Trends in DNA biosensors Teles, F.R.R.; Fonseca, L.P.
Talanta (Oxford),
12/2008, Letnik:
77, Številka:
2
Journal Article, Conference Proceeding
Recenzirano
Odprti dostop
Biosensors have witnessed an escalating interest nowadays, both in the research and commercial fields. Deoxyribonucleic acid (DNA) biosensors (genosensors) have been exploited for their inherent ...physico-chemical stability and suitability to discriminate different organism strains. The main principle of detection among genosensors relies on specific DNA hybridization, directly on the surface of a physical transducer. This review covers the main DNA immobilization techniques reported so far, new micro- and nanotechnological platforms for biosensing and the transduction mechanisms in genosensors. Clinical applications, in particular, demand large-scale and decentralized DNA testing. New schemes for DNA diagnosis include DNA chips and microfluidics, which couples DNA detection with sample pretreatment under in vivo-like hybridization conditions. Higher sensitivity and specificity may arise from nanoengineered structures, like carbon nanotubes (CNTs) and DNA/protein conjugates. A new platform for universal DNA biosensing is also presented, and its implications for the future of molecular diagnosis are argued.
Sarcoendoplasmic reticulum calcium ATPase (SERCA), a member of the P-type ATPase family of ion and lipid pumps, is responsible for the active transport of Ca
from the cytoplasm into the sarcoplasmic ...reticulum lumen of muscle cells, into the endoplasmic reticulum (ER) of non-muscle cells. X-ray crystallography has proven to be an invaluable tool in understanding the structural changes of SERCA, and more than 70 SERCA crystal structures representing major biochemical states (defined by bound ligand) have been deposited in the Protein Data Bank. Consequently, SERCA is one of the best characterized components of the calcium transport machinery in the cell. Emerging approaches in the field, including spectroscopy and molecular simulation, now help integrate and interpret this rich structural information to understand the conformational transitions of SERCA that occur during activation, inhibition, and regulation. In this review, we provide an overview of the crystal structures of SERCA, focusing on identifying metrics that facilitate structure-based categorization of major steps along the catalytic cycle. We examine the integration of crystallographic data with different biophysical approaches and computational methods to link biochemical and structural states of SERCA that are populated in the cell. Finally, we discuss the challenges and new opportunities in the field, including structural elucidation of functionally important and novel regulatory complexes of SERCA, understanding the structural basis of functional divergence among homologous SERCA regulators, and bridging the gap between basic and translational research directed toward therapeutic modulation of SERCA.
Abstract
Thyroid hormone (TH) molecules enter cells via membrane transporters and, depending on the cell type, can be activated (i.e., T4 to T3 conversion) or inactivated (i.e., T3 to ...3,3′-diiodo-l-thyronine or T4 to reverse T3 conversion). These reactions are catalyzed by the deiodinases. The biologically active hormone, T3, eventually binds to intracellular TH receptors (TRs), TRα and TRβ, and initiate TH signaling, that is, regulation of target genes and other metabolic pathways. At least three families of transmembrane transporters, MCT, OATP, and LAT, facilitate the entry of TH into cells, which follow the gradient of free hormone between the extracellular fluid and the cytoplasm. Inactivation or marked downregulation of TH transporters can dampen TH signaling. At the same time, dynamic modifications in the expression or activity of TRs and transcriptional coregulators can affect positively or negatively the intensity of TH signaling. However, the deiodinases are the element that provides greatest amplitude in dynamic control of TH signaling. Cells that express the activating deiodinase DIO2 can rapidly enhance TH signaling due to intracellular buildup of T3. In contrast, TH signaling is dampened in cells that express the inactivating deiodinase DIO3. This explains how THs can regulate pathways in development, metabolism, and growth, despite rather stable levels in the circulation. As a consequence, TH signaling is unique for each cell (tissue or organ), depending on circulating TH levels and on the exclusive blend of transporters, deiodinases, and TRs present in each cell. In this review we explore the key mechanisms underlying customization of TH signaling during development, in health and in disease states.
