Screening and evaluation of antioxidants from lees by micro-injector systems combined with a fluorescent probe,N-borylbenzyloxycarbonyl-3,7-dihydroxyphenoxazine,in living Drosophila

A HPLC-UV-FLD system and a micro-injector imaging system were used in the study of antioxidants from natural products.     

5-Fluorouracil-containing inorganic iron oxide/platinum nanozymes with dual drug delivery and enzyme-like activity for the treatment of breast cancer

Intrinsic enzyme-mimic activity of inorganic nanoparticles has been widely used for nanozymatic anticancer and antibacterial treatment. However, the relatively low peroxidase-mimic activity (PMA) and catalse-mimic activity (CMA) of nanozymes in tumor microenvironment has hampered their potential application in the cancer therapy. Therefore, in this study, we aimed to fabricate platinum (Pt) nanozymes dispersed on the surface of iron oxide (Fe₃O₄) nanosphere that, in addition to boosting the PMA and CMA, resulted in the formation of a pH-sensitive nano-platform for drug delivery in breast cancer therapy. After development of Fe₃O₄ nanospheres containing Pt nanozymes and loading 5-fluorouracil (abbreviated as: Fe₃O₄/Pt-FLU@PEG nanospheres), the physicochemical properties of the nanospheres were examined by electron microscopy, dynamic light scattering, zeta potential, X-ray diffraction, thermogravimetric, BET surface, and PMA/CMA analyses. Then, the cytotoxicity of the Fe₃O₄/Pt-FLU@PEG nanospheres against 4T1 cells was investigated by the cell counting kit-8 assay and flow cytometry. Also, the anticancer effect of fabricated nanoplatform was assessed in mouse bearing 4T1 cancer tumors, in vivo. The results showed that the Fe₃O₄/Pt-FLU@PEG nanospheres provide a platform for optimal FLU loading, continuous pH-sensitive drug release, and potential PMA and CMA to increase the level of ROS and O₂, respectively. Cytotoxicity outputs showed that the Fe₃O₄/Pt-FLU@PEG nanospheres mitigate the proliferation of 4T1 cancer cells mediated by apoptosis and intracellular generation of reactive oxygen species (ROS). Furthermore, in vivo assays indicated a significant reduction in tumor size and overcoming tumor hypoxia. Overall, we believe that the developed nanospheres with dual enzyme-mimic activity and pH-sensitive drug delivery can be used for ROS/chemotherapy double-modality antitumor therapy.     

Antioxidant Activity of Ruthenium Cyclopentadienyl Complexes Bearing Succinimidato and Phthalimidato Ligands

In these studies, we investigated the antioxidant activity of three ruthenium cyclopentadienyl complexes bearing different imidato ligands: (η⁵-cyclopentadienyl)Ru(CO)₂-N-methoxysuccinimidato (1), (η⁵-cyclopentadienyl)Ru(CO)₂-N-ethoxysuccinimidato (2), and (η⁵-cyclopentadienyl)Ru(CO)₂-N-phthalimidato (3). We studied the effects of ruthenium complexes 1–3 at a low concentration of 50 µM on the viability and the cell cycle of peripheral blood mononuclear cells (PBMCs) and HL-60 leukemic cells exposed to oxidative stress induced by hydrogen peroxide (H₂O₂). Moreover, we examined the influence of these complexes on DNA oxidative damage, the level of reactive oxygen species (ROS), and superoxide dismutase (SOD) activity. We have observed that ruthenium complexes 1–3 increase the viability of both normal and cancer cells decreased by H₂O₂ and also alter the HL-60 cell cycle arrested by H₂O₂ in the sub-G1 phase. In addition, we have shown that ruthenium complexes reduce the levels of ROS and oxidative DNA damage in both cell types. They also restore SOD activity reduced by H₂O₂. Our results indicate that ruthenium complexes 1–3 bearing succinimidato and phthalimidato ligands have antioxidant activity without cytotoxic effect at low concentrations. For this reason, the ruthenium complexes studied by us should be considered interesting molecules with clinical potential that require further detailed research.     

First normal stress difference-temperature dependence for polystyrene and high-impact polystyrene melts

The method of reduced variables or superposition is applied to investigate the first normal stress difference (N₁)-temperature dependence over the shear rate (γ) range 10–1000 s^?1 for polystyrene (PS) and high-impact polystyrene (HIPS) melts, at temperatures of 180 C and above. These conditions are similar to those for industrial polymer processing. For PS, the first normal stress differences are obtained using Tanner's equation, leading to good agreement with values obtained by other authors and methods. For HIPS we have not found data in the literature for N₁ at shear rates above 10s^?1. In our case N₁ was obtained by measurements of entrance pressure losses. Correlations, based on master curves, are found for first normal stress difference in terms of shear rate and temperature. All samples follow the power law equation $\text{N}{}_1\text{= k}\text{γ}\text{?}{}^{\mathrm{m}}$, with values of m ranging from 0.50 to 0.64.     

Evaluation of pathogenicity of certain mitosporic ascomycete fungi to the house fly,Musca domestica L. (diptera: muscidae)

The virulence of five fungal strains, Acremonium cephalosporium, Aspergillus niger, Penicillium chrysogenum, Trichoderma viride, and Verticillum albo-atrum by application of 10⁸ conidia mL^?1 to larvae and adults of house flies, Musca domestica was determined. A. niger produced the highest percentage of mortality to larvae, 85.1 ± 1.60, and adults, 98.2 ± 3.10, of house flies. Topical application of the fungal strains to larvae showed concentration-dependent response and caused mortality ranged between 41% and 83% with LT₅₀ values varying between 5.22 and 7.81 days. The highest percentage of mortality, 83.9 ± 3.52, was produced by 10⁷ conidia mL^?1 of A. niger with LT₅₀ 5.22. The lowest mortality percentage, 41.2 ± 3.00 with LT₅₀ 7.81, was produced by 10⁵ conidia mL^?1 of V. alboatrum. On the other hand, the highest percentage of mortality to adults of house flies, 97.3 ± 3.52, was produced by 10⁷ conidia mL^?1 of A. niger with LT₅₀ 3.49. While the lowest mortality percentage, 59.1 ± 2.38 with LT₅₀ 6.91, was produced by 10⁵ conidia mL^?1 of V. albo-atrum.     

