Acid In Supernatant Research Articles

Characterization of a Novel Oncolytic Virus for Cancer Therapy

BackgroundOncolytic viruses (OVs) that selectively replicate (oncoselectivity) and lyse (oncotoxicity) cancer cells are emerging as promising cancer therapeutics. A range of DNA and RNA OVs are in different stages of human clinical trials. The presence of preexisting immunity against OVs and their potential pathogenic effects are significant limitations. Using animal viruses that are non‐pathogenic to humans is considered an attractive strategy. We identified a naturally occurring animal virus with potent oncolytic ability, hereafter referred to as oncolytic virus 1 (OV1). We tested the oncoselectivity and oncotoxicity of OV1 using a range of human cancer cells, immortalized cells, and normal non‐cancerous cells. We further evaluated the oncolytic properties of OV1 in vivo using the murine triple‐negative breast cancer (TNBC) model, 4T1.2.MethodsHuman renal (769P), mammary (HCC 1500), colorectal (HRT‐18G), lung (A549), and pancreatic (BxPC‐3) cancer cells, and leukemia (THP‐1) cells, human immortalized embryonic kidney cells (293T), and human primary bronchial epithelial cells (PBEC) were infected with OV1 at a range of multiplicity of infection (MOI) from 0.01 to 5. After 72h of infection, MTS assay to determine cell viability and Caspase‐Glo 3/7 assay to quantify apoptosis were performed. OV1 replication in cultured 4T1.2 cells was assessed by infecting the cells at an MOI of 0.1 and quantifying viral nucleic acid in the supernatants at 72h. Female BALB/c mice were injected with 4T1.2 cells into the fourth mammary gland on the left side. At 19 days post‐tumor implantation, 105TCID50 units of OV1 in 50µl was injected half intratumorally and half intraperitoneally. Mice in the mock treatment group received 50µl of vehicle control. Tumor sizes were measured regularly and at day 35, mice were sacrificed and the number of live cells in the tumor were quantified by flow cytometry.ResultsThere was a significant decrease in the viability of OV1 infected 769P cells (MOI ≥ 0.1), A549 (MOI ≥ 1), THP‐1 (MOI ≥ 2.5), and HCC 1500 (MOI ≥ 2.5) at 72h compared with controls. Notably, there was no significant change in the viability of primary cells (PBEC) and immortalized 293T infected with OV1 at MOI of 0.1 to 2.5 compared to controls. There was a significant increase in caspase activity in 769P cells (at MOI ≥ 0.1), A549 (at MOI ≥ 1), THP‐1 (at MOI ≥ 1), BxPC3 (at MOI ≥ 2.5), HRT‐18G (at MOI ≥ 2.5) and HCC 1500 (at MOI ≥ 0.1). There was no increase in the caspase activity of PBEC at all the tested MOI. OV1 viral replication was observed in 4T1.2 cells in vitro, as evidenced by increasing viral nucleic acid in culture supernatants. BALB/c mice injected with OV1 showed a significant decrease in tumor size and a decrease in the live cell numbers in the tumor compared with mock treated mice.Conclusions and future directionsThe results established the oncoselectivity and oncotoxicity of OV1 and its oncolytic ability in vivo. Further studies are underway to investigate OV1 replication in cells, immune stimulatory properties of OV1 in vivo, and the generation of a recombinant OV1 with increased oncolytic ability.

Research on the variations of organics and heavy metals in municipal sludge with additive acetic acid and modified phosphogypsum

Concentration and fraction distribution of organics and heavy metals in municipal sludge treated by modified phosphogypsum and acetic acid (signed as MPG/HAC) were studied. The results showed that MPG/HAC conditioning significantly produce synergistic enhancement effect to dissolution of unstable heavy metals wrapped in the stable colloid network. Simultaneously, after conditioning, about 45.16% of organics such as proteins, polysaccharides and humic acid in supernatant was degraded, thus dissociating large amount of active group which accelerated immobilization of dissolved heavy metals and weaken its toxicity. In addition, MPG with a porous structure could adsorb unstable heavy metals and transform them into residual fraction, leading to a considerable decrease in their mobility risk level. Besides, linear regression models showed that a strong oxidizability of sludge, and destruction of colloidal network could greatly promote dissolution of unstable heavy metals. Simultaneously, sludge oxidizability and organics degradation rate, and disintegration of extracellular polymeric substances (EPS) layer highly accelerate immobilization of unstable metals. Excepting Cd, environmental risk of Cr, Cu, Pb, Zn, Ni and As can be effectively weakened after conditioning. Additionally, MPG/HAC conditioning might be appropriate for stabilization of Cd, Cr and Zn in water supply sludge, especially for Zn.

