scholarly journals Photodynamic Action of LED-Activated Curcumin againstStaphylococcus aureusInvolving Intracellular ROS Increase and Membrane Damage

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Yuan Jiang ◽  
Albert Wingnang Leung ◽  
Heyu Hua ◽  
Xiancai Rao ◽  
Chuanshan Xu
Keyword(s):  
Staphylococcus Aureus ◽  
Membrane Permeability ◽  
Blue Light ◽  
Laser Scanning ◽  
Membrane Damage ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Photodynamic Action ◽  
Intracellular Ros

Aim. To investigate the effect of photodynamic action of LED-activated curcumin on cell viability, membrane permeability, and intracellular reactive oxygen species ofStaphylococcus aureus.Methods.Staphylococcus aureuswas incubated with the different concentrations of curcumin for 60 min and then irradiated by blue light with the wavelength of 470 nm and with light dose of 3 J/cm2. The colony forming unit assay was used to investigate photocytotoxicity of curcumin onStaphylococcus aureus, confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) for assaying membrane permeability, FCM analysis with DCFH-DA staining for measuring the intracellular ROS level, and transmission electron microscopy (TEM) for observing morphology and structure.Results. Blue light-activated curcumin significantly killedStaphylococcus aureusin a curcumin dose-dependent manner. TEM observed remarkable structural damages inS. aureusafter light-activated curcumin. More red fluorescence of PI dye was found inS. aureustreated by blue light-activated curcumin than in those of the controlled bacterial cells. Intracellular ROS increase was observed after light-activated curcumin.Conclusion. Blue light-activated curcumin markedly damaged membrane permeability, resulting in cell death ofStaphylococcus aureusand highlighted that intracellular ROS increase might be an important event in photodynamic killing ofStaphylococcus aureusin the presence of curcumin.

scholarly journals Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

1998 ◽  
Vol 330 (2) ◽  
pp. 853-860 ◽  
Author(s):  
N. J. Silvia MORENO ◽  
Li ZHONG ◽  
Hong-Gang LU ◽  
Wanderley DE SOUZA ◽  
Marlene BENCHIMOL
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Laser Scanning ◽  
Membrane Surface ◽  
Surface Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Cytoplasmic Ph ◽  
Bafilomycin A1

Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

scholarly journals Bacterial lipopolysaccharides variably modulate in vitro biofilm formation of Candida species

2010 ◽  
Vol 59 (10) ◽  
pp. 1225-1234 ◽  
Author(s):  
H. M. H. N. Bandara ◽  
O. L. T. Lam ◽  
R. M. Watt ◽  
L. J. Jin ◽  
L. P. Samaranayake
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Significant Inhibition ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Bacterial Lipopolysaccharides ◽  
Species Specific ◽  
Biofilm Development

The objective of this study was to evaluate the effect of the bacterial endotoxin LPS on Candida biofilm formation in vitro. The effect of the LPS of Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens and Salmonella typhimurium on six different species of Candida, comprising Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA 646, was studied using a standard biofilm assay. The metabolic activity of in vitro Candida biofilms treated with LPS at 90 min, 24 h and 48 h was quantified by XTT reduction assay. Viable biofilm-forming cells were qualitatively analysed using confocal laser scanning microscopy (CLSM), while scanning electron microscopy (SEM) was employed to visualize the biofilm structure. Initially, adhesion of C. albicans was significantly stimulated by Pseudomonas and Klebsiella LPS. A significant inhibition of Candida adhesion was noted for the following combinations: C. glabrata with Pseudomonas LPS, C. tropicalis with Serratia LPS, and C. glabrata, C. parapsilosis or C. dubliniensis with Salmonella LPS (P<0.05). After 24 h of incubation, a significant stimulation of initial colonization was noted for the following combinations: C. albicans/C. glabrata with Klebsiella LPS, C. glabrata/C. tropicalis/C. krusei with Salmonella LPS. In contrast, a significant inhibition of biofilm formation was observed in C. glabrata/C. dubliniensis/C. krusei with Pseudomonas LPS, C. krusei with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. parapsilosis/C. dubliniensis /C. krusei with Salmonella LPS (P<0.05). On further incubation for 48 h, a significant enhancement of biofilm maturation was noted for the following combinations: C. glabrata/C. tropicalis with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. glabrata with Salmonella LPS, and a significant retardation was noted for C. parapsilosis/C. dubliniensis/C. krusei with Pseudomonas LPS, C. tropicalis with Serratia LPS, C. glabrata/C. parapsilosis/C. dubliniensis with Klebsiella LPS and C. dubliniensis with Salmonella LPS (P<0.05). These findings were confirmed by SEM and CLSM analyses. In general, the inhibition of the biofilm development of LPS-treated Candida spp. was accompanied by a scanty architecture with a reduced numbers of cells compared with the profuse and densely colonized control biofilms. These data are indicative that bacterial LPSs modulate in vitro Candida biofilm formation in a species-specific and time-dependent manner. The clinical and the biological relevance of these findings have yet to be explored.

