Phototoxicity of BODIPY in long-term imaging can be reduced by intramolecular motion.

For long-term live-cell fluorescence imaging and biosensing, it is crucial to work with a dye that has high fluorescence quantum yield and photostability without being detrimental to the cells. In this paper, we demonstrate that neutral boron-dipyrromethene (BODIPY)-based molecular rotors have great properties for high-light-dosage demanding live-cell fluorescence imaging applications that require repetitive illuminations. In molecular rotors, an intramolecular rotation (IMR) allows an alternative route for the decay of the singlet excited state (S1) via the formation of an intramolecular charge transfer state (CT). The occurrence of IMR reduces the probability of the formation of a triplet state (T1) which could further react with molecular oxygen (3O2) to form cytotoxic reactive oxygen species, e.g., singlet oxygen (1O2). We demonstrate that the oxygen-related nature of the phototoxicity for BODIPY derivatives can be significantly reduced if a neutral molecular rotor is used as a probe. The studied neutral molecular rotor probe shows remarkably lower phototoxicity when compared with both the non-rotating BODIPY derivative and the cationic BODIPY-based molecular rotor in different light dosages and dye concentrations. It is also evident that the charge and localization of the fluorescent probe are as significant as the IMR in terms of the phototoxicity in a long-term live-cell imaging.

Synthesis and Cytotoxicity Evaluation of Spirocyclic Bromotyrosine Clavatadine C Analogs.

Marine-originated spirocyclic bromotyrosines are considered as promising scaffolds for new anticancer drugs. In a continuation of our research to develop potent and more selective anticancer compounds, we synthesized a library of 32 spirocyclic clavatadine analogs by replacing the agmatine, i.e., 4-(aminobutyl)guanidine, side chain with different substituents. These compounds were tested for cytotoxicity against skin cancer using the human melanoma cell line (A-375) and normal human skin fibroblast cell line (Hs27). The highest cytotoxicity against the A-375 cell line was observed for dichloro compound 18 (CC50 0.4 ± 0.3 µM, selectivity index (SI) 2). The variation of selectivity ranged from SI 0.4 to reach 2.4 for the pyridin-2-yl derivative 29 and hydrazide analog of 2-picoline 37. The structure-activity relationships of the compounds in respect to cytotoxicity and selectivity toward cancer cell lines are discussed.

Targeting Quorum Sensing: High-Throughput Screening to Identify Novel LsrK Inhibitors.

Since quorum sensing (QS) is linked to the establishment of bacterial infection, its inactivation represents one of the newest strategies to fight bacterial pathogens. LsrK is a kinase playing a key role in the processing of autoinducer-2 (AI-2), a quorum-sensing mediator in gut enteric bacteria. Inhibition of LsrK might thus impair the quorum-sensing cascade and consequently reduce bacterial pathogenicity. Aiming for the development of a target-based assay for the discovery of LsrK inhibitors, we evaluated different assay set-ups based on ATP detection and optimized an automation-compatible method for the high-throughput screening of chemical libraries. The assay was then used to perform the screening of a 2000-compound library, which provided 12 active compounds with an IC50 ≤ 10 µM confirming the effectiveness and sensitivity of our assay. Follow-up studies on the positive hits led to the identification of two compounds, harpagoside and rosolic acid, active in a cell-based AI-2 QS interference assay, which are at the moment the most promising candidates for the development of a new class of antivirulence agents based on LsrK inhibition.

Synthesis and Antiproliferative Activity of Marine Bromotyrosine Purpurealidin I and Its Derivatives.

