Susceptibility of Candida Species to Photodynamic Effects of Photofrin

ABSTRACT The in vitro susceptibility of pathogenic Candida species to the photodynamic effects of the clinically approved photosensitizing agent Photofrin was examined. Internalization of Photofrin by Candida was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on incubation concentration. Uptake of Photofrin by Candida and subsequent sensitivity to irradiation was influenced by culture conditions. Photofrin uptake was poor in C. albicans blastoconidia grown in nutrient broth. However, conversion of blastoconidia to filamentous forms by incubation in defined tissue culture medium resulted in substantial Photofrin uptake. Under conditions where Photofrin was effectively taken up by Candida, irradiated organisms were damaged in a drug dose- and light-dependent manner. Uptake of Photofrin was not inhibited by azide, indicating that the mechanism of uptake was not dependent on energy provided via electron transport. Fungal damage induced by Photofrin-mediated photodynamic therapy (PDT) was determined by evaluation of metabolic activity after irradiation. A strain of C. glabrata took up Photofrin poorly and was resistant to killing after irradiation. In contrast, two different strains of C. albicans displayed comparable levels of sensitivity to PDT. Furthermore, a reference strain of C. krusei that is relatively resistant to fluconazole compared to C. albicans was equally sensitive to C. albicans at Photofrin concentrations of ≥3 μg/ml. The results indicate that photodynamic therapy may be a useful adjunct or alternative to current anti-Candida therapeutic modalities, particularly for superficial infections on surfaces amenable to illumination.

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Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab136 ◽

2021 ◽

Author(s):

Yu Takahashi ◽

Yu Inoue ◽

Keitaro Kuze ◽

Shintaro Sato ◽

Makoto Shimizu ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Gene Expression Regulation ◽

Intestinal Epithelial Cells ◽

Three Dimensional ◽

Culture Conditions ◽

Dependent Manner ◽

Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

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Enhanced Malignant Phenotypes of Glioblastoma Cells Surviving NPe6-Mediated Photodynamic Therapy are Regulated via ERK1/2 Activation

Cancers ◽

10.3390/cancers12123641 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3641

Author(s):

Tatsuya Kobayashi ◽

Makoto Miyazaki ◽

Nobuyoshi Sasaki ◽

Shun Yamamuro ◽

Eita Uchida ◽

...

Keyword(s):

Cell Death ◽

Photodynamic Therapy ◽

Migration And Invasion ◽

Dependent Manner ◽

Potential Therapeutic Target ◽

Talaporfin Sodium ◽

Polymerase Inhibitor ◽

Human Gbm ◽

Dose Dependent

To manage refractory and invasive glioblastomas (GBM)s, photodynamic therapy (PDT) using talaporfin sodium (NPe6) (NPe6-PDT) was recently approved in clinical practice. However, the molecular machineries regulating resistance against NPe6-PDT in GBMs and mechanisms underlying the changes in GBM phenotypes following NPe6-PDT remain unknown. Herein, we established an in vitro NPe6-mediated PDT model using human GBM cell lines. NPe6-PDT induced GBM cell death in a NPe6 dose-dependent manner. However, this NPe6-PDT-induced GBM cell death was not completely blocked by the pan-caspase inhibitor, suggesting NPe6-PDT induces both caspase-dependent and -independent cell death. Moreover, treatment with poly (ADP-ribose) polymerase inhibitor blocked NPe6-PDT-triggered caspase-independent GBM cell death. Next, it was also revealed resistance to re-NPe6-PDT of GBM cells and GBM stem cells survived following NPe6-PDT (NPe6-PDT-R cells), as well as migration and invasion of NPe6-PDT-R cells were enhanced. Immunoblotting of NPe6-PDT-R cells to assess the behavior of the proteins that are known to be stress-induced revealed that only ERK1/2 activation exhibited the same trend as migration. Importantly, treatment with the MEK1/2 inhibitor trametinib reversed resistance against re-NPe6-PDT and suppressed the enhanced migration and invasion of NPe6-PDT-R cells. Overall, enhanced ERK1/2 activation is suggested as a key regulator of elevated malignant phenotypes of GBM cells surviving NPe6-PDT and is therefore considered as a potential therapeutic target against GBM.

