Identification and characterization of xanthone biosynthetic genes contributing to the vivid red coloration of red-flowered gentian.

Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6'-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.

A covalently bound inhibitor triggers EZH2 degradation through CHIP-mediated ubiquitination.

Enhancer of zeste homolog 2 (EZH2) has been characterized as a critical oncogene and a promising drug target in human malignant tumors. The current EZH2 inhibitors strongly suppress the enhanced enzymatic function of mutant EZH2 in some lymphomas. However, the recent identification of a PRC2- and methyltransferase-independent role of EZH2 indicates that a complete suppression of all oncogenic functions of EZH2 is needed. Here, we report a unique EZH2-targeting strategy by identifying a gambogenic acid (GNA) derivative as a novel agent that specifically and covalently bound to Cys668 within the EZH2-SET domain, triggering EZH2 degradation through COOH terminus of Hsp70-interacting protein (CHIP)-mediated ubiquitination. This class of inhibitors significantly suppressed H3K27Me3 and effectively reactivated polycomb repressor complex 2 (PRC2)-silenced tumor suppressor genes. Moreover, the novel inhibitors significantly suppressed tumor growth in an EZH2-dependent manner, and tumors bearing a non-GNA-interacting C668S-EZH2 mutation exhibited resistance to the inhibitors. Together, our results identify the inhibition of the signaling pathway that governs GNA-mediated destruction of EZH2 as a promising anti-cancer strategy.

Reducing Myosin II and ATP-Dependent Mechanical Activity Increases Order and Stability of Intracellular Organelles.

Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca++ probe as a small and mobile tracer in live T cells. Ca++, highlighted by Fluo-4, is localized in intracellular organelles. Inhibiting intracellular mechanical work by myosin II through blebbistatin treatment increased cellular dis-homogeneity of Ca++-rich features in length scale < 1.1 µm. We detected a similar effect in cells imaged by label-free bright-field (BF) microscopy, in mitochondria-highlighted cells and in ATP-depleted cells. Blebbistatin treatment also reduced the dynamics of the Ca++-rich features and generated prominent negative temporal correlations in their signals. Following Guggenberger et al. and numerical simulations, we suggest that diffusion in the viscoelastic and confined medium of intracellular organelles may promote spatial dis-homogeneity and stability of their content. This may be revealed only after inhibiting intracellular mechanical work and related cell vibrations. Our described mechanisms may allow the cell to control its organization via balancing its viscoelasticity and mechanical activity, with implications to cell physiology in health and disease.

Antioxidant and Cytoprotective Properties of Plant Extract from Dry Flowers as Functional Dyes for Cosmetic Products.

Nowadays, natural dyes are expected by the cosmetic and food industries. In contrast to synthetic dyes, colorants derived from natural sources are more environmentally friendly and safer for human health. In this work, plant extracts from Gomphrena globasa L., Clitoria ternatea L., Carthamus tinctorius L., Punica granatum L. and Papaver rhoeas L. as the natural and functional dyes for the cosmetics industry were assessed. Cytotoxicity on keratinocyte and fibroblast cell lines was determined as well as antioxidant and anti-aging properties by determining their ability to inhibit the activity of collagenase and elastase enzymes. In addition, the composition of the extracts was determined. The obtained extracts were also applied in face cream formulation and color analyses were performed. It has been shown that the obtained extracts were characterized by no cytotoxicity and a high antioxidant potential. The extracts also show strong ability to inhibit the activity of collagenase and moderate ability to inhibit elastase and provide effective and long-lasting hydration after their application on the skin. Application analyses showed that the extracts of P. rhoeas L., C. ternatea L. and C. tinctorius L. can be used as effective cosmetic dyes that allow for attainment of an intense and stable color during the storage of the product. The extracts of P. granatum L. and G. globasa L., despite their beneficial effects as active ingredients, did not work effectively as cosmetic dyes, because cosmetic emulsions with these extracts did not differ significantly in color from emulsions without the extract.

Elastin-derived peptide VGVAPG affects the proliferation of mouse cortical astrocytes with the involvement of aryl hydrocarbon receptor (Ahr), peroxisome proliferator-activated receptor gamma (Pparγ), and elastin-binding protein (EBP).

