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1.
J Clin Immunol ; 44(7): 151, 2024 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-38896336

RESUMEN

A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3' end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.


Asunto(s)
ADN Ligasa (ATP) , Homocigoto , Mutación , Inmunodeficiencia Combinada Grave , Femenino , Humanos , Masculino , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , Fibroblastos , Simulación de Dinámica Molecular , Mutación/genética , Inmunodeficiencia Combinada Grave/genética , Lactante
2.
Int J Mol Sci ; 25(2)2024 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-38279311

RESUMEN

WD40 repeat proteins (WDRs) are present in all eukaryotes and include members that are implicated in numerous cellular activities. They act as scaffold proteins and thus as molecular "hubs" for protein-protein interactions, which mediate the assembly of multifunctional complexes that regulate key developmental processes in Arabidopsis thaliana, such as flowering time, hormonal signaling, and stress responses. Despite their importance, many aspects of their putative functions have not been elucidated yet. Here, we show that the late-flowering phenotype of the anthesis promoting factor 1 (aprf1) mutants is temperature-dependent and can be suppressed when plants are grown under mild heat stress conditions. To gain further insight into the mechanism of APRF1 function, we employed a co-immunoprecipitation (Co-IP) approach to identify its interaction partners. We provide the first interactome of APRF1, which includes proteins that are localized in several subcellular compartments and are implicated in diverse cellular functions. The dual nucleocytoplasmic localization of ARRF1, which was validated through the interaction of APRF1 with HEAT SHOCK PROTEIN 1 (HSP90.1) in the nucleus and with HSP90.2 in the cytoplasm, indicates a dynamic and versatile involvement of APRF1 in multiple biological processes. The specific interaction of APRF1 with the chaperon HSP90.1 in the nucleus expands our knowledge regarding the epigenetic regulation of flowering time in A. thaliana and further suggests the existence of a delicate thermoregulated mechanism during anthesis.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Epigénesis Genética , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Regulación de la Expresión Génica de las Plantas , Flores/metabolismo
3.
Anal Chem ; 2023 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-36627105

RESUMEN

Hematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that takes place through essential interactions with adhesion molecules on an endothelial cell layer. The homing process of HSPCs begins with the tethering and rolling of the cells on the endothelial layer, which is achieved by the interaction between selectins on the endothelium to the ligands on HSPC/leukemic cells under shear stress of the blood flow. Although many studies have been based on in vitro conditions of the cells rolling over recombinant proteins, significant challenges remain when imaging HSPC/leukemic cells on the endothelium, a necessity when considering characterizing cell-to-cell interaction and rolling dynamics during cell migration. Here, we report a new methodology that enables imaging of stem-cell-intrinsic spatiotemporal details during its migration on an endothelium-like cell monolayer. We developed optimized protocols that preserve transiently appearing structures on HSPCs/leukemic cells during its rolling under shear stress for fluorescence and scanning electron microscopy characterization. Our new experimental platform is closer to in vivo conditions and will contribute to indepth understanding of stem-cell behavior during its migration and cell-to-cell interaction during the process of homing.

4.
Am J Hum Genet ; 104(3): 542-552, 2019 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-30827498

RESUMEN

Polyglutamine expansions in the transcriptional co-repressor Atrophin-1, encoded by ATN1, cause the neurodegenerative condition dentatorubral-pallidoluysian atrophy (DRPLA) via a proposed novel toxic gain of function. We present detailed phenotypic information on eight unrelated individuals who have de novo missense and insertion variants within a conserved 16-amino-acid "HX repeat" motif of ATN1. Each of the affected individuals has severe cognitive impairment and hypotonia, a recognizable facial gestalt, and variable congenital anomalies. However, they lack the progressive symptoms typical of DRPLA neurodegeneration. To distinguish this subset of affected individuals from the DRPLA diagnosis, we suggest using the term CHEDDA (congenital hypotonia, epilepsy, developmental delay, digit abnormalities) to classify the condition. CHEDDA-related variants alter the particular structural features of the HX repeat motif, suggesting that CHEDDA results from perturbation of the structural and functional integrity of the HX repeat. We found several non-homologous human genes containing similar motifs of eight to 10 HX repeat sequences, including RERE, where disruptive variants in this motif have also been linked to a separate condition that causes neurocognitive and congenital anomalies. These findings suggest that perturbation of the HX motif might explain other Mendelian human conditions.


