RESUMEN
How disease-associated mutations impair protein activities in the context of biological networks remains mostly undetermined. Although a few renowned alleles are well characterized, functional information is missing for over 100,000 disease-associated variants. Here we functionally profile several thousand missense mutations across a spectrum of Mendelian disorders using various interaction assays. The majority of disease-associated alleles exhibit wild-type chaperone binding profiles, suggesting they preserve protein folding or stability. While common variants from healthy individuals rarely affect interactions, two-thirds of disease-associated alleles perturb protein-protein interactions, with half corresponding to "edgetic" alleles affecting only a subset of interactions while leaving most other interactions unperturbed. With transcription factors, many alleles that leave protein-protein interactions intact affect DNA binding. Different mutations in the same gene leading to different interaction profiles often result in distinct disease phenotypes. Thus disease-associated alleles that perturb distinct protein activities rather than grossly affecting folding and stability are relatively widespread.
Asunto(s)
Enfermedad/genética , Mutación Missense , Mapas de Interacción de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Sistemas de Lectura Abierta , Pliegue de Proteína , Estabilidad ProteicaRESUMEN
Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFß) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue-laminin-111-switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFß signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFß inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFß inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies in vitro.
Asunto(s)
Diferenciación Celular , Técnicas Citológicas , Laminina , Células Madre , Trofoblastos , Humanos , Diferenciación Celular/efectos de los fármacos , Colforsina/farmacología , Colforsina/metabolismo , Antígenos HLA-G/genética , Antígenos HLA-G/metabolismo , Laminina/farmacología , Células Madre/citología , Células Madre/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismo , Medios de Cultivo/química , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas Citológicas/métodosRESUMEN
Aging is characterized by a functional decline in organism fitness over time due to a complex combination of genetic and environmental factors [ 1-4]. With an increasing elderly population at risk of age-associated diseases, there is a pressing need for research dedicated to promoting health and longevity through anti-aging interventions. The roundworm Caenorhabditis elegans is an established model organism for aging studies due to its short life cycle, ease of culture, and conserved aging pathways. These benefits also make the worm well-suited for high-throughput screening (HTS) methods to study biomarkers of the molecular changes, cellular dysfunction, and physiological decline associated with aging. Within this review, we offer a summary of recent advances in HTS techniques to study biomarkers of aging in C. elegans.
Asunto(s)
Envejecimiento , Biomarcadores , Caenorhabditis elegans , Ensayos Analíticos de Alto Rendimiento , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Animales , Envejecimiento/metabolismo , Biomarcadores/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , LongevidadRESUMEN
The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2- stem cell comparable with placenta-derived TS cells-they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development.
Asunto(s)
Factor de Transcripción CDX2/metabolismo , Células Madre Embrionarias/citología , Placenta/citología , Células Madre Pluripotentes/citología , Trofoblastos/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , Linaje de la Célula , Medios de Cultivo , Células Madre Embrionarias/metabolismo , Femenino , Humanos , Placenta/metabolismo , Células Madre Pluripotentes/metabolismo , Embarazo , Trofoblastos/metabolismoRESUMEN
BACKGROUND: Access to quantitative information is crucial to obtain a deeper understanding of biological systems. In addition to being low-throughput, traditional image-based analysis is mostly limited to error-prone qualitative or semi-quantitative assessment of phenotypes, particularly for complex subcellular morphologies. The PVD neuron in Caenorhabditis elegans, which is responsible for harsh touch and thermosensation, undergoes structural degeneration as nematodes age characterized by the appearance of dendritic protrusions. Analysis of these neurodegenerative patterns is labor-intensive and limited to qualitative assessment. RESULTS: In this work, we apply deep learning to perform quantitative image-based analysis of complex neurodegeneration patterns exhibited by the PVD neuron in C. elegans. We apply a convolutional neural network algorithm (Mask R-CNN) to identify neurodegenerative subcellular protrusions that appear after cold-shock or as a result of aging. A multiparametric phenotypic profile captures the unique morphological changes induced by each perturbation. We identify that acute cold-shock-induced neurodegeneration is reversible and depends on rearing temperature and, importantly, that aging and cold-shock induce distinct neuronal beading patterns. CONCLUSION: The results of this work indicate that implementing deep learning for challenging image segmentation of PVD neurodegeneration enables quantitatively tracking subtle morphological changes in an unbiased manner. This analysis revealed that distinct patterns of morphological alteration are induced by aging and cold-shock, suggesting different mechanisms at play. This approach can be used to identify the molecular components involved in orchestrating neurodegeneration and to characterize the effect of other stressors on PVD degeneration.
