scholarly journals In Vitro Cell Death Discrimination and Screening Method by Simple and Cost-Effective Viability Analysis

2017 ◽  
Vol 41 (3) ◽  
pp. 1011-1019 ◽  
Author(s):  
Katharina Helm ◽  
Marlena Beyreis ◽  
Christian Mayr ◽  
Markus Ritter ◽  
Martin Jakab ◽  
...  
Keyword(s):  
Metabolic Activity ◽  
Screening Method ◽  
Cellular Metabolism ◽  
Cost Effective ◽  
Post Treatment ◽  
Time Points ◽  
Viability Analysis ◽  
Starting Point ◽  
Apoptosis And Necrosis

Background/Aims: For in vitro cytotoxicity testing, discrimination of apoptosis and necrosis represents valuable information. Viability analysis performed at two different time points post treatment could serve such a purpose because the dynamics of metabolic activity of apoptotic and necrotic cells is different, i.e. a more rapid decline of cellular metabolism during necrosis whereas cellular metabolism is maintained during the entire execution phase of apoptosis. This study describes a straightforward approach to distinguish apoptosis and necrosis. Methods: A431 human epidermoid carcinoma cells were treated with different concentrations/doses of actinomycin D (Act-D), 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), Ro 31-8220, H2O2 and photodynamic treatment (PDT). The resazurin viability signal was recorded at 2 and 24 hrs post treatment. Apoptosis and necrosis were verified by measuring caspase 3/7 and membrane integrity. Results: Calculation of the difference curve between the 2 and 24 hrs resazurin signals yields the following information: a positive difference signal indicates apoptosis (i.e. high metabolic activity at early time points and low signal at 24 hrs post treatment) while an early reduction of the viability signal indicates necrosis. For all treatments, this dose-dependent sequence of cellular responses could be confirmed by independent assays. Conclusion: Simple and cost-effective viability analysis provides reliable information about the dose ranges of a cytotoxic agent where apoptosis or necrosis occurs. This may serve as a starting point for further in-depth characterisation of cytotoxic treatments.

scholarly journals Identification of Potential Kinase Inhibitors within the PI3K/AKT Pathway of Leishmania Species

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1037
Author(s):  
Rodrigo Ochoa ◽  
Amaya Ortega-Pajares ◽  
Florencia A. Castello ◽  
Federico Serral ◽  
Darío Fernández Do Porto ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Screening Method ◽  
Molecular Targets ◽  
Leishmania Species ◽  
Akt Pathway ◽  
Protein Protein Interaction ◽  
Starting Point ◽  
Binding Pockets ◽  
Protein Protein Interaction Networks

Leishmaniasis is a public health disease that requires the development of more effective treatments and the identification of novel molecular targets. Since blocking the PI3K/AKT pathway has been successfully studied as an effective anticancer strategy for decades, we examined whether the same approach would also be feasible in Leishmania due to their high amount and diverse set of annotated proteins. Here, we used a best reciprocal hits protocol to identify potential protein kinase homologues in an annotated human PI3K/AKT pathway. We calculated their ligandibility based on available bioactivity data of the reported homologues and modelled their 3D structures to estimate the druggability of their binding pockets. The models were used to run a virtual screening method with molecular docking. We found and studied five protein kinases in five different Leishmania species, which are AKT, CDK, AMPK, mTOR and GSK3 homologues from the studied pathways. The compounds found for different enzymes and species were analysed and suggested as starting point scaffolds for the design of inhibitors. We studied the kinases’ participation in protein–protein interaction networks, and the potential deleterious effects, if inhibited, were supported with the literature. In the case of Leishmania GSK3, an inhibitor of its human counterpart, prioritized by our method, was validated in vitro to test its anti-Leishmania activity and indirectly infer the presence of the enzyme in the parasite. The analysis contributes to improving the knowledge about the presence of similar signalling pathways in Leishmania, as well as the discovery of compounds acting against any of these kinases as potential molecular targets in the parasite.