Determination of Perillic Acid in Bioconversion Supernatants by Gas Chromatography

Perillic acid can be obtained from microbial oxidation of the exocyclic methyl group of limonene. Due to the pharmacological potential of such a metabolite, the biotransformation processes leading to its synthesis have been approached in recent studies. A robust analytical method is needed to assess the performance of such studies. An analytical method was developed and validated to determine perillic acid in the supernatants of a yeast-induced bioconversion of limonene, involving gas chromatography (GC) and an acid-induced precipitation during the sample preparation. GC analysis was performed using a column with polyethylene glycol as stationary phase (HP-Innowax) which resulted in higher loads and better peak shape. The sample preparation involved the supernatant initial filtration and precipitation with 0.6 M HCl followed by centrifugation and dissolution in ethyl acetate. GC analysis conditions were oven from 50°C to 250°C at 20°C·min-1, and then held 5 min (total runtime 15 min). Injector was set at 280°C, and detector at 300°C. Helium was the carrier gas at 1 ml·min-1. Injections of 1.0 μl were at the split ratio 25:1. The method was validated: Linearity with R2 of 0.9992, Accuracy of 98.3% in the range 190 - 950 μg·ml-1; Limit of detection of 10.4 μg·ml-1; Repeatability of 2.1% RSD. Thus, a complete methodology to determine perillic acid in a bioconversion supernatant was developed and validated. This overall approach may be useful for bioconversions of monoterpenes by other microorganisms that metabolize limonene.

Biocontrol of post‐harvest Alternaria alternata decay of cherry tomatoes with rhamnolipids and possible mechanisms of action

Rhamnolipids were reported to have evident antifungal activity. The efficacy of rhamnolipids against Alternaria alternata and their possible mechanisms involved were investigated. The decay incidences of A. alternata of cherry tomatoes (Lycopersicon esculentum) treated by rhamnolipids were significantly reduced. The in vitro assays showed that rhamnolipids inhibited fungal growth on solid medium and prevented spore germination and mycelium growth in liquid medium. In addition, the combination of rhamnolipids and essential oil had a synergistic effect leading to the decrease of fungicidal concentrations of laurel oil. Scanning electron microscopy and transmission electron microscopy observations of the pathogen revealed significant morphological and cell structural alterations in the hyphae. Compared to the control, the content of nucleic acid in supernatant of the suspension of A. alternata increased, while the content of DNA and protein of mycelium decreased, which was in agreement with electrolyte leakage experiments. Rhamnolipids could be an alternative to chemicals for controlling post-harvest phytopathogenic fungi on fruits and vegetables.

Uptake of dihomo-γ-linolenic acid by murine macrophages increases series-1 prostaglandin release following lipopolysaccharide treatment

Administration of dihomo-γ-linolenic acid is useful for atopic dermatitis and atherosclerosis in mice; however, the metabolites of dihomo-γ-linolenic acid have been little studied. We employed a method which enabled simultaneous analysis of nine prostaglandins using liquid chromatography–tandem mass spectrometry, and determined the concentrations of prostaglandins in the supernatants of cultures of mouse peritoneal macrophages stimulated with lipopolysaccharide after pre-incubation with dihomo-γ-linolenic acid, arachidonic acid, or eicosapentaenoic acid. Accumulated prostaglandin concentrations from mouse macrophages with dihomo-γ-linolenic acid uptake increased in a dihomo-γ-linolenic acid concentration-dependent fashion. These increases were mainly due to prostaglandin D 1 and prostaglandin E 1. The order of accumulated prostaglandin concentrations was dihomo-γ-linolenic acid>arachidonic acid>eicosapentaenoic acid in supernatants with the same concentration of polyunsaturated fatty acid. Since mouse macrophages can clearly produce series-1 prostaglandins, they must be formed in vivo. These findings suggest that the effects of dihomo-γ-linolenic acid on diseases may be due to series-1 prostaglandins.