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Bovine in vitro oocyte maturation as a model for manipulation of the γ-glutamyl cycle and intraoocyte glutathione

Reproduction Fertility and Development ◽

10.1071/rd08041 ◽

2008 ◽

Vol 20 (5) ◽

pp. 579 ◽

Cited By ~ 21

Author(s):

E. C. Curnow ◽

J. Ryan ◽

D. Saunders ◽

E. S. Hayes

Keyword(s):

Oocyte Maturation ◽

Calf Serum ◽

Cumulus Cell ◽

Buthionine Sulfoximine ◽

Cumulus Cells ◽

Culture Conditions ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Maturation Medium

Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.

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Fibroblast growth factor during mesoderm induction in the early chick embryo

Development ◽

10.1242/dev.109.2.387 ◽

1990 ◽

Vol 109 (2) ◽

pp. 387-393 ◽

Cited By ~ 9

Author(s):

E. Mitrani ◽

Y. Gruenbaum ◽

H. Shohat ◽

T. Ziv

Keyword(s):

Developmental Stages ◽

Culture Conditions ◽

Mesoderm Induction ◽

Dependent Manner ◽

Chick Embryogenesis ◽

Defined Culture ◽

High Degree ◽

Early Developmental ◽

Dose Dependent

A chick genomic clone that reveals a high degree of homology to the mammalian and Xenopus bFGF gene has been isolated. The pattern of expression of bFGF has been examined during early chick embryogenesis. RNA blot analysis revealed that chick bFGF is already transcribed at pregastrula stages. Immunolabeling analysis indicated that bFGF protein is present at these early developmental stages and is distributed evenly in the epiblast, hypoblast and marginal zone of the chick blastula. Substances that can inhibit FGF action were applied to early chick blastoderms grown in vitro under defined culture conditions (DCM). Both heparin and suramin were capable of blocking the formation of mesodermal structures in a dose-dependent manner. Our results indicate that FGF-like substances may need to be present for axial structures to develop although they may be acting earlier during the induction of non-axial mesoderm.

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In vitro differentiation of human granulocyte/macrophage and erythroid progenitors: comparative analysis of the influence of recombinant human erythropoietin, G-CSF, GM-CSF, and IL-3 in serum-supplemented and serum- deprived cultures

Blood ◽

10.1182/blood.v72.1.248.248 ◽

1988 ◽

Vol 72 (1) ◽

pp. 248-256 ◽

Cited By ~ 67

Author(s):

G Migliaccio ◽

AR Migliaccio ◽

JW Adamson

Keyword(s):

Recombinant Human Erythropoietin ◽

Colony Growth ◽

Culture Conditions ◽

Dependent Manner ◽

Erythroid Progenitors ◽

Concentration Dependent Manner ◽

Gm Csf ◽

Human Erythropoietin ◽

Normal Humans

Abstract The effects of recombinant human erythropoietin (Ep), granulocyte/macrophage (GM) and granulocyte (G) colony-stimulating factors (CSF), and interleukin-3 (IL-3) on erythroid burst and GM colony growth have been studied in fetal bovine serum (FBS)- supplemented and FBS-deprived culture. Sources of progenitor cells were nonadherent or nonadherent T-lymphocyte-depleted marrow or peripheral blood cells from normal humans. G-CSF, in concentrations up to 2.3 X 10(-10) mol/L, induced only the formation of neutrophil colonies. In contrast, GM-CSF and IL-3 both induced GM colonies and sustained the formation of erythroid bursts in the presence of Ep. However, the activities of these growth factors were affected by the culture conditions. IL-3 induction of GM colonies depended on the presence of FBS, whereas the degree of GM-CSF induction of GM colonies in FBS- deprived cultures depended on the method by which adherent cells were removed. GM-CSF increased colony numbers in a concentration-dependent manner only if the cells had been prepared by overnight adherence. Both GM-CSF and IL-3 exhibited erythroid burst-promoting activity in FBS- deprived cultures. However, some lineage restriction was evident because GM-CSF was two- to threefold more active than IL-3 in inducing GM colonies but IL-3 was two- to threefold more active in promoting erythroid burst growth. Furthermore, in FBS-deprived cultures, the number of both erythroid bursts and GM colonies reached the maximum only when Ep, GM-CSF, and IL-3 or GM-CSF, IL-3, and G-CSF, respectively, were added together. These results suggest that the colonies induced by IL-3, GM-CSF, and G-CSF are derived from different progenitors.

