•Photodegradation of metoprolol (MET) and propranolol (PRO) under UV-LED radiation.•Kinetics of photolysis/photocatalysis of MET, PRO, and MET-PRO in environmental waters.•Influence of magnesium and ...chloride ions on photodegradation of MET-PRO mixture.•DFT and MD computational studies of MET, PRO, and MET-PRO mixture.•Mineralization efficiency and toxicity of mixture in selected mammalian cell lines.
Phоtоlytic and phоtocatalytic degradatiоn of pharmaceuticals metoprolol (MET) and propranolol (PRO) was investigated in different types of environmental waters (Jegrička, Trbušnica, DTD, and Topli Do) in the presence оf UV–LED radiation, as well as their mixture. During direct photolysis and photocatalysis of MET and PRO, higher degradation efficiency was achieved in MET-PRO mixture compared to MET and PRO alone. This effect was particularly pronounced with PRO, which degradation was higher in the mixture. Computational analysis based on density functiоnal theоry (DFT) calculations and mоlecular dynаmics (MD) was used to understand degradation efficiency better. DFT calculations explained the higher efficiency оf direct phоtоlysis for PRO compared to MET, and also the higher MET-PRO mixture's degradation efficiency compared to MET and PRO alone. Further, phоtolytic and phоtocatalytic degradatiоn оf bоth cоmpounds was more efficient in environmental waters compared to ultrapure water. It was оbserved that the interactiоn оf MET and PRO occurs with water ions. Obtained results indicated that ions such as chloride (anions), and magnesium as (cations) increased the rate of photolysis/photocatalysis. MD simulations were used to consider the influence оf the most critical ions explicitly and have also shown that the presence of ions promotes the interaction between MET and PRO. Higher efficiency of mineralization was observed during photocatalysis compared to photolysis. Toxicity studies showed that some of MET-PRO mixtures obtained after photolysis/photocatalysis induced cell growth inhibition, indicating high hepatotoxicity.
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In bioproduction processes, cellular heterogeneity can cause unpredictable process outcomes or even provoke process failure. Still, cellular heterogeneity is not examined systematically in bioprocess ...research and development. One reason for this shortcoming is the applied average bulk analyses, which are not able to detect cell‐to‐cell differences. In this study, we present a microfluidic tool for mammalian single‐cell cultivation (MaSC) of suspension cells. The design of our platform allows cultivation in highly controllable environments. As a model system, Chinese hamster ovary cells (CHO‐K1) were cultivated over 150 h. Growth behavior was analyzed on a single‐cell level and resulted in growth rates between 0.85 and 1.16 day−1. At the same time, heterogeneous growth and division behavior, for example, unequal division time, as well as rare cellular events like polynucleation or reversed mitosis were observed, which would have remained undetected in a standard population analysis based on average measurements. Therefore, MaSC will open the door for systematic single‐cell analysis of mammalian suspension cells. Possible fields of application represent basic research topics like cell‐to‐cell heterogeneity, clonal stability, pharmaceutical drug screening, and stem cell research, as well as bioprocess related topics such as media development and novel scale‐down approaches.
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By introducing a new microfluidic tool for mammalian single‐cell cultivation, Schmitz and coworkers left common average population measurements behind and made the first step towards systematic growth and heterogeneity analysis of mammalian suspension cells on single‐cell level. Chinese hamster ovary‐K1 suspension cells were cultivated and analyzed by live‐cell imaging for 150 h. The focus of the study was systematic growth analysis that revealed growth rates between 0.85 and 1.16 day−1. Along with that, the study allowed time‐resolved insights into cellular heterogeneities.
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•Stability of β1-blocker pindolol (PIN), detected in river and wastewaters.•Efficiency of photocatalytic degradation of PIN using ZnO and three types of TiO2.•Identification and ...mineralization of degradation intermediates.•In vitro toxicity of PIN and its degradation intermediates to mammalian cell lines.•Computational analysis of PIN and its degradation intermediates: DFT and MD.
