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Transgenic mouse model expressing tdTomato under involucrin promoter as a tool for analysis of epidermal differentiation and wound healing.

The epidermis is a stratified tissue composed of different keratinocyte layers that create a barrier protecting the body from external influences, pathogens, and dehydration. The barrier function is mainly achieved by its outermost layer, the stratum corneum. To create a mouse model to study pathophysiological processes in the outermost layers of the epidermis in vivo and in vitro we prepared a construct containing red fluorescent td-Tomato reporter sequence under the control of involucrin promoter and its first intron. Transgenic mice were generated by pronuclear injection and the expression

[Simultaneous analysis of lymphocyte markers using triple color markers].

The introduction in flow cytometry of the new reagent Streptavidin Duochrome allows the simultaneous three color analysis of biological samples. Streptavidin Duochrome is a phycoerythrin-Texas red fluorochrome complex conjugated to the protein streptavidin. This novel complex exploits the principle fluorescence energy transfer when this reagent is used with FITC, PE and biotin-conjugated monoclonal antibodies; three different emissions of fluorescence are possible simultaneously using an argon laser at 488 nm (FACScan Becton Dickinson). Using Paint-a-gate software which analyses samples and di

Imaging flow cytometry analysis of intracellular pathogens.

Imaging flow cytometry has been applied to address questions in infection biology, in particular, infections induced by intracellular pathogens. This methodology, which utilizes specialized analytic software makes it possible to analyze hundreds of quantified features for hundreds of thousands of individual cellular or subcellular events in a single experiment. Imaging flow cytometry analysis of host cell-pathogen interaction can thus quantitatively addresses a variety of biological questions related to intracellular infection, including cell counting, internalization score, and subcellular pa

Novel discovery of a lymphatic bridge connecting Schlemm's canal to limbal and conjunctival lymphatic pathway.

PURPOSE: Schlemm's canal (SC) is a critical structure regulating aqueous humor (AH) drainage and intraocular pressure (IOP). It is known that in the conventional outflow pathway, AH flows from SC to episcleral veins. We recently reported a high-resolution three-dimensional (3D) imaging technology for intact eyeballs, SC and ocular surface. Using this advanced technology, we herein report the discovery of a new structure, termed lymphatic bridge, that directly connects SC to the limbal and conjunctival lymphatic pathway. Further investigation on this novel outflow pathway may provide new mechan

Fluorescent microbeads for point-of-care testing: a review.

Microbead-based point-of-care testing (POCT) has demonstrated great promise in translating detection modalities from bench-side to bed-side. This is due to the ease of visualization, high surface area-to-volume ratio of beads for efficient target binding, and efficient encoding capability for simultaneous detection of multiple analytes. This review (with 112 references) summarizes the progress made in the field of fluorescent microbead-based POCT. Following an introduction into the field, a first large section sums up techniques and materials for preparing microbeads, typically of dye-labelled

Объективная фотодинамическая виротерапия, вооруженная генетически закодированным фотосенсибилизатором.

Фотодинамическая терапия (PDT) — минимально инвазивная антивизуальная терапия, которая искореняет клетки опухоли с помощью цитотоксичного воздействия на световое облучение. Однако системное управление фотоленсибилизаторами часто затрудняет предотвращение неблагоприятного воздействия фоточувствительности. Красный флуоресцентный белок KillerRed генерирует реактивные виды кислорода (ROS) при зеленом излучении света. Здесь мы показываем терапевтический потенциал новаторского фотодинамического вирусного агента (TelomeKiller), построенного с помощью человека-промоциатора по обратной транскрипции тел

Совпадение в аннотации оригинала
Photodynamic therapy (PDT) is a minimally invasive antitumor therapy that eradicates tumor cells through a photosensitizer-mediated cytotoxic effect upon light irradiation. However, systemic administration of photosensitizer often makes it difficult to avoid a photosensitive adverse effect. The red fluorescent protein KillerRed generates reactive oxygen spec…

Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria.

