AptaShield: A Universal Signal-Transduction System for Fast and High-Throughput Optical Molecular Biosensing.

Biosensing technologies are often described to provide facile, sensitive, and minimally to noninvasive detection of molecular analytes across diverse scientific, environmental, and clinical diagnostic disciplines. However, commercialization has been very limited mostly due to the difficulty of biosensor reconfiguration for different analyte(s) and limited high-throughput capabilities. The immobilization of different biomolecular probes (e.g., antibodies, peptides, and aptamers) requires the sensor surface chemistry to be tailored to provide optimal probe coupling, orientation, and passivation and prevent nonspecific interactions. To overcome these challenges, here we report the development of a solution-phase biosensor consisting of an engineered aptamer, the AptaShield, capable of universally binding to any antigen recognition site (Fab') of fluorescently labeled immunoglobulins (IgG) produced in rabbits. The resulting AptaShield biosensor relies on a low affinity dynamic equilibrium between the fluorescently tagged aptamer and IgG to generate a specific Förster resonance energy transfer (FRET) signal. As the analyte binds to the IgG, the AptaShield DNA aptamer-IgG complex dissociates, leading to an analyte concentration-dependent decrease of the FRET signal. The biosensor demonstrates high selectivity, specificity, and reproducibility for analyte quantification in different biological fluids (e.g., urine and blood serum) in a one-step and low sample volume (0.5-6.25 µL) format. The AptaShield provides a universal signal transduction mechanism as it can be coupled to different rabbit antibodies without the need for aptamer modification, therefore representing a robust high-throughput solution-phase technology suitable for point-of-care applications, overcoming the current limitations of gold standard enzyme-linked immunosorbent assays (ELISA) for molecular profiling.

Engineering a Hybrid Ti6Al4V-Based System for Responsive and Consistent Osteogenesis.

As the aging population increases worldwide, the incidence of musculoskeletal diseases and the need for orthopedic implants also arise. One of the most desirable goals in orthopedic reconstructive therapies is de novo bone formation. Yet, reproducible, long-lasting, and cost-effective strategies for implants that strongly induce osteogenesis are still in need. Nanoengineered titanium substrates (and their alloys) are among the most used materials in orthopedic implants. Although having high biocompatibility, titanium alloys hold a low bioactivity profile. The osteogenic capacity and osseointegration of Ti-based implantable systems are limited, as they critically depend on the body-substrate interactions defined by blood proteins adsorbed into implant surfaces that ultimately lead to the recruitment, proliferation, and differentiation of mesenchymal stem cells (MSCs) to comply bone formation and regeneration. In this work, a hybrid Ti6Al4V system combining micro- and nanoscale modifications induced by hydrothermal treatment followed by functionalization with a bioactive compound (fibronectin derived from human plasma) is proposed, aiming for bioactivity improvement. An evaluation of the biological activity and cellular responses in vitro with respect to bone regeneration indicated that the integration of morphological and chemical modifications into Ti6Al4V surfaces induces the osteogenic differentiation of MSCs to improve bone regeneration by an enhancement of mineral matrix formation that accelerates the osseointegration process. Overall, this hybrid system has numerous competitive advantages over more complex treatments, including reproducibility, low production cost, and potential for improved long-term maintenance of the implant.

MorphoMacro for in vivo and ex vivo quantitative morphometric analysis of microglia.

Microglia cells dynamically survey the central nervous system microenvironment and, in response to tissue damage inflicted by radiation therapy, disease or infection, undergo morphological and functional changes that culminate in microglia activation. Cell shape transformation can be assessed descriptively or, alternatively, it can be quantified as a continuous variable for parameters including total cell size as well as protrusion length, ramification and complexity. The purpose of the MorphoMacro method is to quantitatively profile multiple and single microglia cells using the available ImageJ platform. This method outlines the required steps and ImageJ plugins to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using the MorphoMacro software plugin for multiparametric and multilevel description of microglia cell morphology in vivo and ex vivo. Overall, the protocol provides a quantitative and comprehensive tool that can be used to identify, stratify, and monitor diverse microglia morphologies in homeostatic, different disease conditions and subsequent therapeutic monitoring.

Novel time-domain NMR-based traits for rapid, label-free Olive oils profiling.