scholarly journals Jiedu Sangen Decoction Inhibits Migration and Invasion of Colon Cancer SW480 Cells via Suppressing Epithelial Mesenchymal Transition

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Kai Zhang ◽  
Tao Peng ◽  
Qingying Yan ◽  
Leitao Sun ◽  
Haojun Miao ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Laser Scanning ◽  
Epithelial Mesenchymal Transition ◽  
Laser Scanning Microscopy ◽  
Migration And Invasion ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Matrigel Invasion ◽  
Sw480 Cells

Jiedu Sangen Decoction (JSD), a traditional Chinese medicine (TCM) formula, has been widely used in China to treat gastrointestinal cancer, especially as an adjuvant therapy in colorectal cancer (CRC) patients. This study aimed to evaluate the efficacy of JSD and Jiedu Sangen aqueous extract (JSAE) in colon cancer cells and explored the underlining mechanisms by cytotoxicity assay, scratch assay, transwell migration assay, matrigel invasion assay, confocal laser scanning microscopy, and western blot analysis. We demonstrated that JSAE inhibited the growth of colon cancer SW480 cells in a dose-dependent manner and JSAE repressed cancer cell migration and invasion. Furthermore, epithelial mesenchymal transition (EMT) was reversed by JSAE via enhancing E-cadherin expression and attenuating protein levels of EMT promoting factors such as N-cadherin, Slug, and ZEB1. These findings provided the first experimental evidence confirming the efficacy of JSAE in repressing invasion and metastasis of CRC and paving a way for the broader use of JSD in clinic.

The effect of N-acetylcysteine in a combined antibiofilm treatment against antibiotic-resistant Staphylococcus aureus

2020 ◽  
Vol 75 (7) ◽  
pp. 1787-1798
Author(s):  
Arthika Manoharan ◽  
Theerthankar Das ◽  
Gregory S Whiteley ◽  
Trevor Glasbey ◽  
Frederik H Kriel ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Cleavage ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Combination Treatments ◽  
Resazurin Assay ◽  
Biofilm Architecture ◽  
Biofilm Disruption ◽  
The Uk

Abstract Background The WHO declared Staphylococcus aureus as a ‘pathogen of high importance’ in 2017. One-fifth of all bloodstream-related infections in Australia and 12 000 cases of bacteraemia in the UK (2017–18) were caused by the MRSA variant. To address the need for novel therapies, we investigated several permutations of an innovative combination therapy containing N-acetylcysteine (NAC), an antibiotic and an enzyme of choice in eradicating MRSA and MSSA biofilms. Methods Biofilm viability (resazurin assay) and colony count methods were used to investigate the effect of NAC, antibiotics and enzymes on S. aureus biofilm disruption and killing. The effects of NAC and enzymes on the polysaccharide content of biofilm matrices were analysed using the phenol/sulphuric acid method and the effect of NAC on DNA cleavage was determined using the Qubit fluorometer technique. Changes in biofilm architecture when subjected to NAC and enzymes were visualized using confocal laser scanning microscopy (CLSM). Results NAC alone displayed bacteriostatic effects when tested on planktonic bacterial growth. Combination treatments containing 30 mM NAC resulted in ≥90% disruption of biofilms across all MRSA and MSSA strains with a 2–3 log10 decrease in cfu/mL in treated biofilms. CLSM showed that NAC treatment drastically disrupted S. aureus biofilm architecture. There was also reduced polysaccharide production in MRSA biofilms in the presence of NAC. Conclusions Our results indicate that inclusion of NAC in a combination treatment is a promising strategy for S. aureus biofilm eradication. The intrinsic acidity of NAC was identified as key to maximum biofilm disruption and degradation of matrix components.

