Umbu Fruit Peel as Source of Antioxidant, Antimicrobial and α-Amylase Inhibitor Compounds

Herein, the extraction of bioactive compounds from umbu fruit peel was optimized using thermal-assisted solid–liquid extraction. In parallel, antioxidant, antimicrobial, and inhibitory effects against α-amylase of optimized extract were also evaluated. The combination of operational conditions including the temperature (32–74 °C), ethanol concentration (13–97%), and solid/liquid ratio (1:10–1:60; w/v) was employed using a rotational central composite design for optimization. The extracts were evaluated for total phenolic compounds (TPC), total flavonoid compounds (TFC) and antioxidant capacity by ABTS•+, DPPH• and FRAP assays. The bioactive profile of the optimized extract was obtained by ultra-performance liquid chromatography coupled to quadrupole/time-of-flight mass spectrometry in electrospray ionization in both negative and positive modes. The statistically evaluated results showed that the optimal operational conditions for the recovery of bioactive compounds from umbu fruit peel included 74 °C, 37% ethanol, and a solid–liquid ratio of 1:38. Under these conditions, the obtained values were 1985 mg GAE/100 g, 1364 mg RE/100 g, 122 µmol TE/g, 174 µmol/TE g and 468 µmol Fe2+/g for TPC, TFC, ABTS•+, DPPH•, and FRAP assays, respectively. In addition, the optimized extract was effective against Gram-positive and Gram-negative bacteria (MBC ranged from 0.060 to 0.24 mg GAE/mL), as well as it was effective to inhibit α-amylase (IC50 value of 0.076 mg GAE/mL). The optimized extract showed to be mainly constituted by phenolic acids and flavonoids.

DDRE-10. SCREENING OF EPIGENETIC COMPOUNDS IN GLIOMA AND GLIOBLASTOMA IDENTIFIES NOVEL THERAPEUTICS FOR THEIR POTENTIAL TREATMENT

BACKGROUND 28% of primary central nervous system tumors are glioma and glioblastoma. These tumors are responsible for 80% of malignant brain neoplasms and most brain tumor related deaths. Despite modern therapies, patients with grade II gliomas have an average survival of 8-15 years, while patients with grade III tumors have an average survival of 3-5 years, and patients with glioblastoma have an average survival of 12-15 months. The lack of a curative treatment for this group of tumors supports additional research and novel approaches to identify more effective therapies. METHODS In this study, we developed a high-throughput drug screen and culture system to identify epigenetic inhibitor compounds with the potential to reduce glioma and glioblastoma viability. RESULTS We screened 33 tumors: 18 glioblastoma, 8 oligodendroglioma, and 7 astrocytoma. The top three most effective compounds across the full glioma cohort were all HDAC inhibitors; in order from most effective: panobinostat (average tumor viability = 52.5% +/-14.1SD; p=2.16x10-61), LAQ824 (average tumor viability = 58.1% +/-18SD; p=1.48x10-45), and HC Toxin (average tumor viability = 64% +/-21.1SD; p= 1.16x10-33). Additionally, HDAC inhibition was also the most effective across each histopathological glioma type: astrocytoma, oligodendroglioma, and glioblastoma. UNC0631(G9a inhibitor) and JIB-04(KDM inhibitor) were the most effective compounds in the six recurrent tumors, though HDAC inhibition was still significantly effective in this group. We also evaluated drug sensitivity with respect to tumor grade, prior treatment, de novo vs progressive etiology, EGFR amplification, IDH mutation, MGMT methylation, and patient gender. CONCLUSIONS After screening a large glioma cohort against a panel of epigenetic inhibitors, we found HDAC inhibition most effectively reduced tumor viability across all histopathological types and grades. These findings require further in vivo validation.

Biobanked Glioblastoma Patient-Derived Organoids as a Precision Medicine Model to Study Inhibition of Invasion

Glioblastoma (GBM) is highly resistant to treatment and invasion into the surrounding brain is a cancer hallmark that leads to recurrence despite surgical resection. With the emergence of precision medicine, patient-derived 3D systems are considered potentially robust GBM preclinical models. In this study, we screened a library of 22 anti-invasive compounds (i.e., NF-kB, GSK-3-B, COX-2, and tubulin inhibitors) using glioblastoma U-251 MG cell spheroids. We evaluated toxicity and invasion inhibition using a 3D Matrigel invasion assay. We next selected three compounds that inhibited invasion and screened them in patient-derived glioblastoma organoids (GBOs). We developed a platform using available macros for FIJI/ImageJ to quantify invasion from the outer margin of organoids. Our data demonstrated that a high-throughput invasion screening can be done using both an established cell line and patient-derived 3D model systems. Tubulin inhibitor compounds had the best efficacy with U-251 MG cells, however, in ex vivo patient organoids the results were highly variable. Our results indicate that the efficacy of compounds is highly related to patient intra and inter-tumor heterogeneity. These results indicate that such models can be used to evaluate personal oncology therapeutic strategies.

Sewage Sludge as Inhibitor of the Formation of Persistent Organic Pollutants during Incineration

With the objective of suppressing dioxins and furans (PCDD/Fs) emission in municipal solid waste incineration plants (MSWI), different chemical inhibitors have been tested. Among these inhibitors, nitrogen and sulphur compounds can significantly suppress PCDD/Fs formation via de novo synthesis, which gives very interesting results with very little capital investment. In recent years, the possibility of using waste rich in nitrogen and/or sulphur as a source of inhibitor compounds has been considered, and thus has reduced the emissions of pollutants while the waste is treated. The effect of adding sludge from urban sewage treatment plants in three variants has been specially studied: directly mixing the waste, using the decomposition gas of the previously dried sludge, and using the decomposition gas of the sludge together with other inhibitors such as thiourea. Reduction of emissions in laboratory tests using model samples indicated the efficiency to be higher than 99%, using sewage sludge (SS) as an inhibitor whereas, in actual MSWI plants, the efficiency can be as high as 90%.

