Cytogenetic and oxidative effects of three lichen extracts on human peripheral lymphocytes.

In the present study, we investigated cytogenetic and oxidative [total antioxidant capacity (TAC), total oxidant status (TOS)] effects of methanol and water extracts of Cladonia chlorophaea (Flörke ex Sommerf.) Sprengel, Dermatocarpon miniatum (L.) W.Mann and Parmelia saxatilis (L.) Ach. on cultured human lymphocytes. In addition, different phenolic compounds in the extracts were quantified by high performance liquid chromatography (HPLC) analysis. As a result of HPLC analysis, methanol extracts of all lichen species tested had higher phenolic compounds. Likewise, methanol extracts of each lichen increased TAC levels in lymphocytes more than water extracts. The TOS levels of the cells treated with different concentrations (1-100 mg/L) of the extracts decreased due to the increasing concentration of the extracts. Genotoxicity experiments revealed that the tested lichen extracts did not significantly increase (p > 0.05) the level of genotoxicity on human peripheral lymphocyte culture compared to the negative control group. The results showed that C. chlorophaea, D. miniatum and P. saxatilis lichens, which were found to be a rich source of phenolic compounds, might be of interest in the pharmaceutical and food industries.

Quail (Coturnix japonica) egg attenuated 2-butoxyethanol-induced enzymatic dysregulation, disseminated thrombosis and hemolytic impairment in female wistar rats.

Influence of quail egg on pathologies has increased research interests and series of investigations are currently being done on its influence against these pathologies. The influence of quail egg against 2-butoxyethanol induced hemolysis and disseminated thrombosis was investigated to determine the enzymatic regulations that ensue in the amelioration of deleterious hemolytic and disseminated thrombosis displayed in female Wistar rats. Quail egg was separated into three (3) components (extracts)-quail egg yolk water soluble (QYWS) and fat soluble (QYFS), and albumen extract (QA) and the inorganic and organic compositions were characterized. Depranocytotic assaults was achieved by 250 mg/kg of 2-Butoxyethanol administered for 4 days, the clinical observation revealed a dark purple-red discoloration on the distal tails of the rats and therapeutic applications followed with 1000 mg/kg BWT of QYWS, QYFS and QA, and 15 mg/kg BWT of hydroxyurea. Morphological evaluation, haematological estimations and biochemical evaluations of the influence on the activities of sphingosine kinase-1, RNase, red cell carbonic anhydrase, lactate dehydrogenase, glutathione peroxidase and caspase-3, vis a vis the concentrations of sphingosine-1 phosphate, selenium and zinc (plasma and urine). In vitro anti-inflammatory influence of quail egg components were investigated against hemolysis and key enzymes of inflammation-cycloxygenase, lipoxygenase and ß-glucuronidase. The in vitro anti-inflammatory effects of QYWS, QYFS and QA were concentration dependent from 200 to 800 µg/ml against hemolysis and the key enzymes of inflammation. The characterization of inorganic and organic bioactive composition of the yolk and albumen revealed the presence of folic acid, cobalamin, pyridine, riboflavin, ascorbic acid as well as vitamins D and E, selenium, zinc, iron and calcium. These had reflected in the attenuation of the induced hemolytic and disseminated thrombosis by regulations of enzymes linked to the infarction, apoptosis and oxidative stress characterized in sickle cell index.

Microfluidic encapsulation of Xenopus laevis cell-free extracts using hydrogel photolithography.

Cell-free extract derived from the eggs of the African clawed frog Xenopus laevis is a well-established model system that has been used historically in bulk aliquots. Here, we describe a microfluidic approach for isolating discrete, biologically relevant volumes of cell-free extract, with more expansive and precise control of extract shape compared with extract-oil emulsions. This approach is useful for investigating the mechanics of intracellular processes affected by cell geometry or cytoplasmic volume, including organelle scaling and positioning mechanisms. For complete details on the use and execution of this protocol, please refer to Geisterfer et al. (2020).

Bioactive potential of endophytic fungus Chaetomium globosum and GC-MS analysis of its responsible components.