scholarly journals Powdered Activated Carbon Exacerbates Fouling in MBR Treating Olive Mill Wastewater

Water ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 2498 ◽  
Author(s):  
Noya Ran ◽  
Jack Gilron ◽  
Revital Sharon-Gojman ◽  
Moshe Herzberg
Keyword(s):  
Activated Carbon ◽  
Membrane Permeability ◽  
Membrane Fouling ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Powdered Activated Carbon ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Permeate Flux ◽  
Wastewater Treatment Process

Membrane fouling is a major obstacle in membrane bioreactors (MBRs) that treat wastewater. The addition of powdered activated carbon (PAC) is commonly suggested as a way to improve the MBR wastewater treatment process with respect to membrane fouling and effluent quality. Integrating the PAC addition into the MBR may also improve the stability of the acclimated microbial community for biodegrading the recalcitrant organic compounds that can also enhance membrane fouling. In this study, the ability of the MBR-PAC system to decrease membrane fouling was evaluated. Two pilot-scale reactors were operated: one reactor was supplemented with suspended PAC, and one was operated under similar conditions, without PAC. The feed to the reactors comprised domestic and olive oil mill wastewater. Surprisingly, the permeate flux and the membrane permeability decreased faster in the MBR supplemented with PAC compared to the control reactor. Corroborating these MBR fouling results, soluble microbial products (SMPs), originating from the PAC-supplemented reactor, were found to be more adhesive to an ultrafiltration membrane mimetic surface (polyether sulfone) as analyzed in a quartz crystal microbalance with dissipation monitoring (QCM-D). While the PAC had almost no effect on the dissolved organic carbon in the MBR, it altered the molecular weight distribution of the organic molecules in the SMP as observed with gel permeation chromatography: The fractions of 577–789 kDa and the one bigger than 4 × 103 kDa, were elevated and reduced, respectively, by the addition of PAC. A biofilm formation analysis using a confocal laser scanning microscopy showed a higher amount of biofilm on the membrane taken from the PAC reactor, but this membrane showed no traces of PAC particles when analyzed with a scanning electron microscope (SEM). Taken together, altering the composition of the dissolved organic matter in the MBR by PAC addition promoted its adhesion to the membrane, induced biofilm formation, and more prominently, decreased membrane permeability.

scholarly journals Susceptibility of Mature Staphylococcus Biofilms to Chinese Herbal Decoction Sanhuang Jiedu: An In Vitro Study

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Shaoe Zhang ◽  
Xiao Wang ◽  
Xiaotao Shi ◽  
Honglue Tan ◽  
Himanshu Garg
Keyword(s):  
Laser Scanning ◽  
Multidrug Resistant ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Control Group ◽  
Antibiofilm Activity ◽  
Dependent Manner ◽  
Chinese Herbal ◽  
Titanium Surfaces