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Variation in Resistance of Mycobacterium paratuberculosis to Acid Environments as a Function of Culture Medium

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6833-6840.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6833-6840 ◽

Cited By ~ 31

Author(s):

Nackmoon Sung ◽

Michael T. Collins

Keyword(s):

Growth Rate ◽

Culture Medium ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Acid Resistance ◽

Culture Conditions ◽

Mycobacterium Paratuberculosis ◽

In Vitro Susceptibility ◽

Sds Page

ABSTRACT Acid resistance of Mycobacterium paratuberculosis was examined as a function of growth conditions (i.e., in vitro growth medium and pH). M. paratuberculosis was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8). Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20°C. A radiometric culture method (BACTEC) was used to quantify viable M. paratuberculosis cell data at various acid exposure times, and D values (decimal reduction times, or the times required to kill a 1-log10 concentration of bacteria) were determined. Soluble proteins of M. paratuberculosis grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility. The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD. Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on D values for M. paratuberculosis at any test pH. When grown in 7H9-OADC, M. paratuberculosis was more acid resistant at all test pHs (higher D values) than when grown in WR-GD. Glycerol appeared to be the culture medium component most responsible for lower levels of M. paratuberculosis acid resistance. When glycerol was substituted for OADC in the 7H9 medium, D values were significantly lower than those of 7H9-OADC-grown M. paratuberculosis and were approximately the same as those for M. paratuberculosis grown in WR-GD medium. Comparison of the SDS-PAGE protein profiles for M. paratuberculosis cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of M. paratuberculosis grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance. This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of M. paratuberculosis, and the results emphasize the importance of culture conditions for in vitro susceptibility studies.

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Deregulation of the histone H3K9 di-methylation landscape suppresses canonical Wnt signaling in embryonal rhabdomyosarcoma

10.1101/2020.04.20.050120 ◽

2020 ◽

Author(s):

Ananya Pal ◽

Jia Yu Leung ◽

Gareth Chin Khye Ang ◽

Vinay Kumar Rao ◽

Luca Pignata ◽

...

Keyword(s):

Wnt Signaling ◽

Myogenic Differentiation ◽

Wnt Signaling Pathway ◽

Embryonal Rhabdomyosarcoma ◽

Dependent Manner ◽

Canonical Wnt Signaling ◽

Therapeutic Modalities ◽

Canonical Wnt

AbstractThe Wnt signaling pathway is down-regulated in embryonal rhabdomyosarcoma (ERMS) and contributes to the block of myogenic differentiation. Epigenetic mechanisms leading to its suppression are unknown and could pave the way towards novel therapeutic modalities. In this study, we demonstrate that the H3K9 lysine methyltransferase G9a suppresses canonical Wnt signaling by activating expression of the Wnt antagonist DKK1. Inhibition of G9a expression or activity reduced DKK1 expression and elevated canonical Wnt signaling resulting in myogenic differentiation in vitro and in vivo. Mechanistically, G9a impacted Sp1 and p300 enrichment at the DKK1 promoter in a methylation-dependent manner. The reduced tumor growth upon G9a deficiency was reversed by recombinant DKK1 or LGK974, which also inhibits Wnt signaling. Consistently, among thirteen drugs targeting chromatin modifiers, G9a inhibitors were highly effective in reducing ERMS cell viability. Together, our study demonstrates that ERMS cells are vulnerable to G9a inhibitors and suggest that targeting the G9a-DKK1-β-catenin node holds promise for differentiation therapy.

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Synergistic Antioxidant and Anti-Inflammatory Effects between Modified Citrus Pectin and Honokiol

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/8379843 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9 ◽

Cited By ~ 16

Author(s):

Cheppail Ramachandran ◽

Barry Wilk ◽

Steven J. Melnick ◽

Isaac Eliaz

Keyword(s):

Antioxidant Activity ◽

Synergistic Effects ◽

Dependent Manner ◽

Citrus Pectin ◽

Therapeutic Modalities ◽

Inflammatory Processes ◽

Anti Inflammatory ◽

Dose Dependent Increase ◽

Dose Dependent

Inflammation is a normal physiological process; however, dysregulation of this process may contribute to inflammatory-based chronic disorders and diseases in animals and humans. Therefore, the antioxidant and anti-inflammatory properties of natural products, often recognized in traditional medicine systems, represent therapeutic modalities to reduce or prevent uncontrolled inflammatory processes which in turn potentially ameliorate or prevent sequelae of inflammatory-based symptoms of chronic diseases. We have investigated the antioxidant and anti-inflammatory effects of honokiol (HNK) and modified citrus pectin (MCP) in vitro and examined whether the MCP : HNK combination has synergistic effects on antioxidant and anti-inflammatory properties. Although both HNK and MCP induced a dose-dependent increase in antioxidant activity, the latter has a consistently higher antioxidant effect. The MCP : HNK (9 : 1) combination induced a synergistic effect on antioxidant activity suggesting that the combination is significantly more efficacious than individual compounds. In mouse monocytes, the lipopolysaccharide- (LPS-) induced tumor necrosis-α (TNF-α) synthesis was significantly inhibited by HNK and the MCP : HNK combination in a dose-dependent manner and synergistic effects were clearly demonstrated with the combination on TNF-α inhibition. This combination effect was also evident on inhibition of nuclear factor-kappa B activity, cyclooxygenase-II activity, and lipid peroxidation in mouse monocytes. Further research into the combination is warranted.

