Dietary Intake of Rosmarinic Acid Increases Serum Inhibitory Activity in Amyloid A Aggregation and Suppresses Deposition in the Organs of Mice.

Serum amyloid A (SAA) is one of the most important precursor amyloid proteins and plays a vital step in AA amyloidosis, although the underlying aggregation mechanism has not been elucidated. Since SAA aggregation is a key step in this pathogenesis, inhibitors are useful to prevent and treat AA amyloidosis, serving as tools to investigate the pathogenic mechanism. In this study, we showed that rosmarinic acid (RA), which is a well-known inhibitor of the aggregation of amyloid ß (Aß), displayed inhibitory activity against SAA aggregation in vitro using a microliter-scale high-throughput screening (MSHTS) system with quantum-dot nanoprobes. Therefore, we evaluated the amyloid aggregation inhibitory activity of blood and the deposition of SAA in organs by feeding mice with Melissa officinalis extract (ME) containing RA as an active substance. Interestingly, the inhibitory activity of ME-fed mice sera for SAA and Aß aggregation, measured with the MSHTS system, was higher than that of the control group. The amount of amyloid deposition in the organs of ME-fed mice was lower than that in the control group, suggesting that the SAA aggregation inhibitory activity of serum is associated with SAA deposition. These results suggest that dietary intake of RA-containing ME enhanced amyloid aggregation inhibitory activity of blood and suppressed SAA deposition in organs. This study also demonstrated that the MSHTS system could be applied to in vitro screening and to monitor comprehensive activity of metabolized foods adsorbed by blood.

Air-Drying Time Affects Mortality of Pyrethroid-Susceptible Aedes aegypti Exposed to Transfluthrin-Treated Filter Papers.

Increasing temperature can enhance the geographical spread and behavior of disease vector mosquitoes, exposing vulnerable populations to Aedes-borne viruses and infections. To address this risk, cost-effective and sustained intervention vector control tools are required, such as volatile pyrethroid spatial repellents. This study used a high-throughput screening system toxicity bioassay to determine the discriminating concentrations of transfluthrin-treated filter papers with variable air-drying times exposed to pyrethroid-susceptible Aedes aegypti mosquitoes. At the highest transfluthrin concentration (0.01706%), a significant reduction in mosquito mortality was observed in filter papers air-dried for 24 h compared to those air-dried for 1 h (odds ratio = 0.390, p < 0.001, 95% confidence interval: 0.23-0.66). Conversely, no significant difference in mortality was found between filter papers air-dried for 1 h and those air-dried for 12 h (odds ratio = 0.646, p = 0.107, 95% confidence interval: 0.38-1.10). The discriminating concentration was 2.8-fold higher for transfluthrin-treated filter papers air-dried for 24 h than it was for papers air-dried for 1 h, and it increased 5-fold from 1 h to 336 h of air-drying. These results show that the optimal air-drying period of transfluthrin-treated filter paper is critical, as higher discriminating concentration values may lead to underestimations of insecticide resistance. The instability of transfluthrin-treated papers necessitates the use of the World Health Organization (WHO) bottle bioassay, which is the preferred method for determining mosquito susceptibility to volatile insecticides.

A robust high-throughput screening system to assess bacterial tyrosine ammonia lyase activity in the context of tyrosine inherited metabolic disorders.

