scholarly journals Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the Gardnerella-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis

Antibiotics ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 111
Author(s):  
Agnieszka Latka ◽  
Leen Van Simaey ◽  
Marijke Reynders ◽  
Piet Cools ◽  
Tess Rogier ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Treatment Condition ◽  
Ex Vivo ◽  
Light Exposure ◽  
Living Cells ◽  
Standard Protocol ◽  
Propidium Monoazide ◽  
Planktonic Cells ◽  
Complex Composition ◽  
Pre Treatment

Quantification of the number of living cells in biofilm or after eradication treatments of biofilm, is problematic for different reasons. We assessed the performance of pre-treatment of DNA, planktonic cells and ex vivo vaginal biofilms of Gardnerella with propidium monoazide (PMAxx) to prevent qPCR-based amplification of DNA from killed cells (viability-qPCR). Standard PMAxx treatment did not completely inactivate free DNA and did not affect living cells. While culture indicated that killing of planktonic cells by heat or by endolysin was complete, viability-qPCR assessed only log reductions of 1.73 and 0.32, respectively. Therefore, we improved the standard protocol by comparing different (combinations of) parameters, such as concentration of PMAxx, and repetition, duration and incubation conditions of treatment. The optimized PMAxx treatment condition for further experiments consisted of three cycles, each of: 15 min incubation on ice with 50 µM PMAxx, followed by 15 min-long light exposure. This protocol was validated for use in vaginal samples from women with bacterial vaginosis. Up to log2.2 reduction of Gardnerella cells after treatment with PM-477 was documented, despite the complex composition of the samples, which might have hampered the activity of PM-477 as well as the quantification of low loads by viability-qPCR.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

scholarly journals Preclinical Evaluation of [18F]FACH in Healthy Mice and Piglets: An 18F-Labeled Ligand for Imaging of Monocarboxylate Transporters with PET

2021 ◽  
Vol 22 (4) ◽  
pp. 1645
Author(s):  
Daniel Gündel ◽  
Masoud Sadeghzadeh ◽  
Winnie Deuther-Conrad ◽  
Barbara Wenzel ◽  
Paul Cumming ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Ex Vivo ◽  
Specific Binding ◽  
Monocarboxylate Transporters ◽  
Binding Potential ◽  
Kidney Cortex ◽  
Binding Studies ◽  
Imaging Tool ◽  
Pre Treatment

The expression of monocarboxylate transporters (MCTs) is linked to pathophysiological changes in diseases, including cancer, such that MCTs could potentially serve as diagnostic markers or therapeutic targets. We recently developed [18F]FACH as a radiotracer for non-invasive molecular imaging of MCTs by positron emission tomography (PET). The aim of this study was to evaluate further the specificity, metabolic stability, and pharmacokinetics of [18F]FACH in healthy mice and piglets. We measured the [18F]FACH plasma protein binding fractions in mice and piglets and the specific binding in cryosections of murine kidney and lung. The biodistribution of [18F]FACH was evaluated by tissue sampling ex vivo and by dynamic PET/MRI in vivo, with and without pre-treatment by the MCT inhibitor α-CCA-Na or the reference compound, FACH-Na. Additionally, we performed compartmental modelling of the PET signal in kidney cortex and liver. Saturation binding studies in kidney cortex cryosections indicated a KD of 118 ± 12 nM and Bmax of 6.0 pmol/mg wet weight. The specificity of [18F]FACH uptake in the kidney cortex was confirmed in vivo by reductions in AUC0–60min after pre-treatment with α-CCA-Na in mice (−47%) and in piglets (−66%). [18F]FACH was metabolically stable in mouse, but polar radio-metabolites were present in plasma and tissues of piglets. The [18F]FACH binding potential (BPND) in the kidney cortex was approximately 1.3 in mice. The MCT1 specificity of [18F]FACH uptake was confirmed by displacement studies in 4T1 cells. [18F]FACH has suitable properties for the detection of the MCTs in kidney, and thus has potential as a molecular imaging tool for MCT-related pathologies, which should next be assessed in relevant disease models.

scholarly journals HIV-1 subverts the complement system in semen to enhance viral transmission

