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There is an increasing need for simple-to-use, noninvasive, and rapid tools to identify and separate various cell types or subtypes at the single-cell level with sufficient throughput. Often, the selection of cells based on their direct biological activity would be advantageous. These steps are critical in immune therapy, regenerative medicine, cancer diagnostics, and effective treatment. Today, live cell selection procedures incorporate some kind of biomolecular labeling or other invasive measures, which may impact cellular functionality or cause damage to the cells. In this study, we first introduce a highly accurate single-cell segmentation methodology by combining the high spatial resolution of a phase-contrast microscope with the adhesion kinetic recording capability of a resonant waveguide grating (RWG) biosensor. We present a classification workflow that incorporates the semiautomatic separation and classification of single cells from the measurement data captured by an RWG-based biosensor for adhesion kinetics data and a phase-contrast microscope for highly accurate spatial resolution. The methodology was tested with one healthy and six cancer cell types recorded with two functionalized coatings. The data set contains over 5000 single-cell samples for each surface and over 12,000 samples in total. We compare and evaluate the classification using these two types of surfaces (fibronectin and noncoated) with different segmentation strategies and measurement timespans applied to our classifiers. The overall classification performance reached nearly 95% with the best models showing that our proof-of-concept methodology could be adapted for real-life automatic diagnostics use cases. The label-free measurement technique has no impact on cellular functionality, directly measures cellular activity, and can be easily tuned to a specific application by varying the sensor coating. These features make it suitable for applications requiring further processing of selected cells.
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Selecting and isolating various cell types is a critical procedure in many applications, including immune therapy, regenerative medicine, and cancer research. Usually, these selection processes involve some labeling or another invasive step potentially affecting cellular functionality or damaging the cell. In the current proof of principle study, we first introduce an optical biosensor-based method capable of classification between healthy and numerous cancerous cell types in a label-free setup. We present high classification accuracy based on the monitored single-cell adhesion kinetic signals. We developed a high-throughput data processing pipeline to build a benchmark database of ~ 4500 single-cell adhesion measurements of a normal preosteoblast (MC3T3-E1) and various cancer (HeLa, LCLC-103H, MDA-MB-231, MCF-7) cell types. Several datasets were used with different cell-type selections to test the performance of deep learning-based classification models, reaching above 70-80% depending on the classification task. Beyond testing these models, we aimed to draw interpretable biological insights from their results; thus, we applied a deep neural network visualization method (grad-CAM) to reveal the basis on which these complex models made their decisions. Our proof-of-concept work demonstrated the success of a deep neural network using merely label-free adhesion kinetic data to classify single mammalian cells into different cell types. We propose our method for label-free single-cell profiling and in vitro cancer research involving adhesion. The employed label-free measurement is noninvasive and does not affect cellular functionality. Therefore, it could also be adapted for applications where the selected cells need further processing, such as immune therapy and regenerative medicine.
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Adhesión Celular , Análisis de la Célula Individual , Humanos , Análisis de la Célula Individual/métodos , Cinética , Ratones , Animales , Técnicas Biosensibles/métodos , Línea Celular TumoralRESUMEN
Classification of live or fixed cells based on their unlabeled microscopic images would be a powerful tool for cell biology and pathology. For such software, the first step is the generation of a ground truth database that can be used for training and testing AI classification algorithms. The Application of cells expressing fluorescent reporter proteins allows the building of ground truth datasets in a straightforward way. In this study, we present an automated imaging pipeline utilizing the Cellpose algorithm for the precise cell segmentation and measurement of fluorescent cellular intensities across multiple channels. We analyzed the cell cycle of HeLa-FUCCI cells expressing fluorescent red and green reporter proteins at various levels depending on the cell cycle state. To build the dataset, 37,000 fixed cells were automatically scanned using a standard motorized microscope, capturing phase contrast and fluorescent red/green images. The fluorescent pixel intensity of each cell was integrated to calculate the total fluorescence of cells based on cell segmentation in the phase contrast channel. It resulted in a precise intensity value for each cell in both channels. Furthermore, we conducted a comparative analysis of Cellpose 1.0 and Cellpose 2.0 in cell segmentation performance. Cellpose 2.0 demonstrated notable improvements, achieving a significantly reduced false positive rate of 2.7 % and 1.4 % false negative. The cellular fluorescence was visualized in a 2D plot (map) based on the red and green intensities of the FUCCI construct revealing the continuous distribution of cells in the cell cycle. This 2D map enables the selection and potential isolation of single cells in a specific phase. In the corresponding heatmap, two clusters appeared representing cells in the red and green states. Our pipeline allows the high-throughput and accurate measurement of cellular fluorescence providing extensive statistical information on thousands of cells with potential applications in developmental and cancer biology. Furthermore, our method can be used to build ground truth datasets automatically for training and testing AI cell classification. Our automated pipeline can be used to analyze thousands of cells within 2 h after putting the sample onto the microscope.