Inborn errors of tyrosine metabolism result in patient's inability to degrade tyrosine. Current treatment consists of a phenylalanine and tyrosine restricted diet and nitisinone, causing a block in the tyrosine degradation pathway. However, tyrosine levels will increase, leading to acquired hypertyrosinemia, implying the need for an add-on treatment. Tyrosine ammonia lyases (TAL) can provide such an add-on treatment as they catalyze the deamination of tyrosine into p-coumaric acid and ammonia. In this study, we developed a robust high-throughput screening (HTS) assay to assess the capacity of bacterial TAL enzymes to decrease excessive tyrosine. The assay is based on the spectrophotometric quantification of p-coumaric acid after conversion of tyrosine by bacterial TAL. As a benchmark, TAL from Flavobacterium johnsoniae (FjTAL) was used to optimize the assay. Optimal growth conditions for high-level protein expression were determined by incubating transformed Escherichia coli BL21 (DE3) cells at different temperatures during various incubation times. Subsequently, assay temperature and pH were optimized followed by testing different ratios of tyrosine assay mixes to bacterial lysate. Finally, assay robustness and functionality were evaluated. Optimal FjTAL expression was obtained after incubation for 24 h at 22 °C. Ideal assay conditions consist of a 80/20 ratio of 1 mM tyrosine assay mix to FjTAL lysate performed at pH 9.2 and 37 °C. The robustness test showed Z' values > 0.4 and signal window values > 2 without edge or drift effects. As proof-of-principle, we successfully determined the catalytic activity of two other bacterial TAL enzymes RsTAL (5.718.10-3 ± 0.21.10-3) and SeSAM8 (4.658.10-3 ± 0.37.10-3). A robust, simple and reliable HTS assay was thus developed to evaluate the tyrosine degradation capacity of bacterial TAL enzymes.

Transfluthrin and Metofluthrin as Effective Repellents against Pyrethroid-Susceptible and Pyrethroid-Resistant Aedes aegypti (L.) (Diptera: Culicidae).

Aedes aegypti is a major vector of dengue fever in tropical regions. Spatial repellents (SRs) have shown promise in delaying pesticide resistance. Methods for discriminating concentrations (DCs) are well established using various bioassay tools, while data for high-throughput screening system (HITSS) toxicity bioassay (TOX) are absent. In this study, we compared and optimized lethal (LCs) and sub-lethal concentrations (SLCs) of transfluthrin (TFT) and metofluthrin (MFT) on pyrethroid-susceptible (USDA) and pyrethroid-resistant (Pu-Teuy) Ae. aegypti (L.) strains, using the HITSS-TOX. Mean mortality (MT) was 100% at LC99 and DC, compared to LC50 (45.0 ± 3.7%) and LC75 (65.8 ± 7.0%) for the USDA strain. However, the resistant strain (Pu-Teuy) showed reduced susceptibility against TFT and a significantly lower MT at LC50 (12.5 ± 4.4%; t = 5.665, df = 10, p < 0.001), LC75 (9.2 ± 3.5%; t = 4.844, df = 10, p = 0.001), LC99 (55.0 ± 9.9%; t = 4.538, df = 5, p = 0.006), and DC (75.0 ± 5.2%; U = 3.0, p = 0.007). The DC of TFT (0.15222%) was 4.7-fold higher than for MFT (0.03242%) in USDA strain. The baseline DCs established are useful to better understand susceptibility and the efficacy of various repellents against field populations of Ae. aegypti.

Scar-Degrading Endothelial Cells as a Treatment for Advanced Liver Fibrosis.

Deposition of extracellular matrix (ECM) in the liver is an important feature of liver cirrhosis. Recovery from liver cirrhosis is physiologically challenging, partially due to the ECM in scar tissue showing resistance to cell-mediated degradation by secreted matrix metalloproteinases (MMPs). Here, a cell-mediated ECM-degradation screening system (CEDSS) in vitro is constructed for high-throughput searching for cells with tremendous degradation ability. ECM-degrading liver sinusoidal endothelial cells (dLSECs) are screened using CEDSS, which exhibit 17 times the ability to degrade collagen when compared to other cells. The degradation ability of dLSECs is mediated by the upregulation of MMP9. In particular, mRNA expression of MMP9 shows an 833-fold increase in dLSECs compared to normal endothelial cells (nLSECs), and MMP9 is regulated by transcription factor c-Fos. In vivo, single intrasplenic injection of dLSECs alleviates advanced liver fibrosis in mice, while intraperitoneal administration of liver-targeting peptide-modified dLSECs shows enhanced fibrosis-targeting effects. Degradative human umbilical vein endothelial cells (dHUVECs) prove their enhanced potential of clinical translation. Together, these results highlight the potential of ECM-degrading endothelial cells in alleviating advanced liver fibrosis, thus providing remarkable insights in the development of ECM-targeting therapeutics.

Evaluation of Mosquito Attractant Candidates Using a High-Throughput Screening System for Aedes aegypti (L.), Culex quinquefasciatus Say. and Anopheles minimus Theobald (Diptera: Culicidae).