Olive oil is one of the oldest and essential edible oils in the market. The classification of olive oils (e.g. extra virgin, virgin, refined) is often influenced by factors ranging from its complex inherent physiochemical properties (e.g. fatty acid profiles) to the undisclosed manufacturing processes. Therefore, olive oils have been the target of adulteration due to its profitable margin. In this work, we demonstrate that multi-parametric time-domain NMR relaxometry can be used to rapidly (in minutes) identify and classify olive oils in label-free and non-destructive manner. The subtle differences in molecular microenvironment of the olive oils induce substantial changes in the relaxation mechanism in the time-domain NMR regime. We demonstrated that the proposed NMR-relaxation based detection (AUC = 0.95) is far more sensitive and specific than the current gold-standards in the field i.e. near-infrared spectroscopy (AUC = 0.84) and Ultraviolet-visible spectroscopy (AUC = 0.73), respectively. We further show that, albeit the inherent complexity of olive plant natural phenotypic variations, the proposed NMR-relaxation based traits may be a viable mean (AUC = 0.71) in tracing the regions of origin for olive trees, in agreement with their geographical orientation.

Non-invasive diagnosis and monitoring tool of children's mental health: A point-of-care immunosensor for IL-6 quantification in saliva samples.

Mental disorders are commonly featured as chronic conditions with often onset during childhood. In this context, inflammation has been associated with a higher risk of developing physical and mental health problems. Interleukin (IL)-6 is a key mediator of inflammatory responses and plays a pivotal role in immune and nervous system interaction. High levels of IL-6 during childhood are associated with mental problems, indicating that the IL-6 molecular pathway may represent a new target for monitoring and treating these conditions. Here, we report the detection of IL-6 in saliva samples from children (N = 118, mean age 4.4 years old) with behavioral problems using an immunosensor based on electrochemical impedance spectroscopy. This work demonstrates that the proposed immunosensor requires smaller sample volumes and is significantly faster and more sensitive than conventional ELISA while maintaining comparable levels of specificity and reproducibility. The point-of care immunosensor for detection of IL-6 in saliva samples presented herewith is, therefore, an attractive solution to the clinical practice as a rapid non-invasive, high-sensitive monitoring tool of mental health problems, especially in vulnerable patient populations such as children.

Mechanical actuators in microglia dynamics and function.

Microglia are the most prominent immune resident cell population in the central nervous system (CNS). In the healthy CNS, microglia survey their surrounding microenvironment, through recurrent extension and retraction of filopodia-like membrane protrusions, without evident cell body displacement. Microglia undergo dramatic transcriptomic and shape changes upon brain insults or neurodegenerative disease states and adopt a classical immune effector function (producing an extensive array of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species) to re-establish tissue homeostasis. While the biophysical principles underlying microglia morphological changes remain elusive, several recent studies have highlighted the pivotal role of the actin and non-muscle myosin II filamentous cytoskeleton in this process. In this work, we discuss how subcellular topological patterning of the actin and myosin cytoskeleton can control microglial cell shape dynamics and how it can potentially feedback on their functional specialization, which is of great importance to understanding the mechanisms of microglial action in homeostatic conditions and CNS disease states.

Lab-on-a-chip technologies for minimally invasive molecular sensing of diabetic retinopathy.

Diabetic retinopathy (DR) is the most common diabetic eye disease and the worldwide leading cause of vision loss in working-age adults. It progresses from mild to severe non-proliferative or proliferative DR based on several pathological features including the magnitude of blood-retinal barrier breakdown and neovascularization. Available pharmacological and retinal laser photocoagulation interventions are mostly applied in the advanced stages of DR and are inefficient in halting disease progression in a significantly high percentage of patients. Yet, recent evidence has shown that some therapies could potentially limit DR progression if applied at early stages, highlighting the importance of early disease diagnostics. In the past few decades, different imaging modalities have proved their utility for examining retinal and optic nerve changes in patients with retinal diseases. However, imaging based-methodologies solely rely on morphological examination of the retinal vascularization and are not suitable for recurrent and personalized patient evaluation. This raises the need for new technologies to enable accurate and early diagnosis of DR. In this review, we critically discuss the potential clinical benefit of minimally-invasive molecular biomarker identification and profiling of diabetic patients who are at risk of developing DR. We provide a comparative overview of conventional and recently developed lab-on-a-chip technologies for quantitative assessment of potential DR molecular biomarkers and discuss their advantages, current limitations and challenges for future practical implementation and continuous patient monitoring at the point-of-care.