In this work, we have investigated the stability of pindolol (PIN), a non-selective β1-blocker detected in the river and wastewater of hospitals, in water solution under solar irradiation. Further, detailed insights into the stability of PIN were obtained by the density functional theory (DFT) calculations and molecular dynamics simulations. The kinetics of PIN photocatalytic degradation and mineralization has been studied using four commercial photocatalysts ZnO and TiO2 (P25, Hombikat, and Wackherr). It was found that the major role in degradation of PIN play the reactive hydroxyl radicals. The structures of degradation intermediates were suggested by LC–ESI–MS/MS and DFT calculations. Also, DFT calculations were used to refine molecular structures of intermediates and obtain their geometries. Toxicity of PIN and its mixtures formed during photocatalytic degradation were investigated using mammalian cell lines (H-4-II-E, HT-29, and MRC-5). The H-4-II-E cell line was the most sensitive to PIN and its photodegradation mixtures. The computational results were combined with the experimental data on the amounts of degradation intermediates for determination of the intermediates that were principally responsible for the toxicity. Intermediate with two hydroxyl groups, positioned on indole ring in meta and para positions, was proposed as the one with the highest contribution to toxicity.
Bitter apricot kernels’ extract contains a broad spectrum of biologically active substances with a lot of attention to amygdalin – cyanogenic glycoside. The extract has been used in the ...pharmaceutical industry for years as an ingredient of different pharmaceuticals with anti-inflammatory, antimicrobial, or regenerative properties. In traditional medicine, the bitter apricot kernels are known as a remedy for respiratory disorders and skin diseases. The apricot kernels and amygdalin are often prescribed by practitioners for the prevention and treatment of various medical conditions, including colorectal cancer.
to evaluate the phytochemical composition and the potential antimutagenic, antirecombinogenic, and antitumor effect of apricot kernels’ extract at very low concentrations in yeast cell-based tests and mammalian hepatocellular and colon carcinoma cell lines.
Phytochemical analysis was performed by LC-MS profiling. Reverse-phase HPLC and UV detection were applied for the determination of amygdalin quantity in the extract. Biological activity was evaluated by Zimmermann's mutagenicity and Ty1 retrotransposition test. Cytotoxic/antiproliferative activity of apricot kernels' extract was performed on four types of cell lines – HepG2, HT-29, BALB/3T3, clone A31, and BJ using the standard MTT-dye reduction assay.
Data revealed the presence of more than 1000 compounds and 4 cyanogenic glycosides among them – Amygdalin, Deidaclin, Linamarin and Prulaurasin. The Amygdalin concentration was measured to be 57.8 μg/ml. All extract concentrations demonstrated a strong antigenotoxic, antirecombinogenic, antimutagenic, and anticarcinogenic effect in the yeast cell-based tests. High selectivity of the extract action is established among different mammalian cell lines. Normal cell line BJ is found to be resistant to the extract action. HepG2 was found to be the most sensitive to apricot kernels’ action.
The present study provides the first phytochemical analysis of Bulgarian bitter apricot kernels. Three new cyanogenic glycosides were reported. Evidence is obtained that the apricot kernels’ extract at low concentrations is not able to induce some of the events related to the initial steps of tumorigenesis. Additionally, a high selectivity of the extract action is established among different cell lines. The most sensitive cell line was found to be HepG2.
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•The extract possesses strong dose-dependent antimutagenic potential against MMS.•The hexavalent chromium-induced Ty1 retrotransposition rates are lowered.•High selectivity is established among normal and cancer cell lines.•HepG2 was found to be the most sensitive to apricot kernels action.
