scholarly journals Amphipathic Fluorescent Dyes for Sensitive and Long-Term Monitoring of Plasma Membranes

2020 ◽  
Author(s):  
Masataka Takahashi ◽  
Ryo Seino ◽  
Takatoshi Ezoe ◽  
Munetaka Ishiyama ◽  
Yuichiro Ueno
Keyword(s):  
Fluorescent Probes ◽  
Plasma Membranes ◽  
Fluorescent Dyes ◽  
Imaging Techniques ◽  
Critical Role ◽  
Strong Acid ◽  
Endocytic Pathway ◽  
Polycyclic Aromatic ◽  
Low Cytotoxicity ◽  
High Cytotoxicity

ABSTRACTThe plasma membrane (PM) plays a critical role in many cellular processes, and PM dysfunction is a key biomarker related to the cell status and several diseases. Imaging techniques using small fluorescent probes have become increasingly important tools for visualizing living cells, particularly their PMs. Among the commercially available PM-specific probes, PKH dyes are widely used; however, the utility of these dyes is limited by their short membrane retention times and high cytotoxicity. Herein, PlasMem Bright Green and Red are implemented as new PM-specific fluorescent probes, which employ a polycyclic aromatic fluorophore to improve their retention ability and a strong acid moiety to reduce their transmembrane diffusion and cytotoxicity. We demonstrate that the long retention and low cytotoxicity of the PlasMem Bright dyes enable them to be applied for observing neuronal PMs and monitoring PM dynamics involving the endocytic pathway. Furthermore, we successfully detected mitochondria in nerve axons over long periods using PlasMem Bright dyes. Finally, the combined use of exosome staining probes and PlasMem Bright dyes allowed clear visualization of the endocytic pathway.TOC graphic

scholarly journals Surgical Advances in Osteosarcoma

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 388
Author(s):  
Marcus J. Brookes ◽  
Corey D. Chan ◽  
Bence Baljer ◽  
Sachin Wimalagunaratna ◽  
Timothy P. Crowley ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Imaging Techniques ◽  
Intraoperative Imaging ◽  
Survival Rates ◽  
Computer Navigation ◽  
Bone Cancer ◽  
Technical Difficulty ◽  
Implant Design ◽  
Patient Specific ◽  
Patient Specific Instruments

Osteosarcoma (OS) is the most common primary bone cancer in children and, unfortunately, is associated with poor survival rates. OS most commonly arises around the knee joint, and was traditionally treated with amputation until surgeons began to favour limb-preserving surgery in the 1990s. Whilst improving functional outcomes, this was not without problems, such as implant failure and limb length discrepancies. OS can also arise in areas such as the pelvis, spine, head, and neck, which creates additional technical difficulty given the anatomical complexity of the areas. We reviewed the literature and summarised the recent advances in OS surgery. Improvements have been made in many areas; developments in pre-operative imaging technology have allowed improved planning, whilst the ongoing development of intraoperative imaging techniques, such as fluorescent dyes, offer the possibility of improved surgical margins. Technological developments, such as computer navigation, patient specific instruments, and improved implant design similarly provide the opportunity to improve patient outcomes. Going forward, there are a number of promising avenues currently being pursued, such as targeted fluorescent dyes, robotics, and augmented reality, which bring the prospect of improving these outcomes further.

scholarly journals A Bimodal Fluorescence-Raman Probe for Cellular Imaging

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1699
Author(s):  
Jiarun Lin ◽  
Marcus E. Graziotto ◽  
Peter A. Lay ◽  
Elizabeth J. New
Keyword(s):  
Chemical Composition ◽  
Fluorescent Probes ◽  
Lipid Droplets ◽  
Imaging Techniques ◽  
Spectroscopic Imaging ◽  
Fluorescent Sensors ◽  
Chemical Environment ◽  
Novel Approach ◽  
Health And Disease ◽  
Raman Probe