The recent exploration of various medicinal plants for bioactive potential has led to the growing interest to explore their endophytes for such bioactive potential which may turn out to be better option than the plants. In the present study, Chaetomium globosum, an endophytic fungus isolated from Moringa oleifera Lam has been explored for its various biological activities. The chloroformic extract of C. globosum showed good antimutagenicity against the reactive carcinogenic mutagen, 2-aminofluorene (2-AF) in Ames test. The antiproliferative activity against various cell lines such as HCT-15, HeLa and U87-MG was found to be dose dependent and the viability reduced to 9.26%, 15.7% and 16.3%, respectively. Further, the chloroformic fungal extract was investigated for free radical scavenging activity using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethyl-benzthiazolin-6-sulfonic acid) assay which showed the IC50 value of 45.16 µg/ml and 50.55 µg/ml, respectively. The fungal extract also showed good ferric reducing power. Total phenolic and flavonoid content was found to be in linear relationship with the antioxidant potential of the fungal extract. High performance liquid chromatography showed the presence of phenolics which may help to combat the free radicals. The presence of various bioactive compounds was analysed by GC-MS which endorsed Chaetomium globosum to be a promising candidate for drug development.

A newly isolated strain of Halomonas sp. (HA1) exerts anticancer potential via induction of apoptosis and G2/M arrest in hepatocellular carcinoma (HepG2) cell line.

Marine bacterial strains are of great interest for their ability to produce secondary metabolites with anticancer potentials. Isolation, identification, characterization and anticancer activities of isolated bacteria from El-Hamra Lake, Wadi El-Natrun (Egypt) were the objectives of this study. The isolated bacteria were identified as a moderately halophilic alkaliphilic strain. Ethyl acetate extraction was performed and identified by liquid chromatography-mass spectrophotometry (LC-MS-MS) and nuclear magnetic resonance analysis (NMR). Cytotoxicity of the extract was assessed on the HepG2 cell line and normal human peripheral lymphocytes (HPBL) in vitro. Halomonas sp. HA1 extract analyses revealed anticancer potential. Many compounds have been identified including cyclo-(Leu-Leu), cyclo-(Pro-Phe), C17-sphinganine, hexanedioic acid, bis (2-ethylhexyl) ester, surfactin C14 and C15. The extract exhibited an IC50 of 68 ± 1.8 µg/mL and caused marked morphological changes in treated HepG2 cells. For mechanistic anticancer evaluation, 20 and 40 µg/mL of bacterial extract were examined. The up-regulation of apoptosis-related genes' expression, P53, CASP-3, and BAX/BCL-2 at mRNA and protein levels proved the involvement of P53-dependant mitochondrial apoptotic pathway. The anti-proliferative properties were confirmed by significant G2/M cell cycle arrest and PCNA down-regulation in the treated cells. Low cytotoxicity was observed in HPBL compared to HepG2 cells. In conclusion, results suggest that the apoptotic and anti-proliferative effects of Halomonas sp. HA1 extract on HepG2 cells can provide it as a candidate for future pharmaceutical industries.

Analysis of Base Excision and Single-Strand Break Repair Activities in Trypanosomatid Extracts.

Cellular DNA is inherently unstable, subject to both spontaneous hydrolysis and attack by a range of exogenous and endogenous chemicals as well as physical agents such as ionizing and ultraviolet radiation. For parasitic protists, where an inoculum of infectious parasites is typically small and natural infections are often chronic with low parasitemia, they are also vulnerable to DNA damaging agents arising from innate immune defenses. The majority of DNA damage consists of relatively minor changes to the primary structure of the DNA, such as base deamination, oxidation, or alkylation and scission of the phosphodiester backbone. Yet these small changes can have serious consequences, often being mutagenic or cytotoxic. Cells have therefore evolved efficient mechanisms to repair such damage, with base excision and single strand break repair playing the primary role here. In this chapter we describe a method for analyzing the activity from cell extracts of various enzymes involved in the base excision and single strand break repair pathways of trypanosomatid parasites.

Metabolic Control Analysis for Drug Target Prioritization in Trypanosomatids.