Asunto(s)
Secuencias de Aminoácidos/genética , Variación Genética , Proteínas del Tejido Nervioso/genética , Trastornos Neurocognitivos/etiología , Secuencias Repetitivas de Ácidos Nucleicos , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Trastornos Neurocognitivos/clasificación , Trastornos Neurocognitivos/patología , Fenotipo , Pronóstico , Síndrome
5.
J Nanobiotechnology ; 20(1): 282, 2022 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-35710420

RESUMEN

BACKGROUND: Nanotopographical cues play a critical role as drivers of mesenchymal stem cell differentiation. Nanowire scaffolds, in this regard, provide unique and adaptable nanostructured surfaces with focal points for adhesion and with elastic properties determined by nanowire stiffness. RESULTS: We show that a scaffold of nanowires, which are remotely actuated by a magnetic field, mechanically stimulates mesenchymal stem cells. Osteopontin, a marker of osteogenesis onset, was expressed after cells were cultured for 1 week on top of the scaffold. Applying a magnetic field significantly boosted differentiation due to mechanical stimulation of the cells by the active deflection of the nanowire tips. The onset of differentiation was reduced to 2 days of culture based on the upregulation of several osteogenesis markers. Moreover, this was observed in the absence of any external differentiation factors. CONCLUSIONS: The magneto-mechanically modulated nanosurface enhanced the osteogenic differentiation capabilities of mesenchymal stem cells, and it provides a customizable tool for stem cell research and tissue engineering.


Asunto(s)
Células Madre Mesenquimatosas , Nanocables , Diferenciación Celular , Células Cultivadas , Osteogénesis/fisiología , Ingeniería de Tejidos , Andamios del Tejido
6.
J Biol Chem ; 295(34): 12214-12223, 2020 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-32647010

RESUMEN

The polyhistidine tag (His-tag) is one of the most popular protein tags used in the life sciences. Traditionally, the detection of His-tagged proteins relies on immunoblotting with anti-His antibodies. This approach is laborious for certain applications, such as protein purification, where time and simplicity are critical. The His-tag can also be directly detected by metal ion-loaded nickel-nitrilotriacetic acid-based chelator heads conjugated to fluorophores, which is a convenient alternative method to immunoblotting. Typically, such chelator heads are conjugated to either green or red fluorophores, the detection of which requires specialized excitation sources and detection systems. Here, we demonstrate that post-run staining is ideal for His-tag detection by metal ion-loaded and fluorescently labeled chelator heads in PAGE and blot membranes. Additionally, by comparing the performances of different chelator heads, we show how differences in microscopic affinity constants translate to macroscopic differences in the detection limits in environments with limited diffusion, such as PAGE. On the basis of these results, we devise a simple approach, called UVHis-PAGE, that uses metal ion-loaded and fluorescently labeled chelator heads to detect His-tagged proteins in PAGE and blot membranes. Our method uses a UV transilluminator as an excitation source, and the results can be visually inspected by the naked eye.


Asunto(s)
Electroforesis en Gel de Gradiente Desnaturalizante , Colorantes Fluorescentes/química , Histidina/análisis , Proteínas Recombinantes de Fusión/análisis , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/análisis , Rayos Ultravioleta , Histidina/química , Humanos , Proteínas Recombinantes de Fusión/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética
7.
J Biol Chem ; 295(11): 3719-3733, 2020 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-31949047

RESUMEN

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin-ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin-ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.