The first total synthesis of the marine bromotyrosine purpurealidin I (1) using trifluoroacetoxy protection group and its dimethylated analog (29) is reported along with 16 simplified bromotyrosine derivatives lacking the tyramine moiety. Their cytotoxicity was evaluated against the human malignant melanoma cell line (A-375) and normal skin fibroblast cells (Hs27) together with 33 purpurealidin-inspired simplified amides, and the structure⁻activity relationships were investigated. The synthesized simplified analogs without the tyramine part retained the cytotoxic activity. Purpurealidin I (1) showed no selectivity but its simplified pyridin-2-yl derivative (36) had the best improvement in selectivity (Selectivity index 4.1). This shows that the marine bromotyrosines are promising scaffolds for developing cytotoxic agents and the full understanding of the elements of their SAR and improving the selectivity requires further optimization of simplified bromotyrosine derivatives.

Effect of differentiation on endocytic profiles of endothelial and epithelial cell culture models.

Understanding mechanisms of endocytosis and trafficking of nanoparticles through endothelial and epithelial barriers leads potentially to improved efficacy of nanoparticulate drug delivery systems. Detailed characterizations of cell models with respect to endocytic pathway expression and activity (endocytic profiling) should facilitate data interpretation. We performed endocytic profiling of CaCo-2 and hCMEC/D3 cell lines, widely used as human intestinal and blood-brain barrier permeability models, respectively, during cell differentiation. Furthermore, we compared endocytic profiles of cell lines with those of primary cells. Expression of genes involved in specific endocytic pathways was analyzed at mRNA levels by quantitative real time PCR. Where possible, the respective protein levels were analyzed by Western blotting, and endocytic activities of cells were analyzed by flow cytometry. We showed that differentiated CaCo-2 cells formed tight, well polarized monolayers with reduced endocytic activity accompanied by reduced mRNA expression of most of the endocytosis-related genes. In contrast, hCMEC/D3 cells formed a leaky, less polarized barrier, and in vitro differentiation had little effect on either the expression of endocytosis-related genes or endocytic activity of these cells. Endocytic profiling of in vitro models and comparison with primary cells is an important measure to avoid misleading conclusions in nanoparticle permeation studies.

Highly scalable and standardized organ-on-chip platform with TEER for biological barrier modeling.

The development of new therapies is hampered by the lack of predictive, and patient-relevant in vitro models. Organ-on-chip (OOC) technologies can potentially recreate physiological features and hold great promise for tissue and disease modeling. However, the non-standardized design of these chips and perfusion control systems has been a barrier to quantitative high-throughput screening (HTS). Here we present a scalable OOC microfluidic platform for applied kinetic in vitro assays (AKITA) that is applicable for high, medium, and low throughput. Its standard 96-well plate and 384-well plate layouts ensure compatibility with existing laboratory workflows and high-throughput data collection and analysis tools. The AKITA plate is optimized for the modeling of vascularized biological barriers, primarily the blood-brain barrier, skin, and lung, with precise flow control on a custom rocker. The integration of trans-epithelial electrical resistance (TEER) sensors allows rapid and repeated monitoring of barrier integrity over long time periods. Together with automated liquid handling and compound permeability testing analyses, we demonstrate the flexibility of the AKITA platform for establishing human-relevant models for preclinical drug and precision medicine's efficacy, toxicity, and permeability under near-physiological conditions.

Willow Bark-Derived Material with Antibacterial and Antibiofilm Properties for Potential Wound Dressing Applications.

Tree stems contain wood in addition to 10-20% bark, which remains one of the largest underutilized biomasses on earth. Unique macromolecules (like lignin, suberin, pectin, and tannin), extractives, and sclerenchyma fibers form the main part of the bark. Here, we perform detailed investigation of antibacterial and antibiofilm properties of bark-derived fiber bundles and discuss their potential application as wound dressing for treatment of infected chronic wounds. We show that the yarns containing at least 50% of willow bark fiber bundles significantly inhibit biofilm formation by wound-isolated Staphylococcus aureus strains. We then correlate antibacterial effects of the material to its chemical composition. Lignin plays the major role in antibacterial activity against planktonic bacteria [i.e., minimum inhibitory concentration (MIC) 1.25 mg/mL]. Acetone extract (unsaturated fatty acid-enriched) and tannin-like (dicarboxylic acid-enriched) substances inhibit both bacterial planktonic growth [MIC 1 and 3 mg/mL, respectively] and biofilm formation. The yarn lost its antibacterial activity once its surface lignin reached 20.1%, based on X-ray photoelectron spectroscopy. The proportion of fiber bundles at the fabricated yarn correlates positively with its surface lignin. Overall, this study paves the way to the use of bark-derived fiber bundles as a natural-based material for active (antibacterial and antibiofilm) wound dressings, upgrading this underappreciated bark residue from an energy source into high-value pharmaceutical use.

