Microarray Generation of Thousand-Member Oligonucleotide Libraries

The ability to efficiently and economically generate libraries of defined pieces of DNA would have a myriad of applications, not least in the area of defined or directed sequencing and synthetic biology, but also in applications associated with encoding and tagging. In this manuscript DNA microarrays were used to allow the linear amplification of immobilized DNA sequences from the array followed by PCR amplification. Arrays of increasing sophistication (1, 10, 3,875, 10,000 defined sequences) were used to validate the process, with sequences verified by selective hybridization to a complementary DNA microarray and DNA sequencing, which demonstrated a PCR error rate of 9.7×10−3/site/duplication. This technique offers an economical and efficient way of producing specific DNA libraries of hundreds to thousands of members with the DNA-arrays being used as “factories” allowing specific DNA oligonucleotide pools to be generated. We also found substantial variance observed between the sequence frequencies found via Solexa sequencing and microarray analysis, highlighting the care needed in the interpretation of profiling data

Evaluation of diagnostic and prognostic candidate biomarkers in drug-induced liver injury vs. other forms of acute liver damage

Aims: Detection and characterization of idiosyncratic drug-induced liver injury (DILI) currently rely on standard liver tests, which are suboptimal in terms of specificity, sensitivity and prognosis. Therefore, DILI diagnosis can be delayed, with important consequences for the patient. In this study, we aimed to evaluate the potential of osteopontin, cytokeratin-18 (caspase-cleaved: ccK18 and total: K18), α-glutathione- S-transferase and microRNA-122 as new DILI biomarkers. Methods: Serial blood samples were collected from 32 DILI and 34 non-DILI acute liver injury (ALI) cases and a single sample from 43 population controls without liver injury (HLC) and analysed using enzyme-linked immunosorbent assay (ELISA) or single-molecule arrays. Results: All biomarkers differentiated DILI and ALI from HLC with an area under receiver operator characteristic curve (AUC) value of >0.75 but were less efficient in distinguishing DILI from ALI, with ccK18 (0.79) and K18 (0.76) demonstrating highest potential. However, the AUC improved considerably (0.98) for ccK18 when comparing DILI and a subgroup of autoimmune hepatitis cases. Cytokeratin-18, microRNA- 122 and α-glutathione-S-transferase correlated well with traditional transaminases, while osteopontin correlated most strongly with the international normalized ratio (INR). Conclusions: ccK18 appears promising in distinguishing DILI from autoimmune hepatitis but less so from other forms of acute liver injury. Osteopontin demonstrates prognostic potential with higher levels detected in more severe cases regardless of aetiologyConsejería de Salud y Familia de la Junta de Andalucía, Grant/Award Numbers: PI- 0274-2016, P18-RT-3364Instituto de Salud Carlos III (ISCIII) cofounded by Fondo Europeo de Desarrollo Regional - FEDER, Grant/Award Numbers: PI19/00883, PI18/00901UMA18-FEDERJA-193; Universidad de Málaga/CBU

Spatial Transcriptomics: Emerging Technologies in Tissue Gene Expression Profiling

In this Perspective, we discuss the current status and advances in spatial transcriptomics technologies, which allow high-resolution mapping of gene expression in intact cell and tissue samples. Spatial transcriptomics enables the creation of highresolution maps of gene expression patterns within their native spatial context, adding an extra layer of information to the bulk sequencing data. Spatial transcriptomics has expanded significantly in recent years and is making a notable impact on a range of fields, including tissue architecture, developmental biology, cancer, and neurodegenerative and infectious diseases. The latest advancements in spatial transcriptomics have resulted in the development of highly multiplexed methods, transcriptomic-wide analysis, and single-cell resolution utilizing diverse technological approaches. In this Perspective, we provide a detailed analysis of the molecular foundations behind the main spatial transcriptomics technologies, including methods based on microdissection, in situ sequencing, single-molecule FISH, spatial capturing, selection of regions of interest, and single-cell or nuclei dissociation. We contextualize the detection and capturing efficiency, strengths, limitations, tissue compatibility, and applications of these techniques as well as provide information on data analysis. In addition, this Perspective discusses future directions and potential applications of spatial transcriptomics, highlighting the importance of the continued development to promote widespread adoption of these techniques within the research community.Spanish Ministry of Economy and Competitiveness (Grant Number PID2019.110987RB.I00)European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie actions (MSCA-RISE-101007934, diaRNAgnosis

Characterization and Therapeutic Effect of a pH Stimuli Responsive Polymeric Nanoformulation for Controlled Drug Release