scholarly journals Dietary Intervention Induced Distinct Repercussions in Response to the Individual Gut Microbiota as Demonstrated by the In Vitro Fecal Fermentation of Beef

2021 ◽  
Vol 11 (15) ◽  
pp. 6841
Author(s):  
Vineet Singh ◽  
Youn-Chul Ryu ◽  
Tatsuya Unno
Keyword(s):  
Gut Microbiota ◽  
Metabolic Activity ◽  
Dietary Intervention ◽  
Cost Effective ◽  
Dietary Interventions ◽  
Fecal Microbiome ◽  
Fundamental Information ◽  
The Individual ◽  
The Impact

Animals and humans have very different gut microbiota, and the human microbiota is unique to each individual. For these reasons, it is difficult to find a diet that provides all the nutrients according to individual requirements. In this study, we investigated the possibility of using simple in vitro fecal fermentation of digested food to evaluate fundamental differences in the gut metabolism of individuals with different microbiomes in response to specific dietary interventions. We fermented beef using six human fecal microbiotas, analyzed shifts in these microbiomes, and quantified short-chain fatty acid (SCFA) production in each system. Our results demonstrate that each microbiome responds with a unique shift in composition, SCFA production, and metabolic activity following 90 min of fecal fermentation of beef. Differentially abundant genera and metabolic activities varied among subjects. Only two subjects’ fecal microbiome showed no significant changes in their metabolic activity, while the other subjects’ microbial metagenome showed anywhere between 17 and 60 differences in their metabolism, including several changes associated with heart disease (i.e., depletion of oleate and palmitoleate biosynthesis). This study revealed the varying responses of each microbiome when exposed to digested beef, suggesting that this method could provide fundamental information in understanding personal nutrient requirements and the impact of changes in the individual gut microbiota on human health. Although further studies using larger study populations are required, this study describes a simple and cost-effective protocol for evaluating the interactions between specific dietary interventions and individual gut microbiota differences.

scholarly journals Activation of Human Osteoblasts via Different Bovine Bone Substitute Materials With and Without Injectable Platelet Rich Fibrin in vitro

2021 ◽  
Vol 9 ◽  
Author(s):  
Solomiya Kyyak ◽  
Sebastian Blatt ◽  
Eik Schiegnitz ◽  
Diana Heimes ◽  
Henning Staedt ◽  
...  
Keyword(s):  
Metabolic Activity ◽  
Bone Substitute ◽  
Bone Morphogenetic Protein 2 ◽  
Bovine Bone ◽  
Human Osteoblasts ◽  
Time Points ◽  
Significant Difference ◽  
Bone Substitute Materials ◽  
Substitute Materials

IntroductionThe aim of the in vitro study was to compare the effect of four bovine bone substitute materials (XBSM) with and without injectable platelet-reach fibrin for viability and metabolic activity of human osteoblasts (HOB) as well as expression of alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP-2), and osteonectin (OCN).Materials and MethodsCerabone® (CB), Bio-Oss® (BO), Creos Xenogain® (CX) and MinerOss® X (MO) ± i-PRF were incubated with HOB. At day 3, 7, and 10, cell viability and metabolic activity as well as expression of ALP, OCN, and BMP-2, was examined.ResultsFor non-i-PRF groups, the highest values concerning viability were seen for CB at all time points. Pre-treatment with i-PRF increased viability in all groups with the highest values for CB-i-PRF after 3 and 7 and for CX-i-PRF after 10 days. For metabolic activity, the highest rate among non-i-PRF groups was seen for MO at day 3 and for CB at day 7 and 10. Here, i-PRF groups showed higher values than non-i-PRF groups (highest values: CB + i-PRF) at all time points. There was no difference in ALP-expression between groups. For OCN expression in non-i-PRF groups, CB showed the highest values after day 3, CX after day 7 and 10. Among i-PRF-groups, the highest values were seen for CX + i-PRF. At day 3, the highest BMP-2 expression was observed for CX. Here, for i-PRF groups, the highest increase was seen for CX + i-PRF at day 3. At day 7 and 10, there was no significant difference among groups.ConclusionXBSM sintered under high temperature showed increased HOB viability and metabolic activity through the whole period when compared to XBSM manufactured at lower temperatures. Overall, the combination of XBSM with i-PRF improved all cellular parameters, ALP and BMP-2 expression at earlier stages as well as OCN expression at later stages.

scholarly journals Towards ‘Ancientbiotics’ for Biofilms: How can we bring traditional medicinal remedies out of treatise and into contemporary science?