Antibacterial and antibiofilm activity of permanently ionized quaternary ammonium fluoroquinolones.

A series of quaternary ammonium fluoroquinolones was obtained by exhaustive methylation of the amine groups present at the 7-position of fluoroquinolones, including ciprofloxacin, enoxacin, gatifloxacin, lomefloxacin, and norfloxacin. The synthesized molecules were tested for their antibacterial and antibiofilm activities against Gram-positive and Gram-negative human pathogens, i.e. Staphylococcus aureus and Pseudomonas aeruginosa. The study showed that the synthesized compounds are potent antibacterial agents (MIC values at the lowest 6.25 µM) with low cytotoxicity in vitro as assessed on the BALB 3T3 mouse embryo cell line. Further experiments proved that the tested derivatives are able to bind to the DNA gyrase and topoisomerase IV active sites in a fluoroquinolone-characteristic manner. The most active quaternary ammonium fluoroquinolones, in contrast to ciprofloxacin, reduce the total biomass of P. aeruginosa ATCC 15442 biofilm in post-exposure experiments. The latter effect may be due to the dual mechanism of action of the quaternary fluoroquinolones, which also involves disruption of bacterial cell membranes. IAM-HPLC chromatographic experiments with immobilized artificial membranes (phospholipids) showed that the most active compounds were those with moderate lipophilicity and containing a cyclopropyl group at the N1 nitrogen atom in the fluoroquinolone core.

Effect of Hybrid Type and Harvesting Season on Phytochemistry and Antibacterial Activity of Extracted Metabolites from Salix Bark.

Hundreds of different fast-growing Salix hybrids have been developed mainly for energy crops. In this paper, we studied water extracts from the bark of 15 willow hybrids and species as potential antimicrobial additives. Treatment of ground bark in water under mild conditions extracted 12-25% of the dry material. Preparative high-performance liquid chromatography is proven here as a fast and highly efficient tool in the small-scale recovery of raffinose from Salix bark crude extracts for structural elucidation. Less than half of the dissolved material was assigned by chromatographic (gas chromatography and liquid chromatography) and spectroscopic (mass spectrometry and nuclear magnetic resonance spectroscopy) techniques for low-molecular-weight compounds, including mono- and oligosaccharides (sucrose, raffinose, and stachyose) and aromatic phytochemicals (triandrin, catechin, salicin, and picein). The composition of the extracts varied greatly depending on the hybrid or species and the harvesting season. This information generated new scientific knowledge on the variation in the content and composition of the extracts between Salix hybrids and harvesting season depending on the desired molecule. The extracts showed high antibacterial activity on Staphylococcus aureus with a minimal inhibitory concentration (MIC) of 0.6-0.8 mg/mL; however, no inhibition was observed against Escherichia coli, Enterococcus faecalis, and Salmonella typhimurium. MIC of triandrin (i.e., 1.25 mg/mL) is reported for the first time. Although antibacterial triandrin and (+)-catechin were present in extracts, clear correlation between the antibacterial effect and the chemical composition was not established, which indicates that antibacterial activity of the extracts mainly originates from some not yet elucidated substances. Aquatic toxicity and mutagenicity assessments showed the safe usage of Salix water extracts as possible antibacterial additives.

Development and validation of a high-content screening assay for inhibitors of enteropathogenic E. coli adhesion.