To validate therapeutic targets in metabolic pathways of trypanosomatids, the criterion of enzyme essentiality determined by gene knockout or knockdown is usually being applied. Since, it is often found that most of the enzymes/proteins analyzed are essential, additional criteria have to be implemented for drug target prioritization. Metabolic control analysis (MCA), often in conjunction with kinetic pathway modeling, offers such possibility for prioritization. MCA is a theoretical and experimental approach to analyze how metabolic pathways are controlled. It involves strategies to perform quantitative analyses to determine the degree in which an enzyme controls a pathway flux, a value called flux control coefficient ([Formula: see text]). By determining the [Formula: see text] of individual steps in a metabolic pathway, the distribution of control of the pathway is established, that is, the identification of the main flux-controlling steps. Therefore, MCA can help in ranking pathway enzymes as drug targets from a metabolic perspective. In this chapter, three approaches to determine [Formula: see text] are reviewed: (1) In vitro pathway reconstitution, (2) manipulation of enzyme activities within parasites, and (3) in silico kinetic modeling of the metabolic pathway. To perform these methods, accurate experimental data of enzyme activities, metabolite concentrations and pathway fluxes are necessary. The methodology is illustrated with the example of trypanothione metabolism of Trypanosoma cruzi and protocols to determine such experimental data for this metabolic process are also described. However, the MCA strategy can be applied to any metabolic pathway in the parasite and general directions to perform it are provided in this chapter.

Effect of alerce (Fitzroya cupressoides) cell culture extract on wound healing repair in a human keratinocyte cell line.

BACKGROUND: Fitzroya cupressoides, commonly known as alerce, is an endemic conifer unique to southern South America. Alerce wood is renowned for its durability and resistance to biological degradation due to the presence of a particular class of secondary metabolite. Alerce extracts have been used in traditional medicine for different skin lesion treatments. AIMS: To develop a cell culture system to produce alerce extract and evaluate its cytotoxicity and effects on in vitro wound healing. METHODS: Cell cultures and aqueous extracts were prepared from alerce needles. Cytotoxicity was evaluated in keratinocytes (HaCaT line) and melanocites (C32 line) using the XTT assay. Wound healing was assayed with the scratch test in HaCaT cells, using mitomycin C to evaluate the role of cell division in the wound closure. RESULTS: Alerce cell culture extract has a significant effect on wound healing at different concentrations. No positive effects on the viability of normal and cancerous skin cells were observed. These results suggest that alerce extracts stimulate cell division in human skin epidermal cells in the context of wound repair. CONCLUSIONS: Bioactive compounds extracted from alerce cell cultures show promise as ingredients in dermocosmetic formulations, but further clinical studies are required to support these findings at the tissue level.

Mycelium Polysaccharides from Termitomyces albuminosus Attenuate CCl4-Induced Chronic Liver Injury Via Inhibiting TGFß1/Smad3 and NF-κB Signal Pathways.

A major fraction (MPT-W), eluted by deionized water, was extracted from mycelium polysaccharides of Termitomyces albuminosus (MPT), and its antioxidant, anti-fibrosis, and anti-inflammatory activities in CCl4-induced chronic liver injury mice, as well as preliminary characterizations, were evaluated. The results showed that MPT-W was a polysaccharide of α- and ß-configurations containing xylose (Xyl), fucose (Fuc), mannose (Man), galactose (Gal), and glucose (Glc) with a molar ratio of 0.29:8.67:37.89:35.98:16.60 by gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared (FT-IR) spectroscopy. Its molecular weight (Mw), obtained by high-performance gel permeation chromatography (HPGPC), was 1.30 × 105 Da. The antioxidant assays in vitro showed that MPT-W displayed scavenging free-radical abilities. Based on the data of in vivo experiments, MPT-W could inhibit TGFß1/Smad3 and NF-κB pathways; decrease the level and activity of cytochrome P4502E1 (CYP2E1), malonaldehyde (MDA) and serum enzyme; activate the HO-1/Nrf2 pathway; and increase antioxidant enzymes to protect the liver in CCl4-induced chronic liver injury mice. Therefore, MPT-W could be a potentially natural and functional resource contributing to antioxidant, hepatoprotective, and anti-inflammatory effects with potential health benefits.

Site- and Structure-Specific Quantitative N-Glycoproteomics Using RPLC-pentaHILIC Separation and the Intact N-Glycopeptide Search Engine GPSeeker.

