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1.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804193

RESUMEN

We report here the synthesis and structural characterization of novel cationic (phenothiazinyl)vinyl-pyridinium (PVP) dyes, together with optical (absorption/emission) properties and their potential applicability as fluorescent labels. Convective heating, ultrasound irradiation and mechanochemical synthesis were considered as alternative synthetic methodologies proficient for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent requirements in the synthesis of novel dye (E)-1-(3-chloropropyl)-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium bromide 3d and its N-alkyl-2-methylpyridinium precursor 1c. The trans geometry of the newly synthesized (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium iodide 3b and (E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium tetrafluoroborate 3a' was confirmed by single crystal X-ray diffraction. A negative solvatochromism of the dyes in polar solvents was highlighted by UV-Vis spectroscopy and explanatory insights were supported by molecular modeling which suggested a better stabilization of the lowest unoccupied molecular orbitals (LUMO). The photostability of the dye 3b was investigated by irradiation at 365 nm in different solvents, while the steady-state and time-resolved fluorescence properties of dye 3b and 3a' in solid state were evaluated under one-photon excitation at 485 nm. The in vitro cytotoxicity of the new PVP dyes on B16-F10 melanoma cells was evaluated by WST-1 assay, while their intracellular localization was assessed by epi-fluorescence conventional microscopy imaging as well as one- and two-photon excited confocal fluorescence lifetime imaging microscopy (FLIM). PVP dyes displayed low cytotoxicity, good internalization inside melanoma cells and intense fluorescence emission inside the B16-F10 murine melanoma cells, making them suitable staining agents for imaging applications.


Asunto(s)
Colorantes Fluorescentes/química , Compuestos de Piridinio/química , Coloración y Etiquetado/métodos , Animales , Colorantes Fluorescentes/síntesis química , Ratones , Microscopía Fluorescente , Fenotiazinas/química , Fotones , Compuestos de Piridinio/síntesis química , Solventes/química , Espectrometría de Fluorescencia/métodos
2.
Medicine (Baltimore) ; 100(16): e25566, 2021 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-33879712

RESUMEN

ABSTRACT: This study investigated the feasibility of using immunohistochemistry (IHC) instead of PCR to detect BRAF V600E mutant protein in papillary thyroid carcinoma (PTC), and to determine the value of using preoperative BRAF V600E mutant protein by IHC to assist in the diagnosis of thyroid nodule patients with Hashimoto's thyroiditis (HT).The expression of BRAFV600E mutant protein was measured in 23 cases of HT+PTC, 31 cases of PTC, and 28 cases of HT by IHC, followed by PCR in the same samples for validation. SPSS 19.0 software was used for statistical analysis.The sensitivity and specificity of IHC to detect BRAF V600E mutation were 100% and 42.86%, respectively. In addition, the mutation rate of BRAF V600E protein in the HT+PTC group (34.78%, 8/23) was lower than that in the PTC group (80.65%, 25/31).The application of IHC to detect BRAF V600E mutant protein has good sensitivity but not specificity to diagnose PTC. IHC can be used as a preliminary screening method to detect BRAF V600E mutation. The strongly positive (+++) staining of IHC potently indicated BRAF V600E gene mutation. For suspicious thyroid nodules combined with HT, the detection of BRAF V600E mutant protein with IHC alone is not of great significance for differentiating benign and malignant nodules.


Asunto(s)
Inmunohistoquímica/estadística & datos numéricos , Proteínas Mutantes/análisis , Proteínas Proto-Oncogénicas B-raf/análisis , Cáncer Papilar Tiroideo/diagnóstico , Neoplasias de la Tiroides/diagnóstico , Adulto , Estudios de Factibilidad , Femenino , Enfermedad de Hashimoto/diagnóstico , Humanos , Inmunohistoquímica/métodos , Masculino , Persona de Mediana Edad , Mutación , Adhesión en Parafina , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Periodo Preoperatorio , Sensibilidad y Especificidad , Coloración y Etiquetado , Glándula Tiroides/metabolismo , Nódulo Tiroideo/diagnóstico
3.
J Indian Soc Pedod Prev Dent ; 39(1): 47-52, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33885387