Enteropathogenic E. coli (EPEC) causes intestinal infections leading to severe diarrhea. EPEC attaches to the host cell causing lesions to the intestinal epithelium coupled with the effacement of microvilli. In the process, actin accumulates into a pedestal-like structure under bacterial microcolonies. We designed an automated fluorescence microscopy-based screening method for discovering compounds capable of inhibiting EPEC adhesion and virulence using aurodox, a type three secretion system (T3SS) inhibitor, as a positive control. The screening assay employs an EPEC strain (2348/69) expressing a fluorescent protein and actin staining for monitoring the bacteria and their pedestals respectively, analyzing these with a custom image analysis pipeline. The assay allows for the discovery of compounds capable of preventing the formation of pathogenic actin rearrangements. These compounds may be interfering with virulence-related molecular pathways relevant for developing antivirulence leads.

Miniaturized whole-cell bacterial bioreporter assay for identification of quorum sensing interfering compounds.

The continuing emergence and spread of antibiotic-resistant bacteria is worrisome and new strategies to curb bacterial infections are being sought. The interference of bacterial quorum sensing (QS) signaling has been suggested as a prospective antivirulence strategy. The AI-2 QS system is present in multiple bacterial species and has been shown to be correlated with pathogenicity. To facilitate the discovery of novel compounds interfering with AI-2 QS, we established a high-throughput setup of whole-cell bioreporter assay, which can be performed in either 96- or 384-well format. Agonistic or antagonistic activities of the test compounds against Escherichia coli LsrB-type AI-2 QS system are monitored by measuring the level of ß-galactosidase expression. A control strain expressing ß-galactosidase in quorum sensing-independent manner is included into the assay for false-positive detection.

Hyaluronic Acid Graft Copolymers with Cleavable Arms as Potential Intravitreal Drug Delivery Vehicles.

Treatment of retinal diseases currently demands frequent intravitreal injections due to rapid clearance of the therapeutics. The use of high molecular weight polymers can extend the residence time in the vitreous and prolong the injection intervals. This study reports a water soluble graft copolymer as a potential vehicle for sustained intravitreal drug delivery. The copolymer features a high molecular weight hyaluronic acid (HA) backbone and poly(glyceryl glycerol) (PGG) side chains attached via hydrolysable ester linkers. PGG, a polyether with 1,2-diol groups in every repeating unit available for conjugation, serves as a detachable carrier. The influence of synthesis conditions and incubation in physiological media on the molecular weight of HA is studied. The cleavage of the PGG grafts from the HA backbone is quantified and polymer-from-polymer release kinetics are determined. The biocompatibility of the materials is tested in different cell cultures.

Genetic blockage of endocytic pathways reveals differences in the intracellular processing of non-viral gene delivery systems.

Detailed understanding of the uptake mechanisms and intracellular processing of nonviral gene delivery systems will allow design of more effective carriers. This work gets insight into the intracellular kinetics of pDNA delivered by polyethyleneimine (PEI), cationic lipid DOTAP and calcium phosphate (CaP) precipitates. Amount of cell- and nuclear-associated pDNA was quantified by qRT-PCR at multiple time points after transfection. Moreover, the impact of specific endocytic pathways on the cell entry and intracellular kinetics of pDNA was studied by inhibition (blockage) of either clathrin- or dynamin-mediated endocytosis by using both genetically manipulated cell lines and chemical inhibitors of endocytosis. Quantitative analysis of defined kinetic parameters revealed that neither cellular nor nuclear uptake of pDNA correlated with transgene expression, emphasizing the importance of the post-nuclear processes in overall transfection efficacy. Changes in transgene expression observed upon blockage of endocytosis was carrier dependent and correlated relatively well with the changes at the cellular and nuclear uptake levels but not with the amount of cell-associated pDNA. Due to low specificity of chemical inhibitors and activation of alternative endocytosis pathways after genetic blockage of endocytosis neither of these methods is optimal for studying the role of endocytosis. Therefore, one should be careful when interpreting the obtained results from such studies and not to trust the data obtained only from one method.