Mayaro virus (MAYV) is an arbovirus that circulates in Latin America and is emerging as a potential threat to public health. Infected individuals develop Mayaro fever, a severe inflammatory disease ...characterized by high fever, rash, arthralgia, myalgia and headache. The disease is often associated with a prolonged arthralgia mediated by a chronic inflammation that can last months. Although the immune response against other arboviruses, such as chikungunya virus (CHIKV), dengue virus (DENV) and Zika virus (ZIKV), has been extensively studied, little is known about the pathogenesis of MAYV infection. In this study, we established models of MAYV infection in macrophages and in mice and found that MAYV can replicate in bone marrow-derived macrophages and robustly induce expression of inflammasome proteins, such as NLRP3, ASC, AIM2, and Caspase-1 (CASP1). Infection performed in macrophages derived from Nlrp3-/-, Aim2-/-, Asc-/-and Casp1/11-/-mice indicate that the NLRP3, but not AIM2 inflammasome is essential for production of inflammatory cytokines, such as IL-1β. We also determined that MAYV triggers NLRP3 inflammasome activation by inducing reactive oxygen species (ROS) and potassium efflux. In vivo infections performed in inflammasome-deficient mice indicate that NLRP3 is involved with footpad swelling, inflammation and pain, establishing a role of the NLRP3 inflammasome in the MAYV pathogenesis. Accordingly, we detected higher levels of caspase1-p20, IL-1β and IL-18 in the serum of MAYV-infected patients as compared to healthy individuals, supporting the participation of the NLRP3-inflammasome during MAYV infection in humans.
In the fog computing paradigm, fog nodes are placed on the network edge to meet end-user demands with low latency, providing the possibility of new applications. Although the role of the cloud ...remains unchanged, a new network infrastructure for fog nodes must be created. The design of such an infrastructure must consider user mobility, which causes variations in workload demand over time in different regions. Properly deciding on the location of fog nodes is important to reduce the costs associated with their deployment and maintenance. To meet these demands, this paper discusses the problem of locating fog nodes and proposes a solution which considers time-varying demands, with two classes of workload in terms of latency. The solution was modeled as a mixed-integer linear programming formulation with multiple criteria. An evaluation with real data showed that an improvement in end-user service can be obtained in conjunction with the minimization of the costs by deploying fewer servers in the infrastructure. Furthermore, results show that costs can be further reduced if a limited blocking of requests is tolerated.
Chromosomal instability (CIN) is a hallmark of tumor cells caused by changes in the dynamics and control of microtubules that compromise the mitotic spindle. Thus, CIN cells may respond differently ...than diploid cells to treatments that target mitotic spindle regulation. Here, we test this idea by inhibiting a subset of kinesin motor proteins involved in mitotic spindle control. KIF18A is required for proliferation of CIN cells derived from triple negative breast cancer or colorectal cancer tumors but is not required in near-diploid cells. Following KIF18A inhibition, CIN tumor cells exhibit mitotic delays, multipolar spindles, and increased cell death. Sensitivity to KIF18A knockdown is strongly correlated with centrosome fragmentation, which requires dynamic microtubules but does not depend on bipolar spindle formation or mitotic arrest. Our results indicate the altered spindle microtubule dynamics characteristic of CIN tumor cells can be exploited to reduce the proliferative capacity of CIN cells.