Asunto(s)
Selectina E/química , Selectina E/metabolismo , Animales , Bombyx , Línea Celular Tumoral , Selectina E/aislamiento & purificación , Humanos , Proteínas Inmovilizadas/metabolismo , Cinética , Ligandos , Ratones , Polisacáridos/metabolismo , Dominios Proteicos , Multimerización de Proteína , Relación Estructura-Actividad
8.
Bioinformatics ; 36(4): 1121-1128, 2020 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-31584626

RESUMEN

MOTIVATION: Leucine-aspartic acid (LD) motifs are short linear interaction motifs (SLiMs) that link paxillin family proteins to factors controlling cell adhesion, motility and survival. The existence and importance of LD motifs beyond the paxillin family is poorly understood. RESULTS: To enable a proteome-wide assessment of LD motifs, we developed an active learning based framework (LD motif finder; LDMF) that iteratively integrates computational predictions with experimental validation. Our analysis of the human proteome revealed a dozen new proteins containing LD motifs. We found that LD motif signalling evolved in unicellular eukaryotes more than 800 Myr ago, with paxillin and vinculin as core constituents, and nuclear export signal as a likely source of de novo LD motifs. We show that LD motif proteins form a functionally homogenous group, all being involved in cell morphogenesis and adhesion. This functional focus is recapitulated in cells by GFP-fused LD motifs, suggesting that it is intrinsic to the LD motif sequence, possibly through their effect on binding partners. Our approach elucidated the origin and dynamic adaptations of an ancestral SLiM, and can serve as a guide for the identification of other SLiMs for which only few representatives are known. AVAILABILITY AND IMPLEMENTATION: LDMF is freely available online at www.cbrc.kaust.edu.sa/ldmf; Source code is available at https://github.com/tanviralambd/LD/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
Proteoma , Secuencias de Aminoácidos , Ácido Aspártico , Humanos , Leucina , Prevalencia
9.
Biochemistry ; 59(39): 3757-3771, 2020 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-32901486

RESUMEN

Recruitment of circulating cells toward target sites is primarily dependent on selectin/ligand adhesive interactions. Glycosyltransferases are involved in the creation of selectin ligands on proteins and lipids. α1,3-Fucosylation is imperative for the creation of selectin ligands, and a number of fucosyltransferases (FTs) can modify terminal lactosamines on cells to create these ligands. One FT, fucosyltransferase VI (FTVI), adds a fucose in an α1,3 configuration to N-acetylglucosamine to generate sialyl Lewis X (sLex) epitopes on proteins of live cells and enhances their ability to bind E-selectin. Although a number of recombinant human FTVIs have been purified, apart from limited commercial enzymes, they were not characterized for their activity on live cells. Here we focused on establishing a robust method for producing FTVI that is active on living cells (hematopoietic cells and mesenchymal stromal cells). To this end, we used two expression systems, Bombyx mori (silkworm) and Pichia pastoris (yeast), to produce significant amounts of N-terminally tagged FTVI and demonstrated that these enzymes have superior activity when compared to currently available commercial enzymes that are produced from various expression systems. Overall, we outline a scheme for obtaining large amounts of highly active FTVI that can be used for the application of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.


Asunto(s)
Selectina E/metabolismo , Fucosiltransferasas/metabolismo , Polisacáridos/metabolismo , Células Madre/metabolismo , Animales , Bombyx/genética , Línea Celular , Línea Celular Tumoral , Fucosiltransferasas/genética , Expresión Génica , Humanos , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
J Am Chem Soc ; 142(4): 1715-1720, 2020 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-31931564

RESUMEN

Effective and cell-type-specific delivery of CRISPR/Cas9 gene editing elements remains a challenging open problem. Here we report the development of biomimetic cancer cell coated zeolitic imidazolate frameworks (ZIFs) for targeted and cell-specific delivery of this genome editing machinery. Coating ZIF-8 that is encapsulating CRISPR/Cas9 (CC-ZIF) with a cancer cell membrane resulted in the uniformly covered C3-ZIF(cell membrane type). Incubation of C3-ZIFMCF with MCF-7, HeLa, HDFn, and aTC cell lines showed the highest uptake by MCF-7 cells and negligible uptake by the healthy cells (i.e., HDFn and aTC). As to genome editing, a 3-fold repression in the EGFP expression was observed when MCF-7 were transfected with C3-ZIFMCF compared to 1-fold repression in the EGFP expression when MCF-7 were transfected with C3-ZIFHELA. In vivo testing confirmed the selectivity of C3-ZIFMCF to accumulate in MCF-7 tumor cells. This supports the ability of this biomimetic approach to match the needs of cell-specific targeting, which is unquestionably the most critical step in the future translation of genome editing technologies.