During aging and ischemic and hemorrhagic stroke, elastin molecules are degraded and elastin-derived peptides are released into the brain microenvironment. Val-Gly-Val-Ala-Pro-Gly (VGVAPG) is a repeating hexapeptide in the elastin molecule. It is well documented that the peptide sequence binds with high affinity to elastin-binding protein (EBP) located on the cell surface, thereby transducing a molecular signal into the cell. The aim of our study was to investigate whether EBP, aryl hydrocarbon receptor (Ahr), and peroxisome proliferator-activated receptor gamma (Pparγ) are involved in VGVAPG-stimulated proliferation. Primary astrocytes were maintained in DMEM/F12 medium without phenol red, supplemented with 10 or 1% charcoal/dextran-treated fetal bovine serum (FBS). The cells were exposed to increasing concentrations of VGVAPG peptide, and resazurin reduction was measured. In addition, Glb1, Pparγ, and Ahr genes were silenced. After 48 h of exposure to 10 nM and 1 µM of VGVAPG peptide, the level of estradiol (E2) and the expression of Ki67 and S100B proteins were measured. The results showed that at a wide range of concentrations, VGVAPG peptide increased the metabolism of astrocytes depending on the concentration of FBS. After silencing of Glb1, Pparγ, and Ahr genes, VGVAPG peptide did not affect the cell metabolism which suggests the involvement of all the mentioned receptors in its mechanism of action. Interestingly, in the low-FBS medium, the silencing of Glb1 gene did not result in complete inhibition of VGVAPG-stimulated proliferation. On the other hand, in the medium with 10% FBS VGVAPG increased Ki67 expression after Pparγ silencing, whereas in the medium with 1% FBS VGVAPG decreased Ki67 expression. Following the application of Ahr siRNA, VGVAPG peptide decreased the production of E2 and increased the expression of Ki67 and S100B proteins.

Study of molecular transport through a single nanopore in the membrane of a giant unilamellar vesicle using COMSOL simulation.

The antimicrobial peptide (AMP) magainin 2 induces nanopores in the lipid membranes of giant unilamellar vesicles (GUVs), as observed by the leakage of water-soluble fluorescent probes from the inside to the outside of GUVs through the pores. However, molecular transport through a single nanopore has not been investigated in detail yet and is studied in the present work by simulation. A single pore was designed in the membrane of a GUV using computer-aided design software. Molecular transport, from the outside to the inside of GUV through the nanopore, of various fluorescent probes such as calcein, Texas-Red Dextran 3000 (TRD-3k), TRD-10k and TRD-40k was then simulated. The effect of variation in GUV size (diameter) was also investigated. A single exponential growth function was fitted to the time course of the fluorescence intensity inside the GUV and the corresponding rate constant of molecular transport was calculated, which decreases with an increase in the size of fluorescent probe and also with an increase in the size of GUV. The rate constant found by simulation agrees reasonably well with reported experimental results for inside-to-outside probe leakage. Based on Fick's law of diffusion an analytical treatment is developed for the rate constant of molecular transport that supports the simulation results. These investigations contribute to a better understanding of the mechanism of pore formation using various membrane-active agents in the lipid membranes of vesicles and the biomembranes of cells.

A novel human pluripotent stem cell-based assay to predict developmental toxicity.

There is a great need for novel in vitro methods to predict human developmental toxicity to comply with the 3R principles and to improve human safety. Human-induced pluripotent stem cells (hiPSC) are ideal for the development of such methods, because they are easy to retrieve by conversion of adult somatic cells and can differentiate into most cell types of the body. Advanced three-dimensional (3D) cultures of these cells, so-called embryoid bodies (EBs), moreover mimic the early developing embryo. We took advantage of this to develop a novel human toxicity assay to predict chemically induced developmental toxicity, which we termed the PluriBeat assay. We employed three different hiPSC lines from male and female donors and a robust microtiter plate-based method to produce EBs. We differentiated the cells into cardiomyocytes and introduced a scoring system for a quantitative readout of the assay-cardiomyocyte contractions in the EBs observed on day 7. Finally, we tested the three compounds thalidomide (2.3-36 µM), valproic acid (25-300 µM), and epoxiconazole (1.3-20 µM) on beating and size of the EBs. We were able to detect the human-specific teratogenicity of thalidomide and found the rodent toxicant epoxiconazole as more potent than thalidomide in our assay. We conclude that the PluriBeat assay is a novel method for predicting chemicals' adverse effects on embryonic development.

Effect of Fermentation Time on Antioxidant and Anti-Ageing Properties of Green Coffee Kombucha Ferments.