2021 ◽  
Author(s):  
Bernadien M. Nijmeijer ◽  
Marta Bermejo-Jambrina ◽  
Tanja M. Kaptein ◽  
Carla M. S. Ribeiro ◽  
Doris Wilflingseder ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Virus Transmission ◽  
Sexual Contact ◽  
Target Cells ◽  
People Living With Hiv ◽  
Lectin Receptor ◽  
Pre Treatment ◽  
Living With Hiv ◽  
Hiv 1

AbstractSemen is important in determining HIV-1 susceptibility but it is unclear how it affects virus transmission during sexual contact. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 during sexual contact and have a barrier function as LCs are restrictive to HIV-1. As semen from people living with HIV-1 contains complement-opsonized HIV-1, we investigated the effect of complement on HIV-1 dissemination by human LCs in vitro and ex vivo. Notably, pre-treatment of HIV-1 with semen enhanced LC infection compared to untreated HIV-1 in the ex vivo explant model. Infection of LCs and transmission to target cells by opsonized HIV-1 was efficiently inhibited by blocking complement receptors CR3 and CR4. Complement opsonization of HIV-1 enhanced uptake, fusion, and integration by LCs leading to an increased transmission of HIV-1 to target cells. However, in the absence of both CR3 and CR4, C-type lectin receptor langerin was able to restrict infection of complement-opsonized HIV-1. These data suggest that complement enhances HIV-1 infection of LCs by binding CR3 and CR4, thereby bypassing langerin and changing the restrictive nature of LCs into virus-disseminating cells. Targeting complement factors might be effective in preventing HIV-1 transmission.

scholarly journals Discrimination of Viable and Dead Fecal Bacteroidales Bacteria by Quantitative PCR with Propidium Monoazide

2009 ◽  
Vol 75 (9) ◽  
pp. 2940-2944 ◽  
Author(s):  
Sungwoo Bae ◽  
Stefan Wuertz
Keyword(s):  
Quantitative Pcr ◽  
Light Exposure ◽  
Bacteroides Fragilis ◽  
Extracellular Dna ◽  
Propidium Monoazide ◽  
Human Feces ◽  
Heat Treated ◽  
Pcr Conditions

ABSTRACT Propidium monoazide (PMA) was optimized to discriminate between viable and dead Bacteroides fragilis cells and extracellular DNA at different concentrations of solids using quantitative PCR. Conditions of 100 μM PMA and a 10-min light exposure also excluded DNA from heat-treated cells of nonculturable Bacteroidales in human feces and wastewater influent and effluent.

Dyeing Adsorption Isotherm and Equilibrium Modeling Characteristics of Enzymatic and Caustic Pre-Treated Lyocell Fibers

2011 ◽  
Vol 332-334 ◽  
pp. 568-571
Author(s):  
Yi Jun Pan ◽  
Chien Teng Hsieh ◽  
Chien Kuo Yen
Keyword(s):  
Treatment Condition ◽  
Enzymatic Treatment ◽  
Equilibrium Modeling ◽  
Sorption Ability ◽  
Treatment Stage ◽  
Lyocell Fibers ◽  
Lyocell Fiber ◽  
Consequent Effect ◽  
Pre Treatment ◽  
Dyes Adsorption

The purpose of this study is to achieve a better understanding of the influences of enzymatic and caustic pretreatments on the dying adsorption behavior of Lyocell yarns. The substantivity behavior, dyeing adsorption isotherms and their correlation between the equilibrium modeling and equilibrium isotherm parameters of the pre-treated Lyocell fibers have been studied. Although the conditions of each treatment were the same, changing the processing sequences caused substantially different modifications on the Lyocell fiber fine structure with consequent effect on dyeing behaviors. In general, pretreatment by caustic soda and/or enzyme can improve their dyeability of Lyocell fibers. The results of dyes adsorption show that for the same enzymatic treatment condition if the enzymatic treatment stage is applied before the caustic pre-treatment, the dyeability and sorption ability of small molecule dye on pre-treated Lyocell can be improved over that which can be obtained for caustic pre-treated yarn.

scholarly journals Photoradiation models for the clinical ex vivo treatment of autologous bone marrow grafts