Background. External socking and washing with the Chinese herbal Sanhuang Jiedu decoction (SHJD) can effectively control local limb infections with bone and implant exposure. However, the antibiofilm activities of this decoction in vitro have not yet been investigated. Therefore, the aim of this study was to examine the effects and characteristics of SHJD on the mature biofilms of multidrug-resistant staphylococci on a titanium surface. Methods. Biofilm-forming methicillin-resistant Staphylococcus epidermidis ATCC 35984 and S. aureus ATCC 43330, and non-biofilm-forming S. epidermidis ATCC 12228 were selected as the experimental strains. The mature biofilms were prepared on titanium surfaces. The five experimental groups were based on dilution concentrations (DC) of SHJD: the control group (biofilm incubated with 0.85% NaCl solution), the SHJD (DC:1/8) group (initial SHJD solution was diluted 1/8), the SHJD (DC:1/4) group, the SHJD (DC:1/2) group, and the SHJD (DC:1/1) group (initial SHJD solution). The effects of SHJD on the mature biofilms were observed with the bacterial spread plate method, crystal violet (CV) staining, scanning electron microscopy, and confocal laser scanning microscopy. Results. After culture in tryptic soy broth for 72 h, ATCC 43300 and ATCC 35984 produced mature biofilms and ATCC 12228 did not. The optical density value of ATCC 12228 was 0.11 ± 0.02 , significantly lower than that of ATCC 35984 ( 0.42 ± 0.05 ) or ATCC 43300 ( 0.41 ± 0.03 ) ( P < 0.05 ). The mature biofilms of ATCC 43300 and ATCC 35984 clearly disintegrated when incubated for 12–24 h with SHJD (DC:1/1) or SHJD (DC:1/2), showing only scattered bacterial adhesion. In the SHJD (DC:1/4) group, although many residual bacterial colonies still clustered together, presenting a biofilm structure, it was very looser than that in the SHJD (DC:1/8) group in which the biofilm was similar to that in the control group. For ATCC 12228, only colony adhesion was observed, and the number of colonies decreased as the concentration of SHJD or the culture period increased. The quantitative results for the bacterial spread plate and CV staining showed significant differences between the SHJD groups ( P < 0.05 ). Conclusion. SHJD has antibiofilm activity against multidrug-resistant Staphylococcus strains. It weakens or disrupts already-formed mature biofilms on titanium surfaces in a concentration- and incubation time-dependent manner.

Contribution of Electron and Confocal Microscopy in the Study ofLeishmania–Macrophage Interactions

2004 ◽  
Vol 10 (5) ◽  
pp. 656-661 ◽  
Author(s):  
Birgitta Rasmusson ◽  
Albert Descoteaux
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Protozoan Parasite ◽  
Laser Scanning Microscopy ◽  
Sand Fly ◽  
Confocal Laser ◽  
Mammalian Host ◽  
Dependent Manner ◽  
Phagosome Maturation ◽  
Physical Barrier

Promastigotes of the protozoan parasite genusLeishmaniaare inoculated into a mammalian host when an infected sand fly takes a bloodmeal. Following their opsonization by complement, promastigotes are phagocytosed by macrophages. There, promastigotes differentiate into amastigotes, the form of the parasite that replicates in the phagolysosomal compartments of host macrophages. Although the mechanisms by which promastigotes survive the microbicidal consequence of phagocytosis remain, for the most part, to be elucidated, evidence indicates that glycoconjugates play a role in this process. One such glycoconjugate is lipophosphoglycan, an abundant promastigote surface glycolipid. Using quantitative electron and confocal laser scanning microscopy approaches, evidence was provided thatL. donovanipromastigotes inhibit phagolysosome biogenesis in a lipophosphoglycan-dependent manner. This inhibition correlates with an accumulation of periphagosomal F-actin, which may potentially form a physical barrier that preventsL. donovanipromastigote-containing phagosomes from interacting with endocytic vacuoles. Inhibition of phagosome maturation may constitute a strategy to provide an environment propitious to the promastigote-to-amastigote differentiation.

scholarly journals Synthesis and in vitro biochemical evaluation of oxime bond-linked daunorubicin–GnRH-III conjugates developed for targeted drug delivery

