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1.
PLoS Pathog ; 20(7): e1012392, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-39052670

RESUMEN

Cell migration modes can vary, depending on a number of environmental and intracellular factors. The high motility of the pathogenic amoeba Entamoeba histolytica is a decisive factor in its ability to cross the human colonic barrier. We used quantitative live imaging techniques to study the migration of this parasite on fibronectin, a key tissue component. Entamoeba histolytica amoebae on fibronectin contain abundant podosome-like structures. By using a laminar flow chamber, we determined that the adhesion forces generated on fibronectin were twice those on non-coated glass. When migrating on fibronectin, elongated amoeboid cells converted into fan-shaped cells characterized by the presence of a dorsal column of F-actin and a broad cytoplasmic extension at the front. The fan shape depended on the Arp2/3 complex, and the amoebae moved laterally and more slowly. Intracellular measurements of physical variables related to fluid dynamics revealed that cytoplasmic pressure gradients were weaker within fan-shaped cells; hence, actomyosin motors might be less involved in driving the cell body forward. We also found that the Rho-associated coiled-coil containing protein kinase regulated podosome dynamics. We conclude that E. histolytica spontaneously changes its migration mode as a function of the substrate composition. This adaptive ability might favour E. histolytica's invasion of human colonic tissue. By combining microfluidic experiments, mechanical modelling, and image analysis, our work also introduces a computational pipeline for the study of cell migration.


Asunto(s)
Movimiento Celular , Entamoeba histolytica , Fibronectinas , Entamoeba histolytica/metabolismo , Entamoeba histolytica/fisiología , Fibronectinas/metabolismo , Humanos , Movimiento Celular/fisiología , Entamebiasis/parasitología , Entamebiasis/metabolismo , Actinas/metabolismo , Podosomas/metabolismo , Adhesión Celular/fisiología , Proteínas Protozoarias/metabolismo
2.
Int J Mol Sci ; 24(10)2023 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-37240072

RESUMEN

Entamoeba histolytica is a protozoan parasite and the causative agent of amoebiasis in humans. This amoeba invades human tissues by taking advantage of its actin-rich cytoskeleton to move, enter the tissue matrix, kill and phagocyte the human cells. During tissue invasion, E. histolytica moves from the intestinal lumen across the mucus layer and enters the epithelial parenchyma. Faced with the chemical and physical constraints of these diverse environments, E. histolytica has developed sophisticated systems to integrate internal and external signals and to coordinate cell shape changes and motility. Cell signalling circuits are driven by interactions between the parasite and extracellular matrix, combined with rapid responses from the mechanobiome in which protein phosphorylation plays an important role. To understand the role of phosphorylation events and related signalling mechanisms, we targeted phosphatidylinositol 3-kinases followed by live cell imaging and phosphoproteomics. The results highlight 1150 proteins, out of the 7966 proteins within the amoebic proteome, as members of the phosphoproteome, including signalling and structural molecules involved in cytoskeletal activities. Inhibition of phosphatidylinositol 3-kinases alters phosphorylation in important members of these categories; a finding that correlates with changes in amoeba motility and morphology, as well as a decrease in actin-rich adhesive structures.


Asunto(s)
Amebiasis , Entamoeba histolytica , Humanos , Actinas/metabolismo , Entamoeba histolytica/metabolismo , Citoesqueleto de Actina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Protozoarias/metabolismo
3.
IEEE Trans Med Imaging ; 42(1): 42-54, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36044485

RESUMEN

The method proposed in this paper is a robust combination of multi-task learning and unsupervised domain adaptation for segmenting amoeboid cells in microscopy. A highlight of this work is the manner in which the model's hyperparameters are estimated. The detriments of ad-hoc parameter estimation are well known, but this issue remains largely unaddressed in the context of CNN-based segmentation. Using a novel min-max formulation of the segmentation cost function our proposed method analytically estimates the model's hyperparameters, while simultaneously learning the CNN weights during training. This end-to-end framework provides a consolidated mechanism to harness the potential of multi-task learning to isolate and segment clustered cells from low contrast brightfield images, and it simultaneously leverages deep domain adaptation to segment fluorescent cells without explicit pixel-level re- annotation of the data. Experimental validations on multi-cellular images strongly suggest the effectiveness of the proposed technique, and our quantitative results show at least 15% and 10% improvement in cell segmentation on brightfield and fluorescence images respectively compared to contemporary supervised segmentation methods.