Verminderung der Lipidperoxidation und der Apoptoserate in kornealen Endothelzellen durch Vitamin A

The goal of this study was to determine the effects of lipid peroxidation-mediated toxicity of iron ions on corneal endothelial cells leading to apoptosis. Murine corneal endothelial cells were maintained in tissue culture medium supplemented with free iron ions, known to lead to increased lipid peroxidation. Retinoic acid in the cell supernatant and cytoplasm of these cells was determined using HPLC. The rate of apoptosis was assessed by quantification of caspase-3-like activity. The lipid peroxidation was measured using the malondialdehyde method. Supplementation of retinoic acid was tested in the setting of apoptosis. Free iron ions led to a rapid loss of retinoic acid in the supernatant and the corneal endothelial cells. This was correlated with rising levels of malondialdehyde following oxidative stress and increased apoptosis. Supplementation of retinoic acid alone significantly reduced oxidative stress and apoptosis in the respective cells. In this study the authors present a novel in vitro model to test the direct influence of pro-oxidative species on corneal endothelial cells. The authors also prove that supplementing corneal endothelial cells with retinoic acid sufficiently prevents free radical injury and apoptosis.

Kinetics of soybean lipoxygenase reaction in hydrated reversed micelles

The rate of linoleic acid peroxidation catalysed by soybean lipoxygenase I was studied as a function of the hydration degree of aerosol OT ( bis(2-ethylhexyl)sulfosuccinate sodium salt) reversed micelles in octane. Lipoxygenase reaction parameters for the micelle-bound substrate were spectrophotometrically determined. The linoleic acid distribution between the micelles and octane was detected by the sedimentation method, with the concentration of linoleic acid in supernatant after settling of micelles ( i.e. the concentration of free linoleic acid) being estimated by the enzymatic method. The apparent constant of linoleic acid distribution (the ratio of the bound and free substrate concentrations) was enhanced with increasing hydration of reversed micelles. The dependence of the enzymatic reaction rate on the bound substrate concentration obeyed the empiric Hill equation. The Hill coefficient remained practically constant ( h=1.34) as the hydration degree changed. Parameters of the lipoxygenase reaction, enzyme reaction limiting rate V and semi-saturation substrate concentration [ S] 0.5 increased with increasing degree of hydration and reached the optimum at [H 2O]/[AOT]≈30, where dimensions of the micellar internal cavity coincided with those of the enzyme molecule. Some aspects of kinetic behavior of membrane-bound enzymes participating in chemical transformation of non-polar compounds dispersed in lipid phase are discussed.

High-performance liquid chromatographic determination of metabolic products for fermentation control of mammalian cell culture: analysis of carbohydrates, organic acids and orthophosphate using refractive index and ultraviolet detectors

A method for the determination of carbohydrate substrates and excreted metabolic end-products of cell culture supernatants using a strong cation-exchange column in the H + form has been developed. Organic acids and carbohydrates can be determined in addition to orthophosphoric acid. Pyrrolidone carboxylic acid, resulting from chemical conversion of the amino acid glutamine during the incubation of fresh medium and during the fermentation process, can be determined. The chromatographic method allows the correction of glutamine uptake values for physiological studies. Measured values of pyrrolidone carboxylic acid in supernatants of a human hybridoma cell line show that it cannot be consumed by the cells. This technique allows the separation of major metabolites used in process optimization. Peak Homogeneity is proved by on-line monitoring of the effluent with an ultraviolet (214 nm) and a refractive index detector connected in series.

Influence of pH on organic acid production by Clostridium sporogenes in test tube and fermentor cultures.

The influence of pH on the growth parameters of and the organic acids produced by Clostridium sporogenes 3121 cultured in test tubes and fermentors at 35 degrees C was examined. Specific growth rates in the fermentor maintained at a constant pH ranged from 0.20 h-1 at pH 5.00 to 0.86 h-1 at pH 6.50. Acetic acid was the primary organic acid in supernatants of 24-h cultures; total organic acid levels were 2.0 to 22.0 mumol/ml. Supernatants from pH 5.00 and 5.50 cultures had total organic acid levels less than one-third of those found at pH 6.00 to 7.00. The specific growth rates of the test tube cultures ranged from 0.51 h-1 at pH 5.00 to 0.95 h-1 at pH 6.50. The pH of the medium did not affect the average total organic acid content (51.5 mumol/ml) but did affect the distribution of the organic acids, which included formic, acetic, propionic, butyric, 3-(p-hydroxyphenyl)propionic, and 3-phenylpropionic acids. Butyric acid levels were lower, but formic and propionic acid levels were higher, at pH 5.00 than at other pHs.