Gene regulatory network analysis predicts cooperating transcription factor regulons required for FLT3-ITD+ AML growth.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by different mutations. Previously, we showed that each mutational subtype develops its specific gene regulatory network (GRN) with transcription factors interacting within multiple gene modules, many of which are transcription factor genes themselves. Here, we hypothesize that highly connected nodes within such networks comprise crucial regulators of AML maintenance. We test this hypothesis using FLT3-ITD-mutated AML as a model and conduct an shRNA drop-out screen informed by this analysis. We show that AML-specific GRNs predict crucial regulatory modules required for AML growth. Furthermore, our work shows that all modules are highly connected and regulate each other. The careful multi-omic analysis of the role of one (RUNX1) module by shRNA and chemical inhibition shows that this transcription factor and its target genes stabilize the GRN of FLT3-ITD+ AML and that its removal leads to GRN collapse and cell death.

Generation of an Ovomucoid-Immune scFv Library for the Development of Novel Immunoassays in Hen's Egg Detection.

Hen's egg allergy is the second most common food allergy among infants and young children. The possible presence of undeclared eggs in foods poses a significant risk to sensitized individuals. Therefore, reliable egg allergen detection methods are needed to ensure compliance with food labeling and improve consumer protection. This work describes for the first time the application of phage display technology for the generation of a recombinant antibody aimed at the specific detection of hen's ovomucoid. First, a single-chain variable fragment (scFv) library was constructed from mRNA isolated from the spleen of a rabbit immunized with ovomucoid. After rounds of biopanning, four binding clones were isolated and characterized. Based on the best ovomucoid-binding candidate SR-G1, an indirect phage enzyme-linked immunosorbent assay (phage-ELISA) was developed, reaching limits of detection and quantitation of 43 and 79 ng/mL of ovomucoid, respectively. The developed ELISA was applied to the analysis of a wide variety of food products, obtaining a good correlation with a commercial egg detection assay used as a reference. Finally, in silico modeling of the antigen-antibody complex revealed that the main interactions most likely occur between the scFv heavy chain and the ovomucoid domain-III, the most immunogenic region of this allergen.

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 high-resolution 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.

Development of a new recombinant antibody, selected by phage-display technology from a celiac patient library, for detection of gluten in foods.

Gluten, a group of ethanol-soluble proteins present in the endosperm of cereals, is extensively used in the food industry due to its ability to improve dough properties. However, gluten is also associated with a range of gluten-related diseases (GRDs), such as wheat allergies, celiac disease, and gluten intolerance. The recommended treatment for GRDs patients is a gluten-free diet. To monitor adherence to this diet, it is necessary to develop gluten-detection systems in food products. Among the available methods, immunodetection systems are the most popular due to their simplicity, reproducibility, and accuracy. The aim of this study was to generate novel high-affinity antibodies against gluten to be used as the primary reactant in an enzyme-linked immunosorbent assay (ELISA) test. These antibodies were developed by constructing an immune library from mRNA obtained from two celiac patients with a high humoral response to gluten-related proteins. The resulting library (composed by 1.1x107) was subjected to selection against gliadin using phage display technology. Following several rounds of selection, the Fab-C was selected, and demonstrated good functionality in ELISA tests, presenting a limit of detection of 15 mg/kg for detection of gluten in spiked mixtures and food products. The methodology can discriminate gluten-free products according to the current legislation.

Production and Characterization of Novel Fabs Generated from Different Phage Display Libraries as Probes for Immunoassays for Gluten Detection in Food.

Gluten is the main fraction of wheat proteins. It is widely used in the food industry because of the properties that are generated in the dough, but it is also able to trigger diseases like allergies, autoimmunity processes (such as celiac disease), and intolerances in sensitized persons. The most effective therapy for these diseases is the total avoidance of gluten in the diet because it not only prevents damage but also enhances tissue healing. To ensure the absence of gluten in food products labeled as gluten-free, accurate detection systems, like immunoassays, are required. In this work, four recombinant Fab antibody fragments, selected by phage display technology, were produced and tested for specificity and accuracy against gluten in experimental flour mixtures and commercial food products. A high-affinity probe (Fab-C) was identified and characterized. An indirect ELISA test was developed based on Fab-C that complied with the legal detection limits and could be applied in the assessment of gluten-free diets.

Gene regulatory network analysis predicts cooperating transcription factor regulons required for FLT3-ITD+ AML growth.