Asunto(s)
Envejecimiento/fisiología , Caenorhabditis elegans/fisiología , Respuesta al Choque por Frío/fisiología , Aprendizaje Profundo , Neuronas/fisiología , Fenotipo , AnimalesRESUMEN
The rapid expansion of CRISPR in biotechnology, medicine, and bioprocessing poses an urgent need for advanced manufacturing of Cas nucleases. The lack of Cas-targeting ligands, however, prevents the development of platform processes for purifying this class of molecules. This work represents the first effort at developing short synthetic Cas9-binding peptides and demonstrates their applicability as affinity ligands for the purification of a Cas nuclease. Candidate Cas9-targeting peptides were initially identified by screening a solid-phase peptide library against a model mixture of Streptococcus pyogenes Cas9 spiked in Escherichia coli cell lysate. An ensemble of homologous sequences was identified, conjugated on Toyopearl resin, and evaluated by Cas9 binding studies to identify sequences providing selective Cas9 capture and efficient release. In silico docking studies were also performed to evaluate the binding energy and site of the various peptides on Cas9. Notably, sequences GYYRYSEY and YYHRHGLQ were shown to target the RecII domain of Cas9, which is not involved in nuclease activity and was targeted as an ideal binding site. The peptide ligands were validated by purifying Cas9 from the E. coli lysate in dynamic conditions and through measurements of binding capacity and strength (Qmax and KD). The resulting values of Qmax = 4-5 mg Cas9 per mL of resin and KD â¼ 0.1-0.3 µM, product recovery (86-89%), and purity (91%-93%) indicate that both peptides, and YYHRHGLQ in particular, can serve as capture ligands in a platform purification process of Cas9.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Péptidos/metabolismo , Adsorción , Secuencia de Aminoácidos , Sitios de Unión , Proteína 9 Asociada a CRISPR/aislamiento & purificación , Descubrimiento de Drogas , Ligandos , Simulación del Acoplamiento Molecular , Biblioteca de Péptidos , Resinas Sintéticas/química , Especificidad por SustratoRESUMEN
The nematode Caenorhabditis elegans is a powerful model organism that has been widely used to study molecular biology, cell development, neurobiology, and aging. Despite their use for the past several decades, the conventional techniques for growth, imaging, and behavioral analysis of C. elegans can be cumbersome, and acquiring large data sets in a high-throughput manner can be challenging. Developments in microfluidic "lab-on-a-chip" technologies have improved studies of C. elegans by increasing experimental control and throughput. Microfluidic features such as on-chip control layers, immobilization channels, and chamber arrays have been incorporated to develop increasingly complex platforms that make experimental techniques more powerful. Genetic and chemical screens are performed on C. elegans to determine gene function and phenotypic outcomes of perturbations, to test the effect that chemicals have on health and behavior, and to find drug candidates. In this review, we will discuss microfluidic technologies that have been used to increase the throughput of genetic and chemical screens in C. elegans. We will discuss screens for neurobiology, aging, development, behavior, and many other biological processes. We will also discuss robotic technologies that assist in microfluidic screens, as well as alternate platforms that perform functions similar to microfluidics.
