Fluorescent Vital Dye Research Articles

DeepDOF-SE: affordable deep-learning microscopy platform for slide-free histology

Histopathology plays a critical role in the diagnosis and surgical management of cancer. However, access to histopathology services, especially frozen section pathology during surgery, is limited in resource-constrained settings because preparing slides from resected tissue is time-consuming, labor-intensive, and requires expensive infrastructure. Here, we report a deep-learning-enabled microscope, named DeepDOF-SE, to rapidly scan intact tissue at cellular resolution without the need for physical sectioning. Three key features jointly make DeepDOF-SE practical. First, tissue specimens are stained directly with inexpensive vital fluorescent dyes and optically sectioned with ultra-violet excitation that localizes fluorescent emission to a thin surface layer. Second, a deep-learning algorithm extends the depth-of-field, allowing rapid acquisition of in-focus images from large areas of tissue even when the tissue surface is highly irregular. Finally, a semi-supervised generative adversarial network virtually stains DeepDOF-SE fluorescence images with hematoxylin-and-eosin appearance, facilitating image interpretation by pathologists without significant additional training. We developed the DeepDOF-SE platform using a data-driven approach and validated its performance by imaging surgical resections of suspected oral tumors. Our results show that DeepDOF-SE provides histological information of diagnostic importance, offering a rapid and affordable slide-free histology platform for intraoperative tumor margin assessment and in low-resource settings.

Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Larval Neuromuscular Junction.

Focal recording is an extracellular method for studying synaptic transmission at the Drosophila larval neuromuscular junction (NMJ) designed for the study of synaptic activity of one or a few synaptic boutons rather than the ensemble activity of all the boutons as occurs with intracellular recording or two-electrode voltage-clamp. This is a useful technique for investigating the properties of different motor neurons that innervate the same muscle, applying statistical analysis to discrete synaptic events, and investigating the heterogeneity of synaptic release properties among boutons. A compound microscope with epifluorescent imaging capability is very helpful but not essential; any GFP Drosophila strain that labels the nerve terminal or synaptic boutons can be used to locate the boutons. A particularly useful strain is Mhc-CD8-Sh-GFP, containing a GFP molecule that is expressed in muscle, localizes to the postsynaptic apparatus, and outlines boutons. Vital fluorescent dyes (such as 4-Di-2-Asp) may also be applied to the dissected preparation to help locate boutons. The microscope should be equipped for differential interference contrast (DIC or Nomarski) optics if fluorescence is not used.

Effect of streptococcal arginine deiminase on the function of CD4<sup>+</sup> and CD8<sup>+</sup>T lymphocytes

Arginine metabolism plays an important role in regulating the functions of immune cells in mammals. Pathogenic microbes use the mechanism of arginine depletion to suppress the immune response during infection. Arginine deiminase is a microbial arginine-hydrolyzing enzyme important for survival at low pH in the focus of infection, or in phagolysosomes, as well as under low-glucose conditions. The effect of bacterial arginine deiminase on the functions of adaptive immune cells remains poorly understood. The aim of our study was to evaluate the effect of streptococcal arginine deiminase on the proliferation and autophagy of CD4+ and CD8+ human peripheral blood T lymphocytes.The enzyme effects were studied with supernates of ultrasonic lysates from parental Streptococcus pyogenes M49-16, and its isogenic mutant with inactivated arcA gene (Streptococcus pyogenes M49-16delarcA). The study was performed with blood samples of healthy donors. The fraction of mononuclear leukocytes was isolated by centrifugation in a Ficoll density gradient. To evaluate proliferation levels, a method based on the staining of intracellular proteins with vital fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) was used. The level of autophagy was studied using the fluorescent Lysotracker Green DND-26 dye. To analyze the proliferation and autophagy of T helper cells (CD3+CD4+) and cytotoxic T lymphocytes (CD3+CD4-), the obtained cell suspensions were stained with antibodies against CD4, CD45RA, and CD3. The proportion of necrotic cells was determined by staining with a fluorescent DNA-binding DAPI dye. The normality of the distribution was assessed by Shapiro–Wilk test. The data were analyzed using Kruskal–Wallis criterion, followed by Mann–Whitney criterion for pairwise comparisons and expressed as median and interquartile ranges (Q0.25-Q0.75).When comparing the effects of supernatants from the parental and mutant streptococcal strains, which differed in expression of arginine deiminase gene, we have shown that the bacterial enzyme had no effect on the functions of inactive lymphocytes. However, streptococcal arginine deiminase completely suppressed proliferation of CD4+ and CD8+T lymphocytes stimulated with anti-CD2/CD3/CD28 antibodies. These effects were accompanied by a decrease in the autophagy levels. At the same time, arginine deiminase did not exert cytotoxic effects upon lymphocytes. L-arginine applied at the doses exceeding physiological levels caused restoration of the cellular functions. There were no differences between the studied parameters of CD4+ and CD8+T lymphocyte subsets.The obtained data show that the antiproliferative effect of arginine demimnase may be associated with ability of the enzyme to inhibit autophagy and confirm an opportunity of the bacterial enzyme to suppress host adaptive immune responses.