The eastern Brazilian Amazon contains many isolated ferruginous savanna ecosystem patches (locally known as 'canga vegetation') located on ironstone rocky outcrops on the top of plateaus and ridges, ...surrounded by tropical rainforests. In the Carajás Mineral Province (CMP), these outcrops contain large iron ore reserves that have been exploited by opencast mining since the 1980s. The canga vegetation is particularly impacted by mining, since the iron ores that occur are associated with this type of vegetation and currently, little is known regarding the extent of canga vegetation patches before mining activities began. This information is important for quantifying the impact of mining, in addition to helping plan conservation programmes. Here, land cover changes of the Canga area in the CMP are evaluated by estimating the pre-mining area of canga patches and comparing it to the actual extent of canga patches. We mapped canga vegetation using geographic object-based image analysis (GEOBIA) from 1973 Landsat-1 MSS, 1984 and 2001 Landsat-5 TM, and 2016 Landsat-8 OLI images, and found that canga vegetation originally occupied an area of 144.2 km2 before mining exploitation. By 2016, 19.6% of the canga area was lost in the CMP due to conversion to other land-use types (mining areas, pasturelands). In the Carajás National Forest (CNF), located within the CMP, the original canga vegetation covered 105.2 km2 (2.55% of the CNF total area), and in 2016, canga vegetation occupied an area of 77.2 km2 (1.87%). Therefore, after more than three decades of mineral exploitation, less than 20% of the total canga area was lost. Currently, 21% of the canga area in the CMP is protected by the Campos Ferruginosos National Park. By documenting the initial extent of canga vegetation in the eastern Amazon and the extent to which it has been lost due to mining operations, the results of this work are the first step towards conserving this ecosystem.
The aim of the study was to evaluate efficacy of fractional CO2 vaginal laser treatment (Laser, L) and compare it to local estrogen therapy (Estriol, E) and the combination of both treatments (Laser ...+ Estriol, LE) in the treatment of vulvovaginal atrophy (VVA).
A total of 45 postmenopausal women meeting inclusion criteria were randomized in L, E, or LE groups. Assessments at baseline, 8 and 20 weeks, were conducted using Vaginal Health Index (VHI), Visual Analog Scale for VVA symptoms (dyspareunia, dryness, and burning), Female Sexual Function Index, and maturation value (MV) of Meisels.
Forty-five women were included and 3 women were lost to follow-up. VHI average score was significantly higher at weeks 8 and 20 in all study arms. At week 20, the LE arm also showed incremental improvement of VHI score (P = 0.01). L and LE groups showed a significant improvement of dyspareunia, burning, and dryness, and the E arm only of dryness (P < 0.001). LE group presented significant improvement of total Female Sex Function Index (FSFI) score (P = 0.02) and individual domains of pain, desire, and lubrication. In contrast, the L group showed significant worsening of pain domain in FSFI (P = 0.04), but FSFI total scores were comparable in all treatment arms at week 20.
CO2 vaginal laser alone or in combination with topical estriol is a good treatment option for VVA symptoms. Sexual-related pain with vaginal laser treatment might be of concern.
Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting ..."undruggable" regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand-membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca
pump (Sarcoplasmic reticulum Ca
-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.
In recent years, intrinsically disordered proteins (IDPs) have attracted a lot of attention given the functional importance inherent to their flexible nature. One of the most intriguing features of ...IDPs is their ability to undergo disorder-to-order transitions upon binding in order to perform their function. Although the importance of intermolecular interactions involving IDPs has been widely recognized, there are divergent views on their binding mechanisms. Among the existing mechanistic models, two of them have gained popularity in the IDP field: the ‘conformational selection’ and the ‘coupled folding and binding.’ The first mechanism suggests that folding of IDPs precedes binding, while the second mechanism argues that folding may only take place upon binding. It has been suggested that both models are valid, although they work independently. However, reinterpretation of recent experimental and theoretical data indicates that both models have much more in common that it has been thought. In this manuscript, it is proposed that both mechanistic models should be merged into a single one: the synergistic model. In this model, both ‘conformational selection’ and ‘coupled folding and binding’ will synergistically participate in the binding of IDPs. To what extent each model will contribute to the full binding mechanism will depend on the required rate of binding, IDPs concentration, the native local plasticity of IDPs, the degree of binding degeneracy and the type of disorder-to-order transition. Furthermore, it is proposed that combination of the two mechanisms would bring tremendous advantages to IDP binding. For example, synergy may effectively modulate binding kinetics, balance the delicate interplay between enthalpy and entropy by using the funneled energy landscape more efficiently, thus yielding high specificity with carefully balanced free energy of binding. Given the advantages of the synergistic model, it is proposed that it will provide the basis to fully understand the complex nature of IDP binding.
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