AML is a heterogenous disease caused by different mutations. We have previously shown that each mutational sub-type develops its specific gene regulatory network (GRN) with transcription factors interacting with multiple gene modules, many of which are transcription factor genes themselves. Here we hypothesized that highly connected nodes within such networks comprise crucial regulators of AML maintenance. We tested this hypothesis using FLT3-ITD mutated AML as a model and conducted an shRNA drop-out screen informed by this analysis. We show that AML-specific GRNs predict identifying crucial regulatory modules required for AML but not normal cellular growth. Furthermore, our work shows that all modules are highly connected and regulate each other. The careful multi-omic analysis of the role of one (RUNX1) module by shRNA and chemical inhibition shows that this transcription factor and its target genes stabilize the GRN of FLT3-ITD AML and that its removal leads to GRN collapse and cell death.

N-aryltetrahydroisoquinoline derivatives as HA-CD44 interaction inhibitors: Design, synthesis, computational studies, and antitumor effect.

Hyaluronic acid (HA) plays a crucial role in tumor growth and invasion through its interaction with cluster of differentiation 44 (CD44), a non-kinase transmembrane glycoprotein, among other hyaladherins. CD44 expression is elevated in many solid tumors, and its interaction with HA is associated with cancer and angiogenesis. Despite efforts to inhibit HA-CD44 interaction, there has been limited progress in the development of small molecule inhibitors. As a contribution to this endeavour, we designed and synthesized a series of N-aryltetrahydroisoquinoline derivatives based on existing crystallographic data available for CD44 and HA. Hit 2e was identified within these structures for its antiproliferative effect against two CD44+ cancer cell lines, and two new analogs (5 and 6) were then synthesized and evaluated as CD44-HA inhibitors by applying computational and cell-based CD44 binding studies. Compound 2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinolin-5-ol (5) has an EC50 value of 0.59 µM against MDA-MB-231 cells and is effective to disrupt the integrity of cancer spheroids and reduce the viability of MDA-MB-231 cells in a dose-dependent manner. These results suggest lead 5 as a promising candidate for further investigation in cancer treatment.

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

The 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 (∼3 h/test), cost (∼€1/test manufacturing cost) and simplicity (does not require large equipment). This solution is also promising for developing assays for other infectious diseases.

Direct detection of alpha satellite DNA with single-base resolution by using abasic Peptide Nucleic Acids and Fluorescent in situ Hybridization.

The detection of repetitive sequences with single-base resolution is becoming increasingly important aiming to understand the biological implications of genomic variation in these sequences. However, there is a lack of techniques to experimentally validate sequencing data from repetitive sequences obtained by Next-Generation Sequencing methods, especially in the case of Single-Nucleotide Variations (SNVs). That is one of the reasons why repetitive sequences have been poorly studied and excluded from most genomic studies. Therefore, in addition to sequencing data, there is an urgent need for efficient validation methods of genomic variation in these sequences. Herein we report the development of chemFISH, an alternative method for the detection of SNVs in repetitive sequences. ChemFISH is an innovative method based on dynamic chemistry labelling and abasic Peptide Nucleic Acid (PNA) probes to detect in situ the α-satellite DNA, organized in tandem repeats, with single-base resolution in a direct and rapid reaction. With this approach, we detected by microscopy the α-satellite DNA in a variety of human cell lines, we quantified the detection showing a low coefficient of variation among samples (13.16%-25.33%) and we detected single-base specificity with high sensitivity (82.41%-88.82%). These results indicate that chemFISH can serve as a rapid method to validate previously detected SNVs in sequencing data, as well as to find novel SNVs in repetitive sequences. Furthermore, the versatile chemistry behind chemFISH can lead to develop novel molecular assays for the in situ detection of nucleic acids.

A Multiplex Nanopore Sequencing Approach for the Detection of Multiple Arboviral Species.

The emergence and continued geographic expansion of arboviruses and the growing number of infected people have highlighted the need to develop and improve multiplex methods for rapid and specific detection of pathogens. Sequencing technologies are promising tools that can help in the laboratory diagnosis of conditions that share common symptoms, such as pathologies caused by emerging arboviruses. In this study, we integrated nanopore sequencing and the advantages of reverse transcription polymerase chain reaction (RT-PCR) to develop a multiplex RT-PCR protocol for the detection of Chikungunya virus (CHIKV) and several orthoflaviviruses (such as dengue (Orthoflavivirus dengue), Zika (Orthoflavivirus zikaense), yellow fever (Orthoflavivirus flavi), and West Nile (Orthoflavivirus nilense) viruses) in a single reaction, which provides data for sequence-based differentiation of arbovirus lineages.

Reversal of mitochondrial malate dehydrogenase 2 enables anaplerosis via redox rescue in respiration-deficient cells.

