Microplate-based Assays Research Articles

Multiple online-HPLC methodologies and biological properties of leaves and stem barks extracts of Chrysanthellum indicum

Chrysanthellum indicum, an annual herb with yellow flowers, is used as a functional food in Asia and Africa for health purposes. In this study, we thoroughly investigated the characterization of phytochemicals within C. indicum's ethyl acetate, water, and methanolic extracts from both its bark and leaves. This was achieved through the utilization of the Folin Ciocalteu and aluminum trichloride assays. Moreover, the antioxidant properties of these extracts were comprehensively assessed employing microplate-based assays including ABTS, DPPH, CUPRAC, FRAP, MCA, and PBD, alongside online HPLC-based assays namely ABTS, DPPH, CUPRAC, and FRAP. Furthermore, the inhibitory activity against a range of enzymes, including AChE, BChE, tyrosinase, α-amylase, and α-glucosidase, was ascertained. The tested ethyl acetate extracts from bark and leaves had higher content of total phenolic and flavonoid compared to methanol and water extracts. Additionally, the bark extract exhibited superior antioxidant properties in the in vitro antioxidant assays. Meanwhile, in the realm of online HPLC antioxidant analysis, an effective method has been discovered for swiftly determining the individual antioxidant capacity of compounds within both standard solutions and extract. Within the entirety of the samples subjected to scrutiny, rutin surfaced as the foremost antioxidant, exhibiting its prominence at wavelengths of 280 nm, 595 nm, and 450 nm. Following suit, quercetin emerged as the second most abundant antioxidant, noticeable at wavelengths of 517 nm and 734 nm, both in leaves and bark extracts. Significantly, the extracts derived from both bark and leaves demonstrated noteworthy effectiveness in inhibiting the tyrosinase enzyme. Thus, our study indicates that Chrysanthellum indicum could serve as a promising reservoir of natural bioactive compounds, making it suitable for the formulation of health-promoting applications.

Optimization of Sirius Red-Based Microplate Assay to Investigate Collagen Production In Vitro.

Tissue fibrosis is characterized by chronic fibroblast activation and consequently excessive accumulation of collagen-rich extracellular matrix. In vitro microplate-based assays are essential to investigate the underlying mechanism and the effect of antifibrotic drugs. In this study, in the absence of a gold-standard method, we optimized a simple, cost-effective, Sirius Red-based colorimetric measurement to determine the collagen production of fibroblasts grown on 96-well tissue culture plates. Based on our findings, the use of a serum-free medium is recommended to avoid aspecific signals, while ascorbate supplementation increases the collagen production of fibroblasts. The cell-associated collagens can be quantified by Sirius Red staining in acidic conditions followed by alkaline elution. Immature collagens can be precipitated from the culture medium by acidic Sirius Red solution, and after subsequent centrifugation and washing steps, their amount can be also measured. Increased attention has been paid to optimizing the assay procedure, including incubation time, temperature, and solution concentrations. The resulting assay shows high linearity and sensitivity and could serve as a useful tool in fibrosis-related basic research as well as in preclinical drug screening.

Selective Fluorescent Probes for High-Throughput Functional Diagnostics of the Human Multidrug Transporter P-Glycoprotein (ABCB1).

The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dyes to examine the functional expression and properties of the ABCB1 multidrug transporter, especially in microplate-based high-throughput flow cytometry assays. In addition, we demonstrate the applicability of the TP dyes to efficiently select and separate even a very low number of Pgp-expressing intact cells.

Antibacterial Property and Metagenomic Analysis of Milk Kefir.

Milk kefir fermentation has been used in households for generations. Consumption of milk kefir has been associated with various health benefits, presumably from the probiotics of yeast and bacteria that make up the kefir grains. In addition, many of the microbes are known to produce novel antimicrobial compounds that can be used for other applications. The microbes living inside kefir grains differ significantly depending on geographical location and production methods. In this study, we aimed to use metagenomic analysis of fermented milk by using three different kefir grains (kefir 1, kefir 2, and kefir 3) from different US sources. We analyzed the microbial compositions of the three milk fermentation samples. This study revealed that each sample contains unique and distinct groups of microbes, kefir 1 showed the least diversity, and kefir 3 showed the highest diversity. Kefir 3 is rich in Proteobacteria while kefir 2 is dominated by the Firmicutes. Using bacterial indicator growth analyses carried out by continuous readings from microplate-based bioreactor assays suggested that kefir 2 fermentation filtrate has higher antibacterial property. We have screened 30 purified cultures of kefir 2 sample and isolated two lactic acid bacteria strains with higher antibacterial activities; the two strains were identified as Leuconostoc mesenteroides 28-1 and Lentilactobacillus kefiri 25-2 by 16S genomic PCR with confirmed antibacterial activities of fermentation filtrate after growing under both aerobic and anaerobic conditions.

