Biology subjects

Castro, C.

Publications and source records attributed to Castro, C..

31 records · Page 2Linked to original sources

bioRxiv2023

Versatile Computer Aided Design of Freeform DNA Nanostructures and Assemblies

Recent advances in structural DNA nanotechnology have been facilitated by design tools that continue to push the limits of structural complexity while simplifying an often-tedious design process. We recently introduced the software MagicDNA, which enables design of complex 3D DNA assemblies with many components; however, the design of structures with freeform features like vertices or curvature still required iterative design guided by simulation feedback and user intuition. Here, we present an updated design tool, MagicDNA 2.0, that automates the design of freeform 3D geometries, leveraging design models informed by coarse-grained molecular dynamics simulations. Our GUI-based, stepwise design approach integrates a high level of automation with versatile control over assembly and sub-component design parameters. We experimentally validated this approach by fabricating a range of DNA origami assemblies with complex freeform geometries, including a 3D Nozzle, G-clef, and Hilbert and Trifolium curves, confirming excellent agreement between design input, simulation, and structure formation.

bioengineering↗

bioRxiv2023

Long-term survival and induction of operational tolerance to murine islet allografts through the co-transplantation of cyclosporine A eluting microparticles

One strategy to prevent islet rejection, is to create a favorable immune-protective local environment at the transplant site. Herein, we utilize localized cyclosporine A (CsA) delivery to islet grafts via poly(lactic-co-glycolic acid) (PLGA) microparticles to attenuate allograft rejection. CsA microparticles alone significantly delayed islet allograft rejection compared to islets alone (p<0.05). Over 50% (6/11) of recipients receiving CsA microparticles and short-term cytotoxic T lymphocyte-associated antigen 4-Ig (CTLA4-Ig) therapy displayed prolonged allograft survival for 214 days, compared to 25% (2/8) receiving CTLA4-Ig alone (p>0.05). CsA microparticles + CTLA4-Ig islet allografts exhibited reduced T-cell (CD4+ and CD8+ cells) and macrophage (CD68+ cells) infiltration compared to islets alone. We observed reduced mRNA expression of proinflammatory cytokines (IL-6, IL-10, INF-{gamma} & TNF-; p<0.05) and chemokines (CCL2, CCL5, CCL22, and CXCL10; p<0.05) in CsA microparticles + CTLA4-Ig allografts compared to islets alone. Long-term islet allografts contained insulin+ and intra-graft FoxP3+ T regulatory cells. Rapid rejection of third-party skin grafts (C3H) in islet allograft recipients suggested that CsA microparticles + CTLA4-Ig therapy induced donor specific operational tolerance. This study demonstrates that localized CsA drug delivery plus short-course systemic immunosuppression promotes an immune protective transplant niche for allogeneic islets. Article HighlightsO_LISystemic immunosuppression limits patient inclusion for beta cell replacement therapies C_LIO_LILocalized islet graft immunosuppression may reduce drug toxicity and improve graft survival C_LIO_LICyclosporine eluting microparticles + CTLA4-Ig therapy induced donor specific operational tolerance C_LIO_LIGraft localized drug delivery can create an immune protective transplant niche C_LI

immunology↗

bioRxiv2023

Rescue of neurogenesis and age-associated cognitive decline in SAMP8 mouse: role of transforming growth factor alpha

Neuropathological aging is associated with memory impairment and cognitive decline, and affects several brain areas including the neurogenic niche of the dentate gyrus of the hippocampus (DG). In the healthy brain homeostatic mechanisms regulate neurogenesis in the DG to facilitate the continuous generation of neurons from neural stem cells (NSC). Nevertheless, aging reduces the number of activated neural stem cells, and diminishes the number of newly generated neurons. Strategies that promote neurogenesis in the DG may improve cognitive performance in the elderly resulting in the development of treatments to prevent the progression of neurological disorders in the aged population. Our work is aimed to discover targeting molecules to be used in the design of pharmacological agents to prevent the neurological effects of pathological aging. We study the effect of age on hippocampal neurogenesis using the SAMP8 mouse as a model of pathological aging. Thus, we show that in six-month-old SAMP8 mice, episodic and spatial memory are impaired, concomitantly the generation of neuroblasts and neurons is reduced and the generation of astrocytes is increased in this model. The novelty of our work resides in the fact that treatment of SAMP8 mice with a TGF-alpha targeting molecule, prevents the observed defects, positively regulating neurogenesis and improving cognitive performance. This compound facilitates the release of TGF-alpha in vitro and in vivo and activates signaling pathways initiated by this growth factor. We conclude that targeting the release of TGF-alpha may be the basis of pharmacological drugs to counteract the neurological effects of pathological aging.