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Cell adhesion experiments are important in tissue engineering and for testing new biologically active surfaces, prostheses, and medical devices. Additionally, the initial state of adhesion (referred to as nascent adhesion) plays a key role and is currently being intensively researched. A critical step in handling all adherent cell types is their dissociation from their substrates for further processing. Various cell dissociation methods and reagents are used in most tissue culture laboratories (here, cell dissociation from the culture surface, cell harvesting, and cell detachment are used interchangeably). Typically, the dissociated cells are re-adhered for specific measurements or applications. However, the impact of the choice of dissociation method on cell adhesion in subsequent measurements, especially when comparing the adhesivity of various surfaces, is not well clarified. In this study, we demonstrate that the application of a label-free optical sensor can precisely quantify the effect of cell dissociation methods on cell adhesivity, both at the single-cell and population levels. The optical measurements allow for high-resolution monitoring of cellular adhesion without interfering with the physiological state of the cells. We found that the choice of reagent significantly alters cell adhesion on various surfaces. Our results clearly demonstrate that biological conclusions about cellular adhesion when comparing various surfaces are highly dependent on the employed dissociation method. Neglecting the choice of cellular dissociation can lead to misleading conclusions when evaluating cell adhesion data from various sources and comparing the adhesivity of two different surfaces (i.e., determining which surface is more or less adhesive).
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Adhesión Celular , Humanos , Propiedades de SuperficieRESUMEN
Understanding how plants adapt to changing environments and the potential contribution of transposable elements (TEs) to this process is a key question in evolutionary genomics. While TEs have recently been put forward as active players in the context of adaptation, few studies have thoroughly investigated their precise role in plant evolution. Here, we used the wild Mediterranean grass Brachypodium distachyon as a model species to identify and quantify the forces acting on TEs during the adaptation of this species to various conditions, across its entire geographic range. Using sequencing data from more than 320 natural B. distachyon accessions and a suite of population genomics approaches, we reveal that putatively adaptive TE polymorphisms are rare in wild B. distachyon populations. After accounting for changes in past TE activity, we show that only a small proportion of TE polymorphisms evolved neutrally (<10%), while the vast majority of them are under moderate purifying selection regardless of their distance to genes. TE polymorphisms should not be ignored when conducting evolutionary studies, as they can be linked to adaptation. However, our study clearly shows that while they have a large potential to cause phenotypic variation in B. distachyon, they are not favored during evolution and adaptation over other types of mutations (such as point mutations) in this species.