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 484-489
Author(s):  
J Atzpodien ◽  
SC Gulati ◽  
A Strife ◽  
BD Clarkson
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
White Light ◽  
Ex Vivo ◽  
Light Exposure ◽  
B Cell Lymphoma ◽  
Autologous Bone Marrow ◽  
Neoplastic Cells ◽  
Drug Concentrations ◽  
Autologous Bone

To assess the potential of photoradiation therapy for the in vitro purging of residual tumor cells from autologous bone marrow (BM) transplants, we studied normal marrow and tumor cell clonogenicity in response to different light-activated compounds by using the fluorescent dyes dihematoporphyrin ether (DHE) and merocyanine-540 (MC- 540). After photoradiation of cells with white light, both DHE and MC- 540 showed high cytocidal activity toward lymphoid and myeloid neoplastic cells but had a significantly lesser effect on normal granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and mixed colony- forming (CFU-GEMM) progenitor cells. Acute promyelocytic leukemia (HL- 60), non-B, non-T, CALLA-positive acute lymphoblastic leukemia (Reh), and diffuse histocytic B cell lymphoma (SK-DHL-2) cell lines were exposed to different drug concentrations in combination with white light at a constant illumination rate of 50,000 lux. With DHE doses varying from 2.0 to 2.5 micrograms/mL and MC-540 concentrations of 15 to 20 micrograms/mL, clonogenic tumor cells could be reduced by more than 4 logs when treated alone or in mixtures with normal irradiated human marrow cells. However, preferential cytotoxicity towards neoplastic cells was highly dependent on the mode of light activation. MC-540 had no substantial effect on malignant lymphoid (SK-DHL-2) and myeloid (HL-60) cells and on normal marrow myeloid (CFU-GM) precursors when drug incubation was performed in the dark and followed by light exposure of washed cells. Equal doses of MC-540 (15 to 20 micrograms/mL) could preferentially eliminate tumor cells under conditions of simultaneous light and drug treatment (30 minutes at 37 degrees C). When using DHE (2.5 micrograms/mL), 29.3%, 46.8%, and 27.5% of normal marrow CFU-GM, BFU-E, and CFU-GEMM, respectively, were spared after sequential drug and light exposure of cells, whereas simultaneous treatment reduced both normal (CFU-GM) and neoplastic cells below the limits of detection. In summary, our results indicate the usefulness of various photoradiation models for the ex vivo treatment of leukemic and lymphomatous bone marrow autografts.

PROTECTIVE EFFECT OF A NEW SYNTHETIC COMPOUND: PCA-4230, ON SEVERAL In vivo THROMBOSIS MODELS.

1987 ◽  
Author(s):  
M P Ortega ◽  
C Sunkel ◽  
J G Priego
Keyword(s):  
Protective Effect ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Platelet Activating Factor ◽  
Phase I Trials ◽  
Synthetic Compound ◽  
Prolonged Bleeding Time ◽  
Series Of Experiments ◽  
Pre Treatment

PCA-4230 is a new anti-thrombotic compound which inhibits pla. telet aggregation In vltn.0 and ex. vivo in several species, including man, prolongs the bleeding time and has potent protective ac tivity in several thrombosis models. Phase I trials with different dosage schedules have recently been initiated.In the present study, the effects of PCA-4230 on bleeding time and on several In vivo thrombosis models were studied in mice. Mice were treated with one single oral dose of PCA-4230 (1-10 mg/ kg). One hour after treatment, mice were injected intravenously with four thrombotic challengers {arachidonic acid (AA), thromboxane agonist (U46619), Platelet Activating Factor (PAF) or collagen/epinephrine combination (C/E)} at a dose which induced 80-90% of mortality. The thrombotic agents were prepared in saline. The appropiate doses were dissolved in a volume of 100 μl/mouse. Bleeding time was measured in non-anesthetized mice by the tail transection technique.Effects of compound were recorded from1 to 4 hours after dosage. Acute pre-treatment with PCA-4230 showed a significant dose-depen dent protective effect.Results of each series of experiments are given in the next table.The compound inhibited thrombotic sudden death induced by U46619, PAF or C/E combination, reduced the duration of respiratory distress induced by AA and prolonged bleeding time. Thus, PCA-4230 is protective against a variety of thrombotic stimuli.These results suggest that PCA-4230 may be a promising anti-throm botic drug.