In vitro and in silico Evaluation of ACE2 and LOX Inhibitory Activity of Eucalyptus Essential Oils, 1,8-Cineole, and Citronellal

Eucalyptus essential oils are well-known and used especially in upper respiratory tract pathologies or diseases as herbal drug preparations. In the present study, the in vitro angiotensin-converting enzyme 2 (ACE2) and lipoxygenase (LOX) enzyme inhibitory potentials of commercial Eucalyptus globulus Labill. and Eucalyptus citriodora Hook. essential oils were evaluated for their potential anti-coronavirus disease 2019 (COVID-19), and anti-inflammatory effects. In addition, the major components, 1,8-cineole and citronellal, were evaluated for their ability to bind at the active site of either human ACE2 or human 5-LOX using an in silico setting. Before activity evaluation, Eucalyptus globulus and E citriodora essential oils were analysed by GC/FID and GC/MS, where 1,8-cineole (30%), and citronellal (80%) were identified as the major components, respectively. The in vitro ACE2 inhibition was calculated as 94.9% for E globulus, and that of E citriodora essential oil as 83.4%. In vitro LOX inhibition experiments for essential oils in the same order showed inhibitions of 71.3 and 91.4%, respectively, at 20 µg/mL test concentrations in microplate-based fluorometric assays. In addition, protein–ligand docking, and interaction profiling was used to gain structural and mechanistic insights into the in silico ACE2 and LOX inhibitory potentials of the major Eucalyptus essential oil constituents, 1,8-cineole as well as citronellal. The resulting data supported the in vitro findings; however, further in vivo studies are needed to confirm the activity.

Kinase inhibitor-induced cardiotoxicity assessed in vitro with human pluripotent stem cell derived cardiomyocytes

Many small molecule kinase inhibitors (SMKIs), used predominantly in cancer therapy, have been implicated in serious clinical cardiac adverse events, which means that traditional preclinical drug development assays were not sufficient for identifying these cardiac liabilities. To improve clinical cardiac safety predictions, the effects of SMKIs targeting many different signaling pathways were studied using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) in combined assays designed for the detection of both electrophysiological (proarrhythmic) and non-electrophysiological (non-proarrhythmic) drug-induced cardiotoxicity. Several microplate-based assays were used to quantitate cell death, apoptosis, mitochondrial damage, energy depletion, and oxidative stress as mechanism-based non-electrophysiological cardiomyocyte toxicities. Microelectrode arrays (MEA) were used to quantitate in vitro arrhythmic events (iAEs), field potential duration (FPD) prolongation, and spike amplitude suppression (SAS) as electrophysiological effects. To enhance the clinical relevance, SMKI-induced cardiotoxicities were compared by converting drug concentrations into multiples of reported clinical maximum therapeutic plasma concentration, “FoldCmax”, for each assay. The results support the conclusion that the combination of the hPSC-CM based electrophysiological and non-electrophysiological assays have significantly more predictive value than either assay alone and significantly more than the current FDA-recommended hERG assay. In addition, the combination of these assays provided mechanistic information relevant to cardiomyocyte toxicities, thus providing valuable information on potential drug-induced cardiotoxicities early in drug development prior to animal and clinical testing. We believe that this early information will be helpful to guide the development of safer and more cost-effective drugs.

RpoB Mutations and Effects on Rifampin Resistance in Mycobacterium tuberculosis.

