Microplate Research Articles

Efficacy of CP-COV03 (a niclosamide-based inorganic nanohybrid product) against severe fever with thrombocytopenia syndrome virus in an in vitro model.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease caused by the SFTS virus (SFTSV). CP-COV03 is a novel antiviral candidate that significantly enhanced the bioavailability of niclosamide through inorganic-based drug delivery technology. The active pharmaceutical ingredient of CP-COV03, niclosamide, has been previously shown to possess broad-spectrum antiviral activity against over 30 different viruses in the in vitro tests. The aim of this study is to confirm the antiviral activity of CP-COV03 against the SFTSV in an in vitro model. Vero cells and SFTS viral stock NCCP43270, a 2015 Gangwon Province isolate, were used to obtain the 50% tissue culture infective dose of the virus. Vero cells seeded in 96-well plates were infected with SFTSV for 1 h. SFTSV-infected cells were treated with CP-COV03 at various concentrations of 0.1-100 μM and incubated for 7 days. On the seventh day of the culture, the cytopathic effect (CPE) of SFTSV was checked by microscopy and the cell viability was checked by using Cell Counting Kit-8 assay. The CPE reduced as the CP-COV03 concentration increased. The 50% inhibitory concentration (IC50) range of CP-COV03 was below 0.125 µM, as determined from the viral titers of culture supernatants collected on the third day posttreatment of CP-COV03. The plaque reduction assay showed that the IC50 of CP-COV03 was 1.893 µM, as determined from the percentage reduction of plaque counts for each drug concentration on the second day posttreatment with CP-COV03. This study suggests that CP-COV03 could be used as a potential antiviral agent for SFTS.IMPORTANCEWe demonstrated a concentration-dependent response and identified low a IC50 of CP-COV03. This result is comparable to other antiviral drugs used against viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We believe that our study makes a significant contribution to the literature as our findings suggest that CP-COV03 may serve as a potential treatment for SFTS, highlighting its importance in the field of antiviral research.

Microcavity-assisted cloning (MAC) of hard-to-clone HepG2 cell lines: cloning made easy

Cloning is a key molecular biology procedure for obtaining a genetically homogenous population of organisms or cell lines. It requires the expansion of new cell populations starting from single genetically modified cells. Despite the technical progress, cloning of many cell lines remains difficult. Cloning often fails either due to the strenuous conditions associated with manipulating cells or because many cells don’t tolerate a single-cell state. Here we describe a new cloning method utilizing low adhesion microcavity plates. This new technique, named microcavity-assisted cloning (MAC) was developed to clone difficult-to-clone HepG2 cells. The clones were produced following CRISPR/Cas9 knockout of the GSTA1 gene by a random distribution of 200, 400, and 800 cells into 550 microcavities of a 24-well low adhesion plate originally designed for the culture of spheroids. The knockout of GSTA1 was verified at the protein level using Western blotting. The advantages of the MAC method are its low cost, ease of the procedure, and the possibility of scaling up the throughput and automatization.

Exploring the Antibacterial and Antibiofilm Efficacy of Psammogeton biternatum Edgew and Identification of a Novel Quinoline Alkaloid using X-ray Crystallography.