Inhibition of the electron transport chain (ETC) prevents the regeneration of mitochondrial NAD+, resulting in cessation of the oxidative tricarboxylic acid (TCA) cycle and a consequent dependence upon reductive carboxylation for aspartate synthesis. NAD+ regeneration alone in the cytosol can rescue the viability of ETC-deficient cells. Yet, how this occurs and whether transfer of oxidative equivalents to the mitochondrion is required remain unknown. Here, we show that inhibition of the ETC drives reversal of the mitochondrial aspartate transaminase (GOT2) as well as malate and succinate dehydrogenases (MDH2 and SDH) to transfer oxidative NAD+ equivalents into the mitochondrion. This supports the NAD+-dependent activity of the mitochondrial glutamate dehydrogenase (GDH) and thereby enables anaplerosis-the entry of glutamine-derived carbon into the TCA cycle and connected biosynthetic pathways. Thus, under impaired ETC function, the cytosolic redox state is communicated into the mitochondrion and acts as a rheostat to support GDH activity and cell viability.

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

Barcoding and pooling cells for processing as a composite sample are critical to minimize technical variability in multiplex technologies. Fluorescent cell barcoding has been established as a standard method for multiplexing in flow cytometry analysis. In parallel, mass-tag barcoding is routinely used to label cells for mass cytometry. Barcode reagents currently used label intracellular proteins in fixed and permeabilized cells and, therefore, are not suitable for studies with live cells in long-term culture prior to analysis. In this study, we report the development of fluorescent palladium-based hybrid-tag nanotrackers to barcode live cells for flow and mass cytometry dual-modal readout. We describe the preparation, physicochemical characterization, efficiency of cell internalization, and durability of these nanotrackers in live cells cultured over time. In addition, we demonstrate their compatibility with standardized cytometry reagents and protocols. Finally, we validated these nanotrackers for drug response assays during a long-term coculture experiment with two barcoded cell lines. This method represents a new and widely applicable advance for fluorescent and mass-tag barcoding that is independent of protein expression levels and can be used to label cells before long-term drug studies.

Isogenic GAA-KO Murine Muscle Cell Lines Mimicking Severe Pompe Mutations as Preclinical Models for the Screening of Potential Gene Therapy Strategies.

Pompe disease (PD) is a rare disorder caused by mutations in the acid alpha-glucosidase (GAA) gene. Most gene therapies (GT) partially rely on the cross-correction of unmodified cells through the uptake of the GAA enzyme secreted by corrected cells. In the present study, we generated isogenic murine GAA-KO cell lines resembling severe mutations from Pompe patients. All of the generated GAA-KO cells lacked GAA activity and presented an increased autophagy and increased glycogen content by means of myotube differentiation as well as the downregulation of mannose 6-phosphate receptors (CI-MPRs), validating them as models for PD. Additionally, different chimeric murine GAA proteins (IFG, IFLG and 2G) were designed with the aim to improve their therapeutic activity. Phenotypic rescue analyses using lentiviral vectors point to IFG chimera as the best candidate in restoring GAA activity, normalising the autophagic marker p62 and surface levels of CI-MPRs. Interestingly, in vivo administration of liver-directed AAVs expressing the chimeras further confirmed the good behaviour of IFG, achieving cross-correction in heart tissue. In summary, we generated different isogenic murine muscle cell lines mimicking the severe PD phenotype, as well as validating their applicability as preclinical models in order to reduce animal experimentation.

Selective Anticancer Therapy Based on a HA-CD44 Interaction Inhibitor Loaded on Polymeric Nanoparticles.

Hyaluronic acid (HA), through its interactions with the cluster of differentiation 44 (CD44), acts as a potent modulator of the tumor microenvironment, creating a wide range of extracellular stimuli for tumor growth, angiogenesis, invasion, and metastasis. An innovative antitumor treatment strategy based on the development of a nanodevice for selective release of an inhibitor of the HA-CD44 interaction is presented. Computational analysis was performed to evaluate the interaction of the designed tetrahydroisoquinoline-ketone derivative (JE22) with CD44 binding site. Cell viability, efficiency, and selectivity of drug release under acidic conditions together with CD44 binding capacity, effect on cell migration, and apoptotic activity were successfully evaluated. Remarkably, the conjugation of this CD44 inhibitor to the nanodevice generated a reduction of the dosis required to achieve a significant therapeutic effect.

Nanomedicine as a Promising Tool to Overcome Immune Escape in Breast Cancer.

Breast cancer is the most common type of malignancy and leading cause of cancer death among women worldwide. Despite the current revolutionary advances in the field of cancer immunotherapy, clinical response in breast cancer is frequently below expectations, in part due to various mechanisms of cancer immune escape that produce tumor variants that are resistant to treatment. Thus, a further understanding of the molecular events underlying immune evasion in breast cancer may guarantee a significant improvement in the clinical success of immunotherapy. Furthermore, nanomedicine provides a promising opportunity to enhance the efficacy of cancer immunotherapy by improving the delivery, retention and release of immunostimulatory agents in targeted cells and tumor tissues. Hence, it can be used to overcome tumor immune escape and increase tumor rejection in numerous malignancies, including breast cancer. In this review, we summarize the current status and emerging trends in nanomedicine-based strategies targeting cancer immune evasion and modulating the immunosuppressive tumor microenvironment, including the inhibition of immunosuppressive cells in the tumor area, the activation of dendritic cells and the stimulation of the specific antitumor T-cell response.