Decay-accelerating factor (DAF) is a 70,000 Mr protein that has been isolated from the membrane of red cells. The function of DAF is to inhibit the assembly of amplifying enzymes of the complement cascade on the cell surface, thereby protecting them from damage by autologous complement. We raised monoclonal antibodies to DAF and used them to study its distribution in cells from the peripheral blood of normal individuals and of patients with paroxysmal nocturnal hemoglobinuria (PNH), a disease characterized by the unusual susceptibility of red cells to the hemolytic activity of complement. The

Effect of solid-state fermentation by three different Bacillus species on composition and protein structure of soybean meal.

BACKGROUND: Fermentation efficiency of thermophiles of Bacillus licheniformis YYC4 and Geobacillus stearothermophilus A75, and mesophilic Bacillus subtilis 10 160 on soybean meal (SBM), was evaluated by examining the nutritional and protein structural changes. RESULTS: SBM fermentation by B. licheniformis YYC4, B. subtilis 10 160 and G. stearothemophilus A75 increased significantly the crude and soluble protein from 442.4 to 524.8, 516.1 and 499.9 g kg-1 , and from 53.9 to 203.3, 291.3 and 74.6 g kg-1 , and decreased trypsin inhibitor from 8.19 to 3.19, 2.14 and 5.10 mg g-1 , respectively. Ba

Engineering of CD63 Enables Selective Extracellular Vesicle Cargo Loading and Enhanced Payload Delivery.

Extracellular vesicles (EVs) are mediators of intercellular communication through the transfer of nucleic acids, lipids and proteins between cells. This property makes bioengineered EVs promising therapeutic vectors. However, it remains challenging to isolate EVs with a therapeutic payload due to the heterogeneous nature of cargo loading into EVs. In this study, enrichment of EVs with a desired cargo was possible through engineering of the hallmark CD63 transmembrane protein. E-NoMi refers to engineered CD63 with mCherry on the inside of the EV membrane and a tag (3xFLAG) exposed on the outsid

Intracellular protein and nucleic acid measured in eight cell types using deep-ultraviolet mass mapping.

We present measurements by deep-ultraviolet mass mapping of nucleic acid (NA) and protein for five commonly cultured and three primary cell types. The dry mass distribution at submicron resolution was determined on a single-cell basis for 250-500 cells from each of these types. Since the method carries a direct reference to a spectrophotometric standard (molar extinction coefficient), we are able to calibrate the absolute weight distributions both on a cell-to-cell basis within each type and across types. We also provide a calibration in absolute mass units for fluorescence-based measurements

Direct immobilization and recovery of recombinant proteins from cell lysates by using EctP1-peptide as a short fusion tag for silica and titania supports.

Immobilization of protein, compared to the use of free protein, offers improved stability, easy separation and continuous reusability. Howev … compared to the recovery yield by the salts, NaCl and MgCl2 (about 30-51%). Using BCA and monomeric red fluorescent protein (mRFP) as model proteins, the EctP1 fusion proteins were released in a biologically active form with approximately 80% recovery and 93% purity. Our approach is a simple and reproducible technique for direct immobilization of recombinant proteins fro …

NicE-viewSeq: An Integrative Visualization and Genomics Method to Detect Accessible Chromatin in Fixed Cells.

A novel genome-wide accessible chromatin visualization, quantitation, and sequencing method is described, which allows in situ fluorescence visualization and sequencing of the accessible chromatin in the mammalian cell. The cells are fixed by formaldehyde crosslinking, and processed using a modified nick translation method, where a nicking enzyme nicks one strand of DNA, and DNA polymerase incorporates biotin-conjugated dCTP, 5-methyl-dCTP, Fluorescein-12-dATP or Texas Red-5-dATP, dGTP, and dTTP. This allows accessible chromatin DNA to be labeled for visualization and on bead NGS library prepa

Soft structure formation and cancer cell transport mechanisms of a folic acid-dipeptide conjugate.

Folic acid (FA) is a low-molecular-weight micronutrient, which plays a critical role in the prevention of birth defects and cancers. It is also essential for biochemical pathways responsible for DNA synthesis and maintenance and for the generation of new red blood cells. Cellular trafficking of FA and folate is based on its high-affinity binding to cognate folate receptor, a protein commonly expressed in several human cancers. Thus, folate conjugates of drugs, plasmids, biosensors, contrast, and radiodiagnostic imaging agents have been used for assisted delivery in folate receptor-positive can

Visualization of GLUT1 Trafficking in Live Cancer Cells by the Use of a Dual-Fluorescence Reporter.