The prevalence of resistance to harmful human pathogens is steadily rising, emphasizing the urgent need to identify novel antimicrobial compounds. For this purpose, plants stand out as a significant source of bioactives worthy of exploration. Among these, alkaloids, a vast and structurally diverse category of plant secondary metabolites, have emerged as a foundation for crucial antibacterial medications such as metronidazole and the quinolones. In the current work, the crude methanol leaf extract of Psammogeton biternatum Edgew collected from District Bannu, Pakistan, was subjected to TLC (indirect) bioautography and X-ray crystallography for the isolation of potential antibacterial agents. From the crude extract, a novel quinoline alkaloid called quinoline dione ((3R,3aS,5aR)-3,5a,9-trimethyl-3a,4,5,5a-tetrahydro-2H-isoxazolo[2,3-a] quinoline-2,8(3H)-dione (C14H17NO3)) was isolated. The crystal information (M = 247.296 g/mol) is as follows: orthorhombic, P212121, a = 7.7339(14) Å, b = 10.7254(19) Å, c = 15.730(2) Å, V = 1304.8(4) Å3, Z = 4, T = 296 K, μ(Mo Kα) = 0.088 mm-1, ρ calc = 1.259 g/cm3, 13928 reflections measured (5.86° ≤ 2Θ ≤ 51.98°), 2478 unique (R int = 0.1613, R σ = 0.1335). The final R 1 was 0.1098 (I ≥ 2u(I)), and wR 2 was 0.2183. The antibacterial activity for both crude extract of leaves and quinoline dione was determined by a well diffusion method. The quinoline dione alkaloid demonstrated excellent inhibition zones against methicillin-resistant Staphylococcus aureus (18 mm), Bacillus subtills (17 mm), Escherichia coli (20 mm), and Pseudomonas aeruginosa (23 mm) compared to the crude extract. The antibiofilm potential was recorded against Pseudomonas aeruginosa by the 96-well microtiter plate method. A dose-dependent biofilm inhibition response was recorded, which increased with the increase in concentration. Moreover, quinoline dione showed a greater antibiofilm effect as compared to the crude extract, which may be linked to the presence of a particular active functional group positioned on the compound isolated in its pure form. Through in silico studies, i.e., molecular docking, quinoline dione shows strong binding energies with the LasR transcriptional regulator (6MVN) at -9.3 and LasR transcriptional activator (3IX4) at -9.2 kcal/mol, as well as moderate affinities with other targets such as AHL synthase LasI (PDB ID 1RO5) and OprM channel (PDB ID 3D5K), indicating its potential as a quorum sensing inhibitor. Thus, the antibacterial and antibiofilm potential of quinoline dione was confirmed.

Anticancer Efficacy of Biosynthesized Zinc Oxide and Gold Nanoparticles

Abstract Introduction/Objective Introduction: Advancements in nanoscale materials have led to the exploration of metal oxides for cancer diagnosis and therapy. Zinc oxide nanoparticles (ZnO NPs) and gold nanoparticles (Au NPs) are particularly promising due to their biocompatibility and anticancer properties demonstrated across various cancer cell lines. Objectives This study aimed to isolate fungal endophytes from Eucalyptus sideroxylon leaves and utilize them for the biosynthesis of nanoparticles (ZnO NPs and Au NPs) to assess their anticancer activity. Methods/Case Report Methods: Healthy Eucalyptus sideroxylon samples were collected from a desert research center, and endophytes were isolated and cultivated. The fungal biomass was processed to synthesize ZnO NPs and Au NPs. Cell lines were propagated for cytotoxicity experiments using the MTT assay in a 96-well plate format. Results (if a Case Study enter NA) Results: Biosynthesized ZnO and Au NPs exhibited anticancer activity against HCT-116 and CACO2 cell lines. The IC50 values for ZnO NPs were 5.06 μg/mL for HCT-116 and 6.07 μg/mL for CACO2, while Au NPs showed IC50 values of 5.9 μg/mL for HCT-116 and 1.1 μg/mL for CACO2. ZnO NPs demonstrated dose-dependent toxicity on HCT-116 cells, with increasing concentrations leading to a reduction in cell viability. Similarly, Au NPs exhibited dose-dependent cytotoxicity on both cell lines. Conclusion Conclusion: This study underscores the potential of endophytic fungi for biosynthesizing metal nanoparticles with significant anticancer effects. ZnO NPs displayed superior efficacy against colon cancer cells compared to Au NPs, indicating their potential as therapeutic agents. The findings highlight the importance of exploring natural sources for nanoparticle synthesis and their application in cancer therapy.

Physiological liver microtissue 384-well microplate system for preclinical hepatotoxicity assessment of therapeutic small molecule drugs.

Hepatotoxicity can lead to the discontinuation of approved or investigational drugs. The evaluation of the potential hepatoxicity of drugs in development is challenging because current models assessing this adverse effect are not always predictive of the outcome in human beings. Cell lines are routinely used for early hepatotoxicity screening, but to improve the detection of potential hepatotoxicity, in vitro models that better reflect liver morphology and function are needed. One such promising model is human liver microtissues. These are spheroids made of primary human parenchymal and nonparenchymal liver cells, which are amenable to high throughput screening. To test the predictivity of this model, the cytotoxicity of 152 FDA (US Food & Drug Administration)-approved small molecule drugs was measured as per changes in ATP content in human liver microtissues incubated in 384-well microplates. The results were analyzed with respect to drug label information, drug-induced liver injury (DILI) concern class, and drug class. The threshold IC50ATP-to-Cmax ratio of 176 was used to discriminate between safe and hepatotoxic drugs. "vMost-DILI-concern" drugs were detected with a sensitivity of 72% and a specificity of 89%, and "vMost-DILI-concern" drugs affecting the nervous system were detected with a sensitivity of 92% and a specificity of 91%. The robustness and relevance of this evaluation were assessed using a 5-fold cross-validation. The good predictivity, together with the in vivo-like morphology of the liver microtissues and scalability to a 384-well microplate, makes this method a promising and practical in vitro alternative to 2D cell line cultures for the early hepatotoxicity screening of drug candidates.