Relative survival and cell adhesion of multipotent mesenchymal cells from primary teeth pulp on the surface membranes and sponges produced from collagen

To study the behavior of SHED cell culture on different types of materials for the regeneration of periodontal tissues with different porosity. Porous collagen material Fibro-Gide (Geitstlich Pharma AG, Switzerland), designed to increase the volume of the gum and the barrier collagen membrane Bio-Gide (Geitstlich Pharma AG, Switzerland) were studied in vitro on SHED cultures. As a control sample, a Spongostan sponge made of gelatin (Johnson & Johnson Medical, UK) with the most pronounced porosity and wettability was used. Acute cytotoxicity was determined using a screening method for assessing the number of living cells in a sample (MTT test). SHED cells were sown on the materials to study the attachment of cells to materials and their migration inside the samples. Before seeding, the cells were stained with vital fluorescent dye PKH26 (red fluorescent cell linker kit, Sigma, Germany) for further visualization. Using the MTT test it was shown that they do not have cytotoxic effects. At the same time by the 8th day of the experiment in the presence of Fibro-Gide and Bio-Gide the cells showed an increase in proliferative activity by 19% and 12%, respectively compared with the control group. The cells attached and spread out on the surface of the materials and migrated into the thickness of porous Fibro-Gide and Spongostan. The in vitro study showed that the most favorable material for SHED cell culture is the collagen material Fibro-Gide with sufficient porosity, elasticity and hydrophilicity. SHED cells attach to the collagen matrix and easily penetrate into the sample, filling the entire internal space, while the proliferative capacity of the cell culture increases.

Abstract 2904: Mechanical loading induced osteogenic response does not interfere with the colonization of breast cancer in bone

