Antifungal and Antibiofilm Activities and the Mechanism of Action of Repeating Lysine-Tryptophan Peptides against Candida albicans

The rapid increase in the emergence of antifungal-resistant Candida albicans strains is becoming a serious health concern. Because antimicrobial peptides (AMPs) may provide a potential alternative to conventional antifungal agents, we have synthesized a series of peptides with a varying number of lysine and tryptophan repeats (KWn-NH2). The antifungal activity of these peptides increased with peptide length, but only the longest KW5 peptide displayed cytotoxicity towards a human keratinocyte cell line. The KW4 and KW5 peptides exhibited strong antifungal activity against C. albicans, even under conditions of high-salt and acidic pH, or the addition of fungal cell wall components. Moreover, KW4 inhibited biofilm formation by a fluconazole-resistant C. albicans strain. Circular dichroism and fluorescence spectroscopy indicated that fungal liposomes could interact with the longer peptides but that they did not release the fluorescent dye calcein. Subsequently, fluorescence assays with different dyes revealed that KW4 did not disrupt the membrane integrity of intact fungal cells. Scanning electron microscopy showed no changes in fungal morphology, while laser-scanning confocal microscopy indicated that KW4 can localize into the cytosol of C. albicans. Gel retardation assays revealed that KW4 can bind to fungal RNA as a potential intracellular target. Taken together, our data indicate that KW4 can inhibit cellular functions by binding to RNA and DNA after it has been translocated into the cell, resulting in the eradication of C. albicans.

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Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.00341.2013 ◽

2014 ◽

Vol 306 (7) ◽

pp. C659-C669 ◽

Cited By ~ 7

Author(s):

Krishna P. Subedi ◽

Omkar Paudel ◽

James S. K. Sham

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Laser Scanning ◽

Muscle Cells ◽

Laser Scanning Confocal Microscopy ◽

Cellular Functions ◽

Permeabilized Cells ◽

Scanning Confocal Microscopy ◽

Subcellular Compartments ◽

Transient Elevation

Intracellular calcium (Ca2+) plays pivotal roles in distinct cellular functions through global and local signaling in various subcellular compartments, and subcellular Ca2+ signal is the key factor for independent regulation of different cellular functions. In vascular smooth muscle cells, subsarcolemmal Ca2+ is an important regulator of excitation-contraction coupling, and nucleoplasmic Ca2+ is crucial for excitation-transcription coupling. However, information on Ca2+ signals in these subcellular compartments is limited. To study the regulation of the subcellular Ca2+ signals, genetically encoded Ca2+ indicators (cameleon), D3cpv, targeting the plasma membrane (PM), cytoplasm, and nucleoplasm were transfected into rat pulmonary arterial smooth muscle cells (PASMCs) and Ca2+ signals were monitored using laser scanning confocal microscopy. In situ calibration showed that the Kd for Ca2+ of D3cpv was comparable in the cytoplasm and nucleoplasm, but it was slightly higher in the PM. Stimulation of digitonin-permeabilized cells with 1,4,5-trisphosphate (IP3) elicited a transient elevation of Ca2+ concentration with similar amplitude and kinetics in the nucleoplasm and cytoplasm. Activation of G protein-coupled receptors by endothelin-1 and angiotensin II preferentially elevated the subsarcolemmal Ca2+ signal with higher amplitude in the PM region than the nucleoplasm and cytoplasm. In contrast, the receptor tyrosine kinase activator, platelet-derived growth factor, elicited Ca2+ signals with similar amplitudes in all three regions, except that the rise-time and decay-time were slightly slower in the PM region. These data clearly revealed compartmentalization of Ca2+ signals in the subsarcolemmal regions and provide the basis for further investigations of differential regulation of subcellular Ca2+ signals in PASMCs.

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Antifungal Activity of Mammalian Serum Amyloid A1 against Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01975-19 ◽

2019 ◽

Vol 64 (1) ◽

Cited By ~ 2

Author(s):

Jiao Gong ◽

Jun Wu ◽

Melanie Ikeh ◽

Li Tao ◽

Yulong Zhang ◽

...

Keyword(s):

Candida Albicans ◽

Antifungal Activity ◽

Membrane Integrity ◽

Systemic Infection ◽

Serum Amyloid ◽

Infection Model ◽

Fungal Cell ◽

Content Type ◽

Cell Membrane Integrity ◽

Amyloid A

ABSTRACT Mammalian serum amyloid A (SAA) is a major acute phase protein that shows a massive increase in plasma concentration during inflammation. In the present study, we demonstrate that the expression of mouse SAA1 in serum was increased when infected with Candida albicans, a major human fungal pathogen, in a systemic infection model. We then set out to investigate the antifungal activity of SAA proteins against C. albicans. Recombinant human and mouse SAA1 (rhSAA1 and rmSAA1) were expressed and purified in Escherichia coli. Both rhSAA1 and rmSAA1 exhibited a potent antifungal activity against C. albicans. We further demonstrate that rhSAA1 binds to the cell surface of C. albicans, disrupts cell membrane integrity, and induces rapid fungal cell death in C. albicans. Our finding expands the known functions of SAA1 and provides new insight into host-Candida interactions during fungal infection.

