knowledge. In this context, gene expression meta-analysis (geMAs) is increasingly used in several fields to improve study reproducibility and discovering robust biomarkers. Nevertheless, integrating data is not straightforward without bioinformatics expertise. Here, we present ImaGEO, a web tool for geMAs that implements a complete and comprehensive meta-analysis workflow starting from GEO dataset identifiers. The application integrates GEO datasets, applies different meta-analysis techniques and provides functional analysis results in an easy-to-use environment. ImaGEO is a powerful and useful resource that allows researchers to integrate and perform meta-analysis of GEO datasets to lead robust findings for biomarker discovery studies. AVAILABILITY AND IMPLEMENTATION: ImaGEO is accessible at http://bioinfo.genyo.es/imageo/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Granzyme A Contributes to Inflammatory Arthritis in Mice Through Stimulation of Osteoclastogenesis.

OBJECTIVE: Granzyme A (GzmA) levels are elevated in the plasma and synovium of patients with rheumatoid arthritis (RA), suggesting involvement of this protease in the pathogenesis of the disease. GzmA contributes to sepsis by regulating the production of proinflammatory cytokines. The purpose of this study was to evaluate the contribution of GzmA to the pathogenesis of RA in vivo and to examine the possibility that GzmA acting via tumor necrosis factor (TNF) stimulates osteoclastogenesis. METHODS: Inflammatory arthritis induced by type II collagen was evaluated in wild-type, GzmA-deficient, and perforin-deficient mice. The osteoclastogenic potential of GzmA was examined in vitro using bone marrow cells and colony-forming unit-granulocyte-macrophage (CFU-GM) cells and in vivo using GzmA-deficient mice. RESULTS: Gene deletion of GzmA attenuated collagen-induced arthritis, including serum levels of proinflammatory cytokines, joint damage, and bone erosion in affected mice, suggesting that osteoclast activity is reduced in the absence of GzmA. Accordingly, GzmA-treated bone marrow cells produced multinucleated cells that fulfilled the criteria for mature osteoclasts: tartrate-resistant acid phosphatase (TRAP) activity, ß integrin expression, calcitonin receptor expression, and resorptive activity on dentin slices. GzmA appeared to act without accessory cells, and its activity was not affected by osteoprotegerin, suggesting a minor contribution of RANKL. It also induced the expression and secretion of TNF. Neutralization of TNF or stimulation of CFU-GM cells from TNF-/- mice prevented GzmA-induced osteoclastogenesis. GzmA-deficient mice had reduced osteoclastogenesis in vivo (fewer calcitonin receptor-positive multinucleated cells and fewer transcripts for cathepsin K, matrix metalloproteinase 9, and TRAP in joints) and reduced serum levels of C-terminal telopeptide of type I collagen. CONCLUSION: GzmA contributes to the joint destruction of RA partly by promoting osteoclast differentiation.