Abstract Breast cancer is the most commonly diagnosed cancer and frequently metastasizes to bone. Previous evidence has suggested that cancer cells target the osteoblastic lineage cells to establish colonization of bone. It is well established that mechanical loading (e.g. load bearing exercise) elevates bone remodeling via stimulating osteoblastic activities. Whether mechanical loading facilitates the colonization of breast cancer cells in bone is not fully understood. To test this, we have firstly applied a murine tibial mechanical loading model and determined the effects of mechanical loading on bone remodeling in vivo. Six 10-week-old BALB/c nude female mice were subjected to a right tibial dynamic load (9N) onto a 2N pre-load using the ElectroForce 5500 Test Instrument, 40 load cycles with 10 sec intervals, 3 times a week for 1 week. Mice were then euthanized with additional 4 mice euthanized before mechanical loading as baseline controls. Quantitative micro-CT analysis suggested that bone mass related parameters significantly increased in the loaded tibias compared to contra-lateral non-loaded controls, while data in the baseline group showed no differences between left and right tibias. As previously published 2-week loading regimens, one week of loading was enough to induce a 24.3% increase in trabecular bone volume (P=0.001, paired t-test), a 9.0% increase in trabecular thickness (P= 0.01), and a 17.9% increase in cortical bone volume (P<0.0001). Using this model, we tested whether mechanical loading affects the arrival of breast cancer cells in bone. Human and mouse breast cancer cells (MDA-MB-231 and 4T1) were firstly labelled with vital fluorescent lipophilic dyes (Vybrant DiD) to allow tracking of individual tumor cells arriving in bone ex vivo. Cells were injected into the circulation (intracardiac) after mice (BALB/c nude and BALB/c) subjected to 1-week of mechanical loading. Mice were then loaded 3 times more to maintain bone mass before euthanized 7 days post-injection. DiD labelled cancer cells were examined and quantified in tibial bone marrow using multiphoton microscopy ex vivo. In both xenograft and syngeneic models, there were no significant differences found in the number of breast cancer cells arriving in bone marrow (MDA: P=0.32; 4T1: P=0.67) and their relative location to bone surfaces (MDA: P= 0.45; 4T1: P= 0.91) in loaded vs non-loaded bones. There was no significant difference in size of objectives (indicating size of micro tumors) detected in the MDA model (P= 0.21), comparing loaded to non-loaded control tibias but a significant 17.7% reduction in the 4T1 model in the loaded tibias (P=0.02). These pre-clinical data demonstrate that mechanical loading induced osteoblastic bone formation does not affect the initial colonization of breast cancer cells in bone, and suggest that load bearing exercise will not increase the risk of breast cancer bone metastasis in patients. Citation Format: Alexandria R. Sprules, Norain B. Latif, Janet E. Brown, Alison Gartland, Ning Wang. Mechanical loading induced osteogenic response does not interfere with the colonization of breast cancer in bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2904.

Fine structure of Drosophila larval salivary gland ducts as revealed by laser confocal microscopy and SEM

The functions of the larval salivary glands (SGs) of Drosophila are traditionally associated with the production of a massive secretion during puparium formation; it is exocytosed into a centrally located lumen and subsequently expectorated via ducts, the pharynx and mouth. This so-called proteinaceous glue serves as an adhesive to attach the puparial case to a solid substrate. Great attention has been paid to the secretory cells of SGs, which are famous for their giant polytene chromosomes. However, substantially less attention has been devoted to individual or common ducts that form the most proximal portion of the SG organ via which the glue is released into the pharynx. In the present paper, we describe the organization and fine structure of the taenidia, highly specialized circumferential ring-like extracellular (cuticular) components on the internal side of these tubes. Two chitin-specific probes that have previously been used to recognize taenidia in Drosophila tracheae, Calcofluor White M2R (also known as Fluorescent Brightener 28) and the novel vital fluorescent dye SiR-COOH, show positively stained ductal taenidia in late larval SGs. As seen using scanning electron microscopy (SEM), the interior of the ductal tube contains regular and densely-arranged ridge-like circumferential rings which represent local thickenings of the cuticle in various geometries. The microtubular arrays that optically colocalize with taenidia in both the trachea and SG ducts are specifically and strongly recognized by fluorescently-conjugated colchicine as well as anti-tubulin antibody. In contrast to taenidia in the tracheae, the analogous structures in SG ducts cannot be detected by fluorescently-labeled phalloidin or even actin-GFP fusion protein, suggesting that the ducts lack a cortical network made of filamentous actin. We speculate that these taenidia may serve to reinforce the duct during the secretory processes that SGs undergo during late larval and late prepupal stages.

A Novel Flow Cytometric Method to Study Cytotoxic Activity in Whole Blood Samples.