ObjectiveTo investigate the mutations within the whole rpoB gene of Mycobacterium tuberculosis and analyze their effects on rifampin (RIF) resistance based on crystal structure.MethodsWe sequenced the entire rpoB gene in 175 tuberculosis isolates and quantified their minimum inhibitory concentrations using microplate-based assays. Additionally, the structural interactions between wild-type/mutant RpoB and RIF were also analyzed.ResultsResults revealed that a total of 34 mutations distributed across 17 different sites within the whole rpoB gene were identified. Of the 34 mutations, 25 could alter the structural interaction between RpoB and RIF and contribute to RIF resistance. Statistical analysis showed that S450L, H445D, H445Y and H445R mutations were associated with high-level RIF resistance, while D435V was associated with moderate-level RIF resistance.ConclusionSome mutations within the rpoB gene could affect the interaction between RpoB and RIF and thus are associated with RIF resistance. These findings could be helpful to design new antibiotics and develop novel diagnostic tools for drug resistance in TB.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples.

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Vapors of Volatile Plant-Derived Products Significantly Affect the Results of Antimicrobial, Antioxidative and Cytotoxicity Microplate-Based Assays.

Volatile plant-derived products were observed to exhibit broad spectrum of biological effects. However, due to their volatility, results of conventional microplate-based bioassays can be significantly affected by the vapors. With aim to demonstrate this phenomenon, antimicrobial, antioxidant, and cytotoxic activities of three essential oils (Alpinia elegans, Cinnamomum iners, and Xanthostemon verdugonianus), one supercritical CO2 extract (Nigella sativa), and four plant-derived compounds (capsaicin, caryophyllene oxide, 8-hydroxyquinoline, and thymoquinone) were evaluated in series of experiments including both ethylene vinyl acetate (EVA) Capmat sealed and nonsealed microplates. The results clearly illustrate that vapor transition to adjoining wells causes false-positive results of bioassays performed in nonsealed microtiter plates. The microplate layout and a duration of the assay were demonstrated as the key aspects defining level of the results affection by the vapors of volatile agents. Additionally, we reported biological activities and chemical composition of essential oils from A. elegans seeds and X. verdugonianus leaves, which were, according to our best knowledge, analyzed for the first time. Considering our findings, certain modifications of conventional microplate-based assays are necessary (e.g., using EVA Capmat as vapor barrier) to obtain reliable results when biological properties of volatile agents are evaluated.

Nematocidal Effects of a Coriander Essential Oil and Five Pure Principles on the Infective Larvae of Major Ovine Gastrointestinal Nematodes In Vitro.

The anthelmintic effects of extracted coriander oil and five pure essential oil constituents (geraniol, geranyl acetate, eugenol, methyl iso-eugenol, and linalool) were tested, using larval motility assay, on the third-stage larvae (L3s) of Haemonchus contortus, Trichostrongylus axei, Teladorsagia circumcincta, Trichostrongylus colubriformis, Trichostrongylus vitrinus and Cooperia oncophora. Coriander oil and linalool, a major component of tested coriander oil, showed a strong inhibitory efficacy against all species, except C. oncophora with a half maximal inhibitory concentration (IC50) that ranged from 0.56 to 1.41% for the coriander oil and 0.51 to 1.76% for linalool. The coriander oil and linalool combinations conferred a synergistic anthelmintic effect (combination index [CI] <1) on larval motility comparable to positive control (20 mg/mL levamisole) within 24 h (p < 0.05), reduced IC50 values to 0.11–0.49% and induced a considerable structural damage to L3s. Results of the combined treatment were validated by quantitative fluorometric microplate-based assays using Sytox green, propidium iodide and C12-resazurin, which successfully discriminated live/dead larvae. Only Sytox green staining achieved IC50 values comparable to that of the larval motility assay. The cytotoxicity of the combined coriander oil and linalool on Madin–Darby Canine Kidney cells was evaluated using sulforhodamine-B (SRB) assay and showed no significant cytotoxic effect at concentrations < 1%. These results indicate that testing essential oils and their main components may help to find new potential anthelmintic compounds, while at the same time reducing the reliance on synthetic anthelmintics.