Despite the large number of polymeric nanodelivery systems that have been recently developed, there is still room for improvement in terms of therapeutic efficiency. Most reported nanodevices for controlled release are based on drug encapsulation, which can lead to undesired drug leakage with a consequent reduction in efficacy and an increase in systemic toxicity. Herein, we present a strategy for covalent drug conjugation to the nanodevice to overcome this drawback. In particular, we characterize and evaluate an effective therapeutic polymeric PEGylated nanosystem for controlled pH-sensitive drug release on a breast cancer (MDA-MB-231) and two lung cancer (A549 and H520) cell lines. A significant reduction in the required drug dose to reach its half maximal inhibitory concentration (IC50 value) was achieved by conjugation of the drug to the nanoparticles, which leads to an improvement in the therapeutic index by increasing the efficiency. The genotoxic effect of this nanodevice in cancer cells was confirmed by nucleus histone H2AX specific immunostaining. In summary, we successfully characterized and validated a pH responsive therapeutic polymeric nanodevice in vitro for controlled anticancer drug release.Spanish Ministry of Economy and Competitiveness (MINECO) BIO2016-80519Health Institute Carlos III (ISCIII) DTS18/00121Andalusian Regional Government PAIDI-TC-PVT-PSETC-2.

Hybrid Fluorescent Mass-Tag Nanotrackers as Universal Reagents for Long-Term Live-Cell Barcoding

Cell barcoding and pooling are essential to reduce technical variability in multiplex technologies. While fluorescent barcoding is common in flow cytometry, current mass-tag barcoding methods are unsuitable for live cells. This study presents fluorescent palladium-based nanotrackers for live cell barcoding, compatible with flow and mass cytometry, enabling long-term drug studies with consistent labeling.This research was supported by funding from the Spanish Ministry of Economy and Competitiveness (BIO2016-80519, PID2019.110987RB.I00); the Health Institute Carlos III (DTS18/00121); the Andalusian Regional Government-FEDER (PT18-TP-4160, B-FQM-475-UGR18, A-FQM-760-UGR20); the Department of Defense (W81XWH-12-1-0591); NCI (1R01CA234553, R21CA231280); the 2019 Cancer Innovation Award, supported by the Stanford Cancer Institute, an NCI-designated Comprehensive Cancer Center; BRCA Foundation and the V Foundation for Cancer Research; a gift from the Gray Foundation; the Department of Urology at Stanford University; NHLBI (P01HL10879709); NIAID (U19AI057229); and a PICI Bedside to Bench grant. R.M.S.-M., J.J.D.-M., M.V.C.-C., and J.A.L.-R. are members of the network NANOCARE (RED2018-102469-T) funded by the Spanish State Research Agency. W.J.F. thanks the BRCA Foundation and the V Foundation for Cancer Research, a gift from the Gray Foundation and the Department of Urology at Stanford University. A.D.-G. thanks the Spanish Ministry of Education for PhD funding (scholarship FPU14/02181), the University of Granada for postdoctoral research contract, and the Fundación Alfonso Martín Escudero for his postdoctoral fellowship. J.A.L.-R. thanks the Fundación Benéfica Anticancer San Francisco Javier y Santa Candida for PhD funding. M.V.C.-C. thanks the Andalusian Regional Government for her postdoctoral fellowship (POSTDOC_21_00118). The authors thank the technical support from the Cytometry unit of GENYO. The graphical abstract and Figures 5a and 6a have been created with BioRender.com

An effective polymeric nanocarrier that allows for active targeting and selective drug delivery in cell coculture systems

In this manuscript, we report the development of a versatile, robust, and stable targeting nanocarrier for active delivery. This nanocarrier is based on bifunctionalized polymeric nanoparticles conjugated to a monoclonal antibody that allows for active targeting of either (i) a fluorophore for tracking or (ii) a drug for monitoring specific cell responses. This nanodevice can efficiently discriminate between cells in coculture based on the expression levels of cell surface receptors. As a proof of concept, we have demonstrated efficient delivery using a broadly established cell surface receptor as the target, the epidermal growth factor receptor (EGFR), which is overexpressed in several types of cancers. Additionally, a second validation of this nanodevice was successfully carried out using another cell surface receptor as the target, the cluster of differentiation 147 (CD147). Our results suggest that this versatile nanocarrier can be expanded to other cell receptors and bioactive cargoes, offering remarkable discrimination efficiency between cells with different expression levels of a specific marker. This work supports the ability of nanoplatforms to boost and improve the progress towards personalized medicine.Health Institute Carlos III (ISCIII) DTS18/00121Junta de Andalucia-FEDER, Ministry of Economy, Knowledge, Companies and University (University of Granada) B-FQM-475-UGR18 PT18-TP-1490 AT17_6096- OTRISpanish State Research Agency RED2018-102469-TFundación Benéfica Anticáncer San Francisco Javier y Santa CándidaSpanish Ministry of Economy and Competitiveness PTQ-16- 08597Spanish Government FPU 16/0206