Translocation of TMEM175 Lysosomal Potassium Channel to the Plasma Membrane by Dynasore Compounds

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson’s disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.

Dual Agents: Fungal Macrocidins and Synthetic Analogues with Herbicidal and Antibiofilm Activities

Eight analogues of the bioherbicides macrocidin A (1) and Z (2) with structural variance in the size of the macrocycle, its para- or meta-cyclophane character, and its functional groups were synthesized on two modular routes and tested for herbicidal, antibiotic, and antibiofilm activities. Apart from the lead compounds 1 and 2, the structurally simplified dihydromacrocidin Z (3) and normacrocidin Z (4) showed high herbicidal activity in either thistles, dandelions or in both. The derivatives 2, 3, and dibromide 9 also inhibited the growth of Staphylococcus aureus biofilms by ca 70% when applied at subtoxic concentrations as low as ca 20 µM, which are unlikely to induce bacterial resistance. They also led to the dispersion of preformed biofilms of S. aureus, exceeding a similar effect by microporenic acid A, a known biofilm inhibitor. Compounds 3 and 9 showed no noticeable cytotoxicity against human cancer and endothelial cells at concentrations below 50 µM, making them conceivable candidates for application as anti-biofilm agents in a medicinal context.

Characterization of Compound-Specific, Concentration-Independent Biophysical Properties of Sodium Channel Inhibitor Mechanism of Action Using Automated Patch-Clamp Electrophysiology

We have developed an automated patch-clamp protocol that allows high information content screening of sodium channel inhibitor compounds. We have observed that individual compounds had their specific signature patterns of inhibition, which were manifested irrespective of the concentration. Our aim in this study was to quantify these properties. Primary biophysical data, such as onset rate, the shift of the half inactivation voltage, or the delay of recovery from inactivation, are concentration-dependent. We wanted to derive compound-specific properties, therefore, we had to neutralize the effect of concentration. This study describes how this is done, and shows how compound-specific properties reflect the mechanism of action, including binding dynamics, cooperativity, and interaction with the membrane phase. We illustrate the method using four well-known sodium channel inhibitor compounds, riluzole, lidocaine, benzocaine, and bupivacaine. Compound-specific biophysical properties may also serve as a basis for deriving parameters for kinetic modeling of drug action. We discuss how knowledge about the mechanism of action may help to predict the frequency-dependence of individual compounds, as well as their potential persistent current component selectivity. The analysis method described in this study, together with the experimental protocol described in the accompanying paper, allows screening for inhibitor compounds with specific kinetic properties, or with specific mechanisms of inhibition.

Compound-specific, concentration-independent biophysical properties of sodium channel inhibitor mechanism of action

We have developed an automated patch-clamp protocol that allows high information content screening of sodium channel inhibitor compounds. We have observed that individual compounds had their specific signature patterns of inhibition, which were manifested irrespective of the concentration. Our aim in this study was to quantify these properties. Primary biophysical data, such as onset rate, the shift of the half inactivation voltage, or the delay of recovery from inactivation, are concentration-dependent. We wanted to derive compound-specific properties, therefore, we had to neutralize the effect of concentration. This study describes how this is done, and shows how compound-specific properties reflect the mechanism of action, including binding dynamics, cooperativity, and interaction with the membrane phase. We illustrate the method using four well-known sodium channel inhibitor compounds, riluzole, lidocaine, benzocaine, and bupivacaine. Compound-specific biophysical properties may also serve as a basis for deriving parameters for kinetic modeling of drug action. We discuss how knowledge about the mechanism of action may help to predict the frequency-dependence of individual compounds, as well as their potential persistent current component selectivity. The analysis method described in this study, together with the experimental protocol described in the accompanying paper, allows screening for inhibitor compounds with specific kinetic properties, or with specific mechanisms of inhibition.

Use of Yeast Mannoproteins by Oenococcus oeni during Malolactic Fermentation under Different Oenological Conditions

Oenococcus oeni is the main agent of malolactic fermentation in wine. This fermentation takes place after alcoholic fermentation, in a low nutrient medium where ethanol and other inhibitor compounds are present. In addition, some yeast-derived compounds such as mannoproteins can be stimulatory for O. oeni. The mannoprotein concentration in wine depends on the fermenting yeasts, and non-Saccharomyces in particular can increase it. As a result of the hydrolytic activity of O. oeni, these macromolecules can be degraded, and the released mannose can be taken up and used as an energy source by the bacterium. Here we look at mannoprotein consumption and the expression of four O. oeni genes related to mannose uptake (manA, manB, ptsI, and ptsH) in a wine-like medium supplemented with mannoproteins and in natural wines fermented with different yeasts. We observe a general gene upregulation in response to wine-like conditions and different consumption patterns in the studied media. O. oeni was able to consume mannoproteins in all the wines. This consumption was notably higher in natural wines, especially in T. delbrueckii and S. cerevisiae 3D wines, which presented the highest mannoprotein levels. Regardless of the general upregulation, it seems that mannoprotein degradation is more closely related to the fermenting medium.