Chitosan/Pluronic F127 Thermosensitive Hydrogel as an Injectable Dexamethasone Delivery Carrier

Intra-articular administration of anti-inflammatory drugs is a strategy that allows localized action on damaged articular cartilage and reduces the side effects associated with systemic drug administration. The objective of this work is to prepare injectable thermosensitive hydrogels for the long-term application of dexamethasone. The hydrogels were prepared by mixing chitosan (CS) and Pluronic-F127 (PF) physically. In addition, tripolyphosphate (TPP) was used as a crosslinking agent. Chitosan added to the mix increased the gel time compared to the pluronic gel alone. The incorporation of TPP into the material modified the morphology of the hydrogels formed. Subsequently, MTS and Live/Dead® experiments were performed to investigate the toxicity of hydrogels against human chondrocytes. The in vitro releases of dexamethasone (DMT) from CS-PF and CS-PF-TPP gels had an initial burst and took more time than that from the PF hydrogel. In vivo studies showed that hydrogels retained the fluorescent compound longer in the joint than when administered in PBS alone. These results suggest that the CS-PF and CS-PF-TPP hydrogels loaded with DMT could be a promising drug delivery platform for the treatment of osteoarthritis.

Isolation of Taxol and Flavin-like fluorochrome from Endophytic Fungi of Mangifera indica

Scouting for novel and plant-derived biomolecules from endophytic microbial sources draws greater focus on the discovery of novel bioactive metabolites. With this rationale, we scouted the endophytic fungi for taxol, an anticancer diterpenoid and fluorescent biomolecules. In the present study, about 31 endophytic fungal isolates recovered from the Mangifera indica leaves were screened for taxol production in M1D medium. About five isolates were shortlisted based on the thin layer chromatographic analysis of the fungal extracts. Among them Colletotrichum sp. MIP-5 has been identified as a producer of fungal taxol based on UV, FTIR, TLC and HPLC analysis. The partially purified fungal taxol showed similar spectral and chromatographic features of commercially available paclitaxel. In addition to this, we also report the production of a fluorescent compound by Penicillium sp. MIP-3. The Flavin-like compound exhibited a bright greenish-yellow fluorescence with an emission maximum in the range of 505 – 545nm. GC-MS analysis showed the occurrence of Latia luciferin, primarily associated with the bioluminescence of freshwater limpet Latia neritoides. This is the first report of this compound from Penicillium sp. In addition, therapeutically active steroid (β-Sitosterol, Stigmasterol, Campesterol), quinones (Benzo[h]quinoline, 2,4-dimethyl-) and phloroglucinol (Aspidinol) derivatives were also identified from Penicillium sp. MIP-3 based on GC-MS analysis. These molecules could potentially be used in biological and pharmaceutical applications in future.

Characterization of fluorescent probe substrates to develop an efficient high-throughput assay for neonatal hepatic CYP3A7 inhibition screening

CYP3A7 is a member of the cytochrome P450 (CYP) 3A enzyme sub-family that is expressed in the fetus and neonate. In addition to its role metabolizing retinoic acid and the endogenous steroid dehydroepiandrosterone sulfate (DHEA-S), it also has a critical function in drug metabolism and disposition during the first few weeks of life. Despite this, it is generally ignored in the preclinical testing of new drug candidates. This increases the risk for drug-drug interactions (DDI) and toxicities occurring in the neonate. Therefore, screening drug candidates for CYP3A7 inhibition is essential to identify chemical entities with potential toxicity risks for neonates. Currently, there is no efficient high-throughput screening (HTS) assay to assess CYP3A7 inhibition. Here, we report our testing of various fluorescent probes to assess CYP3A7 activity in a high-throughput manner. We determined that the fluorescent compound dibenzylfluorescein (DBF) is superior to other compounds in meeting the criteria considered for an efficient HTS assay. Furthermore, a preliminary screen of an HIV/HCV antiviral drug mini-library demonstrated the utility of DBF in a HTS assay system. We anticipate that this tool will be of great benefit in screening drugs that may be used in the neonatal population in the future.

Detecting reactive oxygen species in free-living symbiotic dinoflagellates exposed to nanoparticles v1

2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) is a popular fluorescent probe for the detection of oxidative stress in cells. Since the probe can be prone to auto-oxidation, carboxy-2',7'-dichlorodihydrofluorescein-diacetate (carboxy-H2DCF-DA) which is more stable and easily penetrates cell membranes is often reported in the literature instead. Upon crossing the cell membrane, esterases hydrolyse DCFH-DA to DCFH, which remains trapped within cells. The oxidation of DCFH yields DCF, a fluorescent compound which can be measured using excitation/emission wavelengths of 485-495/520-530 nm. Due to several cases of interference when used in cellular systems, DCFH-DA is a general marker of the cellular oxidative stress rather than a specific indicator H2O2 formation or other ROS.