For several decades, cell-mediated cytotoxicity has been measured using the 51 Cr release assay. This assay, however, has several drawbacks and flow cytometry has been used as an alternative to measure cytotoxic activity. Here, we present a quantitative method for cell-mediated cytotoxicity studies, preserving cellular function with minimal sample manipulation. Cytotoxic activity is simply and reproducibly measured as the ability of cytotoxic cells to lyse K562 target cells previously loaded with Calcein-AM vital stain. After spiking a known number of fluorescent viable K562 target cells into whole blood, cell mixtures are incubated for 2 h in a cell incubator and the remaining spiked cells are counted by flow cytometry. In order to discriminate nucleated cells, erythrocytes, and debris, unlysed whole blood is stained with a cell permeable DNA vital fluorescent dye. Cell-mediated lysis is measured by comparing target counts for different effector-to-target ratios. Since the cytotoxicity of these dyes is relatively low, this method can be broadly applied to studies of innate immune response to tumors and infections, especially where target-killing activity might be compromised by small volume samples or low frequency of cytotoxic cells. © 2020 International Society for Advancement of Cytometry.

Rigor and Reproducibility in Confocal Fluorescence Microscopy.

Rigor and Reproducibility in Confocal Fluorescence Microscopy.

Isolation and Characterization of a CD34+ Sub-Clone in B-Cell Lymphoma

Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy in the US. Many types remain incurable despite response to initial therapy and achievement of complete remission (CR). Advanced laboratory techniques like multicolor flow cytometry (MCF) and polymerase chain reaction (PCR) have demonstrated persistence of rare malignant cell population post therapy referred to as minimal residual disease (MRD). However, the functional and biological characteristics of this population have not been fully elucidated. Established B-lymphoma cell lines (B-NHL) and patient-derived samples (PDS) were analyzed using 8-color FCM of leukemia and lymphoma antibody panels (28 antibodies). The CD34+ sub-population was enriched using in vitro exposure to 2-chlorodeoxyadenosine (2-CdA), and a CD34-coated magnetic beads isolation procedure (Miltenyi Biotech). Genetic analysis of CD34+ and CD34-/parent cell fractions was done by karyotyping, and by chromosomal microarray (CMA) using the oligonucleotide-single nucleotide polymorphism (Oligo-SNP), whole genome Agilent 180K GGXChip+SNP (Agilent Technologies, Inc). Sensitivity to chemotherapy was assayed by short-term in vitro exposure to drugs. Clonogenicity was determined by soft agar colony formation assay, and proliferation was determined using DNA staining with propidium iodide and flow cytometry. The side population was determined using the fluorescent vital dye Hoechst 33342 and flow cytometry. Analysis of three B-NHL cell lines revealed the presence of a minute sub-clone (<1%) of monotypic B-cells that expresses CD34. This sub-population enriched several folds in response to exposure of the cells to 2-CdA. Enrichment was highest in the follicular lymphoma cell line, WSU-FSCCL reaching more than 50 folds compared with control (13.2% vs 0.26%), followed by WSU-WM, 10-fold (2.36% vs 0.23%), and least in WSU-DLCL2 (1.25% vs 0.71%). Using a CD34 microbead positive selection technique, we were able to enrich the CD34+ population up to 80% purity. Western blots confirmed higher expression of CD34 protein in the enriched population compared with parent cells. Using StemPro media (Gibco by Life Technologies), CD34+ cell fraction of WSU-WM showed more sustained growth and viability in culture over 9-day period compared with parent cells. This technique allowed the generation of large number of CD34+ cells for further characterization. Except for CD34 expression, this cell fraction expressed identical phenotype, karyotype, SNP and MCA profile to parent cells. However, the CD34+ fraction was more proliferative with an increase in S-phase cells (~60% vs 20% in control cells) and Hoechst 33342-positive cells (~40%). These cells were clonogenic even in presence of the cytotoxic agents Doxorubicin and 2-CdA that completely inhibited colony formation in parent cells. Resistance to chemotherapy was also noticed in liquid culture. A CD34+ population within clonal B-cells was also detected in 8 of 8 PDS. The lowest number of CD34+ cells was detected in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and the highest (5.35%) was seen in Richter's transformation of CLL. The isolated CD34+ cells in this study show many features of what is known as Lymphoma Stem Cells implicated in resistance to therapy and disease recurrence. Its relatedness to MRD cells is uncertain, however, since CD34 is not routinely included in MRD antibody panel. The observation that CD34+ cells are genetically identical to parent cells suggests an epigenetic modification. This conclusion is supported by our finding that Ehd3 expression is dramatically reduced in the CD34+ cell population of WSU-DLCL2 compared with parent cells. Ehd3 was recently identified as an epigenetically-silenced gene in solid tumors and in hematological malignancies including leukemia and lymphoma. The Ehd3-encoded protein belongs to the family of C-terminal Eps15 homology domain-containing (EHD) proteins, implicated in intracellular trafficking of tubular recycling endosomes (TRE), apoptosis, and cell cycle arrest. Further studies are underway to determine the contribution of Ehd3 to the biological and functional characteristics of the CD34+ cells in B-NHL. Utilizing the CD34+ monotypic B-cells in B-NHL as a model for screening new therapeutic agents and to identify potential new therapeutic strategies may lead to a more effective treatment of B-NHL. Disclosures No relevant conflicts of interest to declare.