Biochemical changes in specific organelles underpin cellular function, and studying these changes is crucial to understand health and disease. Fluorescent probes have become important biosensing and imaging tools as they can be targeted to specific organelles and can detect changes in their chemical environment. However, the sensing capacity of fluorescent probes is highly specific and is often limited to a single analyte of interest. A novel approach to imaging organelles is to combine fluorescent sensors with vibrational spectroscopic imaging techniques; the latter provides a comprehensive map of the relative biochemical distributions throughout the cell to gain a more complete picture of the biochemistry of organelles. We have developed NpCN1, a bimodal fluorescence-Raman probe targeted to the lipid droplets, incorporating a nitrile as a Raman tag. NpCN1 was successfully used to image lipid droplets in 3T3-L1 cells in both fluorescence and Raman modalities, reporting on the chemical composition and distribution of the lipid droplets in the cells.

scholarly journals Insights into Bacterial Community Involved in Bioremediation of Aged Oil-Contaminated Soil in Arid Environment

2021 ◽  
Vol 17 ◽  
pp. 117693432110168
Author(s):  
Rita Rahmeh ◽  
Abrar Akbar ◽  
Vinod Kumar ◽  
Hamad Al-Mansour ◽  
Mohamed Kishk ◽  
...  
Keyword(s):  
Oil Spills ◽  
Critical Role ◽  
Arid Environment ◽  
Total Petroleum Hydrocarbons ◽  
Microbial Consortia ◽  
Soil Microbial ◽  
Taxonomic Profiling ◽  
Degradation Pattern ◽  
Polycyclic Aromatic ◽  
Global Concern

Soil contamination by hydrocarbons due to oil spills has become a global concern and it has more implications in oil producing regions. Biostimulation is considered as one of the promising remediation techniques that can be adopted to enhance the rate of degradation of crude oil. The soil microbial consortia play a critical role in governing the biodegradation of total petroleum hydrocarbons (TPHs), in particular polycyclic aromatic hydrocarbons (PAHs). In this study, the degradation pattern of TPHs and PAHs of Kuwait soil biopiles was measured at three-month intervals. Then, the microbial consortium associated with oil degradation at each interval was revealed through 16S rRNA based next generation sequencing. Rapid degradation of TPHs and most of the PAHs was noticed at the first 3 months of biostimulation with a degradation rate of pyrene significantly higher compared to other PAHs counterparts. The taxonomic profiling of individual stages of remediation revealed that, biostimulation of the investigated soil favored the growth of Proteobacteria, Alphaprotobacteria, Chloroflexi, Chlorobi, and Acidobacteria groups. These findings provide a key step towards the restoration of oil-contaminated lands in the arid environment.

scholarly journals Emergence of distinct and heterogeneous strains of amyloid beta as Alzheimer′s disease progresses in Down syndrome

2021 ◽  
Author(s):  
Alison M Maxwell ◽  
Peng Yuan ◽  
Brianna M Rivera ◽  
Wilder Schaaf ◽  
Mihovil Mladinov ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Amyloid Beta ◽  
Fluorescent Probes ◽  
Clinical Symptoms ◽  
Critical Role ◽  
Phenotypic Heterogeneity ◽  
Post Mortem ◽  
Strain Profile ◽  
Post Mortem Brain ◽  
And Control

Amyloid beta (A&#946) is thought to play a critical role in the pathogenesis of Alzheimer&#8242s disease (AD). Prion-like Aβ polymorphs, or strains, can have varying pathogenicity and may underlie the phenotypic heterogeneity of the disease. In order to develop effective AD therapies, it is critical to identify the strains of A&#946 that might arise prior to the onset of clinical symptoms and understand how they may change with progressing disease. Down syndrome (DS), as the most common genetic cause of AD, presents promising opportunities to compare such features between early and advanced AD. In this work, we evaluate the neuropathology and A&#946 strain profile in the post-mortem brain tissues of 210 DS, AD, and control individuals. We assayed the levels of various A&#946 and tau species and used conformation-sensitive fluorescent probes to detect differences in A&#946 strains among individuals and populations. We found that these cohorts have some common but also some distinct strains from one another, with the most heterogeneous populations of A&#946 emerging in subjects with high levels of AD pathology. The emergence of distinct strains in DS at these later stages of disease suggests that the confluence of aging, pathology, and other DS-linked factors may favor conditions that generate strains that are unique from sAD.