Kombucha, also known as the Manchurian mushroom, is a symbiotic culture of bacteria and yeast, the so-called SCOBY. This paper presents a comprehensive evaluation of the ferments obtained from green coffee beans after different fermentation times with kombucha. Results for the ferments were compared to the green coffee extract that was not fermented. In this study, the antioxidant potential of obtained ferments was analyzed by assessing the scavenging of external and intracellular free radicals and the assessment of superoxide dismutase activity. Cytotoxicity of ferments on keratinocyte and fibroblast cell lines was assessed as well as anti-aging properties by determining their ability to inhibit the activity of collagenase and elastase enzymes. In addition, the composition of the obtained ferments and the extract was determined, as well as their influence on skin hydration and transepidermal water loss (TEWL) after application of samples on the skin. It has been shown that the fermentation time has a positive effect on the content of bioactive compounds and antioxidant properties. The highest values were recorded for the tested samples after 28 days of fermentation. After 14 days of the fermentation process, it was observed that the analyzed ferments were characterized by low cytotoxicity to keratinocytes and fibroblasts. On the other hand, the short fermentation time of 7 days had a negative effect on the properties of the analyzed ferments. The obtained results indicate that both green coffee extracts and ferments can be an innovative ingredient of cosmetic products.

Molecular Design Strategy to Construct the Near-Infrared Fluorescent Probe for Selectively Sensing Human Cytochrome P450 2J2.

Cytochrome P450 2J2 (CYP2J2), a key enzyme responsible for oxidative metabolism of various xenobiotics and endogenous compounds, participates in a diverse array of physiological and pathological processes in humans. Its biological role in tumorigenesis and cancer diagnosis remains poorly understood, owing to the lack of molecular tools suitable for real-time monitoring CYP2J2 in complex biological systems. Using molecular design principles, we were able to modify the distance between the catalytic unit and metabolic recognition moiety, allowing us to develop a CYP2J2 selective fluorescent probe using a near-infrared fluorophore ( E)-2-(2-(6-hydroxy-2, 3-dihydro-1 H-xanthen-4-yl)vinyl)-3,3-dimethyl-1-propyl-3 H-indol-1-ium iodide (HXPI). To improve the reactivity and isoform specificity, a self-immolative linker was introduced to the HXPI derivatives in order to better fit the narrow substrate channel of CYP2J2, the modification effectively shortened the spatial distance between the metabolic moiety ( O-alkyl group) and catalytic center of CYP2J2. After screening a panel of O-alkylated HXPI derivatives, BnXPI displayed the best combination of specificity, sensitivity and applicability for detecting CYP2J2 in vitro and in vivo. Upon O-demethylation by CYP2J2, a self-immolative reaction occurred spontaneously via 1,6-elimination of p-hydroxybenzyl resulting in the release of HXPI. Allowing BnXPI to be successfully used to monitor CYP2J2 activity in real-time for various living systems including cells, tumor tissues, and tumor-bearing animals. In summary, our practical strategy could help the development of a highly specific and broadly applicable tool for monitoring CYP2J2, which offers great promise for exploring the biological functions of CYP2J2 in tumorigenesis.

Pyridoxine (VB6 ) restores the down-regulation of serine palmitoyltransferase mRNA expression in keratinocytes cultured in highly oxidative conditions through enhancement of the intracellular antioxidant system.

BACKGROUND: Pyridoxine (VB6 ), which acts as a coenzyme in the biosynthesis of niacin, is formulated in pharmaceuticals to treat skin roughness. However, the mechanism of action of VB6 is not known precisely. OBJECTIVE: This study was conducted to clarify the influence of highly oxidative conditions on the expression of skin moisture-related mRNAs and to evaluate the preventive effects of VB6 focusing on antioxidant behaviour. METHODS: Intracellular levels of reactive oxygen species (ROS) in normal human epidermal keratinocytes (NHEKs) were determined using the 2',7'-dichlorofluorescein diacetate assay. Real-time PCR was employed to investigate the influence of higher oxidative conditions on the expression of mRNAs encoding serine palmitoyl transferase (SPT) and filaggrin, and to characterize the mechanism of the antioxidant effect of VB6 . Intracellular glutathione was quantified using an assay based on the glutathione recycling system with 5,5'-dithiobis (2-nitrobenzoic acid) reagent and glutathione reductase. Carbonylated proteins (CPs) were semi-quantified by detecting aldehyde residues. RESULTS: Treatment of NHEKs with BSO increased the level of intracellular CPs by interfering with intracellular glutathione synthesis. Further, treatment with BSO down-regulated the expression level of SPT mRNA, but VB6 restored SPT mRNA expression in BSO-treated NHEKs. VB6 decreased the level of intracellular CPs with or without BSO treatment in a dose-dependent manner. In addition, VB6 increased levels of intracellular NADH/NADPH and glutathione through the activation of nuclear factor E2-related factor 2 (Nrf2) signalling. CONCLUSION: These results suggest that highly oxidative conditions cause an impaired skin barrier function due to the down-regulation of SPT that results in skin roughness. VB6 improved the down-regulation of SPT mRNA expression initiated by highly oxidative conditions by enhancing the intracellular antioxidant system.