Erythrosin B: a versatile colorimetric and fluorescent vital dye for bacteria.

Rapidly assaying cell viability for diverse bacteria species is not always straightforward. In eukaryotes, cell viability is often determined using colorimetric dyes; however, such dyes have not been identified for bacteria. We screened different dyes and found that erythrosin B (EB), a visibly red dye with fluorescent properties, functions as a vital dye for many Gram-positive and -negative bacteria. EB worked at a similar concentration for all bacteria studied and incubations were as short as 5min. Given EB's spectral properties, diverse experimental approaches are possible to rapidly visualize and/or quantitate dead bacterial cells in a population. As the first broadly applicable colorimetric viability dye for bacteria, EB provides a cost-effective alternative for researchers in academia and industry.

Initial Assessment of Variability of Responses to Toxicants in Donor-Specific Endothelial Colony Forming Cells.

There is increased interest in using high throughput in vitro assays to characterize human population variability in response to toxicants and drugs. Utilizing primary human endothelial colony-forming cells (ECFCs) isolated from blood would be highly useful for this purpose because these cells are involved in neonatal and adult vasculogenesis. We characterized the cytotoxicity of four known toxic chemicals (NaAsO2, CdCl2, tributyltin [TBT], and menadione) and their four relatively nontoxic counterparts (Na2HAsO4, ZnCl2, SnCl2, and phytonadione, respectively) in eight ECFC clones representing four neonatal donors (2 male and 2 female donors, 2 clones per donor). ECFCs were exposed to 9 concentrations of each chemical in duplicate; cell viability was evaluated 48 h later using the fluorescent vital dye fluorescent dye 5-Carboxyfluorescein Diacetate (CFDA), yielding concentration-effect curves from each experiment. Technical (day-to-day) variability of the assay, assessed from three independent experiments, was low: p-values for the differences of results were 0.74 and 0.64 for the comparison of day 2 vs. day 1 and day 3 vs. day 1, respectively. The statistical analysis used to compare the entire concentration-effect curves has revealed significant differences in levels of cytotoxicity induced by the toxic and relatively nontoxic chemical counterparts, demonstrating that donor-specific ECFCs can clearly differentiate between these two groups of chemicals. Partitioning of the total variance in the nested design assessed the contributions of between-clone and between-donor variability for different levels of cytotoxicity. Individual ECFC clones demonstrated highly reproducible responses to the chemicals. The most toxic chemical was TBT, followed by NaAsO2, CdCl2, and Menadione. Nontoxic counterparts exhibited low cytotoxicity at the higher end of concentration ranges tested. Low variability was observed between ECFC clones obtained from the same donor or different donors for CdCl2, NaAsO2, and TBT, but for menadione, the between-donor variability was much greater than the between-clone variability. The low between-clone variability indicates that an ECFC clone may represent an individual donor in cell-based assays, although this finding must be confirmed using a larger number of donors. Such confirmation would demonstrate that an in vitro ECFC-based testing platform can be used to characterize the inter-individual variability of neonatal ECFCs exposed to drugs and/or environmental toxicants.