RESUMEN

Background: Silver diamine fluoride (SDF) is one of the effectual cariostatic agents widely used in minimal intervention dentistry. However, the major drawback of SDF is dark staining after its application. Aim: In the present study, the staining of 38% SDF alone and 38% SDF and potassium iodide (KI) was compared after restoration with glass-ionomer cement (GIC) and resin composite using ImageJ software. Methods and Material: Forty extracted carious primary teeth were sorted into four groups. In Group I and II, SDF was applied and restored with GIC and composite restorations, respectively. In Group III and IV, SDF application was followed by KI and restored with GIC and composite restorations, respectively. Images were captured after initial applications on day 1 and day 14 after restoration. The captured images were imported to ImageJ software and mean gray values were calculated. Statistical Analysis: The mean gray values obtained were subjected to statistical analysis using paired t-test and independent sample t-test. There was statistically significant if P < 0.05. Results: Following the application of SDF and RIVA STAR, the baseline mean gray values showed no statistical significance. On day 1, the mean gray values were highest in Group IV (208.30) and lowest in Group I (178.51). Similarly, on day 14, the highest mean gray values were observed in Group IV (208.45) and lowest in Group I (147.6) which were statistically significant. Conclusions: The restorations after SDF application attained dark stain eventually, whereas with the application of SDF followed by KI (RIVA STAR), the restorations showed the least staining.


Asunto(s)
Caries Dental , Yoduro de Potasio , Cariostáticos , Caries Dental/prevención & control , Fluoruros Tópicos , Humanos , Compuestos de Amonio Cuaternario , Compuestos de Plata , Coloración y Etiquetado
4.
Zhonghua Bing Li Xue Za Zhi ; 50(4): 376-381, 2021 Apr 08.
Artículo en Chino | MEDLINE | ID: mdl-33831998

RESUMEN

Objective: To study the utility of immunohistochemistry (IHC) in differential diagnosis between trichoblastoma (TB) and basal cell carcinoma (BCC). Methods: Fifty-eight cases of TB and 40 cases of BCC were collected at Fudan University Shanghai Cancer Center from January 2009 to December 2019 and retrospectively analyzed by IHC for bcl-2, Ber-EP4, CD10, CK20 and Ki-67. Fisher exact test was performed for statistical analysis. Results: Twenty-five (43.1%) TBs and 5 (12.5%) BCCs showed bcl-2 staining in the outermost layer of the epithelial nests, the difference was statistically significant (P<0.01). The proportion of cases with bcl-2 staining>75% of epithelial cells in BCC group was much higher than that in TB group (40% vs. 12.1%; P<0.01). BCC group showed larger proportions with Ber-EP4 staining>75%, 51%-75% of epithelial cells than TB group (12.5% vs. 1.7%, 37.5% vs. 8.6%;P<0.05). Fifty-five (94.8%) TBs demonstrated CD10 expression in the follicular stroma, while only 16 (40.0%) BCCs showed focal or scattered CD10 expression in reactive fibrous stroma (P<0.01). CK20 expression was present in 37 (63.8%) TBs with scattered pattern, but BCCs exhibited no CK20 staining except for only one case (2.5%) showing focal staining (P<0.01). Compared with TB group, the BCC group included more cases with Ki-67 labeling index ≥15% on average and ≥25% in hotspot areas (P<0.05). Conclusion: IHC is helpful in differential diagnosis between TB and BCC. Scattered CK20 staining pattern and stromal CD10 expression support the diagnosis of TB. Bcl-2 staining limited to the outermost layer of the proliferation is more likely to be found in TB. In contrast, Ber-EP4 positivity and higher Ki-67 labeling index tend to be present in BCC.