Melatonin, a Potential Therapeutic Agent for Preeclampsia, Reduces the Extrusion of Toxic Extracellular Vesicles from Preeclamptic Placentae

Preeclampsia, characterised by maternal endothelial cell activation, is triggered by toxic factors, such as placental extracellular vesicles (EVs) from a dysfunctional placenta. The increased oxidative stress seen in the preeclamptic placenta links to endoplasmic reticulum (ER) stress. The ER regulates protein folding and trafficking. When the ER is stressed, proteins are misfolded, and misfolded proteins are toxic. Misfolded proteins can be exported from cells, via EVs which target to other cells where the misfolded proteins may also be toxic. Melatonin is a hormone and antioxidant produced by the pineal gland and placenta. Levels of melatonin are reduced in preeclampsia. In this study we investigated whether melatonin treatment can change the nature of placental EVs that are released from a preeclamptic placenta. EVs were collected from preeclamptic (n = 6) and normotensive (n = 6) placental explants cultured in the presence or absence of melatonin for 18 h. Misfolded proteins were measured using a fluorescent compound, Thioflavin-T (ThT). Endothelial cells were exposed to placental EVs overnight. Endothelial cell activation was measured by the quantification of cell-surface ICAM-1 using a cell-based ELISA. EVs from preeclamptic placentae carried significantly (p < 0.001) more misfolded proteins than normotensive controls. Incubating preeclamptic placental explants in the presence of melatonin (1 µM and 10 µM) significantly (p < 0.001) reduced the misfolded proteins carried by EVs. Culturing endothelial cells in the presence of preeclamptic EVs significantly increased the expression of ICAM-1. This increased ICAM-1 expression was significantly reduced when the endothelial cells were exposed to preeclamptic EVs cultured in the presence of melatonin. This study demonstrates that melatonin reduces the amount of misfolded proteins carried by EVs from preeclamptic placentae and reduces the ability of these EVs to activate endothelial cells. Our study provides further preclinical support for the use of melatonin as a treatment for preeclampsia.

Senescent Changes and Endoplasmic Reticulum Stress May Be Involved in the Pathogenesis of Missed Miscarriage

BackgroundSenescence is involved in many complications of pregnancy. However, whether senescent changes are also associated with missed miscarriage has not been fully investigated.MethodsThe levels of p16, p21, and γH2AX, markers of senescence, were measured in placentas collected from women with missed miscarriage by immunohistochemistry and Western blotting. Levels of misfolded proteins in missed miscarriage placentas or normal first-trimester placenta that had been treated with H2O2 (100 μM) or extracellular vesicles (EVs) collected from missed miscarriage placental explant culture were measured by fluorescent compound, thioflavin-T. The production of reactive oxygen species (ROS) by missed miscarriage placentas was measured by CellROX® Deep Red.ResultsIncreased levels of p16, p21, and γH2AX were presented in missed miscarriage placentas compared to controls. Increased levels of misfolded proteins were shown in missed miscarriage placentas, but not in EVs that were collected from missed miscarriage placentas. The ROS production was significantly increased in missed miscarriage placental explant cultures. Increased levels of misfolded proteins were seen in the normal first-trimester placenta that had been treated with H2O2 compared to untreated.ConclusionOur data demonstrate that there are increases in senescence and endoplasmic reticulum stress and ROS production in missed miscarriage placenta. Oxidative stress and an accumulation of misfolded proteins in missed miscarriage placentas may contribute to the changes of senescence and endoplasmic reticulum stress seen in missed miscarriage placentas.

Changing the layout of farmers markets can affect cross-contamination

PurposeThe purpose of this study was to investigate whether the layout has an effect on cross-contaminations levels at farmers markets.Design/methodology/approachWe used social cognitive theory's triadic reciprocity model to investigate how influencing the environment could change the behaviors of farmers’ market consumers and reduce the risk of microbial cross-contamination using a Fluorescent Compound (FC). For this purpose, a 3 × 2 experimental between-subject factorial design was utilized in this study: three farmers market layouts (i.e. U-shaped [U-S], L-shaped [L-S] and square-shaped [S–S]) and two different set-ups per market (i.e. produce and non-produce vendors completely separated, and alternating produce and non-produce vendors). FC was utilized to simulate microbial contamination on the participants (n = 54) hands. The participants were allowed to walk through the layout for 3 min and touch items after which a total of 475 swab samples were processed and recorded for absorbance levels.FindingsThe results indicated that the cross-contamination level of the U-S market was significantly lower (p < 0.001) than those of the L-S and S–S markets. The best market layout and set-up based on the average levels of simulated cross-contamination were the U-S market, particularly with the A set-up, where produce and non-produce booths were scattered.Originality/valueThis study is the first to use the quantification of FC to identify the impact of a farmers’ market layout/design on cross-contamination levels. These results can be used to provide guidance to market managers on layout and design from a safety standpoint to reduce the risk of cross-contamination.