neuroscience↗

bioRxiv2022

DNA-caged Nanoparticles via Electrostatic Self-Assembly

DNA-modified nanoparticles enable DNA sensing and therapeutics in nanomedicine and are also crucial for nanoparticle self-assembly with DNA-based materials. However, methods to conjugate DNA to nanoparticle surfaces are limited, inefficient, and lack control. Inspired by DNA tile nanotechnology, we demonstrate a new approach to nanoparticle modification based on electrostatic attraction between negatively charged DNA tiles and positively charged nanoparticles. This approach does not disrupt nanoparticle surfaces and leverages the programmability of DNA nanotechnology to control DNA presentation. We demonstrated this approach using a variety of nanoparticles, including polymeric micelles, polystyrene beads, gold nanoparticles, and superparamagnetic iron oxide nanoparticles with sizes ranging from 5-20 nm in diameter. DNA cage formation was confirmed through transmission electron microscopy (TEM), neutralization of zeta potential, and a series of fluorescence experiments. DNA cages present "handle" sequences that can be used for reversible target attachment or self-assembly. Handle functionality was verified in solution, at the solid-liquid interface, and inside fixed cells, corresponding to applications in biosensing, DNA microarrays, and erasable immunocytochemistry. These experiments demonstrate the versatility of the electrostatic DNA caging approach and provide a new pathway to nanoparticle modification with DNA that will empower further applications of these materials in medicine and materials science.

bioengineering↗

bioRxiv2022

Thermally Reversible Pattern Formation in Arrays of Molecular Rotors

Control over the mesoscale to microscale patterning of materials is of great interest to the soft matter community. Inspired by DNA origami rotors, we introduce a 2D nearest-neighbor lattice of spinning rotors that exhibit discrete orientational states and interactions with their neighbors. Monte Carlo simulations of rotor lattices reveal that they exhibit a variety of interesting ordering behaviors and morphologies that can be modulated through rotor design parameters. The rotor arrays exhibit diverse patterns including closed loops, radiating loops, and bricklayer structures in their ordered states. They exhibit specific heat peaks at very low temperatures for small system sizes, and some systems exhibit multiple order-disorder transitions depending on inter-rotor interaction design. We devise an energy-based order parameter and show via umbrella sampling and histogram reweighting that this order parameter captures well the order-disorder transitions occurring in these systems. We fabricate real DNA origami rotors which themselves can order via programmable DNA base-pairing interactions and demonstrate both ordered and disordered phases, illustrating how rotor lattices may be realized experimentally and used for responsive organization. This work establishes the feasibility of realizing structural nanomaterials that exhibit locally mediated microscale patterns which could have applications in sensing and precision surface patterning.

biophysics↗

bioRxiv2022

Acoustic and Neurophysiological Aspects of Lombard Effect

PurposeThis study aims to describe variations in acoustic and electroencephalography measures when speaking in the presence of background noise (Lombard effect) in participants with typical voice and normal hearing. MethodTwenty-one participants with typical voices and normal hearing uttered simple vocal tasks in three sequential background conditions: Baseline (in quiet), Lombard (in noise), and Recovery (five minutes after removing the noise). Acoustic and electroencephalography signals were recorded in all conditions. The noise used in the Lombard condition consisted of speech-shaped noise at 80 dB SPL sent by headphones. Acoustic measure, and ERP responses were analyzed. ResultsDuring the Lombard condition, the participants increased the intensity of their voice, accompanied by an increase in CPP, and a decrease in H1-H2. The cortical response was characterized by the increased N1-P2 complex amplitude of the ERP elicited by the subjects own vocalizations in noise, The source localization showed neural activities in frontal and temporal cortical regions. ConclusionsThe variation in acoustic measures due to the Lombard Effect could be modulated by temporal, and cortical regions.