Site- and structure-specific quantitative N-glycoproteomics characterization of differentially expressed N-glycosylation at the intact N-glycopeptide level with distinct chromatographic separation and structure-specific fragment ions has become possible with the recent development of RPLC-pentaHILIC 2DLC separation and use of the intact N-glycopeptide search engine GPSeeker. Here we provide a detailed protocol for this GPSeeker-centered structure-specific isotopic-labeling quantitative N-glycoproteomics pipeline. The protocols include sample preparation of a 1:1 mixture of light (-CH3 )2 and heavy (-13 CD2 H)2 dimethylated intact N-glycopeptides from LO2 and HepG2 cells, RPLC-pentaHILIC 2DLC separation of the mixture, intact N-glycopeptide database search and identification using GPSeeker, and quantitation of differentially expressed intact N-glycopeptides using the quantitation module GPSeekerQuan. © 2019 by John Wiley & Sons, Inc.

Assessment of seven protocols to prepare Cutibacterium acnes bacterial lysates to measure its immunogenic potential and review of the literature.

Seven protocols were tested to prepare cell wall extracts from live Cutibacterium acnes. Different parameters were modified: thawing/freezing and sonication/freezing cycles, to impact on mechanical degradation of the bacteria. Finally, the immunogenic potential of the extracts generated was evaluated by measuring IL-8 releases using an in vitro skin explants system. The aim of this article was to compare the existing protocols from the scientific literature, and also propose a standardized method developed in our facilities.

Filopodia-Like Structure Formation from Xenopus Egg Extracts.

The actin cytoskeleton comprises many different architectures of filaments, including branched networks, parallel bundles and antiparallel fibers. A current challenge is to elucidate how the diverse array of actin regulators, which controls the growth, assembly and turnover of actin filaments, is used to orchestrate cytoskeletal organization and in turn cell shape and movement. Long observed to assemble at cell membranes, actin in Xenopus egg extracts recapitulates membrane-triggered assembly at specific lipid and membrane environments. The use of Xenopus egg extracts has contributed greatly to identifying how constitutively autoinhibited regulatory pathways are activated, which converge on activation of the Arp2/3 complex. Here we describe a protocol for making parallel actin bundles using Xenopus egg extracts from supernatants prepared by high-speed centrifugation. These filopodia-like actin bundles emanate from clusters of actin regulators that self-assemble at phosphatidylinositol (4,5)-bisphosphate-containing supported lipid bilayers. Forming a plasma membrane-mimicking bilayer on glass allows easy, optimizable, high signal-to-noise microscopy at high spatial and temporal resolution. The use of Xenopus egg extracts yields large quantities of active material that can be flexibly tailored to address specific questions, for example, by dilution, addition of fluorescent proteins, antibodies or protein fragments, immunodepletion, addition of small molecule inhibitors, or biochemical fractionation.

Influence of Micropatterned Grill Lines on Entamoeba histolytica Trophozoites Morphology and Migration.

Entamoeba histolytica, the causal agent of human amoebiasis, has two morphologically different phases: a resistant cyst and a trophozoite responsible for the invasion of the host tissues such as the colonic mucosa and the intestinal epithelium. During in vitro migration, trophozoites usually produce protuberances such as pseudopods and rarely filopodia, structures that have been observed in the interaction of trophozoites with human colonic epithelial tissue. To study the different membrane projections produced by the trophozoites, including pseudopods, filopodia, uropods, blebs, and others, we designed an induction system using erythrocyte extract or fibronectin (FN) in micropatterned grill lines (each micro-line containing multiple micro-portions of FN or erythrocyte extract) on which the trophozoites were placed in culture for migration assays. Using light, confocal, and scanning electron microscopy, we established that E. histolytica trophozoites frequently produce short and long filopodia, large retractile uropods in the rear, pseudopods, blebs, and others structures, also showing continuous migration periods. The present study provides a simple migration method to induce trophozoites to generate abundant membrane protrusion structures that are rarely obtained in normal or induced cultures, such as long filopodia; this method will allow a-better understanding of the interactions of trophozoites with FN and cell debris. E. histolytica trophozoites motility plays an important role in invasive amoebiasis. It has been proposed that both physical forces and chemical signals are involved in the trophozoite motility and migration. However, the in vivo molecules that drive the chemotactic migration remain to be determined. We propose the present assay to study host molecules that guide chemotactic behavior because the method is highly reproducible, and a live image of cell movement and migration can be quantified.