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Brachypodium , Elementos Transponibles de ADN , Elementos Transponibles de ADN/genética , Brachypodium/genética , Polimorfismo Genético , Genómica , Evolución MolecularRESUMEN
Synchronizing the timing of reproduction with the environment is crucial in the wild. Among the multiple mechanisms, annual plants evolved to sense their environment, the requirement of cold-mediated vernalization is a major process that prevents individuals from flowering during winter. In many annual plants including crops, both a long and short vernalization requirement can be observed within species, resulting in so-called early-(spring) and late-(winter) flowering genotypes. Here, using the grass model Brachypodium distachyon, we explored the link between flowering-time-related traits (vernalization requirement and flowering time), environmental variation, and diversity at flowering-time genes by combining measurements under greenhouse and outdoor conditions. These experiments confirmed that B. distachyon natural accessions display large differences regarding vernalization requirements and ultimately flowering time. We underline significant, albeit quantitative effects of current environmental conditions on flowering-time-related traits. While disentangling the confounding effects of population structure on flowering-time-related traits remains challenging, population genomics analyses indicate that well-characterized flowering-time genes may contribute significantly to flowering-time variation and display signs of polygenic selection. Flowering-time genes, however, do not colocalize with genome-wide association peaks obtained with outdoor measurements, suggesting that additional genetic factors contribute to flowering-time variation in the wild. Altogether, our study fosters our understanding of the polygenic architecture of flowering time in a natural grass system and opens new avenues of research to investigate the gene-by-environment interaction at play for this trait.
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Brachypodium , Flores , Herencia Multifactorial , Brachypodium/genética , Brachypodium/crecimiento & desarrollo , Flores/genética , Flores/crecimiento & desarrollo , Interacción Gen-Ambiente , Ambiente , Fenotipo , Sitios de Carácter CuantitativoRESUMEN
Primary cutaneous follicle center lymphoma (PCFCL) has an excellent prognosis using local treatment, whereas nodal follicular lymphoma (nFL), occasionally presenting with cutaneous spread, often requires systemic therapy. Distinction of the 2 diseases based on histopathology alone might be challenging. Copy number alterations (CNAs) have scarcely been explored on a genome-wide scale in PCFCL; however, they might serve as potential biomarkers during differential diagnosis and risk stratification. Low-coverage whole-genome sequencing is a robust, high-throughput method for genome-wide copy number profiling. In this study, we analyzed 28 PCFCL samples from 20 patients and compared the copy number profiles with a cohort of diagnostic samples of 64 nFL patients. Although the copy number profile of PCFCL was similar to that of nFL, PCFCL lacked amplifications of 18q, with the frequency peaking at 18q21.33 in nFL cases involving the BCL2 locus (PCFCL: 5.0% vs nFL: 31.3%, P = .018, Fisher exact test). Development of distant cutaneous spread was significantly associated with higher genomic instability including the proportion of genome altered (0.02 vs 0.13, P = .033) and number of CNAs (2 vs 9 P = .017), as well as the enrichment of 2p22.2-p15 amplification involving REL and XPO1 (6.3% vs 60.0%, P = .005), 3q23-q24 amplification (0.0% vs 50.0%, P = .004), 6q16.1-q23.3 deletion (6.3% vs 50.0%, P = .018), and 9p21.3 deletion covering CDKN2A and CDKN2B loci (0.0% vs 40.0%, P = .014, all Fisher exact test) in PCFCL. Analysis of sequential tumor samples in 2 cases harboring an unfavorable clinical course pointed to the acquisition of 2p amplification in the earliest common progenitor underlining its pivotal role in malignant transformation. By performing genome-wide copy number profiling on the largest patient cohort to date, we identified distinctive CNA alterations conceivably facilitating the differential diagnosis of PCFCL and secondary cutaneous involvement of nFL and potentially aiding the risk stratification of patients with PCFCL in the future.
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Variaciones en el Número de Copia de ADN , Linfoma Folicular , Neoplasias Cutáneas , Secuenciación Completa del Genoma , Humanos , Linfoma Folicular/genética , Linfoma Folicular/patología , Linfoma Folicular/diagnóstico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/diagnóstico , Femenino , Masculino , Persona de Mediana Edad , Anciano , Diagnóstico Diferencial , Pronóstico , Adulto , Anciano de 80 o más Años , Biomarcadores de Tumor/genéticaRESUMEN
Fluidic force microscopy (FluidFM) fuses the force sensitivity of atomic force microscopy with the manipulation capabilities of microfluidics by using microfabricated cantilevers with embedded fluidic channels. This innovation initiated new research and development directions in biology, biophysics, and material science. To acquire reliable and reproducible data, the calibration of the force sensor is crucial. Importantly, the hollow FluidFM cantilevers contain a row of parallel pillars inside a rectangular beam. The precise spring constant calibration of the internally structured cantilever is far from trivial, and existing methods generally assume simplifications that are not applicable to these special types of cantilevers. In addition, the Sader method, which is currently implemented by the FluidFM community, relies on the precise measurement of the quality factor, which renders the calibration of the spring constant sensitive to noise. In this study, the hydrodynamic function of these special types of hollow cantilevers was experimentally determined with different instruments. Based on the hydrodynamic function, a novel spring constant calibration method was adapted, which relied only on the two resonance frequencies of the cantilever, measured in air and in a liquid. Based on these results, our proposed method can be successfully used for the reliable, noise-free calibration of hollow FluidFM cantilevers.