Adria microplate: a puzzling key stone in west-central Mediterranean geodynamics

The Mediterranean region denotes a special plate tectonic environment as the two big plates, Africa and Eurasia, primarily provide the main frame while the dominant players are the small oceanic basins and the continental micro plates (MP) that for the past 80 Ma was the Adria MP. In this environment slab rollback subduction, orogenic arc formation and rotation of crustal blocks are common. While the tectonic setting and involvement in geodynamic processes of Adria MP might not be unique in global terms, it presents Adria as an ideal natural laboratory to study the geodynamic processes individually and in their combined effects in a generally well-studied and rather wellaccessible region. The current extension of the Adria MP is defined by the continental Moho as far into the bounding orogens as the latter could be confirmed. The slabs surrounding Adria dominate and characterize the geodynamics of this MP and in most cases their geometries and the possible connection with Adria are well defined by tomographic images. The Ionian oceanic lithosphere slab subducting beneath Calabria has been torn off and detached from the very short South Apennines slab remaining attached to Adria. On the southeastern side we observe the Adria slab beneath the Hellenides that seems detached from the Ionian oceanic lithosphere slab of the Hellenic subduction zone. Along the Dinarides a slab gap is observed analogue to the slab window on Adria MP’s western side between the South Apennines and the North Apennines. Encircled by three orogens, the Adria continental lithosphere has been and still is strongly affected by the subductions surrounding it. In the northern part of the Adria MP, the North Apennines slab rollback is the obvious dominant geodynamic force acting locally on the plate while in contrast, the slabs beneath the Eastern Alps and beneath the Hellenides seem stagnant. This study presents a brief overview of the wealth of information aiming to summarize the main constraints on Adria MP available and it discusses some of the questions surrounding this key player in west-central Mediterranean geodynamics.

Towards high throughput analysis using 96-well plate solid phase extraction to determine sedatives and β-blocker residues in food control monitoring

BackgroundVeterinary drugs are widely used in animal production to prevent infections and treat diseases but, this may cause a risk to consumers. Due to the high number of food samples required to monitor yearly, simple, fast, sensitive and selective analytical methods are needed in control laboratories to ensure consumers safety. Nevertheless, many analytical methodologies available in these laboratories include multiple steps and therefore are time-consuming and hinder the analysis throughput requiring significant amounts of solvents and reagents. ResultsThis work developed a 96-well plate solid phase extraction (SPE) method for the extraction of seven sedatives and a β-blocker in animal kidney by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC−MS/MS). The developed method was validated based on Implementing Regulation (EU) 2021/808 in kidney, meat and fish. The performance characteristics of the validation (1–5 μg kg−1) showed good linearity (R2 > 0.998) while decision limits (CCα) were between 1 and 1.2 μg kg−1. Trueness and precision were determined at three levels (n = 7) and the results showed values ranging from 85 to 103 % and from 1 to 9 %, respectively. The feasibility of the method was demonstrated for the residue control requirement established by EU. Method was applied to 201 samples of kidney, meat and fish. SignificanceThis study is the first to present an optimized and validated holistic method for sedatives and β-blocker using 96-well plate SPE and UHPLC−MS/MS. The method showed good performance in kidney, meat and fish samples being an universal method for any species along the same type of sample. The fast, easy, efficient, reliable and universal method showed high throughput and so reduced the analysis time by nine-fold and the required solvent amount by four times fulfilling the green chemistry principals.

Natural compound-induced downregulation of antimicrobial resistance and biofilm-linked genes in wastewater Aeromonas species.