neuroscience↗

bioRxiv2022

Multiplexed Detection of Molecular Interactions with DNA Origami Engineered Cells in 3-D Collagen Matrices

The interactions of cells with signaling molecules present in their local microenvironment maintain cell proliferation, differentiation, and spatial organization, and mediate progression of diseases such as metabolic disorders and cancer. Real-time monitoring of the interactions between cells and their extracellular ligands in a 3-D microenvironment can inform detection and understanding of cell processes and development of effective therapeutic agents. DNA-origami technology allows for the design and fabrication of biocompatible and 3-D functional nanodevices via molecular self-assembly for various applications including molecular sensing. Here, we report a robust method to monitor live cell interactions with molecules in their surrounding environment in a 3-D tissue model using a microfluidic device. We used a DNA origami Cell Sensing Platform (CSP) to detect two specific nucleic acid sequences on the membrane of B cells and dendritic cells. We further demonstrated real-time detection of biomolecules with the DNA sensing platform on the surface of dendritic cells in a 3-D microfluidic tissue model. Our results establish the integration of live cells with membranes engineered with DNA nanodevices into microfluidic chips as a highly capable biosensor approach to investigate subcellular interactions in physiologically relevant 3-D environments under controlled biomolecular transport.

bioengineering↗

bioRxiv2021

Dual targeting of salt inducible kinases and CSF1R uncouples bone formation and bone resorption

Bone formation and resorption are typically coupled, such that the efficacy of anabolic osteoporosis treatments may be limited by bone destruction. The multi-kinase inhibitor YKL-05-099 potently inhibits salt inducible kinases (SIKs) and may represent a promising new class of bone anabolic agents. Here we report that YKL-05-099 increases bone formation in hypogonadal female mice without increasing bone resorption. Postnatal mice with inducible, global deletion of SIK2 and SIK3 show increased bone mass, increased bone formation, and, distinct from the effects of YKL-05-099, increased bone resorption. No cell-intrinsic role of SIKs in osteoclasts was noted. In addition to blocking SIKs, YKL-05-099 also binds and inhibits CSF1R, the receptor for the osteoclastogenic cytokine M-CSF. Modeling reveals that YKL-05-099 binds to SIK2 and CSF1R in a similar manner. Dual targeting of SIK2/3 and CSF1R induces bone formation without concomitantly increasing bone resorption and thereby may overcome limitations of most current anabolic osteoporosis therapies.

physiology↗

bioRxiv2020

Single amino acid-promoted reactions link a non-enzymatic chemical network to the early evolution of enzymatic pentose phosphate pathway

How metabolic pathways emerged in early evolution remains largely unknown. Recently discovered chemical networks driven by iron and sulfur resemble reaction sequences found within glycolysis, gluconeogenesis, the oxidative and reductive Krebs cycle, the Wood Ljungdahl as well as the S-adenosylmethionine pathways, components of the core cellular metabolic network. These findings suggest that the evolution of central metabolism was primed by environmental chemical reactions, implying that non-enzymatic reaction networks served as a "template" in the evolution of enzymatic activities. We speculated that the turning point for this transition would depend on the catalytic properties of the simplest structural components of proteins, single amino acids. Here, we systematically combine constituents of Fe(II)-driven non-enzymatic reactions resembling glycolysis and pentose phosphate pathway (PPP), with single proteinogenic amino acids. Multiple reaction rates are enhanced by amino acids. In particular, cysteine is able to replace (and/or complement) the metal ion Fe(II) in driving the non-enzymatic formation of the RNA-backbone metabolite ribose 5-phosphate from 6-phosphogluconate, a rate-limiting reaction of the oxidative PPP. In the presence of both Fe(II) and cysteine, a complex is formed, enabling the non-enzymatic reaction to proceed at a wide range of temperatures. At mundane temperatures, this minimal enzyme-like complex achieves a much higher specificity in the formation of ribose 5-phosphate than the Fe(II)-driven reaction at high temperatures. Hence, simple amino acids can accelerate key steps within metal-promoted metabolism-like chemical networks. Our results imply a stepwise scenario, in which environmental chemical networks served as primers in the early evolution of the metabolic network structure. Significance StatementThe evolutionary roots of metabolic pathways are barely understood. Here we show results consistent with a stepwise scenario during the evolution of (enzymatic) metabolism, starting from non-enzymatic chemical networks. By systematic screening of metabolic-like reactivities in vitro, and using high-throughput analytical techniques, we identify an iron/cysteine complex to act as a minimal enzymelike complex, which consists of a metal ion, an amino acid, and a sugar phosphate ligand. Integrated in a metal-driven, non-enzymatic pentose phosphate pathway, it promotes the formation of the RNA-backbone precursor ribose 5-phosphate at ambient temperature.