Glucose metabolism is an essential process for energy production and cell survival for both normal and abnormal cellular metabolism. Several glucose transporter/solute carrier 2A (GLUT/SLC2A) superfamily members, including glucose transporter 1 (GLUT1), have been shown to mediate the cellular uptake of glucose in diverse cell types. GLUT1-mediated glucose uptake is a transient and rapid process; thus, the real-time monitoring of GLUT1 trafficking is pivotal for a better understanding of GLUT1 expression and GLUT1-dependent glucose uptake. In the present study, we established a rapid and effect

Exploring new potential role of DDB2 by host cell reactivation assay in human tumorigenic cells.

BACKGROUND: The Host Cell Reactivation assay (HCR) allows studying the DNA repair capability in different types of human cells. This assay was carried out to assess the ability in removing UV-lesions from DNA, thus verifying NER efficiency. Previously we have shown that DDB2, a protein involved in the Global Genome Repair, interacts directly with PCNA and, in human cells, the loss of this interaction affects DNA repair machinery. In addition, a mutant form unable to interact with PCNA (DDB2PCNA-), has shown a reduced ability to interact with a UV-damaged DNA plasmid in vitro. METHODS: In this

Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof.

Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of cl

Light-emitting diode spectral sensitivity relationship with reproductive parameters and ovarian maturation in yellowtail damselfish, Chrysiptera parasema.

The present study investigated the effects of exposure to different light spectra and intensities on ovarian maturation in yellowtail damselfish, Chrysiptera parasema over a 4-months period. We used a white fluorescent bulb and three different light-emitting diodes (LEDs: red, peak at 630nm; green, 530nm; blue, 450nm), at three different intensities each (0.3, 0.6, and 0.9W/m(2)). The effects of different illuminations were assessed by measuring the mRNA and protein expressions of vitellogenin (VTG) and estrogen receptor (ER), gonadosomatic index (GSI), and plasma estradiol-17β (E2) hormone le

[Application of fluorescence probe marked by quantum dots to detect early submandibular lymph node metastasis in a nude mouse model].

OBJECTIVE: A fluorescence probe marked with a specific angle protein antibody by using quantum dots QDs605-CK (AE1/AE3) was utilized to detect metastasis and micrometastasis rates of early submandibular lymph node metastatic carcinoma in a nude mouse model. The method was compared with traditional immunohistochemistry (IHC) staining and hematine-eosin (HE) staining to explore a new method for early diagnosis and treatment of tongue squamous cell carcinoma. METHODS: Human tongue Tca8113 cells were cultured and inoculated in 18 nude mice tongue body tissues (except the midline) to establish sub

Application of JC1 for non-toxic isolation of cells with MDR transporter activity by flow cytometry.

The DNA intercalating dye Hoechst 33342 or its close analog DCV are actively removed from cells by the multidrug resistance transporter ABCG2, a protein overexpressed in metastatic cells and somatic stem cells. In bivariate blue-red flow cytometry fluorescent plots active Hoechst or DCV efflux combined with a concentration dependent bathochromic shifts of these nuclear dyes leads to the segregation of the transporter-rich cells into a distinct cell cohort tilted towards the shorter wavelength axis of the plot, the cohort is generically known as the side population (SP). This feature has facili

Upregulation of FOXO1 contributes to lipopolysaccharide-induced pulmonary endothelial injury by induction of autophagy.

BACKGROUND: Autophagy is activated during the pathogenesis of endothelial dysfunction and sepsis-associated acute lung injury (ALI). This st … ted gene 5 (ATG5) or FOXO1. Endothelial autophagic flux was assessed by transfection of MLVECs with red fluorescent protein (RFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 (RFP-GFP-LC3) adenovirus. Endothelial permeability was analyzed by the diffusion of fluorescein isothiocyanate-carboxymethyl (FITC)-dextran through the endothelial monolayer. Evans …