Addressing the global antimicrobial resistance (AMR) crisis requires a multifaceted innovative approach to mitigate impacts on public health, healthcare and economic systems. In the complex evolution of AMR, biofilms and the acquisition of antimicrobial resistance genes (ARGs) play a pivotal role. Aeromonas is a major AMR player that often forms biofilm, harbors ARGs and is frequently detected in wastewater. Existing wastewater treatment plants (WWTPs) do not have the capacity to totally eliminate antimicrobial-resistant bacteria favoring the evolution of ARGs in wastewater. Besides facilitating the emergence of AMR, biofilms contribute significantly to biofouling process within the activated sludge of WWTP bioreactors. This paper presents the inhibition of biofilm formation, the expression of biofilm-linked genes and ARGs by phytochemicals andrographolide, docosanol, lanosterol, quercetin, rutin and thymohydroquinone. Aeromonas species were isolated and purified from activated sludge samples. The ARGs were detected in the isolated Aeromonas species through PCR. Aeromonas biofilms were quantified following the application of biocompounds through the microtiter plate assay. qPCR analyses of related genes were done for confirmation. Findings showed that the natural compounds inhibited the formation of biofilms and reduced the expression of genes linked to biofilm production as well as ARGs in wastewater Aeromonas. This indicates the efficacy of these compounds in targeting and controlling both ARGs and biofilm formation, highlighting their potential as innovative solutions for combating antimicrobial resistance and biofouling.

Emergence of lineage ST150 and linezolid resistance in Enterococcus faecalis: a molecular epidemiology study of UTIs in Tehran, Iran.

Urinary tract infections (UTIs) represent one of the most prevalent bacterial infections, with Enterococcus species now recognized as the second leading cause of these infections. This study focused on symptomatic UTI cases to investigate the risk factors associated with Enterococcus faecalis clinical isolates in patients from Tehran, Iran. Urine samples were collected from patients presenting with symptomatic UTIs. The identification of E. faecalis isolates was performed using standard microbiological techniques, with confirmation via polymerase chain reaction (PCR). Antibiotic susceptibility testing was conducted using the Kirby-Bauer disc diffusion method. The presence of virulence genes was determined through PCR, and biofilm formation was assessed using the microtiter plate method. Additionally, multi-locus sequence typing (MLST) was utilized to genotype linezolid-resistant isolates. Out of 300 UTI cases, E. faecalis was identified as the causative agent in 160 instances. Notably, a high proportion of these isolates exhibited resistance to tetracycline (83.8%) and minocycline (82.5%). Linezolid resistance was observed in 1.3% (n = 2) of the isolates. Conversely, the highest susceptibility rates were observed for vancomycin, penicillin G, ampicillin, and nitrofurantoin, each demonstrating a 98.8% susceptibility rate. Biofilm formation was detected in 25% of the E. faecalis isolates. A significant majority (93.8%) of the isolates harbored the efbA and ace genes, with varying frequencies of esp (72.5%), asa1 (61.2%), cylA (52.5%), and gelE (88.8%) genes. MLST analysis demonstrated that both linezolid-resistant isolates, characterized by strong biofilm formation and the presence of virulence genes, were assigned to the ST150 lineage, which has not been previously documented in clinical settings. The emergence of the ST150 clonal lineage, underscores its clinical significance, particularly in relation to linezolid resistance in E. faecalis. This study adds to the growing body of evidence linking specific clonal lineages with antibiotic resistance, highlighting the critical need for ongoing surveillance and molecular characterization of resistant pathogens.

A high-throughput protocol for testing heat-stress tolerance in pollen

AbstractViable pollen is crucial for fertilization, but pollen is generally highly susceptible to heat stress. A quick, reliable method for testing the heat-stress tolerance of pollen is needed to improve the heat-stress tolerance in plants, but current methods require considerable space and labor. In addition, many such methods only test tolerance to a single constant temperature, making it time-consuming to screen heat tolerance over a wide temperature range and to examine the dynamics of pollen viability at different temperatures. To address this issue, we aimed to: (1) develop an easy, reliable method for measuring pollen viability at different temperatures; and (2) identify the best temperature range for screening pollen with high heat-stress tolerance. We harvested mature pollen from wheat (Triticum aestivum) plants and transferred it to a 96-well plate filled with liquid medium containing sucrose. We placed the plate in a PCR machine operating under a gradient PCR program to simultaneously test a range of temperatures. After incubating the pollen for 4 h, at temperatures ranging from 21.9 to 47 °C, we examined the pollen grains under a light microscope and employed a specific image analysis pipeline to assess the effects of temperature on pollen morphology, germination, and tube growth. This method facilitated the high-throughput screening of many pollen samples, enabling rapid, reliable, and precise analysis of pollen viability in response to temperature. Our approach should be applicable to other plant species and could be used to identify quantitative trait loci or genes influencing heat stress tolerance in pollen for breeding programs.