biochemistry↗

bioRxiv2019

Ribosome-targeting antibiotics impair T cell effector function and ameliorate autoimmunity by blocking mitochondrial protein synthesis

While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive, for reasons that remain unclear. Here we show that linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of Th17 effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromises the integrity of the electron transport chain (ETC). Ultimately, this leads to loss of mitochondrial metabolism and cytokine production in differentiating Th cells. In accordance, mice lacking Gfm1 in T cells are protected from EAE, demonstrating that this pathway plays a key role in maintaining T cell function and pathogenicity.

immunology↗

bioRxiv2018

Statistical optimization of culture conditions to improve cell density of Komagataeibacter medellinensis NBRC 3288: the first step towards to optimize bacterial cellulose production

The cell density yield of Komagataeibacter medellinensis NBRC 3288 was optimized applying response surface methodology (RSM) for the first time with this strain. Four factors were evaluated: glucose, initial pH, MgSO4 and KH2PO4, these were analyzed in a fractional factorial design to verify which were important to cell density yield. The best treatment assayed showed a yield of 1.09 OD600 and analysis of variance (ANOVA) selected glucose, inital pH and KH2PO4 as the significant factors. Next, a new experimental region with center point at glucose 0.5 %(wt./v), initial pH at 5.58 and KH2PO4 0.058 %(wt./v) was obtained by steepest ascent method and used to optimize biomass yield by Box-Behnken design. The results indicate that the optimum culture medium conditions predicted by the mathematical model (R2adj=72.09 %) were: glucose, 0.54 %(wt./v); peptone, 0.5 %(wt./v); yeast extract, 0.5 %(wt./v); KH2PO4, 0.059 %(wt./v); MgSO4, 0.025 %(wt./v); NaH2PO4, 0.267 %(wt./v) and pH, 5.18 (adjusted with citric acid, 0.2 %(wt./v)) to obtain an optimum cell density yield of 2.85 OD600 with an improve of 68 % respect to the best result obtained at the begin, as well as m improved 34.78 % and a reduction in cost of culture medium due to glucose concentration reduction of 75 %.\n\nImportanceDespite the important role of producer-strain play in bacterial cellulose production, until now there is not anu report about how to improve cell density, as a primary stage, that allows reach higher production of BC in later stages. In this study, was optimized the growth conditions of K. medellinensis (a BC producer in acid pH) in order to increase the cell density, as the primer stage in the whole process to optimize the production of BC, which is a very important supplie in different fields of science such as: food, materials, water treatment, tissue engineering, etc. The optimization process was achieved with savings in production costs of the culture medium, through the reduction of glucose concentration by 75 % and through the reduction of the microorganisms growth time.

microbiology↗

bioRxiv2018

Separation and Loss of Centrioles from Primordidal Germ Cells to Mature Oocytes in the Mouse

Oocytes, including those from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM); concomitantly, a rise in single CETN2 pairs is identified. CETN2 dissolution accelerates following meiosis resumption. Remarkably, a single CETN2 pair is retained in the PCM of most meiotic metaphase-I and -II spindle poles. Partial dissolution of the CETN2 foci occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, much later than previously thought.

developmental biology↗

bioRxiv2018

Bead Enhancement of EV Analysis

Extracellular vesicles (EVs) are recognized cancer biomarkers, however, clinical analysis has been difficult due to a lack of simple and sensitive assays. Here, we describe a bead-enhanced flow cytometry method, BEAD flow, using biotinylated EVs captured on streptavidin particles. With this method, we show analysis of patient-derived EVs using a panel of pancreatic cancer biomarkers. BEAD flow is easily translatable to any biomarker or cancer type and can be run with conventional flow cytometers, making it highly flexible and adaptable to diverse research and clinical needs.

cancer biology↗