Role of AMPA receptor stimulation and TrkB signaling in the antidepressant-like effect of ketamine co-administered with a group II mGlu receptor antagonist, LY341495, in the forced swim test in rats.

Ketamine has been shown to induce a rapid antidepressant effect on patients with depression. In many animal models, both rapid and sustained antidepressant activities were also found in response to an antagonist of group II metabotropic glutamate receptors, LY341495, and its mechanism of action seemed to be similar in many ways to the action of ketamine. It has also been found that LY341495 enhanced the antidepressant-like activity of sub-effective doses of ketamine in rats without inducing adverse effects. Here, we investigated the role of AMPA receptor and TrkB receptor activation in the antidepressant-like effects of ketamine (3 mg/kg) co-administered with LY341495 (0.1 mg/kg), in the forced swim test in rats, at three time points (40 min, 3 h and 24 h) after joint administration of the tested compounds. It was found that the AMPA receptor antagonist NBQX (10 mg/kg) reversed the antidepressant effect of ketamine co-administered with LY341495 at all tested time points, whereas the TrkB receptor antagonist ANA-12 contributed to blockade of the effect of ketamine and LY341495 3 h after their joint administration. These results indicate that activation of AMPA receptor and BDNF-related signaling may play a role in the mechanism of antidepressant action of ketamine co-administered with LY341495.

Screening sediment samples used as anolytes in microbial fuel cells for microbial electron transfer activity using DREAM assay.

OBJECTIVE: The dye reduction-based electron-transfer activity monitoring (DREAM) assay was employed to screen sediment and wastewater samples functioning as anolytes in a microbial fuel cell (MFC) for their microbial electron transfer activity. RESULTS: Electron transfer to redox dyes from microbial activity reduced the dyes and the resulting extent of reduction was measured as DREAM assay coefficient. Methylene blue was decolourised, while resazurin underwent florigenic change from blue to pink to colourless upon formation of resorufin and dihydroxyresorufin. DREAM assay coefficient conformed to power density obtained in the MFC. A correlation was observed between chemical oxygen demand of the sample and the DREAM coefficient (+ 0.934) and also between DREAM coefficient and power density generated (+ 0.976). Highest DREAM coefficient and power density was observed for activated sludge. CONCLUSIONS: The DREAM assay is a rapid, sensitive and low-cost method to assess microbial electron transfer activity for inocula used as anolytes in a MFC.

Four New Insecticidal Xanthene Derivatives from the Mangrove-Derived Fungus Penicillium sp. JY246.

Four new xanthene derivatives, penicixanthenes A-D (1-4), and one known compound 5 were isolated from a marine mangrove endophytic fungus Penicillium sp. JY246 that was obtained from the stem of Ceriops tagal. Their structures were determined by detailed NMR, MS spectroscopic data, modified Mosher's method, and calculated electronic circular dichroism data. All of the isolated compounds were examined for insecticidal activity. Compounds 2 and 3 showed growth inhibition activity against newly hatched larvae of Helicoverpa armigera Hubner with the IC50 values 100 and 200 µg/mL, respectively, and compounds 1, 3, and 4 showed insecticidal activity against newly hatched larvae of Culex quinquefasciatus with LC50 values of 38.5 (±1.16), 11.6 (±0.58), and 20.5 (±1) µg/mL, respectively. The four xanthene derivatives have the potential to be developed as new biopesticides.

Apoptosis Induction Pathway in Human Colorectal Cancer Cell Line SW480 Exposed to Cereal Phenolic Extracts.