Intratumoral VEGF nanotrapper reduces gliobastoma vascularization and tumor cell mass.

Glioblastoma multiforme (GBM) is the most aggressive and invasive malignant brain cancer. GBM is characterized by a dramatic metabolic imbalance leading to increased secretion of the pro-angiogenic factor VEGF and subsequent abnormal tumor vascularization. In 2009, FDA approved the intravenous administration of bevacizumab, an anti-VEGF monoclonal antibody, as a therapeutic agent for patients with GBM. However, the number of systemic side effects and reduced accessibility of bevacizumab to the central nervous system and consequently to the GBM tumor mass limited its effectiveness in improving patient survival. In this study, we combined experimental and computational modelling to quantitatively characterize the dynamics of VEGF secretion and turnover in GBM and in normal brain cells and simultaneous monitoring of vessel growth. We showed that sequestration of VEGF inside GBM cells, can be used as a novel target for improved bevacizumab-based therapy. We have engineered the VEGF nanotrapper, a cargo system that allows cellular uptake of bevacizumab and inhibits VEGF secretion required for angiogenesis activation and development. Here, we show the therapeutic efficacy of this nanocargo in reducing vascularization and tumor cell mass of GBM in vitro and in vivo cancer models.

Morphofunctional programming of microglia requires distinct roles of type II myosins.

The ramified morphology of microglia and the dynamics of their membrane protrusions are essential for their functions in central nervous system development, homeostasis, and disease. Although their ability to change and control shape critically depends on the actin and actomyosin cytoskeleton, the underlying regulatory mechanisms remain largely unknown. In this study, we systematically analyzed the actomyosin cytoskeleton and regulators downstream of the small GTPase RhoA in the control of microglia shape and function. Our results reveal that (i) Myh9 controls cortical tension levels and affects microglia protrusion formation, (ii) cofilin-mediated maintenance of actin turnover regulates microglia protrusion extension, and (iii) Myh10 influences microglia inflammatory activation. Overall we uncover molecular pathways that regulate microglia morphology and identify type-II myosins as important regulators of microglia biology with differential roles in the control of cell shape (Myh9) and functions (Myh10).

Exosome Circuitry During (De)(Re)Myelination of the Central Nervous System.

Reciprocal neuron-glia cell communication is fundamental for the proper function of the nervous system. Oligodendrocytes are the myelinating cells of the central nervous system (CNS) that insulate and provide trophic support to neurons. This effective interaction is crucial not only for myelination but also for long-term axonal survival and neural connectivity. In recent years, exosomes have been portrayed as key players in intercellular interaction in the context of the healthy and diseased CNS. They act as communicating vehicles, true attachés operating between neurons and glial cells. Despite the complex exosome circuitry within the nervous system, experimental evidence supports the role of exosomes in modulating myelination. Oligodendrocytes secrete exosomes in response to neuronal signals in an electric activity-dependent manner. These released exosomes are then internalized by neurons, contributing to their integrity and activity. In turn, neurons secrete exosomes to control the communication between them and with myelinating cells in order to regulate synaptic function in neuronal development, myelin maintenance, and neuroregeneration. In this review, we provide a critical view of the current understanding on how exosomes, either from CNS-resident cells or from the periphery, contribute to the formation and maintenance of myelin and, additionally, on how the differential content of exosomes in normal and pathological conditions foresees the use of these nanovesicles as putative diagnostic and/or therapeutical agents in white matter degeneration-associated diseases.

State of the Art and Future Challenges in Multiple Sclerosis Research and Medical Management: An Insight into the 5th International Porto Congress of Multiple Sclerosis.

The 5th International Porto Congress of Multiple Sclerosis took place between the 14th and 16th of February 2019 in Porto, Portugal. Its intensive programme covered a wide-range of themes-including many of the hot topics, challenges, pitfalls and yet unmet needs in the field of multiple sclerosis (MS)-led by a number of well-acknowledged world experts. This meeting review summarizes the talks that took place during the congress, which focussed on issues in MS as diverse as the development and challenges of progressive MS, epidemiology, differential diagnosis, medical management, molecular research and imaging tools.

Non-invasive molecular assessment of human embryo development and implantation potential.