Fine scale differences within the vagal neural crest for enteric nervous system formation

The enteric nervous system is mostly derived from vagal neural crest (NC) cells adjacent to somites (s)1–7. We used in ovo focal fluorescent vital dyes and focal electroporation of fluorophore-encoding plasmids in quail embryos to investigate NC cell migration to the foregut initially and later throughout the entire gut. NC cells of different somite-level origins were largely separate until reaching the foregut at about QE2.5, when all routes converged. By QE3.5, NC cells of different somite-levels became mixed, although s1-s2 NC cells were mainly confined to rostral foregut. Mid-vagal NC-derived cells (s3 and s4 level) arrived earliest at the foregut, and occurred in greatest number. By QE6.5 ENS was present from foregut to hindgut. Mid-vagal NC-derived cells occurred in greatest numbers from foregut to distal hindgut. NC-derived cells of s2, s5, and s6 levels were fewer and were widely distributed but were never observed in the distal hindgut. Rostro-vagal (s1) and caudo-vagal (s7) levels were few and restricted to the foregut. Single somite levels of quail neural tube/NC from s1 to s8 were combined with chick aneural ChE4.5 midgut and hindgut and the ensemble was grown on the chorio-allantoic membrane for 6 days. This tests ENS-forming competence in the absence of intra-segmental competition between NC cells, of differential influences of segmental paraxial tissues, and of positional advantage. All vagal NC-levels, but not s8 level, furnished enteric plexuses in the recipient gut, but the density of both ENS cells in total and neurons was highest from mid-vagal level donors, as was the length colonised. We conclude that the fate and competence for ENS formation of vagal NC sub-levels is not uniform over the vagal level but is biased to favour mid-vagal levels. Overviewing this and prior studies suggests the vagal region is, as in its traditional sense, a natural unit but with complex sub-divisions.

Multispectral Live-Cell Imaging.

Fluorescent proteins and vital dyes are invaluable tools for studying dynamic processes within living cells. However, the ability to distinguish more than a few different fluorescent reporters in a single sample is limited by the spectral overlap of available fluorophores. Here, we present a protocol for imaging live cells labeled with six fluorophores simultaneously. A confocal microscope with a spectral detector is used to acquire images, and linear unmixing algorithms are applied to identify the fluorophores present in each pixel of the image. We describe the application of this method to visualize the dynamics of six different organelles, and to quantify the contacts between organelles. However, this method can be used to image any molecule amenable to tagging with a fluorescent probe. Thus, multispectral live-cell imaging is a powerful tool for systems-level analysis of cellular organization and dynamics. © 2018 by John Wiley & Sons, Inc.

Transepithelial migration of mucosal hemocytes in Crassostrea virginica and potential role in Perkinsus marinus pathogenesis.