Cereal phenolic extracts have previously been investigated for their potential anticancer properties; however, the exact mechanisms involved in the inhibition of tumour growth are unclear. One possible mechanism is the induction of apoptosis which is characterised by cell shrinkage, protein fragmentation, and DNA degradation followed by rapid engulfment of cell debris by macrophages. This study examines the ability of phenolic extracts from four cereals: rice, barley, oats and sorghum to induce apoptosis on colorectal cancer cells SW480. Wholegrain extracts from pigmented varieties of red rice, purple rice, black sorghum, and brown sorghum showed a significant reduction in cancer cell proliferation. Morphological observation using APOPercentage™ dye indicated positive for apoptosis. Further analyses of Yunlu29 (rice), Shawaya Short Black 1 and IS1136 (sorghum) showed expression of p53 and confirmed activation of multiple caspases, specifically for caspase 3 and 7. Purple rice, on the other hand, did not upregulate caspase 3 and 7, hence, suggestive of cell cycle arrest. Therefore, phenolic compounds present in cereals such as pigmented rice and sorghum may suppress cancer cell proliferation through the activation of the apoptosis.

Mag-Fluo4 in T cells: Imaging of intra-organelle free Ca2+ concentrations.

Ca2+ signaling is a major signal transduction pathway involved in T cell activation, but also in apoptosis of T cells. Since T cells make use of several Ca2+-mobilizing second messengers, such as nicotinic acid adenine dinucleotide phosphate, d-myo-inositol 1,4,5-trisphosphate, and cyclic ADP-ribose, we intended to analyze luminal Ca2+ concentration upon cell activation. Mag-Fluo4/AM, a low-affinity Ca2+ dye known to localize to the endoplasmic reticular lumen in many cell types, showed superior brightness and bleaching stability, but, surprisingly, co-localized with mito-tracker, but not with ER-tracker in Jurkat T cells. Thus, we used Mag-Fluo4/AM to monitor the free luminal mitochondrial Ca2+ concentration ([Ca2+]mito) in these cells. Simultaneous analysis of the free cytosolic Ca2+ concentration ([Ca2+]i) and [Ca2+]mito upon cell stimulation revealed that Ca2+ signals in the majority of mitochondria were initiated at [Ca2+ ]i≥approx. 400 to 550nM. In primary murine CD4+ T cells, Mag-Fluo4 showed two different localization patterns: either co-localization with mito-tracker, as in Jurkat T cells, or with ER-tracker. Thus, in single primary murine CD4+ T cells, either decreases of [Ca2+ ]ER or increases of [Ca2+ ]mito were observed upon cell stimulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Evidence against a Role of Elevated Intracellular Ca2+ during Plasmodium falciparum Preinvasion.

Severe malaria is primarily caused by Plasmodium falciparum parasites during their asexual reproduction cycle within red blood cells. One of the least understood stages in this cycle is the brief preinvasion period during which merozoite-red cell contacts lead to apical alignment of the merozoite in readiness for penetration, a stage of major relevance in the control of invasion efficiency. Red blood cell deformations associated with this process were suggested to be active plasma membrane responses mediated by transients of elevated intracellular calcium. Few studies have addressed this hypothesis because of technical challenges, and the results remained inconclusive. Here, Fluo-4 was used as a fluorescent calcium indicator with optimized protocols to investigate the distribution of the dye in red blood cell populations used as P. falciparum invasion targets in egress-invasion assays. Preinvasion dynamics was observed simultaneously under bright-field and fluorescence microscopy by recording egress-invasion events. All the egress-invasion sequences showed red blood cell deformations of varied intensities during the preinvasion period and the echinocytic changes that follow during invasion. Intraerythrocytic calcium signals were absent throughout this interval in over half the records and totally absent during the preinvasion period, regardless of deformation strength. When present, calcium signals were of a punctate modality, initiated within merozoites already poised for invasion. These results argue against a role of elevated intracellular calcium during the preinvasion stage. We suggest an alternative mechanism of merozoite-induced preinvasion deformations based on passive red cell responses to transient agonist-receptor interactions associated with the formation of adhesive coat filaments.

Small Molecule Interferences in Resazurin and MTT-Based Metabolic Assays in the Absence of Cells.