Several types of olfactometers have been used to evaluate mosquito responses to agents that mimic natural volatiles that repel or attract. The Y-tube olfactometer has been widely used to study repellents and attractants, while the high-throughput screening system assay has only been used to study repellents. Whether the high-throughput screening system assay is suitable for evaluating attractants is unknown. We evaluated the responses to four lactic-acid-based mixtures and two non-lactic-acid-based chemical lure candidates using the high-throughput screening system (HITSS) for three mosquito species (laboratory strains and field populations of both Aedes aegypti (L.) and Culex quinquefasciatus Say.; laboratory strain of Anopheles minimus Theobald) under laboratory-controlled conditions. HITSS assay results showed that KU-lure #1 elicited the greatest percent attraction for pyrethroid-resistant and -susceptible Ae. aegypti. KU-lure #6 elicited the strongest attractive response for pyrethroid-susceptible and -resistant Cx. quinquefasciatus and pyrethroid-susceptible An. minimus. The response to the lures from each species was independent of the pyrethroid susceptibility status (Ae. aegypti, p = 0.825; Cx. quinquefasciatus, p = 0.056). However, a significant difference in attraction to KU-lure #6 was observed between diurnal and nocturnal mosquitoes (Cx. quinquefasciatus vs. Ae. aegypti, p = 0.014; An. minimus vs. Ae. aegypti, p = 0.001). The laboratory-level HITSS assay effectively selects potential lure candidates. Because the host-seeking behavior differs between mosquito species, further studies are needed to develop species-specific attractants. Additional studies in semi-field screen houses using commercial traps are necessary to evaluate the accuracy of these laboratory assay results.

Dose-Response Assay for Synthetic Mosquito (Diptera: Culicidae) Attractant Using a High-Throughput Screening System.

Natural volatile host cues play a critical role for mosquito orientation and locating a blood source for egg production. Similar olfactory activation responses have allowed the use and development of artificial chemical attractants to lure mosquitoes to trapping devices. Using a pre-formulated commercial product mixture of different attractant chemicals, a high-throughput screening system (HITSS) is used to screen varying doses of chemical required to activate behavioral responses. Two strains of Aedes aegypti (L.): permethrin-susceptible (USDA) and -resistant (Pu Teuy) phenotypes and one Culex quinquefasciatus Say. (NIH) laboratory strain were tested. Overall, mosquitoes showed repellency between 1.0 g and to 10.0 g dose of each compound. However, by progressively reducing the dose, Cx. quinquefasciatus showed a greater positive percent attraction (88.9%) at 0.025 g, whereas the USDA and Pu Teuy Ae. aegypti produced optimum attractant activation at 0.005 g (72.6% and 58.9%, respectively) without significant difference within species (p > 0.05). In parallel control assays, Cx. quinquefasciatus was significantly attracted to 1 g of dry ice (carbon dioxide) (76%) more than Ae. aegypti (USDA) (12.2%). The HITSS was originally designed to measure three chemical actions to sublethal concentrations of chemicals by mosquitoes: toxicity and the two primary behavior avoidance responses (contact excitation and spatial repellency). These findings demonstrate that the HITSS assay, with only minor modifications, allows comparison screening of candidate compounds as potential attractants for anemotactic responses under laboratory-controlled conditions. Further investigations will be required to equate measurements obtained from controlled laboratory assays to more varied field conditions for attracting natural mosquito populations.

The Antifungal Test: An Efficient Screening Tool for the Discovery of Microbial Metabolites with Respiratory Inhibitory Activity.