Mitochondrial pH Nanosensors for Metabolic Profiling of Breast Cancer Cell Lines

We thank the company Nanogetic S.L. (Granada, Spain) for support with the synthesis of the SS peptides and the Centro de Instrumentacion Cientifica (CIC) of the Universidad de Granada for use of the TEM facilities.The main role of mitochondria, as pivotal organelles for cellular metabolism, is the production of energy (ATP) through an oxidative phosphorylation system. During this process, the electron transport chain creates a proton gradient that drives the synthesis of ATP. One of the main features of tumoral cells is their altered metabolism, providing alternative routes to enhance proliferation and survival. Hence, it is of utmost importance to understand the relationship between mitochondrial pH, tumoral metabolism, and cancer. In this manuscript, we develop a highly specific nanosensor to accurately measure the intramitochondrial pH using fluorescence lifetime imaging microscopy (FLIM). Importantly, we have applied this nanosensor to establish differences that may be hallmarks of different metabolic pathways in breast cancer cell models, leading to the characterization of different metabophenotypes.Spanish Ministerio de Ciencia, Innovacion y Universidades CTQ2014-56370-R CTQ2017-86568-REuropean Union (EU)Spanish Agencia Estatal de InvestigacionFundacion Ramon Arece

Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast

In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction, and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast cells, which is due to the higher endocytic activity of tumor cells compared to normal cells. In this way, we demonstrate that biosynthesized Ag NPs can be an alternative for the treatment of tumors.Support from Universidad Nacional de San Luis, to the Agencia Nacional de Promoción Científica y Tecnológica, from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (Argentina), and from GENYO, Centre for Genomics and Oncological Research: Pfizer-University of Granada, Andalusian Regional Government, Granada, Spain are acknowledged

Circulating epithelial cell as viral infection and tissue origin marker in patients with severe COVID-19

Liquid biopsy (LB) is a minimally invasive procedure that detects biomarkers in body fluids for real-time monitoring of patients. This study developed a new LB approach to analyze Circulating Epithelial Cells (CECs) in Intensive Care Unit (ICU) patients with severe COVID-19 and High-Exposure Negative Population to COVID-19 (HENPC) as the control group. The CECs were characterized by multispectral imaging flow cytometry, and an anti-SARS-CoV-2 Spike S1 protein (ProtS) antibody was used to detect infection. The results showed that CECs were present in most ICU patients (p = 0.0412), and their median number was significantly higher (p = 0.0004) than in controls. CEC clusters were only identified in patients, and high positive ProtS expression was observed in CECs from ICU patients compared to negative controls. In conclusion, LB could be a minimally invasive tool for detecting tissue damage caused by infectious agents and could provide real-time biological information about disease status and evolution. However, further validation in a larger population of patients is needed

SARS-CoV-2 viral RNA detection using the novel CoVradar device associated with the CoVreader smartphone app

Supplementary data to this article can be found online at https://doi. org/10.1016/j.bios.2023.115268The COVID-19 pandemic has highlighted the need for innovative approaches to its diagnosis. Here we present CoVradar, a novel and simple colorimetric method that combines nucleic acid analysis with dynamic chemical labeling (DCL) technology and the Spin-Tube device to detect SARS-CoV-2 RNA in saliva samples. The assay includes a fragmentation step to increase the number of RNA templates for analysis, using abasic peptide nucleic acid probes (DGL probes) immobilized to nylon membranes in a specific dot pattern to capture RNA fragments. Duplexes are formed by labeling complementary RNA fragments with biotinylated SMART bases, which act as templates for DCL. Signals are generated by recognizing biotin with streptavidin alkaline phosphatase and incubating with a chromogenic substrate to produce a blue precipitate. CoVradar results are analysed by CoVreader, a smartphone-based image processing system that can display and interpret the blotch pattern. CoVradar and CoVreader provide a unique molecular assay capable of detecting SARS-CoV-2 viral RNA without the need for extraction, preamplification, or pre-labeling steps, offering advantages in terms of time (similar to 3 h/test), cost (similar to epsilon 1/test manufacturing cost) and simplicity (does not require large equipment). This solution is also promising for developing assays for other infectious diseases.FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento CV20-77741, A-FQM-760-UGR20, PID 2019-110987RB-I00, PID 2019-103938RB-I00Spanish MCIN/AEI P18-RT-2961, P18-TP-4160FEDER/Junta de Andalucia-Consejeria de Salud y Familias PIP-0232-2021European CommissionMCIN/AEI PTQ 2020-011388, IJC 2020-043307-IEuropean Union NextGenerationEU/PRTR PTQ 2020-011388, IJC 2020-043307-