Measuring GPCR-Induced Intracellular Calcium Signaling Using a Quantitative High-Throughput Assay.

Alterations in intracellular calcium are integral to signal transduction pathways for many G-protein-coupled receptors, but this signaling is not well studied. This is mostly due to a lack of reliable, robust, high-throughput, quantitative methods to monitor intracellular calcium concentrations in live cells. Recently, we developed a reliable, robust, quantitative method to measure intracellular calcium levels in which HEK293 cell suspensions loaded with Fura-2/AM are placed in 96-well plates. Minimum and maximum intracellular calcium levels, which are required for converting fluorescence into calcium concentrations, are calibrated using EGTA to chelate calcium and ionomycin to load calcium into cells, respectively. Fluorescence is monitored with a PHERAstar FS plate reader. We provide a detailed method for this high-throughput assay that can be used to quantitate intracellular calcium in endogenous and exogenously (stable or transient) expressed GPCRs in HEK293 cells.

Development of a Bioactive Titanium Surface via Alkalinization and Naringenin Coating for Peri-Implant Repair: In Vitro Study

This study assessed the effects of titanium (Ti) surface modification with sodium hydroxide (NaOH) associated or not with Naringenin (NA) citrus flavonoid-coating on osteoblastic-like cells (Ob) metabolism. Ti discs were submitted to alkalinization by NaOH solution (5 M, 60 °C) for 24 h; then, the discs were impregnated or not with 100 µg/mL of NA and dried for 1 h at room temperature. The chemical composition, surface topography, and NA release were evaluated. For the biological assays, the discs were placed on 24-well cell culture plates and Ob (Saos-2; ATCC HTB-85) was seeded onto the discs. After different periods, cell adhesion and viability, alkaline phosphatase activity (ALP), and mineralized nodules deposition (MND) were assessed. In addition, cells stimulated with tumor necrosis factor-alpha (TNF-α) were submitted to matrix metalloproteinase (MMP)-2 synthesis and ALP gene expression assessment. Since data presented normal distribution and homogeneity (Shapiro-Wilk; Levene), Student’s t-test or one-way ANOVA/post-hoc tests were selected for data analysis (α = 0.05). Higher roughness was observed on Ti discs submitted to NaOH treatment, while the chemical and NA release evaluations indicated the successful adsorption of NA to alkali-treated Ti surface. Higher cell adhesion, cell viability (after 7 days of culture), ALP activity, and MND were observed on Ti NaOH coated with NA compared to the control group (Ti NaOH) (p < 0.05). Moreover, NA coating also promoted decreased MMP-2 synthesis and increased ALP gene expression in the presence of the inflammatory stimulus TNF-α (p < 0.05). The modification of Ti disks with NaOH associated with NA-coating enhanced bone cell metabolism, suggesting that this type of surface modification has a promising potential to accelerate bone repair and formation around dental implants.

Evaluation of Antimicrobial Activity of Selected Plant Extracts against Staphylococcus Strains Isolated from Wound Infections.

Wound infections are a significant medical problem, with Staphylococcus species being one of the most common etiological factors. Treatment is complex due to bacterial antibiotic resistance and the ability to form a biofilm. The aim of this study was to determine the drug susceptibility of the clinical isolates of Staphylococcus species obtained from wound infections, assess their ability to form a biofilm in vitro, evaluate the level of extracellular slime synthesis, and test the antistaphylococcal properties of selected plant extracts against plate-cultured bacteria and activity against mature biofilms. A total of 20 Staphylococcus strains were evaluated. The antibiotic susceptibility and sensitivity of the strains to the tested extracts were determined using the disc diffusion method. The production of extracellular bacterial slime was assessed using Congo Red agar plates. The biofilm formation and the effect of plant extracts on the biofilm layer were examined using the MTT method on polystyrene microtiter plates. Diverse drug susceptibility profiles, slime production ability, and in vitro biofilm formation were observed among the tested strains. It was found that aqueous extracts from the Serpylli herba, Arctii lappae folium, Taraxaci folium, and Galii aparini herba showed antibacterial activity against some of the tested strains. In contrast, their antibiofilm activity was not confirmed. Among all tested preparations, the most promising antimicrobial extracts in both planktonic cultures and biofilm were Thymus serpyllum herb and Taraxacum officinale leaf extracts. These results allowed us to conclude their potential application in medicine to support the treatment of challenging wound conditions.