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Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158376 ◽

1990 ◽

Vol 48 (3) ◽

pp. 166-167

Author(s):

J. Holy ◽

G. Schatten

Keyword(s):

Confocal Microscopy ◽

Mitotic Spindle ◽

Laser Scanning ◽

Three Dimensional ◽

Laser Scanning Confocal Microscopy ◽

Two Dimensions ◽

Embryonic Cells ◽

Mitotic Spindles ◽

Cleavage Division ◽

Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

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Automated 3-D cell population analysis in thick tissue sections using laser-scanning confocal-microscopy data

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100148642 ◽

1993 ◽

Vol 51 ◽

pp. 562-563

Author(s):

Hakan Ancin

Keyword(s):

Laser Scanning ◽

Population Analysis ◽

Three Dimensional ◽

Large Data ◽

Laser Scanning Confocal Microscopy ◽

Tissue Slices ◽

Scanning Confocal Microscopy ◽

Tissue Sections ◽

Spatially Adaptive ◽

Microscopy Data

This paper presents methods for performing detailed quantitative automated three dimensional (3-D) analysis of cell populations in thick tissue sections while preserving the relative 3-D locations of cells. Specifically, the method disambiguates overlapping clusters of cells, and accurately measures the volume, 3-D location, and shape parameters for each cell. Finally, the entire population of cells is analyzed to detect patterns and groupings with respect to various combinations of cell properties. All of the above is accomplished with zero subjective bias.In this method, a laser-scanning confocal light microscope (LSCM) is used to collect optical sections through the entire thickness (100 - 500μm) of fluorescently-labelled tissue slices. The acquired stack of optical slices is first subjected to axial deblurring using the expectation maximization (EM) algorithm. The resulting isotropic 3-D image is segmented using a spatially-adaptive Poisson based image segmentation algorithm with region-dependent smoothing parameters. Extracting the voxels that were labelled as "foreground" into an active voxel data structure results in a large data reduction.

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The newly synthesized thiazole derivatives as potential antifungal compounds against Candida albicans

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11477-7 ◽

2021 ◽

Author(s):

Anna Biernasiuk ◽

Anna Berecka-Rycerz ◽

Anna Gumieniczek ◽

Maria Malm ◽

Krzysztof Z. Łączkowski ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Antifungal Activity ◽

Cell Membrane ◽

Mode Of Action ◽

Thiazole Derivatives ◽

Fungal Cell ◽

Erythrocyte Lysis ◽

Low Cytotoxicity ◽

High Antifungal Activity

Abstract Recently, the occurrence of candidiasis has increased dramatically, especially in immunocompromised patients. Additionally, their treatment is often ineffective due to the resistance of yeasts to antimycotics. Therefore, there is a need to search for new antifungals. A series of nine newly synthesized thiazole derivatives containing the cyclopropane system, showing promising activity against Candida spp., has been further investigated. We decided to verify their antifungal activity towards clinical Candida albicans isolated from the oral cavity of patients with hematological malignancies and investigate the mode of action on fungal cell, the effect of combination with the selected antimycotics, toxicity to erythrocytes, and lipophilicity. These studies were performed by the broth microdilution method, test with sorbitol and ergosterol, checkerboard technique, erythrocyte lysis assay, and reversed phase thin-layer chromatography, respectively. All derivatives showed very strong activity (similar and even higher than nystatin) against all C. albicans isolates with minimal inhibitory concentration (MIC) = 0.008–7.81 µg/mL Their mechanism of action may be related to action within the fungal cell wall structure and/or within the cell membrane. The interactions between the derivatives and the selected antimycotics (nystatin, chlorhexidine, and thymol) showed additive effect only in the case of combination some of them and thymol. The erythrocyte lysis assay confirmed the low cytotoxicity of these compounds as compared to nystatin. The high lipophilicity of the derivatives was related with their high antifungal activity. The present studies confirm that the studied thiazole derivatives containing the cyclopropane system appear to be a very promising group of compounds in treatment of infections caused by C. albicans. However, this requires further studies in vivo. Key points • The newly thiazoles showed high antifungal activity and some of them — additive effect in combination with thymol. • Their mode of action may be related with the influence on the structure of the fungal cell wall and/or the cell membrane. • The low cytotoxicity against erythrocytes and high lipophilicity of these derivatives are their additional good properties. Graphical abstract