Asunto(s)
Biomimética , Sistemas CRISPR-Cas , Estructuras Metalorgánicas/química , Animales , Células HeLa , Xenoinjertos , Humanos , Células MCF-7 , Ratones
11.
Anal Chem ; 92(9): 6200-6206, 2020 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-32264668

RESUMEN

The parallel plate flow chamber assay is widely utilized to study physiological cell-cell adhesive interactions under dynamic flow that mimics the bloodstream. In this technique, the cells are perfused under defined shear stresses over a monolayer of endothelial cells (expressing homing molecules, e.g., selectins) or a surface (expressing recombinant homing molecules). However, with the need to study multiple samples and multiple parameters per sample, using a traditional bright-field microscope-based flow assay allows only one sample at a time to be analyzed, resulting in high interexperiment variability, the need for normalization, waste of materials, and significant consumption of time. We developed a multiplexing approach using a three-color fluorescence staining method, which allowed for up to seven different combination signatures to be run at one time. Using this fluorescent multiplex cell rolling (FMCR) assay, each sample is labeled with a different signature of emission wavelengths and mixed with other samples just minutes before the flow run. Subsequently, real-time images are acquired in a single pass using a line-scanning spectral confocal microscope. To illustrate the glycan-dependent binding of E-selectin, a central molecule in cell migration, to its glycosylated ligands expressed on myeloid-leukemic cells in flow, the FMCR assay was used to analyze E-selectin-ligand interactions following the addition (fucosyltransferase-treatment) or removal (deglycosylation) of key glycans on the flowing cells. The FMCR assay allowed us to analyze the cell-adhesion events from these different treatment conditions simultaneously in a competitive manner and to calculate differences in rolling frequency, velocity, and tethering capability of cells under study.


Asunto(s)
Colorantes Fluorescentes/química , Microscopía Confocal/métodos , Animales , Anticuerpos/química , Anticuerpos/inmunología , Células CHO , Línea Celular , Cricetinae , Cricetulus , Selectina E/inmunología , Selectina E/metabolismo , Humanos , Inmunoensayo , Células Madre/citología , Células Madre/metabolismo , Imagen de Lapso de Tiempo
12.
J Nanobiotechnology ; 18(1): 42, 2020 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-32164746

RESUMEN

BACKGROUND: Identifying the precise location of cells and their migration dynamics is of utmost importance for achieving the therapeutic potential of cells after implantation into a host. Magnetic resonance imaging is a suitable, non-invasive technique for cell monitoring when used in combination with contrast agents. RESULTS: This work shows that nanowires with an iron core and an iron oxide shell are excellent materials for this application, due to their customizable magnetic properties and biocompatibility. The longitudinal and transverse magnetic relaxivities of the core-shell nanowires were evaluated at 1.5 T, revealing a high performance as T2 contrast agents. Different levels of oxidation and various surface coatings were tested at 7 T. Their effects on the T2 contrast were reflected in the tailored transverse relaxivities. Finally, the detection of nanowire-labeled breast cancer cells was demonstrated in T2-weighted images of cells implanted in both, in vitro in tissue-mimicking phantoms and in vivo in mouse brain. Labeling the cells with a nanowire concentration of 0.8 µg of Fe/mL allowed the detection of 25 cells/µL in vitro, diminishing the possibility of side effects. This performance enabled an efficient labelling for high-resolution cell detection after in vivo implantation (~ 10 nanowire-labeled cells) over a minimum of 40 days. CONCLUSIONS: Iron-iron oxide core-shell nanowires enabled the efficient and longitudinal cellular detection through magnetic resonance imaging acting as T2 contrast agents. Combined with the possibility of magnetic guidance as well as triggering of cellular responses, for instance by the recently discovered strong photothermal response, opens the door to new horizons in cell therapy and make iron-iron oxide core-shell nanowires a promising theranostic platform.