Morphological Observation of the Cashmere Goat Fetal Fibroblasts after mTOR Kinase Inhibition with Combination of Fluorescent Dyes and Confocal Cell Imaging

2011 ◽  
Vol 343-344 ◽  
pp. 590-593
Author(s):  
Yan Liang ◽  
Xiao Jing Wang ◽  
Shu Yu Li ◽  
Cheberi ◽  
Zhi Gang Wang ◽  
...  
Keyword(s):  
Inner Mongolia ◽  
Mammalian Cells ◽  
Fluorescent Dyes ◽  
Cell Structure ◽  
Critical Role ◽  
Laser Scanning Microscopy ◽  
Morphological Observation ◽  
Cashmere Goat ◽  
Mtor Kinase ◽  
Fetal Fibroblasts

The mammalian target of rapamycin（mTOR）is a kind of Ser/Thr kinase in mammalian cells. It can recruit and integrate input signals from nutrients, growth factors, energy and environmental stress to regulate cell growth and proliferation via different cellular processes. This study uses the fetal fibroblasts of Inner Mongolia Cashmere goat (Capra hircas) to prove that the mTOR plays a critical role in formation of the cell cytoskeleton structure. The mTOR kinase activity was inhibited in Inner Mongolia Cashmere goat fetal fibroblasts (GFb) after treatment with CCI-779 (temsirolimus), an mTOR specific inhibitor for 48 h. The results showed that GFb cells were sensitive to CCI-779. GFb cells morphology and its cytoskeleton structure changed under confocal laser scanning microscopy stained with the Fluorescent phalloidin (50µg/ml, Phalloidin-FITC5282) which combines with F-actin. In summary, mTOR signaling pathway was proved to be functional in GFb cells and acts as a key regulator to form cell structure. The Morphological results indicated that perhaps the synthesis of microfilament or organization of cytoskeleton was disrupted in GFb cells when mTOR was inhibited.

scholarly journals A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast

2011 ◽  
Vol 437 (2) ◽  
pp. 269-278 ◽  
Author(s):  
José R. Pérez-Castiñeira ◽  
Agustín Hernández ◽  
Rocío Drake ◽  
Aurelio Serrano
Keyword(s):  
Fluorescent Dyes ◽  
Experimental System ◽  
Proton Pumping ◽  
Endocytic Pathway ◽  
Inorganic Pyrophosphatase ◽  
Proton Pumps ◽  
Specific Class ◽  
Ph Gradients ◽  
Atpase Subunit ◽  
Relative Contribution

V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.

How Neurons Communicate: Gap Junctions and Neurosecretion

The Neuron ◽  
2015 ◽  
pp. 153-186
Author(s):  
Irwin B. Levitan ◽  
Leonard K. Kaczmarek
Keyword(s):  
Synaptic Vesicles ◽  
Fluorescent Dyes ◽  
Imaging Techniques ◽  
Electrical Coupling ◽  
Snare Complex ◽  
Biologically Active ◽  
Nerve Terminals ◽  
Capacitance Measurements ◽  
Complex Process ◽  
Cytoplasmic Vesicles

Two ways that neurons communicate with one another are by direct electrical coupling and by the secretion of neurotransmitters. Electrical coupling arises from the existence of proteins, known as connexins, that form pores linking the cytoplasm of adjacent cells. Ions and small molecules can carry signals from one cell to another through these pores. Neurosecretion is a more complex process whereby different categories of molecules are sorted into cytoplasmic vesicles. Chemical processes within these vesicles ensure that they contain biologically active transmitters or hormones. SNARE complex proteins cooperate with other proteins to allow synaptic vesicles containing neurotransmitter to release their components into the external medium following calcium entry into nerve terminals. Such exocytosis of synaptic vesicles can be monitored with imaging techniques using fluorescent dyes or proteins, or by capacitance measurements. A second set of molecules retrieves the membrane of synaptic vesicles back from the plasma membrane through endocytosis.

scholarly journals The Ovistatic Effect of Saprotrophic Soil Fungi on Ascaris suum Eggs