In vitro fertilization (IVF) is the most common assisted reproductive technology used to treat infertility. Embryo selection for transfer in IVF cycles relies on the morphological evaluation by embryologists, either by conventional microscopic assessment or more recently by time-lapse imaging systems. Despite the introduction of time-lapse imaging improvements in IVF success rates have failed to materialize, therefore alternative approaches are needed. Recent studies have shown that embryos resulting in successful pregnancy differ in their secretome and metabolism compared to embryos that fail to implant, suggesting that molecular analysis of embryo culture medium could assist in non-invasive single embryo selection. However, this approach has yet to be adopted clinically due to the lack of appropriate highly sensitive screening technologies needed to assess volume-limited samples. Here we report the detection of hCGß, IL-8 and TNFα from conditioned culture media of single human embryos using electrochemical impedance spectroscopy. The impedimetric immunosensors revealed that morphologically non-viable embryos produce higher levels of IL-8 and TNFα, associated with abnormal cell division and cell death, respectively. More importantly, hCGß detection was able to discriminate apparently morphologically identical viable embryos. This work brings an objective dimension to embryo selection, which could overcome the major limitations of morphology-based embryo selection for implantation. Future work should include the validation of these biomarkers in a large patient cohort.

The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction.

Neurons have a membrane periodic skeleton (MPS) composed of actin rings interconnected by spectrin. Here, combining chemical and genetic gain- and loss-of-function assays, we show that in rat hippocampal neurons the MPS is an actomyosin network that controls axonal expansion and contraction. Using super-resolution microscopy, we analyzed the localization of axonal non-muscle myosin II (NMII). We show that active NMII light chains are colocalized with actin rings and organized in a circular periodic manner throughout the axon shaft. In contrast, NMII heavy chains are mostly positioned along the longitudinal axonal axis, being able to crosslink adjacent rings. NMII filaments can play contractile or scaffolding roles determined by their position relative to actin rings and activation state. We also show that MPS destabilization through NMII inactivation affects axonal electrophysiology, increasing action potential conduction velocity. In summary, our findings open new perspectives on axon diameter regulation, with important implications in neuronal biology.

Electrochemical Immunosensor for TNFα-Mediated Inflammatory Disease Screening.

Inflammation associated with cancer, neurodegenerative, ocular, and autoimmune diseases has a considerable impact on public health. Tumor necrosis factor alpha (TNFα) is a key mediator of inflammatory responses, responsible for many of the systemic manifestations during the inflammatory process. Thus, inhibition of TNFα is a commonplace practice in the treatment of these disorders. Successful therapy requires the ability to determine the appropriate dose of anti-TNFα drugs to be administered in a timely manner, based on circulating TNFα levels. In this Letter, we report the development of an immunosensor technology able to quantify TNFα at the picogram level in relevant human body fluids, holding the potential to early detect inflammation  and monitor TNFα levels during treatment, enabling TNFα-targeted treatments to be tailored according to the immune status of an individual patient. This immunosensor technology is significantly more rapid and sensitive than conventional enzyme linked immunosorbent assays, maintaining high specificity and requiring small sample volumes. These features might also be advantageous in the context of personalized medicine, as this analytical platform can deliver advanced diagnostics and reduce clinical burden.

Evolvability of the actin cytoskeleton in oligodendrocytes during central nervous system development and aging.

The organization of actin filaments into a wide range of subcellular structures is a defining feature of cell shape and dynamics, important for tissue development and homeostasis. Nervous system function requires morphological and functional plasticity of neurons and glial cells, which is largely determined by the dynamic reorganization of the actin cytoskeleton in response to intrinsic and extracellular signals. Oligodendrocytes are specialized glia that extend multiple actin-based protrusions to form the multilayered myelin membrane that spirally wraps around axons, increasing conduction speed and promoting long-term axonal integrity. Myelination is a remarkable biological paradigm in development, and maintenance of myelin is essential for a healthy adult nervous system. In this review, we discuss how structure and dynamics of the actin cytoskeleton is a defining feature of myelinating oligodendrocytes' biology and function. We also review "old and new" concepts to reflect on the potential role of the cytoskeleton in balancing life and death of myelin membranes and oligodendrocytes in the aging central nervous system.

Emerging Biosensing Technologies for Neuroinflammatory and Neurodegenerative Disease Diagnostics.