Asunto(s)
Carcinoma Basocelular , Neoplasias Cutáneas , Biomarcadores de Tumor , Carcinoma Basocelular/diagnóstico , China , Diagnóstico Diferencial , Humanos , Antígeno Ki-67 , Estudios Retrospectivos , Neoplasias Cutáneas/diagnóstico , Coloración y Etiquetado
5.
Adv Exp Med Biol ; 1310: 1-30, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33834430

RESUMEN

Confocal laser scanning microscopy (CLSM) and related microscopic techniques allow a unique and versatile approach to image and analyze living cells due to their specificity and high sensitivity. Among confocal related techniques, fluorescence correlation methods, such as fluorescence correlation spectroscopy (FCS) and dual-color fluorescence cross-correlation spectroscopy (FCCS), are highly sensitive biophysical methods for analyzing the complex dynamic events of molecular diffusion and interaction change in live cells as well as in solution by exploiting the characteristics of fluorescence signals. Analytical and quantitative information from FCS and FCCS coupled with fluorescence images obtained from CLSM can now be applied in convergence science such as drug delivery and nanomedicine, as well as in basic cell biology. In this chapter, a brief introduction into the physical parameters that can be obtained from FCS and FCCS is first provided. Secondly, experimental examples of the methods for evaluating the parameters is presented. Finally, two potential FCS and FCCS applications for convergence science are introduced in more detail.


Asunto(s)
Microscopía Confocal , Color , Difusión , Espectrometría de Fluorescencia , Coloración y Etiquetado
6.
Adv Exp Med Biol ; 1310: 133-152, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33834436

RESUMEN

Since their development in the 1960s, immuno-gold techniques have been steadily used in biomedical science, because these techniques are applicable to all kinds of antigens, from viruses to animal tissues. Immuno-gold staining exploits antigen-antibody reactions and is used to investigate locations and interactions of components in the ultrastructure of tissues, cells, and particles. These methods are increasingly used with advanced technologies, such as correlative light and electron microscopy and cryo-techniques. In this protocol, we introduce the principles and technical details of recent advances in this area and discuss their advantages and limitations.


Asunto(s)
Antígenos , Oro , Animales , Inmunohistoquímica , Microscopía Electrónica , Coloración y Etiquetado
7.
J Vis Exp ; (168)2021 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-33682851

RESUMEN

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.


Asunto(s)
Citometría de Flujo/métodos , Leucemia Mieloide Aguda/patología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Anticuerpos Antineoplásicos/metabolismo , Biotinilación , Recuento de Células , Humanos , Ligandos , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Coloración y Etiquetado , Células Tumorales Cultivadas
8.
J Vis Exp ; (168)2021 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-33682862

RESUMEN

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.


Asunto(s)
Modelos Biológicos , Piel/patología , Coloración y Etiquetado , Cicatrización de Heridas , Anciano , Animales , Anticuerpos/metabolismo , Medios de Cultivo , Diabetes Mellitus/patología , Humanos , Procesamiento de Imagen Asistido por Computador , Indicadores y Reactivos , Reproducibilidad de los Resultados , Piel/lesiones , Cicatrización de Heridas/fisiología
9.
J Vis Exp ; (168)2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33720124

RESUMEN

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.


Asunto(s)
Neoplasias/patología , Células de Población Lateral/patología , Bencimidazoles/metabolismo , Línea Celular Tumoral , Análisis de Datos , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factor de Transcripción STAT3/metabolismo , Células de Población Lateral/metabolismo , Coloración y Etiquetado
10.
J Vis Exp ; (169)2021 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-33749674

RESUMEN

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 µm (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.


Asunto(s)
Retina/metabolismo , Coloración y Etiquetado/métodos , Células Amacrinas/fisiología , Animales , Dendritas/fisiología , Neuronas Dopaminérgicas/fisiología , Procesamiento de Imagen Asistido por Computador , Ratones , Microscopía Confocal/métodos , Proteínas del Tejido Nervioso/metabolismo , Receptores AMPA/metabolismo , Refractometría
11.
J Vis Exp ; (168)2021 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-33645584

RESUMEN

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.


Asunto(s)
Neovascularización Fisiológica , Piel Artificial , Piel/irrigación sanguínea , Ingeniería de Tejidos/métodos , Animales , Biomarcadores/metabolismo , Células Cultivadas , Colágeno/metabolismo , Dermis/metabolismo , Epidermis/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Imagenología Tridimensional , Imagen Óptica , Permeabilidad , Ratas , Coloración y Etiquetado , Suspensiones
12.
J Vis Exp ; (168)2021 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-33645587

RESUMEN

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.