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Small molecule natural compounds are gaining popularity in biomedicine due to their easy access to wide structural diversity and their proven health benefits in several case studies. Affinity measurements of small molecules below 100 Da molecular weight in a label-free and automatized manner using small amounts of samples have now become a possibility and reviewed in the present work. We also highlight novel label-free setups with excellent time resolution, which is important for kinetic measurements of biomolecules and living cells. We summarize how molecular-scale affinity data can be obtained from the in-depth analysis of cellular kinetic signals. Unlike traditional measurements, label-free biosensors have made such measurements possible, even without the isolation of specific cellular receptors of interest. Throughout this review, we consider epigallocatechin gallate (EGCG) as an exemplary compound. EGCG, a catechin found in green tea, is a well-established anti-inflammatory and anti-cancer agent. It has undergone extensive examination in numerous studies, which typically rely on fluorescent-based methods to explore its effects on both healthy and tumor cells. The summarized research topics range from molecular interactions with proteins and biological films to the kinetics of cellular adhesion and movement on novel biomimetic interfaces in the presence of EGCG. While the direct impact of small molecules on living cells and biomolecules is relatively well investigated in the literature using traditional biological measurements, this review also highlights the indirect influence of these molecules on the cells by modifying their nano-environment. Moreover, we underscore the significance of novel high-throughput label-free techniques in small molecular measurements, facilitating the investigation of both molecular-scale interactions and cellular processes in one single experiment. This advancement opens the door to exploring more complex multicomponent models that were previously beyond the reach of traditional assays.
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In the past decade, extracellular vesicles (EVs) have attracted substantial interest in biomedicine. With progress in the field, we have an increasing understanding of cellular responses to EVs. In this Technical Report, we describe the direct nanoinjection of EVs into the cytoplasm of single cells of different cell lines. By using robotic fluidic force microscopy (robotic FluidFM), nanoinjection of GFP positive EVs and EV-like particles into single live HeLa, H9c2, MDA-MB-231 and LCLC-103H cells proved to be feasible. This injection platform offered the advantage of high cell selectivity and efficiency. The nanoinjected EVs were initially localized in concentrated spot-like regions within the cytoplasm. Later, they were transported towards the periphery of the cells. Based on our proof-of-principle data, robotic FluidFM is suitable for targeting single living cells by EVs and may lead to information about intracellular EV cargo delivery at a single-cell level.
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Vesículas Extracelulares , Procedimientos Quirúrgicos Robotizados , Humanos , Microscopía de Fuerza Atómica , Transporte Biológico , Células HeLaRESUMEN
We report on a new approach of a low phase noise electro-optomechanical oscillator directly working in the GHz frequency range. The developed nanoscale oscillator is a one-dimensional photonic crystal made of gallium phosphide (GaP), heterogeneously integrated on silicon-on-insulator circuitry. Based on the strong interaction between the optical mode at the telecommunication wavelength and the mechanical mode in GHz, ultra-pure mechanical oscillations are enabled and directly imprinted on an optical carrier. Further stabilization is achieved with a delayed optoelectronic feedback loop using integrated electro-mechanical self-injection. We achieve a short-term stability of 0.7 Hz linewidth and a long-term stability with an Allan deviation below 10-7 Hz/Hz at 10 s averaging time, which represents an important step toward fully integrated optomechanical oscillators. Integrability and the low phase noise of this oscillator address some of the most important needs of optoelectronic oscillators and pave the way toward on-chip integrated microwave oscillators for microwave applications such as RADARs.