2020 ◽  
Author(s):  
Madhusoodhanan Vandana ◽  
Snehal Kadam ◽  
Anuradha Bandgar ◽  
Karishma S Kaushik
Keyword(s):  
Metabolic Activity ◽  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Cynodon Dactylon ◽  
Test Phase ◽  
Scientific Practices ◽  
Ancient Medicine ◽  
Traditional Remedies ◽  
Starting Point

AbstractIntroductionTraditional medicinal remedies hold vast potential as novel antimicrobial agents, particularly for recalcitrant infection states such as biofilms. To explore their potential, it is important to bring these remedies out of ancient treatise and into present-day scientific evaluation. For traditional medical practices, this ‘development pipeline’ starts with probing treatise for potential remedies and testing them for anti-biofilm effects, or the ‘treatise to test’ phase.AimThe aim of this work is to present a primer for developing ‘ancientbiotics’ against biofilms, that focuses on the ‘treatise to test’ phase of the pipeline. Based on our approach and results, we provide insights into the considerations and challenges relevant to evaluating traditional remedies as anti-biofilm agents.MethodologyWe identified and reconstituted plant-based medicinal formulations from historical treatises of Indian traditional medicine, and analyzed their efficacy using widely-employed microtiter based assays, that constitute the cornerstone of biofilm studies. Measuring biomass and metabolic activity, we evaluated effects on biofilm formation and eradication of pre-formed biofilms, of Pseudomonas aeruginosa and Staphylococcus aureus.ResultsBased on recipes and preparation practices across several texts, and with modifications to ensure compatibility with modern scientific practices, three plant-based traditional remedies were identified and formulated in sesame oil (Bryophyllum pinnatum, Cynodon dactylon, and Ocimum tenuiflorum). We observed differential effects on biomass and metabolic activity on the biofilm formation and eradication of P. aeruginosa and S. aureus; highlighting the value of the microtiter-based assays as an initial screening tool for traditional remedies.ConclusionThrough this study, we provide insights into considerations relevant to the ‘treatise to test’ phase of the ‘ancientbiotics’ pipeline, such as identifying ancient remedies, reconstituting them with present-day modifications, and using in vitro assay formats for evaluation. The learnings in this primer will be relevant to both contemporary scientists and practitioners of ancient medicine, and will serve as a starting point for future studies exploring anti-biofilm approaches at the interface of historical and modern medicine.

scholarly journals Preclinical therapeutics ex ovo quail eggs as a biomimetic automation-ready xenograft platform

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samuel V. Rasmussen ◽  
Noah E. Berlow ◽  
Lisa Hudson Price ◽  
Atiya Mansoor ◽  
Stefano Cairo ◽  
...  
Keyword(s):  
Drug Toxicity ◽  
Screening Method ◽  
Chorioallantoic Membrane ◽  
Drug Efficacy ◽  
Cost Effective ◽  
Coturnix Japonica ◽  
In Vivo Studies ◽  
Anti Cancer

AbstractPreclinical cancer research ranges from in vitro studies that are inexpensive and not necessarily reflective of the tumor microenvironment to mouse studies that are better models but prohibitively expensive at scale. Chorioallantoic membrane (CAM) assays utilizing Japanese quail (Coturnix japonica) are a cost-effective screening method to precede and minimize the scope of murine studies for anti-cancer efficacy and drug toxicity. To increase the throughput of CAM assays we have built and optimized an 11-day platform for processing up to 200 quail eggs per screening to evaluate drug efficacy and drug toxicity caused by a therapeutic. We demonstrate ex ovo concordance with murine in vivo studies, even when the in vitro and in vivo studies diverge, suggesting a role for this quail shell-free CAM xenograft assay in the validation of new anti-cancer agents.