2018 ◽  
Vol 14 ◽  
pp. 756-771 ◽  
Author(s):  
Sabine Schuster ◽  
Beáta Biri-Kovács ◽  
Bálint Szeder ◽  
Viktor Farkas ◽  
László Buday ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Cancer Cells ◽  
Cellular Uptake ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Binding Studies ◽  
Gnrh Receptors

Gonadotropin releasing hormone-III (GnRH-III), a native isoform of the human GnRH isolated from sea lamprey, specifically binds to GnRH receptors on cancer cells enabling its application as targeting moieties for anticancer drugs. Recently, we reported on the identification of a novel daunorubicin–GnRH-III conjugate (GnRH-III–[4Lys(Bu), 8Lys(Dau=Aoa)] with efficient in vitro and in vivo antitumor activity. To get a deeper insight into the mechanism of action of our lead compound, the cellular uptake was followed by confocal laser scanning microscopy. Hereby, the drug daunorubicin could be visualized in different subcellular compartments by following the localization of the drug in a time-dependent manner. Colocalization studies were carried out to prove the presence of the drug in lysosomes (early stage) and on its site of action (nuclei after 10 min). Additional flow cytometry studies demonstrated that the cellular uptake of the bioconjugate was inhibited in the presence of the competitive ligand triptorelin indicating a receptor-mediated pathway. For comparative purpose, six novel daunorubicin–GnRH-III bioconjugates have been synthesized and biochemically characterized in which 6Asp was replaced by D-Asp, D-Glu and D-Trp. In addition to the analysis of the in vitro cytostatic effect and cellular uptake, receptor binding studies with 125I-triptorelin as radiotracer and degradation of the GnRH-III conjugates in the presence of rat liver lysosomal homogenate have been performed. All derivatives showed high binding affinities to GnRH receptors and displayed in vitro cytostatic effects on HT-29 and MCF-7 cancer cells with IC50 values in a low micromolar range. Moreover, we found that the release of the active drug metabolite and the cellular uptake of the bioconjugates were strongly affected by the amino acid exchange which in turn had an impact on the antitumor activity of the bioconjugates.

scholarly journals Ivermectin Inhibits Bovine Herpesvirus 1 DNA Polymerase Nuclear Import and Interferes With Viral Replication

2020 ◽  
Vol 8 (3) ◽  
pp. 409 ◽  
Author(s):  
Sohail Raza ◽  
Farzana Shahin ◽  
Wenjun Zhai ◽  
Hanxiong Li ◽  
Gualtiero Alvisi ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Nuclear Import ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Physical Interaction ◽  
Genome Replication ◽  
Dependent Manner ◽  
Virus Attachment ◽  
Dsdna Molecule

Bovine herpesvirus1 (BoHV-1) is a major bovine pathogen. Despite several vaccines being available to prevent viral infection, outbreaks are frequent and cause important economic consequences worldwide. The development of new antiviral drugs is therefore highly desirable. In this context, viral genome replication represents a potential target for therapeutic intervention. BoHV-1 genome is a dsDNA molecule whose replication takes place in the nuclei of infected cells and is mediated by a viral encoded DNA polymerase holoenzyme. Here, we studied the physical interaction and subcellular localization of BoHV-1 DNA polymerase subunits in cells for the first time. By means of co-immunoprecipitation and confocal laser scanning microscopy (CLSM) experiments, we could show that the processivity factor of the DNA polymerase pUL42 is capable of being autonomously transported into the nucleus, whereas the catalytic subunit pUL30 is not. Accordingly, a putative classic NLS (cNLS) was identified on pUL42 but not on pUL30. Importantly, both proteins could interact in the absence of other viral proteins and their co-expression resulted in accumulation of UL30 to the cell nucleus. Treatment of cells with Ivermectin, an anti-parasitic drug which has been recently identified as an inhibitor of importin α/β-dependent nuclear transport, reduced UL42 nuclear import and specifically reduced BoHV-1 replication in a dose-dependent manner, while virus attachment and entry into cells were not affected. Therefore, this study provides a new option of antiviral therapy for BoHV-1 infection with Ivermectin.

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