Augmentation of Hyperglycemia‐Induced Damage in a Diabetic Rat Endothelium is Potentially Regulated by the Nfκβ Signaling Pathway

Endothelial dysfunction is a key factor for the development of organ failure in type 2 diabetes mellitus (T2DM). While hyperglycemia is a well‐established factor in the development of microvascular complications in T2DM, specific mechanisms of endothelial damage remains equivocal. In a previous study, we observed that hyperglycemic states resulted in distinct alterations in proteins involved in metabolic and angiogenic processes in endothelial cells isolated from diabetic when compared to non‐diabetic rats. In the present study, we identified and validated diverse NFκβ‐regulated cellular functions that are involved in augmenting hyperglycemia‐induced damage in the diabetic endothelium. Bioinformatics and functional studies were carried out in the primary cardiac microvascular endothelial cells (RCMVECs) derived from the spontaneously T2DM Goto‐Kakizaki (GK) rat model in comparison to the control Wistar Kyoto (WKY) rat model under normal and hyperglycemic conditions. GK and WKY RCMVECs were cultured under both normal (NG; 4.5 mM) and high glucose (HG; 25 mM) conditions for two weeks, followed by tandem mass spectrometry (MS/MS), qPCR, western blotting, microplate‐based cell proliferation, metabolic and REDOX assays. Evidence of hyperglycemia‐induced cytotoxicity in GK RCMVECs was determined by employing various microplate‐based assays, wherein a 20% reduction in cell proliferation (p&lt;0.05) and a 33% increase in PMA‐induced ROS production (p&lt;0.05) was observed. Enrichment and pathway analyses of the proteomic tandem MS/MS and qPCR datasets, revealed induction of inflammatory, metabolic and apoptotic pathways, which are tightly regulated by NFκβ, in GK RCMVECs (HG vs NG). Platelet‐activating factor receptor (LogFC = 3.5, p &lt;0.01) and beta‐catenin (LogFC= 1.3, p &lt;0.01), both key inductors of NFκβ signaling, were significantly upregulated in GK RCMVECs (HG vs NG). Western blotting analysis revealed significant upregulation of NFκβ in GK RCMVECs (HG vs NG) (FC=1.6, p&lt;0.05) and in GK NG vs WKY NG RCMVECs (FC=2.5, p&lt;0.05), suggesting a potential increase in NFκβ activity in the diabetic endothelium. Additionally, the activity of hexokinase‐2, which is reported to be a transcriptional target of NFκβ, was observed to be reduced in GK NG vs WKY NG RCMVECs (20% reduction, p&lt;0.05). Furthermore, qPCR analysis identified an upregulation of several inflammatory transcripts that are that are regulated by NFκβ such as CXCR4 (FC=7.5, p&lt;0.01) and NLRP3 (FC=5.8, p&lt;0.01) expression in GK RCMVECs (HG vs NG). By the integration of high‐throughput tandem MS/MS analysis, bioinformatics analyses and in vitro functional assays, we identified a potential induction of NFκβ signaling in GK (HG vs NG) RCMVECs, resulting in metabolic reprogramming and a heightened inflammatory and apoptotic response.Support or Funding InformationSupport for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University, Milwaukee, WI, USA.

A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway.

DNA repair is essential for the maintenance of genomic integrity, and evidence suggest that inter-individual variation in DNA repair efficiency may contribute to disease risk. However, robust assays suitable for quantitative determination of DNA repair capacity in large cohort and clinical trials are needed to evaluate these apparent associations fully. We describe here a set of microplate-based oligonucleotide assays for high-throughput, non-radioactive and quantitative determination of repair enzyme activity at individual steps and over multiple steps of the DNA base excision repair pathway. The assays are highly sensitive: using HepG2 nuclear extract, enzyme activities were quantifiable at concentrations of 0.0002 to 0.181 μg per reaction, depending on the enzyme being measured. Assay coefficients of variation are comparable with other microplate-based assays. The assay format requires no specialist equipment and has the potential to be extended for analysis of a wide range of DNA repair enzyme activities. As such, these assays hold considerable promise for gaining new mechanistic insights into how DNA repair is related to individual genetics, disease status or progression and other environmental factors and investigating whether DNA repair activities can be used a biomarker of disease risk.

Assessing Calcium-Stimulated Mitochondrial Bioenergetics Using the Seahorse XF96 Analyzer.

The development of fluorescence-based oxygen sensors coupled with microplate-based assays for quantitative bioenergetics analyses enables screening multiple experimental conditions at once with small biological material and in a timely manner. In this chapter, we outline detailed protocols and practical tips to design and perform controlled measurements of (a) respiratory and glycolytic metabolism of intact cells, (b) substrate-dependent respiration in permeabilized cells and isolated mitochondria, and (c) calcium-dependent regulation of mitochondrial bioenergetics with Seahorse XF Flux Analyzers.