INITIAL STUDIES IN MAN WITH A NOVEL THROMBOXANE RECEPTOR BLOCKING DRUG GR32191

1987 ◽  
Author(s):  
M Thomas ◽  
P Lumley ◽  
P Ballard ◽  
J R O'Brien
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Plasma Concentrations ◽  
Blocking Drug ◽  
Double Blind ◽  
Receptor Blocking ◽  
Thromboxane Receptor ◽  
Pre Treatment ◽  
Induced Aggregation

In-vitro GR32191 is a potent and specific thromboxane receptor blocking drug on platelets, and vascular and airways smooth muscle (Lumley et al this meeting). We have undertaken studies in healthy male subjects (n) to examine the effects of oral GR32191 upon platelet aggregation ex-vivo and template bleeding time. Platelet aggregation was monitored in whole blood by counting platelets electronically. Concentration-effect curves to U-46619 and ADP were constructed prior to and following drug or placebo. The degree of rightward displacement of a curve due to treatment was expressed as a concentration-ratio (CR) which was calculated at the 50% aggregation level (ECso post-treatment ECso pre-treatment). Plasma concentrations of GR32191 were determined by h.p.l.c. After single doses of GR32191 mean peak CR's of 8 and 80 were achieved with 0.125 and 0.25mg/kg (n=4) and values of 74 and 234 with 0.5 and lmg/kg (n=4). Peak effects were seen within 2 hours of dosing while activity was still present between 8 and 24 hours. ADP-induced aggregation was unaffected by drug (CR<2) and placebo was without significant effect upon the sensitivity to either aggregating agent (CR<2). GR32191 was rapidly absorbed and the plasma elimination half-life was about 2 hours. GR32191 17.5mg 12-hourly for 10 days (n=6) produced a progressive antagonism of U-46619 induced aggregation which resulted in a large continuous blockade in all subjects (range of 12htrough CR's 85 to 287). However, plasma concentrations of GR32191 did not accumulate on repeated administration. In a double-blind, placebo-controlled, cross-over study (n=16), a statistically significant (p= 0.002) increase in bleeding time was seen following treatment with GR32191 40mg twice daily for 7 days (pre-treatment mean 3.79 min, post-placebo mean 3.47 min, post-GR32191 mean 5.42 min). Rectal bleeding (n=l) has occurred with GR32191 but otherwise tolerability has been good. No drug related changes have been seen in routine laboratory safety screens. Clinical studies are in progress.

scholarly journals The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Erika Günther ◽  
André Klauß ◽  
Mauricio Toro-Nahuelpan ◽  
Dirk Schüler ◽  
Carsten Hille ◽  
...  
Keyword(s):  
Magnetic Particles ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Stimulated Emission ◽  
Fluorescence Lifetime Imaging ◽  
Sted Microscopy

AbstractProtein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Förster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.

scholarly journals SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells

2016 ◽  
Vol 113 (37) ◽  
pp. 10322-10327 ◽  
Author(s):  
Matthew J. Smola ◽  
Thomas W. Christy ◽  
Kaoru Inoue ◽  
Cindo O. Nicholson ◽  
Matthew Friedersdorf ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Rna Structure ◽  
Noncoding Rna ◽  
Ex Vivo ◽  
Living Cells ◽  
Mammalian Development ◽  
Cellular Environment ◽  
Nucleotide Resolution ◽  
In Cells

The 18-kb Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation during female eutherian mammalian development. Global structural architecture, cell-induced conformational changes, and protein–RNA interactions within Xist are poorly understood. We used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to examine these features of Xist at single-nucleotide resolution both in living cells and ex vivo. The Xist RNA forms complex well-defined secondary structure domains and the cellular environment strongly modulates the RNA structure, via motifs spanning one-half of all Xist nucleotides. The Xist RNA structure modulates protein interactions in cells via multiple mechanisms. For example, repeat-containing elements adopt accessible and dynamic structures that function as landing pads for protein cofactors. Structured RNA motifs create interaction domains for specific proteins and also sequester other motifs, such that only a subset of potential binding sites forms stable interactions. This work creates a broad quantitative framework for understanding structure–function interrelationships for Xist and other lncRNAs in cells.

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