Chemical synthesis of NiO nanoparticles from Solanum trilobatum leaf extract for antibacterial and cytotoxic properties

The chemical precipitation approach was employed to synthesize Nickel oxide nanoparticles (NiONPs) using Solanum trilobatum leaf extract as the stimulant and Nickel nitrate as the precursor. The Nickel oxide is examined using a range of characterization methods including X-ray diffraction, Fourier Transform Infrared spectroscopy, High-Resolution Transmission Electron Microscopy, High-Resolution Scanning Electron Microscopy, X-ray photoelectron spectroscopy, Thermo gravimetric Analysis/Derivative Thermo gravimetric Analysis, Diffuse Reflectance Spectroscopy, cytotoxicity and antimicrobial investigations The X-ray diffraction examination determined that the average size of the crystals increases as the quantities of leaf extract in the NiO2 composites rise. The decrease in line broadening (β) value and the increase in leaf extract concentrations may be the cause of this phenomenon. The FTIR spectrum confirms that the as-synthesized NiO-NPs are of great purity and match well with the XRD pattern. The thermal stability of the synthesized samples was determined using TGA/DTG analysis. The analysis was conducted in an air atmosphere, with the temperature increasing at a rate of 10°C per minute. The temperature range for the analysis was from room temperature to 750°C. The optical properties are determined by the use of Diffuse Reflectance Spectroscopy, which examines the coordinated movement of electrons in the conduction band when exposed to electromagnetic waves. Rat skeletal muscle cell line and SKMEL cancer cells were cultured on 96-well plates and incubated at 37°C and 5 % CO2 for 24 hours to allow them to adapt to the culture conditions. An investigation was conducted to assess the antibacterial properties of synthesized nanocomposites against two types of bacteria: gram-positive Staphylococcus aureus (MTCC No: 87) and gram-negative Escherichia coli (MTCC No: 443), in order to explore their potential for biological applications.

Demonstration of potential DNA contamination introduced by laboratory consumables using Fluorescein

The development of increasingly efficient DNA extraction and profiling kits has increased the amount of allelic information obtained from trace DNA samples, but also inadvertently, increased the detection of DNA contamination. This study aimed to evaluate the potential of DNA transfer using fluorescein, fluorescent under an alternate light source, in the use of a range of forensically relevant DNA profiling consumables. An evaluation of two pre-lysis methods adopting three different sample tubes, some with deliberate seal damage, showed the PrepFiler™ Automated Forensic DNA Extraction Kit caused leakage and crusting when the rim of the PrepFiler™ LySep column was compromised, but no leakage was observed under the same conditions using the Investigator STAR Lyse&Prep kit. The AutoLys tube showed minimal leakage using the PrepFiler™ chemistry. A DNA extract tube with an external thread, similar to the AutoLys tube, showed no leakage after fridge or freezer storage. However, it highlighted that a centrifugal spin does not guarantee all the DNA will pool at the base of the tube. A comparison of adhesive plate sealing films to 8-well strip caps for sealing 96-well PCR plates showed the adhesive plate sealing films presented a lower risk of DNA transfer, largely due to the adhesion of dispersed liquid on the sticky surface of the film. Overall, this study highlighted a number of variables that may be considered in the development of more refined contamination minimisation protocols in respect to increased sensitivities of DNA profiling.