Peptide Extract from Olivancillaria hiatula Exhibits Broad-Spectrum Antibacterial Activity.

Increasing reports of infectious diseases worldwide have become a global concern in recent times. Depleted antibiotic pipelines, rapid and complex cases of antimicrobial resistance, and emergence and re-emergence of infectious disease have necessitated an urgent need for the development of new antimicrobial therapeutics, preferably with novel modes of action. Due to their distinct mode of action, antimicrobial peptides offer an interesting alternative to conventional antibiotics to deal with the problems enumerated. In this study, the antimicrobial potential of the peptide extract from the marine mollusc, Olivancillaria hiatula, was evaluated in vitro. Agar diffusion and broth dilution techniques were used to evaluate microbial susceptibility to the peptide extract. Microplate-based assays were also used to investigate time-dependent growth inhibition profiles of microbes in the presence of peptide and evaluate the peptide's ability to modulate the activities of standard antibiotics. Both Gram-positive and Gram-negative bacteria were inhibited by the peptide extract in the agar diffusion assay. The minimum inhibitory concentration (MIC) of peptide against test microorganisms was between 0.039 and 2.5 mg/mL. At the MIC, the peptide extract was bacteriostatic towards all tested microorganisms but bactericidal to Staphylococcus aureus. In the presence of the peptide extract, a prolonged lag phase was observed for all microbes, similar to standard ciprofloxacin. When administered together, peptide extracts enhanced the activities of ciprofloxacin and cefotaxime and were antagonistic towards erythromycin but indifferent towards metronidazole. Taken together, these results show the broad-spectrum antibacterial activity of peptide extract from Olivancillaria hiatula and demonstrate that antimicrobial peptides can be employed in combination with some conventional antibiotics for improved effects.

High-Throughput Microplate-Based Fluorescence Assays for Studying Stochastic Aggregation of Superoxide Dismutase-1.

Investigating in vitro kinetics of superoxide dismutase-1 (SOD1) aggregation with high-throughput microplate-based assays provides valuable information regarding SOD1 pathogenesis in amyotrophic lateral sclerosis (ALS) and opens venues for the development of effective therapies. In this chapter, we first explain the step-by-step purification and demetallation of wild-type (WT) and ALS-variant SOD1 proteins from Saccharomyces cerevisiae (baker's yeast). We then describe the methodology for a microplate-based fluorescence assay that is used to study real-time kinetics of metal-free (apo)-SOD1 aggregation. This technique is highly sensitive, semiautomated, requires minimum modifications to protein, and produces a plethora of data in a short period of time. We also describe a new approach for extracting clinically relevant information from SOD1 aggregation data using Kaplan-Meier estimators.

A Quantum Dot-Protein Bioconjugate That Provides for Extracellular Control of Intracellular Drug Release.

The ability to control the intracellular release of drug cargos from nanobioconjugate delivery scaffolds is critical for the successful implementation of nanoparticle (NP)-mediated drug delivery. This is particularly true for hard NP carriers such as semiconductor quantum dots (QDs) and gold NPs. Here, we report the development of a QD-based multicomponent drug release system that, when delivered to the cytosol of mammalian cells, is triggered to release its drug cargo by the simple addition of a competitive ligand to the extracellular medium. The ensemble construct consists of the central QD scaffold that is decorated with a fixed number of maltose binding proteins (MBPs). The MBP binding site is loaded with dye or drug conjugates of the maltose analogue beta-cyclodextrin (βCD) to yield a QD-MBP-βCD ensemble conjugate. The fidelity of conjugate assembly is monitored by Förster resonance energy transfer (FRET) from the QD donor to the dye/drug acceptor. Microplate-based FRET assays demonstrated that the βCD conjugate was released from the MBP binding pocket by maltose addition with an affinity that matched native MBP-maltose binding interactions. In COS-1 cells, the microinjected assembled conjugates remained stably intact in the cytosol until the addition of maltose to the extracellular medium, which underwent facilitated uptake into the cell. Live cell FRET-based confocal microscopy imaging captured the kinetics of realtime release of the βCD ligand as a function of extracellular maltose concentration. Our results demonstrate the utility of the self-assembled QD-MBP-βCD system to facilitate intracellular drug release that is triggered extracellularly through the simple addition of a well-tolerated nutrient and is not dependent on the use of light, magnetic field, ultrasound, or other traditional methods of stimulated drug release. We expect this extracellularly triggered drug release modality to be useful for the in vitro characterization of new drug candidates intended for systemic delivery/actuation and potentially for on-demand drug release in vivo.