We have recently described the presence of hemocytes associated with mucus covering the pallial organs (mantle, gills, and body wall) 3 of the eastern oyster Crassostrea virginica. These hemocytes, hereby designated "pallial hemocytes" share common general characteristics with circulating hemocytes but also display significant differences particularly in their cell surface epitopes. The specific location of pallial hemocytes as peripheral cells exposed directly to the marine environment confers them a putative sentinel role. The purpose of this study was to gain a better understanding of the source of these pallial hemocytes by evaluating possible exchanges between circulatory and pallial hemocyte populations and whether these exchanges are regulated by pathogen exposure. Bi-directional transepithelial migrations of hemocytes between pallial surfaces and the circulatory system were monitored using standard cell tracking approaches after staining with the vital fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in conjunction with fluorescent microscopy and flow cytometry. Results showed bi-directional migration of hemocytes between both compartments and suggest that hemocyte migration from the pallial mucus layer to the circulatory system may occur at a greater rate compared to migration from the circulatory system to the pallial mucus layer, further supporting the role of pallial hemocytes as sentinel cells. Subsequently, the effect of the obligate parasite Perkinsus marinus and the opportunistic pathogen Vibrio alginolyticus on transepithelial migration of oyster hemocytes was investigated. Results showed an increase in hemocyte migration in response to P. marinus exposure. Furthermore, P. marinus cells were acquired by pallial hemocytes before being visible in underlying tissues and the circulatory system suggesting that this parasite could use pallial hemocytes as a vehicle facilitating its access to oyster tissues. These results are discussed in light of new evidence highlighting the role of oyster pallial organs as a portal for the initiation of P. marinus infections in oysters.

Vital fluorescent dyes for study of silicifying organisms

The mini-review describes application of vital fluorescent dyes to study silica biosynthesis in diatom algae and siliceous sponges. Mechanisms of these processes are not clear and study of growing siliceous constructions in living organisms is complicated with silica transparency and presence of mature elements (siliceous valves of diatoms and spicules of sponges). Specifc fluorescence dyes stain growing siliceous elements of skeletons and also primary siliceous particles which are formed during silicon capture from the environment. Vital fluorescent dyes based on 7-nitro-2,1,3-benzoxadiazole, pyridyl oxazole and coumarin fluorophores are described as well as examples of application of these dyes to study diatom algae and siliceous sponges.

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins.

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro antifungal activity against a broad-spectrum of fungal pathogens and therefore have the potential to be used as antifungal agents in transgenic crops. In order to harness the full potential of plant defensins for diseases control, it is crucial to elucidate their mechanisms of action (MOA). With the advent of advanced microscopy techniques, live-cell imaging has become a powerful tool for understanding the dynamics of the antifungal MOA of plant defensins. Here, a confocal microscopy based live-cell imaging method is described using two fluorescently labeled plant defensins (MtDef4 and MtDef5) in combination with vital fluorescent dyes. This technique enables real-time visualization and analysis of the dynamic events of MtDef4 and MtDef5 internalization into fungal cells. Importantly, this assay generates a wealth of information including internalization kinetics, mode of entry and subcellular localization of these peptides. Along with other cell biological tools, these methods have provided critical insights into the dynamics and complexity of the MOA of these peptides. These tools can also be used to compare the MOA of these peptides against different fungi.

Migration of normal stem CD34 + cells to the brain in experimental models of ischemic stroke and glioblastoma