Asunto(s)
Rastreo Celular/métodos , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita , Nanocables , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Línea Celular , Compuestos Férricos , Hierro , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Fantasmas de Imagen , Nanomedicina Teranóstica
13.
FASEB J ; 32(6): 3346-3360, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29401622

RESUMEN

The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, anoxic water, and elevated concentrations of salt and heavy metals. This environment provides a rare system to study the interplay between halophilic and thermophilic adaptation in biologic macromolecules. The present article reports the first DNA polymerase with halophilic and thermophilic features. Biochemical and structural analysis by Raman and circular dichroism spectroscopy showed that the charge distribution on the protein's surface mediates the structural balance between stability for thermal adaptation and flexibility for counteracting the salt-induced rigid and nonfunctional hydrophobic packing. Salt bridge interactions via increased negative and positive charges contribute to structural stability. Salt tolerance, conversely, is mediated by a dynamic structure that becomes more fixed and functional with increasing salt concentration. We propose that repulsive forces among excess negative charges, in addition to a high percentage of negatively charged random coils, mediate this structural dynamism. This knowledge enabled us to engineer a halophilic version of Thermococcus kodakarensis DNA polymerase.-Takahashi, M., Takahashi, E., Joudeh, L. I., Marini, M., Das, G., Elshenawy, M. M., Akal, A., Sakashita, K., Alam, I., Tehseen, M., Sobhy, M. A., Stingl, U., Merzaban, J. S., Di Fabrizio, E., Hamdan, S. M. Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.


Asunto(s)
Proteínas Arqueales/química , ADN Polimerasa Dirigida por ADN/química , Simulación de Dinámica Molecular , Thermococcus/enzimología , Océano Índico
14.
J Am Chem Soc ; 140(1): 143-146, 2018 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-29272114

RESUMEN

CRISPR/Cas9 is a combined protein (Cas9) and an engineered single guide RNA (sgRNA) genome editing platform that offers revolutionary solutions to genetic diseases. It has, however, a double delivery problem owning to the large protein size and the highly charged RNA component. In this work, we report the first example of CRISPR/Cas9 encapsulated by nanoscale zeolitic imidazole frameworks (ZIFs) with a loading efficiency of 17% and enhanced endosomal escape promoted by the protonated imidazole moieties. The gene editing potential of CRISPR/Cas9 encapsulated by ZIF-8 (CC-ZIFs) is further verified by knocking down the gene expression of green fluorescent protein by 37% over 4 days. The nanoscale CC-ZIFs are biocompatible and easily scaled-up offering excellent loading capacity and controlled codelivery of intact Cas9 protein and sgRNA.


Asunto(s)
Sistemas CRISPR-Cas/fisiología , Endosomas/metabolismo , Edición Génica , Imidazoles/química , Nanopartículas/química , Zeolitas/química , Animales , Células CHO , Cricetulus , Tamaño de la Partícula
16.
Small ; 13(7)2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27879037

RESUMEN

Knowledge of materials' thermal-transport properties, conductivity and diffusivity, is crucial for several applications within areas of biology, material science and engineering. Specifically, a microsized, flexible, biologically integrated thermal transport sensor is beneficial to a plethora of applications, ranging across plants physiological ecology and thermal imaging and treatment of cancerous cells, to thermal dissipation in flexible semiconductors and thermoelectrics. Living cells pose extra challenges, due to their small volumes and irregular curvilinear shapes. Here a novel approach of simultaneously measuring thermal conductivity and diffusivity of different materials and its applicability to single cells is demonstrated. This technique is based on increasing phonon-boundary-scattering rate in nanomembranes, having extremely low flexural rigidities, to induce a considerable spectral dependence of the bandgap-emission over excitation-laser intensity. It is demonstrated that once in contact with organic or inorganic materials, the nanomembranes' emission spectrally shift based on the material's thermal diffusivity and conductivity. This NM-based technique is further applied to differentiate between different types and subtypes of cancer cells, based on their thermal-transport properties. It is anticipated that this novel technique to enable an efficient single-cell thermal targeting, allow better modeling of cellular thermal distribution and enable novel diagnostic techniques based on variations of single-cell thermal-transport properties.