Exposure to xenobiotics can increase the production of reactive oxygen species (ROS). When detoxification organs such as the intestines and liver cannot neutralise these xenobiotics, it can induce ...oxidative stress and cause damage to tissues. Therefore, cell-based bioassays that indicate intracellular ROS production are a useful screening tool to evaluate the effect of these chemicals. Although flow cytometry is commonly used to measure ROS in cells, many research laboratories in the Global South do not always have access to such specialised instrumentation. Therefore, we describe a sensitive but low-cost method that can easily be used to determine ROS production in vitro. This method employs the fluorogenic dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), which emits fluorescence after being oxidised to a fluorescent derivative. Since the H2DCF-DA bioassay indicates non-specific ROS production it can be used as a marker of overall oxidative stress. This method was validated by exposing human duodenum epithelial adenocarcinoma (HuTu-80) and rat liver epithelial hepatoma (H4IIE-luc) cells to agricultural soil samples.•Production of ROS can be determined in vitro in intestinal and liver cells.•This method is inexpensive and can be easily performed in standard laboratories.•The method provides a tool for the high-throughput screening of environmental samples.
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Serum-free suspension cultures are preferably required for recombinant protein production due to its readiness in upstream/downstream processing and scale-up, therefore increasing process ...productivity and competitiveness. This type of culture replaces traditional cell culturing as the presence of animal-derived components may introduce lot-a-lot variability and adventitious pathogens to the process. However, adapting cells to serum-free conditions is challenging, time-consuming, and cell line and medium dependent. In this chapter, we present different approaches that can be used to adapt mammalian cell lines from an anchorage-dependent serum supplemented culture to a suspension serum-free culture.
The function of a protein within a cell critically depends on its interaction with other proteins as well as its subcellular localization. The expression of mutants of a particular protein that have ...selective perturbation of specific protein interaction motifs is a very useful strategy for resolving a protein's mechanism of action in a cellular process. In addition, expression of fluorescent protein fusions is a key strategy for determining the subcellular localization of a protein. These strategies require tight regulation to avoid potential alterations in protein interactions or localizations that can result from protein overexpression. Previous work led to the development of a Sleeping Beauty transposon system that allows doxycycline-inducible expression of protein mutants or fusions; titration of doxycycline allows expression of protein fusions or mutants at near endogenous levels. When used in combination with siRNA gene silencing, this strategy allows for knockdown-rescue experiments to assess the function of specific protein mutants. In this protocol, we describe the use of this Sleeping Beauty strategy for expression of eGFP fusion or mutant proteins in ARPE-19 and MDA-MB-231 cells. This includes design of expression plasmids, transfection, and selection to obtain stable engineered cells, as well as doxycycline treatment for controlled induction of protein expression, either alone or in combination with siRNA silencing for knockdown-rescue experiments. This strategy is advantageous as it allows rapid generation of stable cells for controlled protein expression, suitable for functional studies that require knockdown-rescue as well as various forms of live cell fluorescence imaging. Key features • Highly versatile doxycycline-inducible expression system that can be used in various mammalian cell lines. • Stable integration of transgene allows for sustained and stable expression. • Titration of doxycycline levels allows expression of transgene at near endogenous levels.
In this work
Talaromyces australis
and
Penicillium murcianum
pigment production in liquid cultures and the cytotoxic effect of such pigments on skin model cells were studied. Response surface ...methodology (RSM) was used to optimize culture conditions aiming to increase pigment production in malt extract and peptone-glucose-yeast extract medium. Cytotoxicity of fungal pigments and also from lixiviates of wool fabrics dyed with
T. australis
and
P. murcianum
pigment was evaluated on mammalian cell lines HEK293 and NIH/3T3. Results showed that variations on initial pH, NaCl and peptone, resulted in increments up to 188.2% for red pigment of
T. australis
and 107.4% for yellow pigment of
P. murcianum
, regarding non-optimized conditions. Tested fungi also showed great differences in culture conditions for the maximum pigment production, with
P. murcianum
requiring an alkaline medium (initial pH 9) supplemented with NaCl and
T. australis
an acidic medium (initial pH 5) without addition of salt. The cytotoxicity assays provided evidences on the safe nature of these natural pigments when used for textile applications. The cytotoxicity assay showed that the threshold of toxicity, given by the lowest IC
50
value (0.21 g L
−1
) was more than double of the concentration of pigment required to dye the wool samples. In addition, cytotoxicity of lixiviates depicted no toxic effect over tested cells.
Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) ...or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2-3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage. Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.
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