Asunto(s)
Amoeba , Microscopía , Algoritmos , Procesamiento de Imagen Asistido por Computador/métodos
4.
Sci Adv ; 8(42): eabo5767, 2022 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-36269830

RESUMEN

Physical forces are essential to biological function, but their impact at the tissue level is not fully understood. The gut is under continuous mechanical stress because of peristalsis. To assess the influence of mechanical cues on enteropathogen invasion, we combine computational imaging with a mechanically active gut-on-a-chip. After infecting the device with either of two microbes, we image their behavior in real time while mapping the mechanical stress within the tissue. This is achieved by reconstructing three-dimensional videos of the ongoing invasion and leveraging on-manifold inverse problems together with viscoelastic rheology. Our results show that peristalsis accelerates the destruction and invasion of intestinal tissue by Entamoeba histolytica and colonization by Shigella flexneri. Local tension facilitates parasite penetration and activates virulence genes in the bacteria. Overall, our work highlights the fundamental role of physical cues during host-pathogen interactions and introduces a framework that opens the door to study mechanobiology on deformable tissues.


Asunto(s)
Entamoeba histolytica , Peristaltismo , Dispositivos Laboratorio en un Chip , Simulación por Computador , Análisis de Secuencia por Matrices de Oligonucleótidos
5.
PLoS Pathog ; 18(5): e1010550, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35594320

RESUMEN

Entamoeba histolytica is a protozoan responsible for several pathologies in humans. Trophozoites breach the intestinal site to enter the bloodstream and thus traverse to a secondary site. Macropinocytosis and phagocytosis, collectively accounting for heterophagy, are the two major processes responsible for sustenance of Entamoeba histolytica within the host. Both of these processes require significant rearrangements in the structure to entrap the target. Rho GTPases play an indispensable role in mustering proteins that regulate cytoskeletal remodelling. Unlike phagocytosis which has been studied in extensive detail, information on machinery of macropinocytosis in E. histolytica is still limited. In the current study, using site directed mutagenesis and RNAi based silencing, coupled with functional studies, we have demonstrated the involvement of EhRho5 in constitutive and LPA stimulated macropinocytosis. We also report that LPA, a bioactive phospholipid present in the bloodstream of the host, activates EhRho5 and translocates it from cytosol to plasma membrane and endomembrane compartments. Using biochemical and FRAP studies, we established that a PI Kinase acts upstream of EhRho5 in LPA mediated signalling. We further identified EhGEF2 as a guanine nucleotide exchange factor of EhRho5. In the amoebic trophozoites, EhGEF2 depletion leads to reduced macropinocytic efficiency of trophozoites, thus phenocopying its substrate. Upon LPA stimulation, EhGEF2 is found to sequester near the plasma membrane in a wortmannin sensitive fashion, explaining a possible mode for activation of EhRho5 in the amoebic trophozoites. Collectively, we propose that LPA stimulated macropinocytosis in E. histolytica is driven by the PI Kinase-EhGEF2-EhRho5 axis.


Asunto(s)
Entamoeba histolytica , Animales , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Humanos , Lipopolisacáridos , Fagocitosis , Pinocitosis , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trofozoítos/metabolismo , Proteínas de Unión al GTP rho/metabolismo
6.
Front Immunol ; 11: 582061, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33193389

RESUMEN

Zika virus (ZIKV) dramatically emerged in French Polynesia and subsequently in the Americas where it has been associated with severe neurological complications in adults and newborns, respectively. Although plasmacytoid dendritic cells (pDCs) are a key sensor of viral infection and are critical for initiating an antiviral response, little is known about the impact of ZIKV infection on pDCs. Here, we investigated the susceptibility of human pDCs to infection with multiple strains of ZIKV and further investigated the impact of infection on pDCs functions. We observed that pDCs were refractory to cell-free ZIKV virions but were effectively infected when co-cultured with ZIKV-infected cells. However, exposure of pDCs to ZIKV-infected cells resulted in limited maturation/activation with significant down regulation of CD303 expression, a severe impairment of inflammatory cytokine production, and an inability to mount an IFN-α response. We show that ZIKV developed a strategy to inhibit the IFN-α response in primary human pDCs likely mediated through NS1-dependent CD303 signaling, thus suggesting a new mechanism of immune evasion.


Asunto(s)
Células Dendríticas/inmunología , Interferón-alfa/inmunología , Lectinas Tipo C/inmunología , Glicoproteínas de Membrana/inmunología , Receptores Inmunológicos/inmunología , Transducción de Señal/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Animales , Línea Celular , Chlorocebus aethiops , Citocinas/inmunología , Regulación hacia Abajo/inmunología , Humanos , Inflamación/inmunología , Células Vero
7.
Artículo en Inglés | MEDLINE | ID: mdl-29896453