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Controlling cellular adhesion is a critical step in the development of biomaterials, and in cell- based biosensing assays. Usually, the adhesivity of cells is tuned by an appropriate biocompatible layer. Here, synthetic poly(diallyldimethylammonium chloride) (PDADMAC), natural chitosan, and heparin (existing in an extracellular matrix) were selected to assembly PDADMAC/heparin and chitosan/heparin films. The physicochemical properties of macroion multilayers were determined by streaming potential measurements (SPM), quartz crystal microbalance (QCM-D), and optical waveguide lightmode spectroscopy (OWLS). The topography of the wet films was imaged using atomic force microscopy (AFM). The adhesion of preosteoblastic cell line MC3T3-E1 on those well-characterized polysaccharide-based multilayers was evaluated using a resonant waveguide grating (RWG) based optical biosensor and digital holographic microscopy. The latter method was engaged to investigate long-term cellular behavior on the fabricated multilayers. (PDADMAC/heparin) films were proved to be the most effective in inducing cellular adhesion. The cell attachment to chitosan/heparin-based multilayers was negligible. It was found that efficient adhesion of the cells occurs onto homogeneous and rigid multilayers (PDADMAC/heparin), whereas the macroion films forming "sponge-like" structures (chitosan/heparin) are less effective, and could be employed when reduced adhesion is needed. Polysaccharide-based multilayers can be considered versatile systems for medical applications. One can postulate that the presented results are relevant not only for modeling studies but also for applied research.
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Técnicas Biosensibles , Quitosano , Quitosano/química , Polisacáridos/farmacología , Heparina/farmacología , Heparina/química , Adhesión Celular , Propiedades de SuperficieRESUMEN
Capnocytophaga canimorsus is a fastidious, capnophilic and facultative anaerobic Gram-negative rod found commonly in the oral flora of dogs that may cause zoonotic infections such as cellulitis and eye infections. In immunocompromised patients, it may cause fulminant sepsis. Meningitis due to C. canimorsus is, however, a rare manifestation. This is the first reported case of C. canimorsus meningitis in Australia in an immunocompetent veterinarian diagnosed by 16s ribosomal RNA polymerase chain reaction.
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Mordeduras y Picaduras , Infecciones por Bacterias Gramnegativas , Meningitis , Veterinarios , Humanos , Animales , Perros , ARN Ribosómico 16S/genética , Capnocytophaga/genética , Reacción en Cadena de la Polimerasa , Infecciones por Bacterias Gramnegativas/diagnóstico , Mordeduras y Picaduras/complicacionesRESUMEN
Objective: Response to treatment in multiple myeloma (MM) is routinely measured by serum and urine M-protein and free light chain (FLC), as described by the International Myeloma Working Group (IMWG) consensus statement. A non-negligible subgroup of patients however present without measurable biomarkers, others become oligo or non-secretory during recurrent relapses. The aim of our research was to evaluate soluble B-cell maturation antigen (sBCMA) as a monitoring marker measured concurrent with the standard monitoring in MM patients at diagnosis, at relapse and during follow up, in order to establish its potential usefulness in oligo and non-secretory disease. Method: sBCMA levels were measured in 149 patients treated for plasma cell dyscrasia (3 monoclonal gammopathy of unknown significance, 5 smoldering myeloma, 7 plasmacytoma, 8 AL amyloidosis and 126 MM) and 16 control subjects using a commercial ELISA kit. In 43 newly diagnosed patients sBCMA levels were measured at multiple timepoints during treatment, and compared to conventional IMWG response and progression free survival (PFS). Results: sBCMA levels among control subjects were significantly lower than among newly diagnosed or relapsed MM patients [20.8 (14.7-38.7) ng/mL vs. 676 (89.5-1,650) and 264 (20.7-1,603) ng/mL, respectively]. Significant correlations were found between sBCMA and the degree of bone marrow plasma cell infiltration. Out of the 37 newly diagnosed patients who have reached partial response or better per IMWG criteria, 33 (89%) have had at least a 50% drop in sBCMA level by therapy week 4. Cohorts made similarly to IMWG response criteria-achieving a 50% or 90% drop in sBCMA levels compared to level at diagnosis-had statistically significant differences in PFS. Conclusion: Our results confirmed that sBCMA levels are prognostic at important decision points in myeloma, and the percentage of BCMA change is predictive for PFS. This highlights the great potential use of sBCMA in oligo- and non-secretory myeloma.