Asunto(s)
Coloración y Etiquetado , Papilas Gustativas/citología , Animales , Recuento de Células , Disección , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Ratones , Microscopía Confocal , Neuronas/citología , Paladar (Hueso)/citología , Paladar (Hueso)/inervación
13.
J Vis Exp ; (168)2021 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-33720126

RESUMEN

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.


Asunto(s)
ADN/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Análisis de la Célula Individual/métodos , Aldehídos/química , Adhesión Celular , Comunicación Celular , Supervivencia Celular , Colesterol/metabolismo , Dimetilpolisiloxanos/química , Compuestos Epoxi/química , Humanos , Hidrogeles/química , Interacciones Hidrofóbicas e Hidrofílicas , Oligonucleótidos/metabolismo , Polímeros/química , Coloración y Etiquetado
14.
Exp Parasitol ; 224: 108099, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33713660

RESUMEN

BACKGROUND: Trichinellosis is caused by consumption of raw or undercooked meat containing infective Trichinella muscle larvae (ML). Only few studies on heat-inactivation of Trichinella ML are available in literature and more validated data concerning heat inactivation is needed to improve the risk estimation. OBJECTIVE AND METHODS: The aim of the present study was to evaluate the two in vitro methods "staining" and "morphological examination" as proxies for Trichinella ML heat inactivation in comparison with the mouse bioassay method to get more insight in the relationship between heat, heating time and inactivation of Trichinella ML. The second aim was to evaluate whether these methods could replace the bioassay in the light of ongoing animal use reduction in lifescience research. Tubes containing quantified live Trichinella ML were exposed to heat profiles ranging from 40 to 80 °C. Subsequently, inactivation was evaluated using both methylene blue staining and morphological examination, which was validated by bioassay. Results were used to model Trichinella inactivation. RESULTS: Trichinella muscle larvae exposed to 60 °C or higher for 12-12.5 min were not infective to mice. We found that morphological examination was more consistent with the bioassay than methylene blue staining. Modelled inactivation fitted experimental data consistently. Moreover, this study shows that larval Trichinella morphology may be used in situations where bioassays are not possible or prohibited. CONCLUSIONS: The relationship between heat and inactivation of larvae obtained from this study could be used in Trichinella QMRA models to improve quantification of the risk of Trichinella infection.


Asunto(s)
Culinaria/métodos , Músculos/parasitología , Trichinella/fisiología , Animales , Bioensayo , Culinaria/normas , Calor , Azul de Metileno , Ratones , Coloración y Etiquetado , Factores de Tiempo
15.
Nat Commun ; 12(1): 1681, 2021 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-33727561

RESUMEN

Site-selective modification of oligonucleotides serves as an indispensable tool in many fields of research including research of fundamental biological processes, biotechnology, and nanotechnology. Here we report chemo- and regioselective modification of oligonucleotides based on rhodium(I)-carbene catalysis in a programmable fashion. Extensive screening identifies a rhodium(I)-catalyst that displays robust chemoselectivity toward base-unpaired guanosines in single and double-strand oligonucleotides with structurally complex secondary structures. Moreover, high regioselectivity among multiple guanosines in a substrate is achieved by introducing guanosine-bulge loops in a duplex. This approach allows the introduction of multiple unique functional handles in an iterative fashion, the utility of which is exemplified in DNA-protein cross-linking in cell lysates.