Asunto(s)
Caenorhabditis elegans/genética , Ensayos Analíticos de Alto Rendimiento , Dispositivos Laboratorio en un Chip , Transporte de Proteínas/genética , Animales , Movimiento Celular/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
During synapse development, synaptic proteins must be targeted to sites of presynaptic release. Directed transport as well as local sequestration of synaptic vesicle precursors (SVPs), membranous organelles containing many synaptic proteins, might contribute to this process. Using neuron-wide time-lapse microscopy, we studied SVP dynamics in the DA9 motor neuron in Caenorhabditis elegans. SVP transport was highly dynamic and bi-directional throughout the entire neuron, including the dendrite. While SVP trafficking was anterogradely biased in axonal segments prior to the synaptic domain, directionality of SVP movement was stochastic in the dendrite and distal axon. Furthermore, frequency of movement and speed were variable between different compartments. These data provide evidence that SVP transport is differentially regulated in distinct neuronal domains. It also suggests that polarized SVP transport in concert with local vesicle capturing is necessary for accurate presynapse formation and maintenance. SVP trafficking analysis of two hypomorphs for UNC-104/KIF1A in combination with mathematical modeling identified directionality of movement, entry of SVPs into the axon as well as axonal speeds as the important determinants of steady-state SVP distributions. Furthermore, detailed dissection of speed distributions for wild-type and unc-104/kif1a mutant animals revealed an unexpected role for UNC-104/KIF1A in dendritic SVP trafficking.
Asunto(s)
Transporte Axonal , Caenorhabditis elegans/metabolismo , Neuronas Motoras/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Caenorhabditis elegans (C. elegans) is an excellent model system to study neurodegenerative diseases, such as Parkinson's disease, as it enables analysis of both neuron morphology and function in live animals. Multiple structural changes in neurons, such as cephalic dendrite morphological abnormalities, have been considered hallmarks of neurodegeneration in this model, but their relevance to changes in neuron function are not entirely clear. We sought to test whether hallmark morphological changes associated with chemically induced dopaminergic neuron degeneration, such as dendrite blebbing, breakage, and loss, are indicative of neuronal malfunction and result in changes in behavior. We adapted an established dopaminergic neuronal function assay by measuring paralysis in the presence of exogenous dopamine, which revealed clear differences between cat-2 dopamine deficient mutants, wildtype worms, and dat-1 dopamine abundant mutants. Next, we integrated an automated image processing algorithm and a microfluidic device to segregate worm populations by their cephalic dendrite morphologies. We show that nematodes with dopaminergic dendrite degeneration markers, such as blebbing or breakage, paralyze at higher rates in a dopamine solution, providing evidence that dopaminergic neurodegeneration morphologies are correlated with functional neuronal outputs.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Dopamina , Animales Modificados Genéticamente , Neuronas Dopaminérgicas , Modelos Animales de EnfermedadRESUMEN
Saccharomyces boulardii (Sb) is an emerging probiotic chassis for delivering biomolecules to the mammalian gut, offering unique advantages as the only eukaryotic probiotic. However, precise control over gene expression and gut residence time in Sb have remained challenging. To address this, we developed five ligand-responsive gene expression systems and repaired galactose metabolism in Sb, enabling inducible gene expression in this strain. Engineering these systems allowed us to construct AND logic gates, control the surface display of proteins, and turn on protein production in the mouse gut in response to dietary sugar. Additionally, repairing galactose metabolism expanded Sb's habitat within the intestines and resulted in galactose-responsive control over gut residence time. This work opens new avenues for precise dosing of therapeutics by Sb via control over its in vivo gene expression levels and localization within the gastrointestinal tract.