Siamese Crocodile White Blood Cell Extract Inhibits Cell Proliferation and Promotes Autophagy in Multiple Cancer Cell Lines.

Cancer represents one of the most significant threats to human health on a global scale. Hence, the development of effective cancer prevention strategies, as well as the discovery of novel therapeutic agents against cancer, is urgently required. In light of this challenge, this research aimed to evaluate the effects of several potent bioactive peptides and proteins contained in crocodile white blood cell extract (cWBC) against LU-1, LNCaP, PC-3, MCF-7, and CaCo-2 cancer cell lines. The results demonstrate that 25, 50, 100, and 200 µg/ml cWBC exhibits a strong cytotoxic effect against all investigated cell lines (IC50 70.34-101.0 µg/ml), while showing no signs of cytotoxicity towards noncancerous Vero and HaCaT cells. Specifically, cWBC treatment caused a significant reduction in the cancerous cells' colony forming ability. A remarkable suppression of cancerous cell migration was observed after treatment with cWBC, indicating potent antimetastatic properties. The mechanism involved in the cancer cell cytotoxicity of cWBC may be related to apoptosis induction, as evidenced by typical apoptotic morphology features. Moreover, certain cWBC concentrations induced significant overproduction of ROS and significantly inhibited the S-G2/M transition in the cancer cell. The molecular mechanisms of cWBC in apoptosis induction were to decrease Bcl-2 and XIAP expression levels and increase the expression levels of caspase-3, caspase-8, and p53. These led to a decrease in the expression level of the cell cycle-associated gene cyclin-B1 and the arrest of cell population growth. Consequently, these findings demonstrate the prospect of the use of cWBC for cancer therapy.

Antioxidative Activity of Methanolic and Water Extracts from the Hyperthermophilic Archaeon Aeropyrum pernix K1.

The hyperthermophilic archaeon Aeropyrum pernix has adapted to optimal growth under high temperatures in saline environments and under oxidizing conditions. In the present study, we focused on the antioxidative activity of proteins from A. pernix K1. Following high temperature methanol and water extractions of the protein from the biomass of A. pernix K1, the total sulphydryl groups and radical scavenging activities were investigated. The total protein in the methanolic extract was 36% lower and showed 10% fewer sulphydryl groups than that from the water extract. However, the radical scavenging activity of the water extract was four-fold greater than for the methanolic extract. The proteins of both of these extracts were separated by two-dimensional electrophoresis, and selected proteins were identified using mass spectrometry. The majority of these identified proteins were intracellular proteins, such as those involved in oxidative stress responses and osmotic stress responses, and proteins with hydrolase and dehydrogenase activities. These proteins are also common to most organisms, and included putative uncharacterized proteins.

Chaga ( Inonotus obliquus), a Future Potential Medicinal Fungus in Oncology? A Chemical Study and a Comparison of the Cytotoxicity Against Human Lung Adenocarcinoma Cells (A549) and Human Bronchial Epithelial Cells (BEAS-2B).

BACKGROUND: Inonotus obliquus, also known as Chaga, is a parasitic fungus growing on birches and used in traditional medicine (especially by Khanty people) to treat various health problems. In this study, we aimed to quantify the 3 metabolites frequently cited in literature, that is, betulin, betulinic acid, and inotodiol in the Chaga recently discovered in forests located in Normandy (France), and to compare their concentrations with Ukrainian and Canadian Chaga. This study also explores the cytotoxicity of the French Chaga against cancer-derived cells and transformed cells. METHODS: A quantification method by HPLC-MS-MS (high-performance liquid chromatography-tandem mass spectrometry) of betulin, betulinic acid, and inotodiol was developed to study the French Chaga and compare the concentration of these metabolites with extracts provided from Chaga growing in Canada and Ukraine. This method was also used to identify and quantify those 3 compounds in other traditional preparations of Chaga (aqueous extract, infusion, and decoction). Among these preparations, the aqueous extract that contains betulin, betulinic acid, and inotodiol was chosen to evaluate and compare its cytotoxic activity toward human lung adenocarcinoma cells (A549 line) and human bronchial epithelial cells (BEAS-2B line). RESULTS: French Chaga contains betulin and betulinic acid at higher levels than in other Chaga, whereas the concentration of inotodiol is greater in the Canadian Chaga. Moreover, the results highlighted a cytotoxic activity of the Chaga's aqueous extract after 48 and 72 hours of exposure with a higher effect on cancer-derived cells A549 than on normal transformed cells BEAS-2B ( P = 0.025 after 48 hours of exposure and P = 0.004 after 72 hours of exposure).