Asunto(s)
Metano/análogos & derivados , Oligonucleótidos/química , Coloración y Etiquetado , Secuencia de Bases , Espectroscopía de Resonancia Magnética con Carbono-13 , Catálisis , Reactivos de Enlaces Cruzados/química , ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Metano/química , Regiones Promotoras Genéticas/genética , Espectroscopía de Protones por Resonancia Magnética , Rodio/química , Especificidad por Sustrato , Proteínas Virales/metabolismo
16.
J Vis Exp ; (169)2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33749668

RESUMEN

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the tumor to support its growth. Their accumulation in tumor and secondary lymphoid tissue leads to the suppression of antitumor immune responses and is thus a target for therapeutic intervention. As it is known that the local cytokine milieu can dictate the functional programming of tumor-infiltrating myeloid cells, strategies have been devised to manipulate the tumor microenvironment (TME) to express a cytokine landscape more conducive to antitumor myeloid cell activity. To evaluate therapy-induced changes in tumor-infiltrating myeloid cells, this paper will outline the procedure to dissociate intradermal/subcutaneous tumor tissue from solid tumor-bearing mice in preparation for leukocyte recovery. Strategies for flow cytometric analysis will be provided to enable the identification of heterogeneous myeloid populations within isolated leukocytes and the characterization of unique myeloid phenotypes. Lastly, this paper will describe a means of purifying viable myeloid cells for functional assays and determining their therapeutic value in the context of adoptive transfer.


Asunto(s)
Traslado Adoptivo , Citometría de Flujo/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Células Mieloides/inmunología , Animales , Línea Celular Tumoral , Femenino , Ratones Endogámicos C57BL , Coloración y Etiquetado , Microambiente Tumoral/inmunología
17.
J Vis Exp ; (169)2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33749676

RESUMEN

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins - α-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) - and one M-line protein - Myomesin-2 (Myom2) - were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.


Asunto(s)
Colorantes Fluorescentes/metabolismo , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Sarcómeros/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Dependovirus/metabolismo , Cuerpos Embrioides/citología , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Miocitos Cardíacos/citología , Coloración y Etiquetado , Imagen de Lapso de Tiempo
18.
J Vis Exp ; (169)2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33749679

RESUMEN

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.


Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular , Proteínas Bacterianas/metabolismo , Cuerpos de Inclusión/metabolismo , Marcaje Isotópico , Mutación Puntual/genética , Canales de Potasio/metabolismo , Replegamiento Proteico , Proteolípidos/aislamiento & purificación , Protones , Coloración y Etiquetado
20.
Nature ; 590(7846): 457-462, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33568812

RESUMEN

In contrast to nearly all other tissues, the anatomy of cell differentiation in the bone marrow remains unknown. This is owing to a lack of strategies for examining myelopoiesis-the differentiation of myeloid progenitors into a large variety of innate immune cells-in situ in the bone marrow. Such strategies are required to understand differentiation and lineage-commitment decisions, and to define how spatial organizing cues inform tissue function. Here we develop approaches for imaging myelopoiesis in mice, and generate atlases showing the differentiation of granulocytes, monocytes and dendritic cells. The generation of granulocytes and dendritic cells-monocytes localizes to different blood-vessel structures known as sinusoids, and displays lineage-specific spatial and clonal architectures. Acute systemic infection with Listeria monocytogenes induces lineage-specific progenitor clusters to undergo increased self-renewal of progenitors, but the different lineages remain spatially separated. Monocyte-dendritic cell progenitors (MDPs) map with nonclassical monocytes and conventional dendritic cells; these localize to a subset of blood vessels expressing a major regulator of myelopoiesis, colony-stimulating factor 1 (CSF1, also known as M-CSF)1. Specific deletion of Csf1 in endothelium disrupts the architecture around MDPs and their localization to sinusoids. Subsequently, there are fewer MDPs and their ability to differentiate is reduced, leading to a loss of nonclassical monocytes and dendritic cells during both homeostasis and infection. These data indicate that local cues produced by distinct blood vessels are responsible for the spatial organization of definitive blood cell differentiation.


Asunto(s)
Rastreo Celular/métodos , Células Mieloides/citología , Mielopoyesis , Coloración y Etiquetado/métodos , Animales , Atlas como Asunto , Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Linaje de la Célula , Autorrenovación de las Células , Células Dendríticas/citología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Femenino , Granulocitos/citología , Listeria monocytogenes/patogenicidad , Listeriosis/microbiología , Factor Estimulante de Colonias de Macrófagos/deficiencia , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Masculino , Ratones , Monocitos/citología , Células Mieloides/metabolismo
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