An integrated screening method for evaluating the pulmonary toxicity of inhaled particulates: Role of microscopic techniques in assessing pulmonary macrophage clearance functions

1990 ◽  
Vol 48 (3) ◽  
pp. 826-827
Author(s):  
David B. Warheit ◽  
Lena Achinko ◽  
Mark A. Hartsky
Keyword(s):  
Bronchoalveolar Lavage ◽  
Pulmonary Toxicity ◽  
Screening Method ◽  
Pulmonary Response ◽  
Inhaled Particles ◽  
Cytochemical Analysis ◽  
Pulmonary Macrophages ◽  
Integrated Screening

There is a great need for the development of a rapid and reliable bioassay to evaluate the pulmonary toxicity of inhaled particles. A number of methods have been proposed, including lung clearance studies, bronchoalveolar lavage analysis, and in vitro cytotoxicity tests. These methods are often limited in scope inasmuch as they measure only one dimension of the pulmonary response to inhaled, instilled or incubated dusts. Accordingly, a comprehensive approach to lung toxicity studies has been developed.To validate the method, rats were exposed for 6 hours or 3 days to various concentrations of either aerosolized alpha quartz silica (Si) or carbonyl iron (CI) particles. Cells and fluids from groups of sham and dust-exposed animals were recovered by bronchoalveolar lavage (BAL). Alkaline phosphatase, LDH and protein values were measured in BAL fluids at several time points postexposure. Cells were counted and evaluated for viability, as well as differential and cytochemical analysis. In addition, pulmonary macrophages (PM) were cultured and studied for morphology, chemotaxis, and phagocytosis by scanning electron microscopy.

Mechanisms involved in effects of antimicrobial peptides, bactenectins ChBac3.4, ChBac5, and mini-ChBac7.5Nb, on bacterial cells

2017 ◽  
pp. 43-50
Author(s):  
О.В. Шамова ◽  
М.С. Жаркова ◽  
П.М. Копейкин ◽  
Д.С. Орлов ◽  
Е.А. Корнева
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Innate Immunity ◽  
Infectious Diseases ◽  
Metabolic Activity ◽  
Capra Hircus ◽  
Bacterial Cells ◽  
Antibiotic Resistant ◽  
Resistant Strains