Background: Glyoblastoma is one of the most common brain tumors, which is characterized by high aggressiveness and unfavorable prognosis. One of the leading causes of the therapeutic resistance of glioblastoma is associated with tumor stem cells (TSC). One of the promising ways to solve this problem is the creation of biomedical products based on stem and progenitor cells of the human bone marrow. We have established, in vitro, that the stem CD34 + cells of the bone marrow are able to migrate toward the cells of different types. In vivo, stem cells of bone marrow CD 34+ actively migrate to the tumor tissue, where they accumulate in the area of invasion and penetrate into the hypoxic zones of the tumor. We suggested that the appearance of CD34 + cells in the neoplastic tissue is associated with the processes of neoplastic angiogenesis, and the role of hypoxia in this case is secondary. The study of migration processes and the homing of stem cells in the pathological focus in the brain on experimental models of acute ischemia and glioblastoma. Methods: The work was performed on 60 mature Wistar female rats divided into 2 groups (N = 30). The animals of the first group underwent stereotactic implantation of C6 glioma cells in the brain, tumor development was confirmed by magnetic resonance imaging (MRI) data; the second group - the ligation of the common and internal carotid artery on the left was performed surgically. The development of the stroke was confirmed by MRI data. The haploidentical CD34 + stem cells stained with the fluorescent vital dye Vybrant® CFDA SE (V12883, Molecular Probes, USA) were injected into the tail vein. The animals were removed from the experiment on days 4 and 8, the material was subjected to morphological and immunohistochemical analysis. Results: Four days after the infusion of cells on the glioblastoma model, the fluorescent label was detected at the periphery of the tumor site, which was associated with localized blood vessels in this region. On day 8, the area of fluorescing objects was 12.7 ± 9.2% at the tumor boundary, 47.6 ± 9.3% in the parenchyma of the tumor tissue. In the experimental model of ischemia, fluorescent substances in the spectrum of characteristic CFDA on days 4 and 8 were not detected. Conclusions: Hypoxic brain damage is not enough to ensure the effective homing of cells in the tumor. With a glioblastome formed a specific type of microglia, this circumstance allows one to assume the possibility of increasing the life expectancy of patients with glioblastoma. Legal entity responsible for the study: School of Biomedicine, Far Eastern Federal University, Russian Federation Funding: Russian Scientific Fund (14-15-00084) Disclosure: All authors have declared no conflicts of interest.

New “old” polylactides for tissue engineering constructions

New methods for the synthesis of aliphatic polyesters were proposed and realized with highly effective and low-toxic 1,5,7-triazabicyclo[4.4.0]decene-5 and (2,6-di-tert-butyl-4-methylphenoxy)-butyl-magnesium as catalysts and ethanol as an initiator. The interaction processes between D,L-polylactides of different molecular weights, obtained through these methods, and supercritical carbon dioxide were studied to provide “soft and dry” formation (without high temperatures and toxic organic solvents) of highly porous (up to 90 vol %) bioresorbable matrices for tissue engineering constructions. The chemical structure, molecular weight characteristics, morphology, cytotoxicity, and matrix properties of the samples synthesized from D,L-polylactides were studied with nuclear magnetic resonance spectroscopy, gel permeation chromatography, scanning electron microscopy, colorimetric testing to estimate the metabolic activity of cells (MTT test), and coloration with a vital fluorescent dye of in vitro cultures of multipotent stromal cells of human adipose tissue. A comparative analysis for biocompatibility of these matrices with control samples made from “medical grade” polylactide analog produced industrially indicates that the materials developed can be recommended for tissue engineering.

Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging

Although having great potential for live cell imaging to address numerous cell biological questions with high spatial and temporal resolution, primary cell cultures of zebrafish embryos are not widely used. We present an easy-to-use protocol for preparing primary cell cultures of 2 dpf zebrafish embryos allowing for live cell imaging of fully differentiated cells such as neurons and myocytes. We demonstrate that different cell types can be identified by morphology and expression of transgenic cell type-specific fluorescent reporters and that fluorescent cells can be sorted by flow cytometry to prepare an enriched culture. To facilitate subcellular imaging in live primary cells, we successfully tested a selection of fluorescent vital dyes. Most importantly, we demonstrate that zebrafish primary cells can be transfected efficiently with expression constructs allowing for visualizing subcellular structures with fluorescent marker proteins for time lapse imaging. We propose zebrafish primary cell culture as a versatile tool to address cell biological questions in combination with a powerful in vivo model.

Live Cell Imaging in Fission Yeast.

Live cell imaging complements the array of biochemical and molecular genetic approaches to provide a comprehensive insight into functional dependencies and molecular interactions in fission yeast. Fluorescent proteins and vital dyes reveal dynamic changes in the spatial distribution of organelles and the proteome and how each alters in response to changes in environmental and genetic composition. This introduction discusses key issues and basic image analysis for live cell imaging of fission yeast.