Asunto(s)
Galactosa , Probióticos , Saccharomyces boulardii , Animales , Ratones , Galactosa/metabolismo , Saccharomyces boulardii/genética , Saccharomyces boulardii/metabolismo , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/metabolismo , DietaRESUMEN
Any modern information system is expected to feature a set of primordial features and functions: a substrate stably carrying data; the ability to repeatedly write, read, erase, reload and compute on specific data from that substrate; and the overall ability to execute such functions in a seamless and programmable manner. For nascent molecular information technologies, proof-of-principle realization of this set of primordial capabilities would advance the vision for their continued development. Here we present a DNA-based store and compute engine that captures these primordial capabilities. This system comprises multiple image files encoded into DNA and adsorbed onto ~50-µm-diameter, highly porous, hierarchically branched, colloidal substrate particles comprised of naturally abundant cellulose acetate. Their surface areas are over 200 cm2 mg-1 with binding capacities of over 1012 DNA oligos mg-1, 10 TB mg-1 or 104 TB cm-3. This 'dendricolloid' stably holds DNA files better than bare DNA with an extrapolated ability to be repeatedly lyophilized and rehydrated over 170 times compared with 60 times, respectively. Accelerated ageing studies project half-lives of ~6,000 and 2 million years at 4 °C and -18 °C, respectively. The data can also be erased and replaced, and non-destructive file access is achieved through transcribing from distinct synthetic promoters. The resultant RNA molecules can be directly read via nanopore sequencing and can also be enzymatically computed to solve simplified 3 × 3 chess and sudoku problems. Our study establishes a feasible route for utilizing the high information density and parallel computational advantages of nucleic acids.
RESUMEN
In many organisms, dietary restriction (DR) leads to lifespan extension through the activation of cell protection and pro-longevity gene expression programs. In the nematode C. elegans, the DAF-16 transcription factor is a key aging regulator that governs the Insulin/IGF-1 signaling pathway and undergoes translocation from the cytoplasm to the nucleus of cells when animals are exposed to food limitation. However, how large is the influence of DR on DAF-16 activity, and its subsequent impact on lifespan has not been quantitatively determined. In this work, we assess the endogenous activity of DAF-16 under various DR regimes by coupling CRISPR/Cas9-enabled fluorescent tagging of DAF-16 with quantitative image analysis and machine learning. Our results indicate that DR regimes induce strong endogenous DAF-16 activity, although DAF-16 is less responsive in aged individuals. DAF-16 activity is in turn a robust predictor of mean lifespan in C. elegans, accounting for 78% of its variability under DR. Analysis of tissue-specific expression aided by a machine learning tissue classifier reveals that, under DR, the largest contribution to DAF-16 nuclear intensity originates from the intestine and neurons. DR also drives DAF-16 activity in unexpected locations such as the germline and intestinal nucleoli.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Longevidad/genética , Proteínas de Caenorhabditis elegans/metabolismo , Transducción de Señal , Insulina/metabolismo , Factores de Transcripción Forkhead/metabolismoRESUMEN
Caenorhabditis elegans ( C. elegans ) has served as a simple model organism to study dopaminergic neurodegeneration, as it enables quantitative analysis of cellular and sub-cellular morphologies in live animals. These isogenic nematodes have a rapid life cycle and transparent body, making high-throughput imaging and evaluation of fluorescently tagged neurons possible. However, the current state-of-the-art method for quantifying dopaminergic degeneration requires researchers to manually examine images and score dendrites into groups of varying levels of neurodegeneration severity, which is time consuming, subject to bias, and limited in data sensitivity. We aim to overcome the pitfalls of manual neuron scoring by developing an automated, unbiased image processing algorithm to quantify dopaminergic neurodegeneration in C. elegans . The algorithm can be used on images acquired with different microscopy setups and only requires two inputs: a maximum projection image of the four cephalic neurons in the C. elegans head and the pixel size of the userâ™s camera. We validate the platform by detecting and quantifying neurodegeneration in nematodes exposed to rotenone, cold shock, and 6-hydroxydopamine using 63x epifluorescence, 63x confocal, and 40x epifluorescence microscopy, respectively. Analysis of tubby mutant worms with altered fat storage showed that, contrary to our hypothesis, increased adiposity did not sensitize to stressor-induced neurodegeneration. We further verify the accuracy of the algorithm by comparing code-generated, categorical degeneration results with manually scored dendrites of the same experiments. The platform, which detects 19 different metrics of neurodegeneration, can provide comparative insight into how each exposure affects dopaminergic neurodegeneration patterns.