Construction of a Fab Library Merging Chains from Semisynthetic and Immune Origin, Suitable for Developing New Tools for Gluten Immunodetection in Food.

The observed increase in the prevalence of gluten-related disorders has prompted the development of novel immunological systems for gluten detection in foodstuff. The innovation on these methods relies on the generation of new antibodies, which might alternatively be obtained by molecular evolution methods such as phage display. This work presents a novel approach for the generation of a Fab library by merging semi-synthetic heavy chains built-up from a pre-existent recombinant antibody fragment (dAb8E) with an immune light chain set derived from celiac donors. From the initial phage population (107 candidates) and after three rounds of selection and amplification, four different clones were isolated for further characterization. The phage Fab8E-4 presented the best features to be applied in an indirect ELISA for the detection of gluten in foods, resulting in improved specificity and sensitivity.

IFN-γ Drives Human Monocyte Differentiation into Highly Proinflammatory Macrophages That Resemble a Phenotype Relevant to Psoriasis.

As key cells of the immune system, macrophages coordinate the activation and regulation of the immune response. Macrophages present a complex phenotype that can vary from homeostatic, proinflammatory, and profibrotic to anti-inflammatory phenotypes. The factors that drive the differentiation from monocyte to macrophage largely define the resultant phenotype, as has been shown by the differences found in M-CSF- and GM-CSF-derived macrophages. We explored alternative inflammatory mediators that could be used for in vitro differentiation of human monocytes into macrophages. IFN-γ is a potent inflammatory mediator produced by lymphocytes in disease and infections. We used IFN-γ to differentiate human monocytes into macrophages and characterized the cells at a functional and proteomic level. IFN-γ alone was sufficient to generate macrophages (IFN-γ Mϕ) that were phagocytic and responsive to polarization. We demonstrate that IFN-γ Mϕ are potent activators of T lymphocytes that produce IL-17 and IFN-γ. We identified potential markers (GBP-1, IP-10, IL-12p70, and IL-23) of IFN-γ Mϕ and demonstrate that these markers are enriched in the skin of patients with inflamed psoriasis. Collectively, we show that IFN-γ can drive human monocyte to macrophage differentiation, leading to bona fide macrophages with inflammatory characteristics.

Survey of Commercial Food Products for Detection of Walnut (Juglans regia) by Two ELISA Methods and Real Time PCR.

Labeling of food allergens in accordance with legal regulations is important to protect the health of allergic consumers. The requirements for detecting allergens in foods involve adequate specificity and sensitivity to identify very small amounts of the target allergens in complex food matrices and processed foods. In this work, one hundred commercial samples were analyzed for walnut detection using three different methods: a sandwich enzyme-linked immunosorbent assay (ELISA) kit based on polyclonal antibodies, a direct ELISA using a recombinant multimeric scFv, and a real time PCR. The most sensitive method was real time PCR followed by sandwich ELISA kit and multimeric scFv ELISA. There was agreement between the three methods for walnut detection in commercial products, except for some heat-treated samples or those that contained pecan. The walnut ELISA kit was less affected by sample processing than was the multimeric scFv ELISA, but there was cross-reactivity with pecan, producing some false positives that must be confirmed by real time PCR. According to the results obtained, 7.0 to 12.6% of samples (depending on the analytical method) contained walnut but did not declare it, confirming there is a risk for allergic consumers. Moreover, there was one sample (3.7%) labelled as containing walnut but that tested negative for this tree nut. Genetic and immunoenzymatic techniques offer complementary approaches to develop a reliable verification for walnut allergen labeling.

Development of a nanotechnology-based approach for capturing and detecting nucleic acids by using flow cytometry.

Nucleic acid-based molecular diagnosis has gained special importance for the detection and early diagnosis of genetic diseases as well as for the control of infectious disease outbreaks. The development of systems that allow for the detection and analysis of nucleic acids in a low-cost and easy-to-use way is of great importance. In this context, we present a combination of a nanotechnology-based approach with the already validated dynamic chemical labeling (DCL) technology, capable of reading nucleic acids with single-base resolution. This system allows for the detection of biotinylated molecular products followed by simple detection using a standard flow cytometer, a widely used platform in clinical and molecular laboratories, and therefore, is easy to implement. This proof-of-concept assay has been developed to detect mutations in KRAS codon 12, as these mutations are highly important in cancer development and cancer treatments.

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.