RESUMEN

Entamoeba histolytica is the anaerobic protozoan parasite responsible for human amoebiasis, the third most deadly parasitic disease worldwide. This highly motile eukaryotic cell invades human tissues and constitutes an excellent experimental model of cell motility and cell shape deformation. The absence of extranuclear microtubules in Entamoeba histolytica means that the actin-rich cytoskeleton takes on a crucial role in not only amoebic motility but also other processes sustaining pathogenesis, such as the phagocytosis of human cells and the parasite's resistance of host immune responses. Actin is highly conserved among eukaryotes, although diverse isoforms exist in almost all organisms studied to date. However, E. histolytica has a single actin protein, the structure of which differs significantly from those of its human homologs. Here, we studied the expression, structure and dynamics of actin in E. histolytica. We used molecular and cellular approaches to evaluate actin gene expression during intestinal invasion by E. histolytica trophozoites. Based on a three-dimensional structural bioinformatics analysis, we characterized protein domains differences between amoebic actin and human actin. Fine-tuned molecular dynamics simulations enabled us to examine protein motion and refine the three-dimensional structures of both actins, including elements potentially accounting for differences changes in the affinity properties of amoebic actin and deoxyribonuclease I. The dynamic, multifunctional nature of the amoebic cytoskeleton prompted us to examine the pleiotropic forms of actin structures within live E. histolytica cells; we observed the cortical cytoskeleton, stress fibers, "dot-like" structures, adhesion plates, and macropinosomes. In line with these data, a proteomics study of actin-binding proteins highlighted the Arp2/3 protein complex as a crucial element for the development of macropinosomes and adhesion plaques.


Asunto(s)
Citoesqueleto de Actina/química , Movimiento Celular/fisiología , Forma de la Célula/fisiología , Entamoeba histolytica/citología , Entamoeba histolytica/fisiología , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/química , Actinas/genética , Secuencia de Aminoácidos , Desoxirribonucleasa I/metabolismo , Entamoeba histolytica/genética , Entamebiasis/inmunología , Entamebiasis/parasitología , Expresión Génica , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/fisiología , Humanos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Fagocitosis , Proteómica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes , Alineación de Secuencia , Trofozoítos/metabolismo
8.
Sci Rep ; 7(1): 9178, 2017 08 23.
Artículo en Inglés | MEDLINE | ID: mdl-28835648

RESUMEN

Cell motility is governed by a complex molecular machinery that converts physico-chemical cues into whole-cell movement. Understanding the underlying biophysical mechanisms requires the ability to measure physical quantities inside the cell in a simple, reproducible and preferably non-invasive manner. To this end, we developed BioFlow, a computational mechano-imaging method and associated software able to extract intracellular measurements including pressure, forces and velocity everywhere inside freely moving cells in two and three dimensions with high spatial resolution in a non-invasive manner. This is achieved by extracting the motion of intracellular material observed using fluorescence microscopy, while simultaneously inferring the parameters of a given theoretical model of the cell interior. We illustrate the power of BioFlow in the context of amoeboid cell migration, by modelling the intracellular actin bulk flow of the parasite Entamoeba histolytica using fluid dynamics, and report unique experimental measures that complement and extend both theoretical estimations and invasive experimental measures. Thanks to its flexibility, BioFlow is easily adaptable to other theoretical models of the cell, and alleviates the need for complex or invasive experimental conditions, thus constituting a powerful tool-kit for mechano-biology studies. BioFlow is open-source and freely available via the Icy software.


Asunto(s)
Modelos Teóricos , Imagen Molecular , Programas Informáticos , Algoritmos , Movimiento Celular , Fenómenos Mecánicos , Microscopía Fluorescente , Imagen Molecular/métodos , Fenómenos Físicos
9.
Cell Microbiol ; 18(8): 1134-52, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-26857352

RESUMEN

The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well-defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER-Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin-rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica.


Asunto(s)
Entamoeba histolytica/metabolismo , Proteínas Protozoarias/metabolismo , Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/ultraestructura , Entamoeba histolytica/ultraestructura , Transporte de Proteínas , Vesículas Transportadoras/metabolismo , Factores de Virulencia/metabolismo
10.
PLoS Pathog ; 8(12): e1003087, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23300443

RESUMEN

The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT) is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs) block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes from nerve terminals to the soma, and represents a general gateway for the delivery of virulence factors and pathogens to the central nervous system.