RESUMEN
Caenorhabditis elegans (C. elegans) is an excellent model system to study neurodegenerative diseases, such as Parkinson's disease, as it enables analysis of both neuron morphology and function in live animals. Multiple structural changes in neurons, such as cephalic dendrite morphological abnormalities, have been considered hallmarks of neurodegeneration in this model, but their relevance to changes in neuron function are not entirely clear. We sought to test whether hallmark morphological changes associated with chemically induced dopaminergic neuron degeneration, such as dendrite blebbing, breakage, and loss, are indicative of neuronal malfunction and result in changes in behavior. We adapted an established dopaminergic neuronal function assay by measuring paralysis in the presence of exogenous dopamine, which revealed clear differences between cat-2 dopamine deficient mutants, wildtype worms, and dat-1 dopamine abundant mutants. Next, we integrated an automated image processing algorithm and a microfluidic device to segregate worm populations by their cephalic dendrite morphologies. We show that nematodes with dopaminergic dendrite degeneration markers, such as blebbing or breakage, paralyze at higher rates in a dopamine solution, providing evidence that dopaminergic neurodegeneration morphologies are correlated with functional neuronal outputs.
RESUMEN
Caenorhabditis elegans (C. elegans) has served as a simple model organism to study dopaminergic neurodegeneration, as it enables quantitative analysis of cellular and sub-cellular morphologies in live animals. These isogenic nematodes have a rapid life cycle and transparent body, making high-throughput imaging and evaluation of fluorescently tagged neurons possible. However, the current state-of-the-art method for quantifying dopaminergic degeneration requires researchers to manually examine images and score dendrites into groups of varying levels of neurodegeneration severity, which is time consuming, subject to bias, and limited in data sensitivity. We aim to overcome the pitfalls of manual neuron scoring by developing an automated, unbiased image processing algorithm to quantify dopaminergic neurodegeneration in C. elegans. The algorithm can be used on images acquired with different microscopy setups and only requires two inputs: a maximum projection image of the four cephalic neurons in the C. elegans head and the pixel size of the user's camera. We validate the platform by detecting and quantifying neurodegeneration in nematodes exposed to rotenone, cold shock, and 6-hydroxydopamine using 63x epifluorescence, 63x confocal, and 40x epifluorescence microscopy, respectively. Analysis of tubby mutant worms with altered fat storage showed that, contrary to our hypothesis, increased adiposity did not sensitize to stressor-induced neurodegeneration. We further verify the accuracy of the algorithm by comparing code-generated, categorical degeneration results with manually scored dendrites of the same experiments. The platform, which detects 20 different metrics of neurodegeneration, can provide comparative insight into how each exposure affects dopaminergic neurodegeneration patterns.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/fisiología , Animales Modificados Genéticamente , Dopamina , Oxidopamina , Neuronas Dopaminérgicas/fisiologíaRESUMEN
The wetting behavior of solid surfaces can be altered dramatically by introducing surface roughness on the nanometer scale. Some of nature's most fascinating wetting phenomena are associated with surface roughness; they have inspired both fundamental research and the adoption of surface roughness as a design parameter for man-made functional coatings. So far the attention has focused primarily on macroscopic surfaces, but one should expect the wetting properties of colloidal particles to be strongly affected by roughness, too. Particle wettability, in turn, is a key parameter for the adsorption of particles at liquid interfaces and for the industrially important use of particles as emulsion stabilizers; yet, the consequence of particle roughness for emulsion stability remains poorly understood. In order to investigate the matter systematically, we have developed a surface treatment, applicable to micrometer-sized particles and macroscopic surfaces alike, that produces surface coatings with finely tunable nanoscale roughness and identical surface chemistry. Coatings with different degrees of roughness were characterized with regard to their morphology, charging, and wetting properties, and the results were correlated with the stability of emulsions prepared with coated particles of different roughness. We find that the maximum capillary pressure, a metric of the emulsions' resistance to droplet coalescence, varies significantly and in a nonmonotonic fashion with particle roughness. Surface topography and contact angle hysteresis suggest that particle roughness benefits the stability of our emulsions as long as wetting occurs homogeneously (Wenzel regime), whereas the transition toward heterogeneous wetting (Cassie-Baxter regime) is associated with a loss of stability.