Антимикробные пептиды (АМП) системы врожденного иммунитета - соединения, играющие важную роль в патогенезе инфекционных заболеваний, так как обладают свойством инактивировать широкий спектр патогенных бактерий, обеспечивая противомикробную защиту живых организмов. В настоящее время АМП рассматриваются как потенциальные соединения-корректоры инфекционной патологии, вызываемой антибиотикорезистентными бактериями (АБР). Цель данной работы состояла в изученим механизмов антибактериального действия трех пептидов, принадлежащих к семейству бактенецинов - ChBac3.4, ChBac5 и mini-ChBac7.5Nb. Эти химически синтезированные пептиды являются аналогами природных пролин-богатых АМП, обнаруженных в лейкоцитах домашней козы Capra hircus и проявляющих высокую антимикробную активность, в том числе и в отношении грамотрицательных АБР. Методы. Минимальные ингибирующие и минимальные бактерицидные концентрации пептидов (МИК и МБК) определяли методом серийных разведений в жидкой питательной среде с последующим высевом на плотную питательную среду. Эффекты пептидов на проницаемость цитоплазматической мембраны бактерий для хромогенного маркера исследовали с использованием генетически модифицированного штамма Escherichia coli ML35p. Действие бактенецинов на метаболическую активность бактерий изучали с применением маркера резазурина. Результаты. Показано, что все исследованные пептиды проявляют высокую антимикробную активность в отношении Escherichia coli ML35p и антибиотикоустойчивых штаммов Escherichia coli ESBL и Acinetobacter baumannii in vitro, но их действие на бактериальные клетки разное. Использован комплекс методик, позволяющих наблюдать в режиме реального времени динамику действия бактенецинов в различных концентрациях (включая их МИК и МБК) на барьерную функцию цитоплазматической мембраны и на интенсивность метаболизма бактериальных клеток, что дало возможность выявить различия в характере воздействия бактенецинов, отличающихся по структуре молекулы, на исследуемые микроорганизмы. Установлено, что действие каждого из трех исследованных бактенецинов в бактерицидных концентрациях отличается по эффективности нарушения целостности бактериальных мембран и в скорости подавления метаболизма клеток. Заключение. Полученная информация дополнит существующие фундаментальные представления о механизмах действия пролин-богатых пептидов врожденного иммунитета, а также послужит основой для биотехнологических исследований, направленных на разработку на базе этих соединений новых антибиотических препаратов для коррекции инфекционных заболеваний, вызываемых АБР и являющимися причинами тяжелых внутрибольничных инфекций. Antimicrobial peptides (AMPs) of the innate immunity are compounds that play an important role in pathogenesis of infectious diseases due to their ability to inactivate a broad array of pathogenic bacteria, thereby providing anti-microbial host defense. AMPs are currently considered promising compounds for treatment of infectious diseases caused by antibiotic-resistant bacteria. The aim of this study was to investigate molecular mechanisms of the antibacterial action of three peptides from the bactenecin family, ChBac3.4, ChBac5, and mini-ChBac7.5Nb. These chemically synthesized peptides are analogues of natural proline-rich AMPs previously discovered by the authors of the present study in leukocytes of the domestic goat, Capra hircus. These peptides exhibit a high antimicrobial activity, in particular, against antibiotic-resistant gram-negative bacteria. Methods. Minimum inhibitory and minimum bactericidal concentrations of the peptides (MIC and MBC) were determined using the broth microdilution assay followed by subculturing on agar plates. Effects of the AMPs on bacterial cytoplasmic membrane permeability for a chromogenic marker were explored using a genetically modified strain, Escherichia coli ML35p. The effect of bactenecins on bacterial metabolic activity was studied using a resazurin marker. Results. All the studied peptides showed a high in vitro antimicrobial activity against Escherichia coli ML35p and antibiotic-resistant strains, Escherichia coli ESBL and Acinetobacter baumannii, but differed in features of their action on bacterial cells. The used combination of techniques allowed the real-time monitoring of effects of bactenecin at different concentrations (including their MIC and MBC) on the cell membrane barrier function and metabolic activity of bacteria. The differences in effects of these three structurally different bactenecins on the studied microorganisms implied that these peptides at bactericidal concentrations differed in their capability for disintegrating bacterial cell membranes and rate of inhibiting bacterial metabolism. Conclusion. The obtained information will supplement the existing basic concepts on mechanisms involved in effects of proline-rich peptides of the innate immunity. This information will also stimulate biotechnological research aimed at development of new antibiotics for treatment of infectious diseases, such as severe in-hospital infections, caused by antibiotic-resistant strains.

IMPACT OF SORBITOL- INDUCED OSMOTIC STRESS ON SOME BIOCHEMICAL TRAITS OF POTATO IN VITRO

2020 ◽  
Vol 51 (4) ◽  
pp. 1038-1047
Author(s):  
Mawia & et al.
Keyword(s):  
Ascorbic Acid ◽  
Osmotic Stress ◽  
Cytoplasmic Membrane ◽  
Genotypic Variation ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Culture Collection ◽  
Nutritive Medium ◽  
Starting Point