2019 ◽  
Vol 67 (3) ◽  
pp. 109-118
Author(s):  
Lidia Kołodziejczyk ◽  
Kinga Mazurkiewicz-Zapałowicz ◽  
Magdalena Twarużek ◽  
Jan Grajewski ◽  
Łukasz Łopusiewicz ◽  
...  
Keyword(s):  
Mtt Assay ◽  
Soil Fungi ◽  
Developmental Stages ◽  
Ascaris Suum ◽  
Hydrolytic Activity ◽  
Inhibitory Effect ◽  
Penicillium Expansum ◽  
Fungal Enzymes ◽  
Low Cytotoxicity ◽  
High Cytotoxicity

The aim of the study was to evaluate the potential use of selected species of soil fungi (Fusarium oxysporum, F. sulphureum, F. verticillioides, and Penicillium expansum) for the bioregulation of the dispersive stages of a parasitic nematode – the large roundworm of pig (Ascaris suum). Experimental cultures containing A. suum eggs with soil fungi and control cultures without fungi were incubated at 26°C for 28 days. Microscopic observations of the developmental stages of the A. suum eggs (zygote, 2-8 blastomeres, morula/blastula, gastrula, and larva) were performed at 7, 14, 21, and 28 days. The API-ZYM test was used to semi-quantitatively determine the activity of 19 hydrolytic fungal enzymes. The cytotoxicity of the fungi was determined with a tetrazole salt MTT assay. Microscopic observations of A. suum eggs incubated in the presence of fungi up to day 28 did not show any signs of destruction to egg shells and/or penetration of the fungi into the eggs. The ovistatic effect of all tested fungi (F. sulphureum, P. expansum, F. verticillioides, and F. oxysporum; p<0.05) was seen only on the 7th day of incubation, whereas on the 14th day, only F. verticillioides and F. oxysporum showed an inhibitory effect on the embryogenesis of A. suum, and by the 28th day, only P. expansum. The API-ZYM test showed differences in the hydrolytic activity of the tested strains, while the MTT assay showed the high cytotoxicity of F. sulphureum, the moderate cytotoxicity of F. verticillioides and P. expansum, and the low cytotoxicity of F. oxysporum. Among the fungal strains studied, F. sulphureum showed the highest ovistatic effect, which may be related to its enzymatic activity and cytotoxicity.

scholarly journals CD33 (Siglec-3) Inhibitory Function: Role in the NKG2D/DAP10 Activating Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Trinidad Hernández-Caselles ◽  
Rubén Corral-San Miguel ◽  
Antonio José Ruiz-Alcaraz ◽  
Pilar García-Peñarrubia
Keyword(s):  
Nk Cells ◽  
Innate Immune ◽  
Inhibitory Receptor ◽  
Inhibitory Function ◽  
Activating Receptors ◽  
Self Tolerance ◽  
Lower Extent ◽  
Low Cytotoxicity ◽  
High Cytotoxicity

CD33 (siglec-3), a well-known target in leukemia therapy, is an inhibitory sialoadhesin expressed in human leukocytes of the myeloid lineage and some lymphoid subsets, including NK cells. It may constitute a control mechanism of the innate immune system; nevertheless, its role as an inhibitory receptor remains elusive. Using human NK cells as a cellular model, we analyzed CD33 inhibitory function upon different activating receptors. In high-cytotoxicity NKL cells, CD33 displayed a prominent inhibition on cytotoxicity triggered by the activating receptors NKG2D and, in a lower extent, 2B4, whereas it did not inhibit NKp46-induced cytotoxicity. NKp46 was partially inhibited by CD33 only when low-cytotoxicity NKL cells were tested. CD33 triggering did not inhibit IFN-γsecretion, contrasting with ILT-2 and CD94/NKG2A inhibitory receptors that inhibited cytotoxicity and IFN-γsecretion induced by all activating receptors tested. CD33-mediated inhibition of NKG2D-induced triggering involved Vav1 dephosphorylation. Our results support the role of CD33 as an inhibitory receptor preferentially regulating the NKG2D/DAP10 cytotoxic signaling pathway, which could be involved in self-tolerance and tumor and infected cell recognition.

Sign in / Sign up

Export Citation Format

Share Document