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Advanced Static and Dynamic Fluorescence Microscopy Techniques to Investigate Drug Delivery Systems

Pharmaceutics ◽

10.3390/pharmaceutics13060861 ◽

2021 ◽

Vol 13 (6) ◽

pp. 861

Author(s):

Jacopo Cardellini ◽

Arianna Balestri ◽

Costanza Montis ◽

Debora Berti

Keyword(s):

Drug Delivery ◽

Fluorescence Microscopy ◽

Drug Delivery Systems ◽

Laser Scanning ◽

Super Resolution ◽

Laser Scanning Confocal Microscopy ◽

Delivery Systems ◽

Scanning Confocal Microscopy ◽

Biological Phenomena

In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.

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Observation of Fine Structure in Bicontinuous Phase-Separated Domains of a Polymer Blend by Laser Scanning Confocal Microscopy

Macromolecules ◽

10.1021/ma010190d ◽

2001 ◽

Vol 34 (15) ◽

pp. 5186-5191 ◽

Cited By ~ 11

Author(s):

Hiroshi Jinnai ◽

Hiroshi Yoshida ◽

Kohtaro Kimishima ◽

Yoshinori Funaki ◽

Yoshitsugu Hirokawa ◽

...

Keyword(s):

Fine Structure ◽

Confocal Microscopy ◽

Polymer Blend ◽

Laser Scanning ◽

Laser Scanning Confocal Microscopy ◽

Scanning Confocal Microscopy

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The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy.

Journal of Histochemistry & Cytochemistry ◽

10.1177/42.11.7930524 ◽

1994 ◽

Vol 42 (11) ◽

pp. 1413-1416 ◽

Cited By ~ 24

Author(s):

S L Erlandsen ◽

E M Rasch

Keyword(s):

Confocal Microscopy ◽

Genome Size ◽

Dna Content ◽

Giardia Lamblia ◽

Laser Scanning ◽

Laser Scanning Confocal Microscopy ◽

Evolutionary Development ◽

Scanning Confocal Microscopy ◽

Dividing Cells ◽

C Value

We investigated direct measurement of the DNA content of the parasitic intestinal flagellate Giardia lamblia through quantitation by Feulgen microspectrophotometry and also by visualization of Feulgen-stained DNA chromosomes within dividing cells by laser scanning confocal microscopy. Individual trophozoites of Giardia (binucleate) contained 0.144 +/- 0.018 pg of DNA/cell or 0.072 pg DNA/nucleus. Giardia lamblia cysts (quadranucleate) contained 0.313 +/- 0.003 pg DNA or 0.078 pg DNA/nucleus. The genome size (C) value per nucleus ranged between 6.5-7.1 x 10(7) BP for trophozoites and cysts, respectively. Confocal microscopic examination of Giardia trophozoites undergoing binary fission revealed five chromosome-like bodies within each nucleus. Further information about genome size and DNA content within different Giardia species may help to clarify the pivotal role of these primitive eukaryotic cells in evolutionary development.

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The Effect of Chloroquine on the Apoptosis Induced by Cisplatin in Human Gastric Cancer BGC823 Cells

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.926-930.1124 ◽

2014 ◽

Vol 926-930 ◽

pp. 1124-1127

Author(s):

Zhen Xun Jin ◽

Li Li Zhang ◽

Yan Wang ◽

Lin Chuan Zeng ◽

Yang Yu ◽

...

Keyword(s):

Gastric Cancer ◽

Confocal Microscopy ◽

Laser Scanning ◽

Laser Scanning Confocal Microscopy ◽

Combination Group ◽

Human Gastric Cancer ◽

Apoptotic Cells ◽

Toxic Dose ◽

Scanning Confocal Microscopy ◽

Mtt Tests

The aim of this study is to investigate the effects and mechanism of chloroquine (CQ) on the apoptosis induced by cisplatin in human gastric cancer BGC823 cells. MTT assay was used to detect the state of cell growth. The appearances of cellular apoptosis were detected by laser scanning confocal microscopy and light microscopy. The expressions of LC3 and p62 were detected by laser scanning confocal microscopy. MTT tests showed that the non-toxic dose of CQ could increase the inhibition rate of BGC823 cells induced by cisplatin. Under the light microscope, the ratio of apoptotic cells in the group treated with non-toxic dose of CQ combined with cisplatin was higher than that in the group treated with cisplatin alone. Hoechst33342 staining showed that the ratio of apoptotic cells in the combination group was higher than that in the cisplatin group. The expression and colocalization of LC3 and p62 proteins were significantly increased in the combination group. These results indicate that CQ can enhance the cell apoptosis induced by cisplatin in BGC823 cells, which is through the inhibition of autophagy.

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