Asunto(s)
Transporte Axonal/efectos de los fármacos , Toxinas Botulínicas Tipo A/farmacología , Toxinas Botulínicas/farmacología , Neuronas Motoras/efectos de los fármacos , Neurotransmisores/antagonistas & inhibidores , Acetilcolina/metabolismo , Animales , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas Tipo A/metabolismo , Células Cultivadas , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Endocitosis/efectos de los fármacos , Ratones , Neuronas Motoras/metabolismo , Unión Neuromuscular/metabolismo , Parálisis/metabolismo , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Transmisión Sináptica/efectos de los fármacos , Toxina Tetánica/metabolismo , Toxina Tetánica/farmacología
11.
Cell Microbiol ; 12(2): 217-32, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19840028

RESUMEN

Inactivation of different small GTPases upon their glucosylation by lethal toxin from Clostridium sordellii strain IP82 (LT-82) is already known to lead to cell rounding, adherens junction (AJ) disorganization and actin depolymerization. In the present work, we observed that LT-82 induces a rapid dephosphorylation of paxillin, a protein regulating focal adhesion (FA), independently of inactivation of paxillin kinases such as Src, Fak and Pyk2. Among the small GTPases inactivated by this toxin, including Rac, Ras, Rap and Ral, we identified Rac1, as responsible for paxillin dephosphorylation using cells overexpressing Rac1(V12). Rac1 inactivation by LT-82 modifies interactions between proteins from AJ and FA complexes as shown by pull-down assays. We showed that in Triton X-100-insoluble membrane proteins from these complexes, namely E-cadherin, beta-catenin, p120-catenin and talin, are decreased upon LT-82 intoxication, a treatment that also induces a rapid decrease in cell phosphoinositide content. Therefore, we proposed that Rac inactivation by LT-82 alters phosphoinositide metabolism leading to FA and AJ complex disorganization and actin depolymerization.


Asunto(s)
Actinas/metabolismo , Toxinas Bacterianas/farmacología , Clostridium sordellii/efectos de los fármacos , Clostridium sordellii/metabolismo , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Microscopía Confocal , Microscopía de Contraste de Fase , Unión Proteica/efectos de los fármacos
12.
PLoS One ; 3(11): e3764, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19018299

RESUMEN

Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside G(M2) in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), with C. perfringens NetB (40% identity) and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to G(M2), in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes G(M2) as receptor and forms anion-selective channels.


Asunto(s)
Toxinas Bacterianas/química , Enterotoxinas/química , Proteínas Hemolisinas/química , Secuencia de Aminoácidos , Animales , Toxinas Bacterianas/metabolismo , Clonación Molecular , Clostridium perfringens/metabolismo , Enterotoxinas/fisiología , Eritrocitos/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Datos de Secuencia Molecular , Oligonucleótidos/química , Homología de Secuencia de Aminoácido , Ovinos , Staphylococcus aureus/metabolismo
13.
J Clin Microbiol ; 44(10): 3842-4, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17021125

RESUMEN

We describe a case of osteitis caused by a new and unusual Clostridium species, Clostridium amygdalinum, an environmental, moderately thermophilic bacterium. This is the first documented report of human infection caused by this organism.


Asunto(s)
Enfermedad Crónica , Infecciones por Clostridium/microbiología , Clostridium/clasificación , Clostridium/aislamiento & purificación , Osteítis/microbiología , Acetamidas/uso terapéutico , Adulto , Antibacterianos , Clostridium/genética , Infecciones por Clostridium/tratamiento farmacológico , Humanos , Linezolid , Masculino , Datos de Secuencia Molecular , Oxazolidinonas/uso terapéutico , Filogenia
14.
FEBS Lett ; 572(1-3): 299-306, 2004 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-15304366

RESUMEN

The botulinum neurotoxin A C-terminal fragment (Hc), which mediates the binding of the toxin to neuronal cell surface receptors, comprises two subdomains, Hc-N (amino acids 873-1095) and Hc-C (amino acids 1096-1296). In order to define the minimal fragment of Hc carrying protective antigenic properties, Hc, Hc-N and Hc-C have been produced as recombinant proteins in Escherichia coli, and have been tested for their antigenicity in mouse protection assays. Hc, Hc-N and Hc-C induced similar antibody levels as shown by ELISA. However, a single immunization with Hc (10 microg) fully protected mice challenged with 10(3) mouse lethal dose 50 of toxin, whereas Hc-N, Hc-C, or Hc-N plus Hc-C did not give any protection. Triple immunizations with Hc-N or Hc-C were necessary to induce a higher level of protection. Circular dichroism and fluorescence studies showed that the isolated subdomains were folded and stable. However, an intense near-UV dichroic signal was only observed in the Hc spectrum, revealing a highly structured interface between both subdomains. Taken together, the results show that the generation of protective antibodies requires the whole Hc domain and especially the native structure of the interfacial region between Hc-N and Hc-C.


Asunto(s)
Formación de Anticuerpos , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Animales , Toxinas Botulínicas Tipo A/toxicidad , Dicroismo Circular , Clonación Molecular , Cartilla de ADN , Escherichia coli , Masculino , Ratones , Modelos Moleculares , Fragmentos de Péptidos/toxicidad , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología
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