In vitro assays (such as resazurin and MTT) provide an opportunity to determine the cytotoxicity of novel therapeutics before moving forward with expensive and resource-intensive in vivo studies. A concern with using these assays, however, is the production of false responses in the presence of particular chemical functionalities. To better understand this phenomenon, 19 small molecules at 6 concentrations (1 µM-100 mM) were tested in the presence of resazurin and MTT reagents to highlight potential interfering species. Through the use of absorbance measurements (using well-plate assays and UV-vis spectroscopy) with parallel MS analysis, we have shown that significant conversion of the assay reagents readily occurs in the presence of many tested interfering species without the need for any cellular activity. The most attributable sources of interference seem to arise from the presence of thiol and carboxylic acid moieties. Interestingly, the detectable interferences were more prevalent and larger in the presence of MTT (19 species with some deviations >3000%) compared to resazurin (16 species with largest deviation of ∼150%). Additionally, those deviations in the presence of resazurin were only substantial at high concentrations, while MTT showed deviations across the tested concentrations. This comprehensive study gives insight into chemical functional groups (thiols, amines, amides, carboxylic acids) that may interfere with resazurin and MTT assays in the absence of metabolic activity and indicates that proper control studies must be performed to obtain accurate data from these in vitro assays.

Metabolic reduction of resazurin; location within the cell for cytotoxicity assays.

Resazurin is widely used as a metabolic indicator for living cells, however, there has been considerable debate in the literature with regards to the specific location in the cell where the non-fluorescent resazurin is reduced to the strongly fluorescent resorufin. This lack of clarity about the reduction site makes the use of resazurin reduction data in cytotoxicity studies difficult to interpret. In this study, E. faecalis, a Gram-positive and facultative anaerobic bacterial strain, and the most toxic chlorophenol, pentachlorophenol (PCP), were chosen as models for an anaerobe and toxicant, respectively. By studying the kinetics of resazurin reduction by E. faecalis after different treatments (cell disruption, bacterial filtration, and pre-exposure to toxicant), we confirmed that resazurin reduction to resorufin by live Gram-positive and facultative anaerobic bacterial cells can only happen intracellularly under anaerobic conditions, while resorufin reduction to dihydroresorufin can happen both intracellularly and extracellularly. Based on the understanding of these fundamental mechanisms, we suggest that resazurin reduction can be used as a quick bioassay for measuring cytotoxicity.

Development of a Xanthene-Based Red-Emissive Fluorescent Probe for Visualizing H2O2 in Living Cells, Tissues and Animals.

Hydrogen peroxide (H2O2) plays important roles in the regulation of many biological processes, and the abnormal level of H2O2 has close relation with the initiation and progression of many diseases. Herein, we describe a novel red-emissive fluorescence probe (RhoB) for the visualization of H2O2 in living cells, tissues and animals. RhoB was constructed on the basis of a xanthene-based red-emissive dye, and displayed nearly no fluorescence. After the treatment with H2O2, RhoB can exhibit red fluorescence with the emission wavelength at 638 nm. RhoB exhibited highly sensitive and selective response to H2O2. Density functional theory (DFT) calculations were conducted to shed light on the optical properties of RhoB, and natural bond orbital (NBO) calculations demonstrate that the boron atom shows the highest positive electricity and further support the response mechanism. RhoB was successfully applied for imaging of exogenous and endogenous H2O2 in living cells, and also can be utilized for visualizing H2O2 in living tissues and animals.

Characterization and mapping of secondary metabolites of Streptomyces sp. from caatinga by desorption electrospray ionization mass spectrometry (DESI-MS).

The discovery of new secondary metabolites is a challenge to biotechnologists due to the emergence of superbugs and drug resistance. Knowledge about biodiversity and the discovery of new microorganisms have become major objectives; thus, new habitats like extreme ecosystems have become places of interest to research. In this context, caatinga is an unexplored biome. The ecosystem caatinga is a rich habitat for thermophilic microbes. Its high temperature and dry climate cause selective microbes to flourish and become established. Actinobacteria (Caat 1-54 genus Streptomyces sp.) isolated from the soil of caatinga was investigated to characterize and map its secondary metabolites by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). With this technique, the production of bioactive metabolites was detected and associated with the different morphological differentiation stages within a typical Streptomyces sp. life cycle. High-resolution mass spectrometry, tandem mass spectrometry, UV-Vis profiling and NMR analysis were also performed to characterize the metabolite ions detected by DESI-MS. A novel compound, which is presumed to be an analogue of the antifungal agent lienomycin, along with the antimicrobial compound lysolipin I were identified in this study to be produced by the bacterium. The potency of these bioactive compounds was further studied by disc diffusion assays and their minimum inhibitory concentrations (MIC) against Bacillus and Penicillium were determined. These bioactive metabolites could be useful to the pharmaceutical industry as candidate compounds, especially given growing concern about increasing resistance to available drugs with the emergence of superbugs. Consequently, the unexplored habitat caatinga affords new possibilities for novel bioactive compound discovery. Graphical Abstract ᅟ.