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Genetic screens are widely used to identify genes that control specific biological functions. In Caenorhabditis elegans, forward genetic screens rely on the isolation of reproductively active mutants that can self-propagate clonal populations. Screens that target post-reproductive phenotypes, such as lifespan, are thus challenging. We combine microfluidic technologies and image processing to perform high-throughput automated screening for short-lived mutants using protein aggregation as a marker for aging. We take advantage of microfluidics for maintaining a reproductively active adult mutagenized population and for performing serial high-throughput analysis and sorting of animals with increased protein aggregation, using fluorescently-labeled PAB-1 as a readout. We demonstrate that lifespan mutants can be identified by screening for accelerated protein aggregation through quantitative analysis of fluorescently labeled aggregates while avoiding conditional sterilization or manual separation of parental and progeny populations. We also show that aged wildtypes and premature aggregation mutants differ in aggregate morphology, suggesting aggregate growth is time-dependent.
RESUMEN
Analysis of Caenorhabditis elegans natural movement and optogenetic control of its muscle cells enable controlled locomotion.
Asunto(s)
Proteínas de Caenorhabditis elegans , Optogenética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , LocomociónRESUMEN
Size-based microfluidic filtration systems can be affected by clogging, which prevents their use in high-throughput and continuous applications. To address these concerns, we have developed two microfluidic lobe filters bioinspired by the filtration mechanism of two species of manta ray. These chips enable filtration of particles around 10-30 µm with precise control and high throughput by using two arrays of equally spaced filter lobes. For each filter design, we investigated multiple inlet flow rates and particle sizes to identify successful operational parameters. Filtration efficiency increases with fluid flow rate, suggesting that particle inertial effects play a key role in lobe filter separation. Microparticle filtration efficiencies up to 99% were obtainable with inlet flow rates of 20 mL min-1. Each filter design successfully increased microparticle concentrations by a factor of two or greater at different inlet flow rates ranging from 6-16 mL min-1. At higher inlet flow rates, ANSYS Fluent simulations of each device revealed a complex velocity profile that contains three local maxima and two inflection points. Ultimately, we show that distances from the lobe array to the closest local maxima and inflection point of the velocity profile can be used to successfully estimate lobe filtration efficiency at each operational flow rate.
Asunto(s)
Técnicas Analíticas Microfluídicas , Microfluídica , Filtración , Tamaño de la PartículaRESUMEN
In Caenorhabditis elegans, optogenetic stimulation has been widely used to assess neuronal function, control animal movement, or assay circuit responses to controlled stimuli. Most studies are performed on single animals and require high-end components such as lasers and shutters. We present an accessible platform that enables controlled optogenetic stimulation of C. elegans in two modes: single animal stimulation with locomotion tracking and entire population stimulation for neuronal exercise regimens. The system consists of accessible electronic components: a high-power light-emitting diode, Arduino board, and relay are integrated with MATLAB to enable programmable optogenetic stimulation regimens. This system provides flexibility in optogenetic stimulation in freely moving animals while providing quantitative information of optogenetic-driven locomotion responses. We show the applicability of this platform in single animals by stimulation of cholinergic motor neurons in C. elegans and quantitative assessment of contractile responses. In addition, we tested synaptic plasticity by coupling the entire-population stimulation mode with measurements of synaptic strength using an aldicarb assay, where clear changes in synaptic strength were observed after regimens of neuronal exercise. This platform is composed of inexpensive components, while providing the illumination strength of high-end systems, which require expensive lasers, shutters, or automated stages. This platform requires no moving parts but provides flexibility in stimulation regimens.