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Antígeno de Maduración de Linfocitos B , Mieloma Múltiple , Humanos , Biomarcadores , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/terapia , PronósticoRESUMEN
The label-free interaction analysis of macromolecules and small molecules has increasing importance nowadays, both in diagnostics and therapeutics. In the blood vascular system, human serum albumin (HSA) is a vital globular transport protein with potential multiple ligand binding sites. Characterizing the binding affinity of compounds to HSA is essential in pharmaceutics and in developing new compounds for clinical application. Aryltetralin lignans from the roots of Anthriscus sylvestris are potential antitumor therapeutic candidates, but their molecular scale interactions with specific biomolecules are unrevealed. Here, we applied the label-free grating-coupled interferometry (GCI) biosensing method with a polycarboxylate-based hydrogel layer with immobilized HSA on top of it. With this engineered model surface, we could determine the binding parameters of two novel aryltetralin lignans, deoxypodophyllotoxin (DPT), and angeloyl podophyllotoxin (APT) to HSA. Exploiting the multi-channel referencing ability, the unique surface sensitivity, and the throughput of GCI, we first revealed the specific biomolecular interactions. Traditional label-free kinetic measurements were also compared with a novel, fast way of measuring affinity kinetics using less sample material (repeated analyte pulses of increasing duration (RAPID)). Experiments with well-characterized molecular interactions (furosemide to carbonic-anhydrase (CAII) and warfarin, norfloxacin to HSA) were performed to prove the reliability of the RAPID method. In all investigated cases, the RAPID and traditional measurement gave similar affinity values. In the case of DPT, the measurements and relevant modeling suggested two binding sites on HSA, with dissociation constant values of Kd1 = 1.8 ± 0.01 µM, Kd2 = 3 ± 0.02 µM. In the case of APT, the experiments resulted in Kd1 = 9 ± 1.7 µM, Kd2 = 28 ± 0.3 µM. The obtained binding values might suggest the potential medical application of DPT and APT without further optimization of their binding affinity to HSA. These results could be also adapted to other biomolecules and applications where sample consumption and the rapidity of the measurements are critical.
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Lignanos , Albúmina Sérica , Humanos , Albúmina Sérica/química , Unión Proteica , Reproducibilidad de los Resultados , Sitios de Unión , Albúmina Sérica Humana/metabolismoRESUMEN
The purpose of the recent work is to give a better explanation of how Dean vortices affect lateral focusing, and to understand how cell morphology can alter the focusing position compared to spherical particles. The position and extent of the focused region were investigated using polystyrene fluorescent beads with different bead diameters (Ø = 0.5, 1.1, 1.97, 2.9, 4.8, 5.4, 6.08, 10.2, 15.8, 16.5 µm) at different flow rates (0.5, 1, 2 µL/s). Size-dependent focusing generated a precise map of the equilibrium positions of the spherical beads at the end of the periodically altering channels, which gave a good benchmark for focusing multi-dimensional particles and cells. The biological samples used for experiments were rod-shaped Escherichia coli (E. coli), discoid biconcave-shaped red blood cells (RBC), round or ovoid-shaped yeast, Saccharomyces cerevisiae, and soft-irregular-shaped HeLa cancer-cell-line cells to understand how the shape of the cells affects the focusing position at the end of the channel.