Unbiased high-throughput screening of drug-repurposing libraries identifies small-molecule inhibitors of clot retraction

AbstractPlatelet clot retraction, the ultimate phase of platelet thrombus formation, is critical for clot stabilization. It requires functional αIIbβ3 receptors, fibrin, and the integrated actions of the actin-myosin contractile and cytoskeletal systems. Disturbances in clot retraction have been associated with both bleeding and thrombosis. We recently demonstrated that platelets treated with the αIIbβ3 antagonist peptide RGDW, which eliminates fibrinogen-mediated platelet aggregation, are still able to retract clots. We have exploited this observation to develop an unbiased, functional high-throughput assay to identify small-molecule inhibitors of fibrin-mediated clot retraction adapted for a 384-well plate format. We tested 9710 compounds from drug-repurposing libraries (DRLs). These libraries contain compounds that are either US Food and Drug Administration approved or have undergone preclinical/clinical development. We identified 27 compounds from the Library of Pharmacologically Active Compounds library as inhibitors of clot retraction, of which 14 are known inhibitors of platelet function. From the DRLs, we identified 135 compounds (1.6% hit rate). After extensive curation, these compounds were categorized based on the activity of their reported target. Multiple kinase and phosphodiesterase inhibitors with known antiplatelet effects were identified, along with multiple deubiquitination and receptor inhibitors, as well as compounds that have not previously been reported to have antiplatelet activity. Studies of 1 of the deubiquitination inhibitors (degrasyn) suggest that its effects are downstream of thrombin-induced platelet-fibrinogen interactions and thus may permit the separation of platelet thrombin-induced aggregation-mediated events from clot retraction. Additional studies of the identified compounds may lead to novel mechanisms of inhibiting thrombosis.

Purification Of Bacteriocin Produced By Staphylococcus Aureus And Its Effect On Some Candida Spp.

Candida is the most implicated fungal pathogen in the clinical setting. Virulence factors, such as biofilms play important roles in its pathogenesis. Hence, this study aimed to investigate role of staphylococcal bacteriocin in inhibit biofilm and viability of Candida spp. Seventy-two isolates were collected from abscess, wounds and burns . All isolates were reidentified using different examinations. In addition, eight species of previously isolated clinical Candida species, from oral and vaginal cavities of patients with candidiasis, including C. albicans, C. glabrata, C. parasilosis, C. krusei, C. lusitaniae, C. kefyr, C. tropicalis and C. ciferrii, were utilized to check the antimicrobial activity of S. aureus on Candida spp. Agar well diffusion method were used to detect bacteriocin production by S. aureus, whereas only 5 isolates were selected. The antibacterial effect of selected S. aureus isolates were tested against indicators (Pseudomonas aeruginosa, Escherichia coli and Candida albicans). Isolate of S. aureus no.2 was showed the best bacteriocin production. Bacteriocin was purified using ammonium sulfate and dialysis. Different concentrations was utilized to determine sub-minimal inhibitory concentration (sub-MIC) of bacteriocin. Sub-MIC of bacteriocin was utilized to estimate its inhibitory effect on viability of Candida spp. Bacteriocin showed the maximal inhibition percentage against C. parapsilosis CV26 with 85.42%, followed by C. tropicalis CO49 with 80.49%. The viability of C. kefyr CO45, C. krusei CO41, C. glabrata CO38, C. albicans CV5 and C. lusitaniae was inhibited by 77.78, 75.41, 74.67, 71.43 and 70.18%, respectively. In addition, 69.84 and 64.62% of C. albicans CV1 and C. ciferrii. Using microtiter plate, sub-MIC of bacteriocin showed maximal inhibitory effect against C. ciferrii with 82.26%, followed by C. lusitaniae with 80.4%, the biofilm formation of C. parapsilosis CV26 and C. tropicalis CO49 was inhibited by 80.49% and 79.82%, respectively.

Standardisation of high throughput microdilution antifungal susceptibility testing for Candida albicans and Cryptococcus neoformans.

The Clinical and Laboratory Standards Institute (CLSI) M27 guidelines are the recommended and most commonly used protocols for broth microdilution antifungal susceptibility testing of yeasts. However, these guidelines are limited to the use of 96-well assay plates, limiting assay capacity. With the increased risk of fungal resistance emerging in the community, it is important to have alternative protocols available, that offer higher throughput and can screen more than eight to ten potential antifungal compounds per plate. This study presents an optimised broth microdilution minimum inhibitory concentration (MIC) method for testing the susceptibility of yeasts in an efficient high throughput screening setup, with minimal growth variability and maximum reproducibility. We extend the M27 guidelines and optimise the conditions for 384-well plates. Validation of the assay was performed with ten clinically used antifungals (fluconazole, amphotericin B, 5-fluorocytosine, posaconazole, voriconazole, ketoconazole, itraconazole, caspofungin diacetate, anidulafungin and micafungin) against Candida albicans and Cryptococcus neoformans.