Neuroinflammation plays a critical role in the onset and progression of many neurological disorders, including Multiple Sclerosis, Alzheimer's and Parkinson's diseases. In these clinical conditions the underlying neuroinflammatory processes are significantly heterogeneous. Nevertheless, a common link is the chronic activation of innate immune responses and imbalanced secretion of pro and anti-inflammatory mediators. In light of this, the discovery of robust biomarkers is crucial for screening, early diagnosis, and monitoring of neurological diseases. However, the difficulty to investigate biochemical processes directly in the central nervous system (CNS) is challenging. In recent years, biomarkers of CNS inflammatory responses have been identified in different body fluids, such as blood, cerebrospinal fluid, and tears. In addition, progress in micro and nanotechnology has enabled the development of biosensing platforms capable of detecting in real-time, multiple biomarkers in clinically relevant samples. Biosensing technologies are approaching maturity where they will become deployed in community settings, at which point screening programs and personalized medicine will become a reality. In this multidisciplinary review, our goal is to highlight both clinical and recent technological advances toward the development of multiplex-based solutions for effective neuroinflammatory and neurodegenerative disease diagnostics and monitoring.

Collar occupancy: A new quantitative imaging tool for morphometric analysis of oligodendrocytes.

BACKGROUND: Oligodendrocytes (OL) are the myelinating cells of the central nervous system. OL differentiation from oligodendrocyte progenitor cells (OPC) is accompanied by characteristic stereotypical morphological changes. Quantitative imaging of those morphological alterations during OPC differentiation is commonly used for characterization of new molecules in cell differentiation and myelination and screening of new pro-myelinating drugs. Current available imaging analysis methods imply a non-automated morphology assessment, which is time-consuming and prone to user subjective evaluation. NEW METHOD: Here, we describe an automated high-throughput quantitative image analysis method entitled collar occupancy that allows morphometric ranking of different stages of in vitro OL differentiation in a high-content analysis format. Collar occupancy is based on the determination of the percentage of area occupied by OPC/OL cytoplasmic protrusions within a defined region that contains the protrusion network, the collar. RESULTS: We observed that more differentiated cells have higher collar occupancy and, therefore, this parameter correlates with the degree of OL differentiation. COMPARISON WITH EXISTING METHODS: In comparison with the method of manual categorization, we found the collar occupancy to be more robust and unbiased. Moreover, when coupled with myelin basic protein (MBP) staining to quantify the percentage of myelinating cells, we were able to evaluate the role of new molecules in OL differentiation and myelination, such as Dusp19 and Kank2. CONCLUSIONS: Altogether, we have successfully developed an automated and quantitative method to morphologically characterize OL differentiation in vitro that can be used in multiple studies of OL biology.

Exosomes as Reconfigurable Therapeutic Systems.

Historically, small molecules, including steroid hormones and cytokines, have been attributed a role in paracrine and endocrine signaling, and now include a new player: biological nanoparticles, or 'exosomes'. Generated intracellularly, and defined simply as nanoparticulate packages of signaling moieties, exosomes have emerged as vehicles for highly specialized local and distant intercellular communication. Exosomes are increasingly being recognized as contributing factors in many diseases, and their potential as biomarkers and in therapeutics is rapidly emerging. This review highlights recent advances in the exploitation of exosomes in diagnostic and therapeutic applications. We discuss various facets of nanoparticles, namely the isolation and manipulation of exosomes, the construction of synthetic exosome-like particles in vivo, and their potential use in the treatment of various diseases.

Independence of symmetry breaking on Bem1-mediated autocatalytic activation of Cdc42.

The ability to break symmetry and polarize through self-organization is a fundamental feature of cellular systems. A prevailing theory in yeast posits that symmetry breaking occurs via a positive feedback loop, wherein the adaptor protein Bem1 promotes local activation and accumulation of Cdc42 by directly tethering Cdc42(GTP) with its guanine nucleotide exchange factor (GEF) Cdc24. In this paper, we find that neither Bem1 nor the ability of Bem1 to bind Cdc42(GTP) is required for cell polarization. Instead, Bem1 functions primarily by boosting GEF activity, a role critical for polarization without actin filaments. In the absence of actin-based transport, polarization of Cdc42 is accomplished through Rdi1, the Cdc42 guanine nucleotide dissociation inhibitor. A mathematical model is constructed describing cell polarization as a product of distinct pathways controlling Cdc42 activation and protein localization. The model predicts a nonmonotonic dependence of cell polarization on the concentration of Rdi1 relative to that of Cdc42.