Asunto(s)
Técnicas Biosensibles/métodos , Membranas Artificiales , Nanopartículas/química , Temperatura , Línea Celular Tumoral , Supervivencia Celular , Humanos , Mediciones Luminiscentes
17.
J Biol Chem ; 290(35): 21213-30, 2015 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-26124272

RESUMEN

Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling.


Asunto(s)
Selectina E/metabolismo , Receptores de Hialuranos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mapas de Interacción de Proteínas , Línea Celular Tumoral , Movimiento Celular , Humanos , Inmunoprecipitación , Unión Proteica , Mapeo de Interacción de Proteínas
18.
Plant J ; 78(6): 916-26, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24654847

RESUMEN

Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Resistencia a los Herbicidas/genética , Fotorreceptores de Plantas/genética , Fitocromo B/genética , 3-Fosfoshikimato 1-Carboxiviniltransferasa/antagonistas & inhibidores , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Ritmo Circadiano , Análisis Mutacional de ADN , Glicina/análogos & derivados , Glicina/toxicidad , Mutación , Fenotipo , Fotorreceptores de Plantas/metabolismo , Fotorreceptores de Plantas/fisiología , Fitocromo B/metabolismo , Fitocromo B/fisiología , Glifosato
19.
Glycobiology ; 25(12): 1392-409, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26153105

RESUMEN

Neural stem cell (NSC)-based therapies offer potential for neural repair in central nervous system (CNS) inflammatory and degenerative disorders. Typically, these conditions present with multifocal CNS lesions making it impractical to inject NSCs locally, thus mandating optimization of vascular delivery of the cells to involved sites. Here, we analyzed NSCs for expression of molecular effectors of cell migration and found that these cells are natively devoid of E-selectin ligands. Using glycosyltransferase-programmed stereosubstitution (GPS), we glycan engineered the cell surface of NSCs ("GPS-NSCs") with resultant enforced expression of the potent E-selectin ligand HCELL (hematopoietic cell E-/L-selectin ligand) and of an E-selectin-binding glycoform of neural cell adhesion molecule ("NCAM-E"). Following intravenous (i.v.) injection, short-term homing studies demonstrated that, compared with buffer-treated (control) NSCs, GPS-NSCs showed greater neurotropism. Administration of GPS-NSC significantly attenuated the clinical course of experimental autoimmune encephalomyelitis (EAE), with markedly decreased inflammation and improved oligodendroglial and axonal integrity, but without evidence of long-term stem cell engraftment. Notably, this effect of NSC is not a universal property of adult stem cells, as administration of GPS-engineered mouse hematopoietic stem/progenitor cells did not improve EAE clinical course. These findings highlight the utility of cell surface glycan engineering to boost stem cell delivery in neuroinflammatory conditions and indicate that, despite the use of a neural tissue-specific progenitor cell population, neural repair in EAE results from endogenous repair and not from direct, NSC-derived cell replacement.


Asunto(s)
Movimiento Celular , Encefalomielitis Autoinmune Experimental/terapia , Células-Madre Neurales/metabolismo , Polisacáridos/metabolismo , Animales , Terapia Genética , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Ratones , Ratones Endogámicos C57BL , Regeneración Nerviosa , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Células-Madre Neurales/trasplante , Selectinas/metabolismo
20.
PLoS One ; 19(5): e0301761, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38718025

RESUMEN

Tracking small extracellular vesicles (sEVs), such as exosomes, requires staining them with dyes that penetrate their lipid bilayer, a process that leaves excess dye that needs to be mopped up to achieve high specificity. Current methods to remove superfluous dye have limitations, among them that they are time-intensive, carry the risk of losing sample and can require specialized equipment and materials. Here we present a fast, easy-to-use, and cost-free protocol for cleaning excess dye from stained sEV samples by adding their parental cells to the mixture to absorb the extra dye much like sponges do. Since sEVs are considered a next-generation drug delivery system, we further show the success of our approach at removing excess chemotherapeutic drug, daunorubicin, from the sEV solution.


Asunto(s)
Vesículas Extracelulares , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Humanos , Daunorrubicina/economía , Colorantes/química , Coloración y Etiquetado/métodos , Coloración y Etiquetado/economía
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