Environmentally Controlled Microfluidic System Enabling Immune Cell Flow and Activation in an Endothelialised Skin-On-Chip.

Integration of reconstructed human skin (RhS) into organ-on-chip (OoC) platforms addresses current limitations imposed by static culturing. This innovation, however, is not without challenges. Microfluidic devices, while powerful, often encounter usability, robustness, and gas bubble issues that hinder large-scale high-throughput setups. This study aims to develop a novel re-usable multi-well microfluidic adaptor (MMA) with the objective to provide a flexible tool for biologists implementing complex 3D biological models (e.g., skin) while enabling simultaneous user control over temperature, medium flow, oxigen (O2), nitrogen (N2), and carbon dioxide (CO2) without the need for an incubator. The presented MMA device is designed to be compatible with standard, commercially available 6-well multi-well plates (6MWPs) and 12-well transwells. This MMA-6MWP setup is employed to generate a skin-on-chip (SoC). RhS viability is maintained under flow for three days and their morphology closely resembles that of native human skin. A proof-of-concept study demonstrates the system's potential in toxicology applications by combining endothelialised RhS with flowing immune cells. This dynamic setting activates the monocyte-like MUTZ-3 cells (CD83 and CD86 upregulation) upon topical exposure of RhS to a sensitizer, revealing the MMA-6MWP's unique capabilities compared to static culturing, where such activation is absent.

6651 A Traceable Clinical Free Thyroxine Assay Based on Equilibrium Dialysis ID-LC/MS/MS

Abstract Disclosure: D. Spector: None. L. Zhang: None. A. Ribera: None. A. Doty: None. C. Tse: None. O. Sugahara: None. N. Vazquez: None. U. Danilenko: None. H.W. Vesper: None. Accurate diagnosis and effective treatment of thyroid diseases rely on reliable thyroid function tests. Serum free thyroxine (FT4) immunoassays (IAs) are most commonly used for FT4 measurements in clinical practice. However, FT4 IAs have not been standardized with large variations between different assay platforms. A reference measurement procedure (RMP) for FT4 based on well-defined equilibrium dialysis (ED) of serum has been established for CDC standardization program to support the standardization of clinical assays for patient care. In the current study, we aim to develop a routine clinical FT4 assay based on ED-LC/MS/MS that is traceable to the RMP. ED-LC/MS/MS analysis was performed using a commercially available micro-ED plate. FT4 in dialysate was extracted with a 96-well C18 SPE plate. FT4 in the samples was quantitated by using LC/MS/MS in positive ion mode. The assay was calibrated with the certified primary reference material. By using RMP as a reference, we compared the analysis of 40 single donor sera between the ED-LC/MS/MS assay and FT4 IA on Roche e401platform. ED-LC/MS/MS assay could resolve T4, T3, reverse triiodothyronine (rT3), and other interferences in the samples with C18 chromatography in 4 min. The linear range of the routine FT4 assay was from 0.5-100 pg/mL covering clinically relevant FT4 concentrations including hypo-, hyperthyroid patient samples. The intra and inter-run CV% of the assay in 20 days was 3.72 and 5.25%, respectively. The effect of minor changes to the method conditions was studied. It was determined that serum/buffer ratios from 1:1 to 1:2 in the ED inserts, speeds of shaker for ED, and ED incubation time, did not influence the FT4 measurement. However, elevated ED temperature at 38°C could result in a more than 10% increase in FT4 values. A microplate format for the procedures of ED and SPE is amiable to the application of an automation system which significantly improves the throughput of the procedure. Method comparison showed good agreement between ED-LC/MS/MS assay and RMP in Deming regression analysis with a slope close to 1, while there was significant bias of IA from RMP. ED-LC/MS/MS assay was also applied for pregnancy serum samples with minimal difference on the FT4 measurement from RMP. We have developed a routine clinical FT4 assay that is traceable to RMP. The accuracy, precision, and sensitivity of the assay are suitable for high-throughput FT4 measurements in clinical laboratories and large epidemiologic studies. We demonstrate that the developed method can provide accurate FT4 measurements in patients including pregnant women. Presentation: 6/3/2024