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Técnicas Analíticas Microfluídicas , Microfluídica , Humanos , Microfluídica/métodos , Escherichia coli , Eritrocitos , Saccharomyces cerevisiae , Células HeLa , Técnicas Analíticas Microfluídicas/métodosRESUMEN
Functionalized nanoparticles (NPs) are widely used in targeted drug delivery and biomedical imaging due to their penetration into living cells. The outer coating of most cells is a sugar-rich layer of the cellular glycocalyx, presumably playing an important part in any uptake processes. However, the exact role of the cellular glycocalyx in NP uptake is still uncovered. Here, we in situ monitored the cellular uptake of gold NPsâfunctionalized with positively charged alkaline thiol (TMA)âinto adhered cancer cells with or without preliminary glycocalyx digestion. Proteoglycan (PG) components of the glycocalyx were treated by the chondroitinase ABC enzyme. It acts on chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate and slowly on hyaluronate. The uptake measurements of HeLa cells were performed by applying a high-throughput label-free optical biosensor based on resonant waveguide gratings. The positively charged gold NPs were used with different sizes [d = 2.6, 4.2, and 7.0 nm, small (S), medium (M), and large(L), respectively]. Negatively charged citrate-capped tannic acid (CTA, d = 5.5 nm) NPs were also used in control experiments. Real-time biosensor data confirmed the cellular uptake of the functionalized NPs, which was visually proved by transmission electron microscopy. It was found that the enzymatic digestion facilitated the entry of the positively charged S- and M-sized NPs, being more pronounced for the M-sized. Other enzymes digesting different components of the glycocalyx were also employed, and the results were compared. Glycosaminoglycan digesting heparinase III treatment also increased, while glycoprotein and glycolipid modifying neuraminidase decreased the NP uptake by HeLa cells. This suggests that the sialic acid residues increase, while heparan sulfate decreases the uptake of positively charged NPs. Our results raise the hypothesis that cellular uptake of 2-4 nm positively charged NPs is facilitated by glycoprotein and glycolipid components of the glycocalyx but inhibited by PGs.
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Glicocálix , Nanopartículas del Metal , Humanos , Oro/química , Células HeLa , Nanopartículas del Metal/química , Glicosaminoglicanos , Sulfatos de CondroitinaAsunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Pandemias , Secuenciación Completa del Genoma , HospitalesRESUMEN
Transmembrane glycoprotein integrins play crucial roles in biochemical processes, and by their inhibition or activation, different signal pathways can be disrupted, leading to abnormal physiological functions. We have previously demonstrated the inhibitory effect of glyphosate herbicide's active ingredient on cell adhesion and its αvß3 integrin antagonist effect. Therefore, it appeared particularly exciting to investigate inhibition of glyphosate and its metabolites on a wider range of Arg-Gly-Asp (RGD) binding integrins, namely αvß3, α5ß1 and αllbß3. Thus, the purpose of this study was to assess how extended the inhibitory effect observed for glyphosate on the integrin αvß3 is in terms of other RGD integrins and other structurally or metabolically related derivatives of glyphosate. Five different experimental setups using enzyme-linked immunosorbent assays were applied: (i) αvß3 binding to a synthetic polymer containing RGD; (ii) αvß3 binding to its extracellular matrix (ECM) protein, vitronectin; (iii) α5ß1 binding to the above polymer containing RGD; (iv) αllbß3 binding to its ECM protein, fibrinogen and (v) αvß3 binding to the SARS-CoV-2 spike protein receptor binding domain. Total inhibition of αvß3 binding to RGD was detected for glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), as well as for acetylglycine on α5ß1 binding to RGD.
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COVID-19 , Herbicidas , Humanos , Integrina alfaVbeta3/metabolismo , Vitronectina , Herbicidas/farmacología , SARS-CoV-2 , Oligopéptidos/química , Ensayo de Inmunoadsorción Enzimática , Fibrinógeno , PolímerosRESUMEN
In a recent issue in Nature, Chen et al. present Live-seq, a single-cell transcriptomic profiling method using picoliter scale single-cell cytoplasmic biopsies instead of complete cell lysis. Since the cells quickly recover and basically remain unaffected after the cytoplasmic extraction, the authors transform single-cell RNA sequencing (scRNA-seq) from an end point to a temporal analysis platform.