This study had as principal objective identification of osmotic-tolerant potato genotypes by using "in vitro" tissue culture and sorbitol as a stimulating agent, to induce water stress, which was added to the  culture nutritive medium in different concentration (0,50, 110, 220, 330 and 440 mM).  The starting point was represented by plantlets culture collection, belonging to eleven potato genotypes: Barcelona, Nectar, Alison, Jelly, Malice, Nazca, Toronto, Farida, Fabulla, Colomba and Spunta. Plantlets were multiplied between two internodes to obtain microcuttings (in sterile condition), which were inoculated on medium. Sorbitol-induced osmotic stress caused a significant reduction in the ascorbic acid, while the concentration of proline, H2O2 and solutes leakage increased compared with the control. Increased the proline content prevented lipid peroxidation, which played a pivotal role in the maintenance of membrane integrity under osmotic stress conditions. The extent of the cytoplasmic membrane damage depends on osmotic stress severity and the genotypic variation in the maintenance of membranes stability was highly associated with the ability of producing more amounts of osmoprotectants (proline) and the non-enzymic antioxidant ascorbic acid in response to osmotic stress level. The results showed that the genotypes Jelly, Nectar, Allison, Toronto, and Colomba are classified as highly osmotic stress tolerant genotypes, while the genotypes Nazca and Farida are classified as osmotic stress susceptible ones.

Enzyme Promiscuous Activity: How to Define it and its Evolutionary Aspects

2020 ◽  
Vol 27 (5) ◽  
pp. 400-410
Author(s):  
Valentina De Luca ◽  
Luigi Mandrich
Keyword(s):  
Gene Evolution ◽  
Sequence Divergence ◽  
High Specificity ◽  
Industrial Applications ◽  
Point Of View ◽  
Enzyme Promiscuity ◽  
Primary Activity ◽  
Starting Point ◽  
Main Activity

: Enzymes are among the most studied biological molecules because better understanding enzymes structure and activity will shed more light on their biological processes and regulation; from a biotechnological point of view there are many examples of enzymes used with the aim to obtain new products and/or to make industrial processes less invasive towards the environment. Enzymes are known for their high specificity in the recognition of a substrate but considering the particular features of an increasing number of enzymes this is not completely true, in fact, many enzymes are active on different substrates: this ability is called enzyme promiscuity. Usually, promiscuous activities have significantly lower kinetic parameters than to that of primary activity, but they have a crucial role in gene evolution. It is accepted that gene duplication followed by sequence divergence is considered a key evolutionary mechanism to generate new enzyme functions. In this way, promiscuous activities are the starting point to increase a secondary activity in the main activity and then get a new enzyme. The primary activity can be lost or reduced to a promiscuous activity. In this review we describe the differences between substrate and enzyme promiscuity, and its rule in gene evolution. From a practical point of view the knowledge of promiscuity can facilitate the in vitro progress of proteins engineering, both for biomedical and industrial applications. In particular, we report cases regarding esterases, phosphotriesterases and cytochrome P450.

Proliferative Effect of Tilapia Fish (Oreochromis niloticus) Lectin on BALB/c Mice Splenocytes

2019 ◽  
Vol 26 (12) ◽  
pp. 887-892
Author(s):  
Cynarha Daysy Cardoso da Silva ◽  
Cristiane Moutinho Lagos de Melo ◽  
Elba Verônica Matoso Maciel Carvalho ◽  
Mércia Andréa Lino da Silva ◽  
Rosiely Félix Bezerra ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Oreochromis Niloticus ◽  
Cytokine Production ◽  
Thymidine Incorporation ◽  
Con A ◽  
Cell Viability Assay ◽  
Proliferative Effect ◽  
Viability Assay ◽  
Apoptosis And Necrosis

Background: Lectins have been studied in recent years due to their immunomodulatory activities. Objective: We purified a lectin named OniL from tilapia fish (Oreochromis niloticus) and here we analyzed the cell proliferation and cytokine production in Balb/c mice splenocytes. Methods: Cells were stimulated in vitro in 24, 48, 72 hours and 6 days with different concentrations of OniL and Con A. Evaluation of cell proliferation was performed through [3H]-thymidine incorporation, cytokines were investigated using ELISA assay and cell viability assay was performed by investigation of damage through signals of apoptosis and necrosis. Results: OniL did not promote significant cell death, induced high mitogenic activity in relation to control and Con A and stimulated the cells to release high IL-2 and IL-6 cytokines. Conclusion: These findings suggest that, like Con A, OniL lectin can be used as a mitogenic agent in immunostimulatory assays.

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