Protective Effect of Fucoidan Extract from Ecklonia cava on Hydrogen Peroxide-Induced Neurotoxicity.

We evaluated the antioxidant activity and neuronal cell-protective effect of fucoidan extract from Ecklonia cava (FEC) on hydrogen peroxide (H2O2)-induced cytotoxicity in PC-12 and MC-IXC cells to assess its protective effect against oxidative stress. Antioxidant activities were examined using the ABTS radical scavenging activity and malondialdehyde-inhibitory effect, and the results showed that FEC had significant antioxidant activity. Intracellular ROS contents and neuronal cell viability were investigated using the DCF-DA assay and MTT reduction assay. FEC also showed remarkable neuronal cell-protective effect compared with vitamin C as a positive control for both H2O2-treated PC-12 and MC-IXC cells. Based on the neuronal cell-protective effects, mitochondrial function was analyzed in PC-12 cells, and FEC significantly restored mitochondrial damage by increasing the mitochondrial membrane potential (Δψm) and ATP levels and regulating mitochondrial-mediated proteins (p-AMPK and BAX). Finally, the inhibitory effects against acetylcholinesterase (AChE), which is a critical hydrolyzing enzyme of the neurotransmitter acetylcholine in the cholinergic system, were investigated (IC50 value = 1.3 mg/ml) and showed a mixed (competitive and noncompetitive) pattern of inhibition. Our findings suggest that FEC may be used as a potential material for alleviating oxidative stress-induced neuronal damage by regulating mitochondrial function and AChE inhibition.

A highly efficient method for extracting next-generation sequencing quality RNA from adipose tissue of recalcitrant animal species.

The next-generation sequencing (NGS) based RNA sequencing (RNA-Seq) and transcriptome profiling offers an opportunity to unveil complex biological processes. Successful RNA-Seq and transcriptome profiling requires a large amount of high-quality RNA. However, NGS-quality RNA isolation is extremely difficult from recalcitrant adipose tissue (AT) with high lipid content and low cell numbers. Further, the amount and biochemical composition of AT lipid varies depending upon the animal species which can pose different degree of resistance to RNA extraction. Currently available approaches may work effectively in one species but can be almost unproductive in another species. Herein, we report a two step protocol for the extraction of NGS quality RNA from AT across a broad range of animal species.

Toxoplasma gondii tachyzoite-extract acts as a potent immunomodulator against allergic sensitization and airway inflammation.

Epidemiological and experimental studies have shown an inverse relationship between infections with certain parasites and a reduced incidence of allergic diseases. We and others have shown that infection with Toxoplasma gondii prevents the development of allergy in mice. To establish whether this beneficial effect could be recapitulated by soluble products of this parasite, we tested an extract derived from T. gondii tachyzoites. Immunization of BALB/c mice with tachyzoites lysate antigen (TLA) elicited mixed Th1/Th2 responses. When TLA was applied together with the sensitizing ovalbumin (OVA), the development of allergic airway inflammation was reduced, with decreased airway hyperresponsiveness associated with reduced peribronchial and perivascular cellular infiltration, reduced production of OVA-specific Th2 cytokines in lungs and spleens and reduced levels of serum OVA-specific IgG1 as well as IgE-dependent basophil degranulation. Of note, TLA retained its immunomodulatory properties, inducing high levels of IL-6, TNFα, IL-10 and IL-12p70 in bone marrow-derived dendritic cells after heat-inactivation or proteinase K-treatment for disruption of proteins, but not after sodium metaperiodate-treatment that degrades carbohydrate structures, suggesting that carbohydrates may play a role in immunomodulatory properties of TLA. Here we show that extracts derived from parasites may replicate the benefits of parasitic infection, offering new therapies for immune-mediated disorders.

Assay for ß-Galactosidase in Extracts of Mammalian Cells.

This protocol contains a method for the detection of ß-galactosidase expressed from reporter vectors that have been transfected into mammalian cells. The assay is both simple and rapid and can be performed using a visible light spectrophotometer.