Bioprospecting Fluorescent Plant Growth Regulators from Arabidopsis to Vegetable Crops

The phytohormone auxin is involved in almost every process of a plant’s life, from germination to plant development. Nowadays, auxin research connects synthetic chemistry, plant biology and computational chemistry in order to develop innovative and safe compounds to be used in sustainable agricultural practice. In this framework, we developed new fluorescent compounds, ethanolammonium p-aminobenzoate (HEA-pABA) and p-nitrobenzoate (HEA-pNBA), and investigated their auxin-like behavior on two main commercial vegetables cultivated in Europe, cucumber (Cucumis sativus) and tomato (Solanumlycopersicum), in comparison to the model plant Arabidopsis (Arabidopsis thaliana). Moreover, the binding modes and affinities of two organic salts in relation to the natural auxin indole-3-acetic acid (IAA) into TIR1 auxin receptor were investigated by computational approaches (homology modeling and molecular docking). Both experimental and theoretical results highlight HEA-pABA as a fluorescent compound with auxin-like activity both in Arabidopsis and the commercial cucumber and tomato. Therefore, alkanolammonium benzoates have a great potential as promising sustainable plant growth stimulators to be efficiently used in vegetable crops.

Bioprospecting Fluorescent Plant Growth Regulators from Arabidopsis to Vegetable Crops

The phytohormone auxin is involved in almost every process of a plant&rsquo;s life, from germination to plant development. Nowadays, auxin research connects synthetic chemistry, plant biology, and computational chemistry in order to develop innovative and safe compounds to be used in sustainable agricultural practice. In this framework, we developed new environmentally-friendly fluorescent compounds, ethanolammonium p-aminobenzoate (HEA-pABA) and p-nitrobenzoate (HEA-pNBA), and investigated their auxin-like behavior on two main commercial vegetables cultivated in Europe, cucumber (Cucumis sativus) and tomato (Solanum lycopersicum), in comparison to the model plant Arabidopsis (Arabidopsis thaliana). Moreover, the binding modes and affinities of two organic salts in relation to the natural auxin indole-3-acetic acid (IAA) into TIR1 auxin receptor were investigated by computational approaches (homology modeling and molecular docking). Both experimental and theoretical results highlight HEA-pABA as a fluorescent compound with auxin-like activity both in Arabidopsis and the commercial cucumber and tomato. Therefore, alkanolammonium benzoates have a great potential as promising sustainable plant growth stimulators to be efficiently used in vegetable crops.

ChemFLuo: a web-server for structure analysis and identification of fluorescent compounds

Background Fluorescent detection methods are indispensable tools for chemical biology. However, the frequent appearance of potential fluorescent compound has greatly interfered with the recognition of compounds with genuine activity. Such fluorescence interference is especially difficult to identify as it is reproducible and possesses concentration-dependent characteristic. Therefore, the development of a credible screening tool to detect fluorescent compounds from chemical libraries is urgently needed in early stages of drug discovery. Results In this study, we developed a webserver ChemFLuo for fluorescent compound detection, based on two large and high-quality training datasets containing 4906 blue and 8632 green fluorescent compounds. These molecules were used to construct a group of prediction models based on the combination of three machine learning algorithms and seven types of molecular representations. The best blue fluorescence prediction model achieved with balanced accuracy (BA) = 0.858 and area under the receiver operating characteristic curve (AUC) = 0.931 for the validation set, and BA = 0.823 and AUC = 0.903 for the test set. The best green fluorescence prediction model achieved the prediction accuracy with BA = 0.810 and AUC = 0.887 for the validation set, and BA = 0.771 and AUC = 0.852 for the test set. Besides prediction model, 22 blue and 16 green representative fluorescent substructures were summarized for the screening of potential fluorescent compounds. The comparison with other fluorescence detection tools and theapplication to external validation sets and large molecule libraries have demonstrated the reliability of prediction model for fluorescent compound detection. Conclusion ChemFLuo is a public webserver to filter out compounds with undesirable fluorescent properties, which will benefit the design of high-quality chemical libraries for drug discovery. It is freely available at http://admet.scbdd.com/chemfluo/index/.