Robust Microplate-Based Methods for Culturing and in Vivo Phenotypic Screening of Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii (Cr), a unicellular alga, is routinely utilized to study photosynthetic biochemistry, ciliary motility, and cellular reproduction. Its minimal culture requirements, unicellular morphology, and ease of transformation have made it a popular model system. Despite its relatively slow doubling time, compared with many bacteria, it is an ideal eukaryotic system for microplate-based studies utilizing either, or both, absorbance as well as fluorescence assays. Such microplate assays are powerful tools for researchers in the areas of toxicology, pharmacology, chemical genetics, biotechnology, and more. However, while microplate-based assays are valuable tools for screening biological systems, these methodologies can significantly alter the conditions in which the organisms are cultured and their subsequent physiology or morphology. Herein we describe a novel method for the microplate culture and in vivo phenotypic analysis of growth, viability, and photosynthetic pigments of C. reinhardtii. We evaluated the utility of our assay by screening silver nanoparticles for their effects on growth and viability. These methods are amenable to a wide assortment of studies and present a significant advancement in the methodologies available for research involving this model organism.

An insight into the antibiofilm properties of Costa Rican stingless bee honeys.

There is an increasing search for antibiofilm agents that either have specific activity against biofilms or may act in synergy with antimicrobials. Our objective is to examine the the antibiofilm properties of stingless bee honeys. Meliponini honeys from Costa Rica were examined along with Medihoney as a reference. All honeys were submitted to a screening composed of minimum inhibitory concentration, inhibition of biofilm formation and biofilm destruction microplate-based assays against a Staphylococcus aureus biofilm forming strain. Dialysis led to the isolation of an antibiofilm fraction in Tetragonisca angustula honeys. The honey antibiofilm fraction was evaluated for protease activity and for any synergistic effect with antibiotics on a Staphylococcus aureus biofilm. The active fraction was then separated through activity guided isolation techniques involving SDS-PAGEs, anion exchange and size exclusion fast protein liquid chromatographies. The fractions obtained and the isolated antibiofilm constituents were tested for amylase and DNase activity. A total of 57 Meliponini honeys from Costa Rica were studied in this research. The honeys studied belonged to the Tetragonisca angustula (n=36) and Melipona beecheii (n=21) species. Costa Rican Tetragonisca angustula honeys can inhibit the planktonic growth, biofilm formation, and are capable of destroying a Staphylococcus aureus biofilm. The antibiofilm effect was observed in the protein fraction of Tetragonisca angustula honeys. The biofilm destruction proteins allowed ampicillin and vancomycin to recover their antimicrobial activity over a Staphylococcus aureus biofilm. The antibiofilm proteins are of bee origin, and their activity was not due to serine, cysteine or metalloproteases. There were 2 proteins causing the antibiofilm action; these were named the Tetragonisca angustula biofilm destruction factors (TABDFs). TABDF-1 is a monomeric protein of approximately 50kDa that is responsible of the amylase activity of Tetragonisca angustula honeys. TABDF-2 is a protein monomer of approximately 75kDa. Tetragonisca angustula honeys from Costa Rica are a promising candidate for research and development of novel wound dressings focused on the treatment of acute and chronic Staphylococcus aureus biofilm wound infections.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP).

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

Review of lignocellulolytic enzyme activity analyses and scale-down to microplate-based assays

With the increasing use of enzymes in environmental applications, there is a need for analytical methods adapted to large factorial experiments. Existing reference methods are chemical and labor intensive and unsuitable to analyze in parallel a large number of samples. Based on an extensive literature review and on experimental results, this work compares reference and microplate adapted methods to define the most adequate filter paper, carboxymethylcellulase, β-glucosidase and xylanase activity tests. In the adapted methods, the total reaction volume was reduced from 2.2–24.5mL to 0.21–0.24mL. Statistical analysis of the activities measured on enzyme mixtures by applying the 96-well plate reduced methods showed that they were not significantly different to the activities obtained with reference tests.