Valuable natural compounds produced by a variety of microorganisms can be used as lead molecules for development of new agrochemicals. Furthermore, high-throughput in vitro screening systems with specific modes of action can increase the probability of discovery of new fungicides. In the current study, a rapid assay tested with various microbes was developed to determine the degree of respiratory inhibition of Saccharomyces cerevisiae in two different liquid media, YG (containing a fermentable carbon source) and NFYG (containing a non-fermentable carbon source). Based on this system, we screened 100 fungal isolates that were classified into basidiomycetes, to find microbial secondary metabolites that act as respiratory inhibitors. Consequently, of the 100 fungal species tested, the culture broth of an IUM04881 isolate inhibited growth of S. cerevisiae in NFYG medium, but not in YG medium. The result is comparable to that from treatment with kresoxim-methyl used as a control, suggesting that the culture broth of IUM04881 isolate might contain active compounds showing the inhibition activity for respiratory chain. Based on the assay developed in this study and spectroscopic analysis, we isolated and identified an antifungal compound (-)-oudemansin A from culture broth of IUM04881 that is identified as Oudemansiella venosolamellata. This is the first report that (-)-oudemansin A is identified from O. venosolamellata in Korea. Taken together, the development of this assay will accelerate efforts to find and identify natural respiratory inhibitors from various microbes.

Machine learning linked evolutionary biosensor array for highly sensitive and specific molecular identification.

Bacteria initiate complicated signaling cascades from the detection of intracellular metabolites or exogenous substances by hundreds of transcription factors, which have been widely investigated as genetically-encoded biosensors for molecular recognition. However, the limited number of transcription factors and their broad substrate specificity result in ambiguity in small molecule identification. This study presents a new small molecule fingerprinting technique using evolutionary biosensor arrays with a machine learning technique that can capture highly specific substrate signals. Employing multiple mutant transcription factors derived from a single transcription factor has effectively circumvented the limited availability of transcription factors induced by a small molecule of our interest. This method achieved up to 95.3% true positive rate for identifying small molecules, and the high-resolution protein engineering technique improved the limit of detection 75-fold. The signal trade-offs with background noises caused by the complex cellular biochemistry of mutant transcription factors enable the biosensor arrays to be more informative in terms of statistical variance. The machine learning technology, coupled with the single transcription factor-driven evolutionary biosensor array, will open new avenues for molecular fingerprinting technologies.

Spatial repellency and other effects of transfluthrin and linalool on Aedes aegypti and Aedes albopictus.

In the present study, the effects of two spatial repellents (SR) were determined for Aedes aegypti and Ae albopictus, the main vectors of dengue, Chikungunya, and Zika fever. The modular high-throughput screening system (HITSS) was used to evaluate the response of both species to transfluthrin and linalool SR at different concentrations. The highest spatial repellency results for Ae. aegypti were obtained by transfluthrin to 0.001% with 37.50 ± 4.33%, and for linalool to 10% with 77.50 ± 3.90%. For Ae. albopictus, the highest spatial repellency percentages for transfluthrin 0.01% were 45.00 ± 3.78%, and linalool at 1% and 10% were 56.25 ± 7.06% and 56.25 ± 6.46%, respectively. Transfluthrin caused high levels of mortality with 71.25 ± 6.66%, 79.75 ± 8.65%, and 100% to Ae. aegypti and 70.00 ± 5.98% and 98.75 ± 0.82% to Aedes albopcitus. With the results of this study, we concluded that both the transfluthrin and linalool could be used as protection measures against the bite of Ae. aegypti and Ae. albopictus in the integral strategies for the control of vectors in Mexico.

Synthetic polyphosphate inhibits endogenous coagulation and platelet aggregation in vitro.

Platelet-derived polyphosphate has previously been indicated to induce coagulation. However, industrially synthesized polyphosphate has been found to have different effects from those of the platelet-derived form. The present study investigated whether synthetic sodium polyphosphate inhibits coagulation using routine coagulation tests and thromboelastography. Synthetic polyphosphate was found to inhibit adenosine diphosphate-, epinephrine-, arachidonic acid-, ristocetin-, thrombin-, oxytocin- and pituitrin-induced platelet aggregation. The effects of synthetic polyphosphate in clotting inhibition were revealed by the analysis of clotting factor activity and platelet aggregation tests. Synthetic polyphosphate may inhibit platelet aggregation by reducing platelet calcium levels, as indicated by the results of flow cytometric analysis and high-throughput fluorescent screening. Furthermore, analysis of thromboxane (TX)B2 by ELISA indicated that synthetic polyphosphate reduces platelet aggregation by inhibiting the TXA2 signaling pathway. In conclusion, synthetic polyphosphate inhibits clotting factor activity and endogenous coagulation by reducing the levels of calcium ions and TXA2 to curb platelet aggregation.