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Drug Resistance Assessment Following Administration of Respiratory Syncytial Virus (RSV) Fusion Inhibitor Presatovir to Participants Experimentally Infected With RSV

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa028 ◽

2020 ◽

Vol 222 (9) ◽

pp. 1468-1477 ◽

Cited By ~ 4

Author(s):

Kirsten Stray ◽

Michel Perron ◽

Danielle P Porter ◽

Francisco Anderson ◽

Sandra A Lewis ◽

...

Keyword(s):

Viral Load ◽

Respiratory Syncytial Virus ◽

Clinical Manifestations ◽

Viral Fitness ◽

Controlled Study ◽

Fusion Inhibitor ◽

Dependent Manner ◽

In Vitro Susceptibility ◽

Syncytial Virus

Abstract Background Presatovir is an oral respiratory syncytial virus (RSV) fusion inhibitor targeting RSV F protein. In a double-blind, placebo-controlled study in healthy adults experimentally infected with RSV (Memphis-37b), presatovir significantly reduced viral load and clinical disease severity in a dose-dependent manner. Methods Viral RNA from nasal wash samples was amplified and the F gene sequenced to monitor presatovir resistance. Effects of identified amino acid substitutions on in vitro susceptibility to presatovir, viral fitness, and clinical outcome were assessed. Results Twenty-eight treatment-emergent F substitutions were identified. Of these, 26 were tested in vitro; 2 were not due to lack of recombinant virus recovery. Ten substitutions did not affect presatovir susceptibility, and 16 substitutions reduced RSV susceptibility to presatovir (2.9- to 410-fold). No substitutions altered RSV susceptibility to palivizumab or ribavirin. Frequency of phenotypically resistant substitutions was higher with regimens containing lower presatovir dose and shorter treatment duration. Participants with phenotypic presatovir resistance had significantly higher nasal viral load area under the curve relative to those without, but substitutions did not significantly affect peak viral load or clinical manifestations of RSV disease. Conclusions Emergence of presatovir-resistant RSV occurred during therapy but did not significantly affect clinical efficacy in participants with experimental RSV infection.

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S. aureus Colonization, Biofilm Production, and Phage Susceptibility in Peritoneal Dialysis Patients

Antibiotics ◽

10.3390/antibiotics9090582 ◽

2020 ◽

Vol 9 (9) ◽

pp. 582

Author(s):

Karlis Racenis ◽

Juta Kroica ◽

Dace Rezevska ◽

Lauris Avotins ◽

Edgars Skuditis ◽

...

Keyword(s):

Peritoneal Dialysis ◽

Biofilm Formation ◽

Bacterial Resistance ◽

In Vitro Susceptibility ◽

Phenotypic Differences ◽

Biofilm Production ◽

Adaptation Procedure ◽

Different Strains ◽

Vitro Effect

Peritonitis caused by Staphylococcusaureus is of major importance in peritoneal dialysis (PD) patients due to its great virulence profile and biofilm formation ability. Bacteriophages are a potential tool to treat peritonitis resulting from biofilm-associated infections. We screened S. aureus colonization in 71 PD patients from the nasal cavity, groin, and PD exit-site regions and analyzed clinical outcomes in these patients. We performed biofilm-formation testing of different strains and compared the isolates of one patient to detect phenotypic differences in S. aureus. Phage cocktails were used to detect S. aureus in vitro susceptibility. An adaptation procedure was performed in cases of bacterial resistance. Around 30% of PD patients (n = 21) were found to be S. aureus carriers; from these, a total of 34 S. aureus strains were isolated, of which 61.8% (n = 21) produced a strong biofilm. Phenotypic differences in strain biofilm production were detected in eight patients out of ten. All strains were sensitive to commonly used antibiotics. Broadly positive phage lytic activity (100%) was observed in six cocktails out of seven, and bacterial resistance towards phages was overcome using adaptation. Overall phages showed a promising in vitro effect in biofilm-forming S. aureus strains.

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