Mitochondrial ROS production under cellular stress: comparison of different detection methods

Reactive oxygen species (ROS) are involved in the regulation of many physiological processes. However, overproduction of ROS under various cellular stresses results in cell death and organ injury and thus contributes to a broad spectrum of diseases and pathological conditions. The existence of different cellular sources for ROS and the distinct properties of individual ROS (their reactivity, lifetime, etc.) require adequate detection methods. We therefore compared different models of cellular stress and various ROS-sensitive dyes—2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), MitoSOX?, and MitoTracker® red CM-H₂XRos—using a confocal fluorescent imaging approach, which has the advantage of not only detecting but also of localizing intracellular sources for ROS. Confocal acquisition of DCF-DA fluorescence can be combined with ROS detection by the mitochondria-specific probes MitoSOX? and MitoTracker® red CM-H₂XRos. Specificity was controlled using various antioxidants such as Trolox and N-acetylcysteine. Using different fluorescent ROS-sensitive probes, we detected higher ROS production equally under cell starvation (IL-3 or serum depletion), hypoxia–reoxygenation, or treatment of cells with prooxidants. The detected increase in ROS was approximately threefold in IL-3-depleted 32D cells, approximately 3.5-fold in serum-deprived NIH cells, and 2.5-fold to threefold in hypoxic HL-1 cells, and these findings agree well with previously published spectrofluorometric measurements. In some cases, electron spin resonance (ESR) spectroscopy was used for the validation of results from confocal fluorescent imaging. Our data show that confocal fluorescent imaging and ESR data are in good agreement. Under cellular stress, mitochondrial ROS are released into the cytoplasm and may participate in many processes, but they do not escape from the cell.

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Online abstract Mitochondrial ROS production under cellular stress

     

Stress Tolerance of the Endemic Antarctic Brown Alga Desmarestia anceps to UV Radiation and Temperature is Mediated by High Concentrations of Phlorotannins

The endemic Antarctic brown macroalga Desmarestia anceps is strongly shade‐adapted, but shows also a high capacity to cope with different environmental stressors, e.g. UV radiation and temperature. Therefore, this species colonizes wide depth gradients, which are characterized by changing environmental conditions. In this study, we examine whether the different physiological abilities allowing D. anceps to grow across a wide depth range is determined by high levels of phlorotannins. Photosynthesis, measured by PAM‐fluorometry, the contents of soluble phlorotannins, antioxidant capacities of field grown were analyzed in response to different conditions of radiation (PAR and PAR + UV) and temperature (2, 7 and 12°C). The results show that maximal quantum of fluorescence (F_v/F_m) decreased with increasing doses of UV radiation, but remained unaffected by temperature. High levels of soluble phlorotannins were detected and confirmed by microscopic observation revealing the abundance of large physodes. Exposure to UV radiation and elevated temperature showed that phlorotannins were not inducible by UV but increased at 12°C. ROS scavenging capacity was positively correlated with the contents of phlorotannins. In general, highest contents of phlorotannins were correlated with the lowest inhibition of F_v/F_m in all experimental treatments, highlighting the UV‐protective role of these compounds in D. anceps.     

Application of stress induces ascorbate peroxidases of S. polyrhiza for green-synthesis Cu nanoparticles

The objective of this study was to assess the effects of stress on physiology/biochemical component of S. polyrhiza and its impact on CuNPs synthesis and bioethanol production. NaCl with RV5 provokes oxidative stress in S. polyrhiza and significantly increase MAD, Proline, H₂O₂, ROS, SOD and APX activity compare to control condition. Starch accumulation in S. polyrhiza was found 354% higher and correspond 4.4 times higher ethanol yield under stress condition compare to control. CuNPs were synthesized with an average size of 23–26 nm by purified fraction of APX having 37 KDa MW, 1.44 IU specific activity. Synthesized CuNPs were stable up to 15 consecutive cycles and potency against wide range of reactive dyes. The maximum remedial efficiency of synthesized CuNPs for COD and BOD was 55263.3 ± 3298.5 mg/m³min. and 30560.3 ± 1987.5 mg/m³min. respectively for RV5 wastewater. 0.072 mg/g of bioethanol was produced from the wet pulp remaining after nanoparticles synthesis. High efficiency of CuNPs and significant production of Ethanol, indicate that the feasibility for circular model for continuous industrial wastewater treatment.     

Dunaliella tertiolecta (Chlorophyta) Avoids Cell Death Under Ultraviolet Radiation By Triggering Alternative Photoprotective Mechanisms

The effect of different ultraviolet radiation (UVR) treatments combining PAR (P), UVA (A) and UVB (B) on the molecular physiology of Dunaliella tertiolecta was studied during 6 days to assess the response to chronic UVR exposure. UVR reduced cell growth but did not cause cell death, as shown by the absence of SYTOX Green labeling and cellular morphology. However, caspase‐like enzymatic activities (CLs), (regarded as cell death proteases), were active even though the cells were not dying. Maximal quantum yield of fluorescence (F_v/F_m) and photosynthetic electron transport rate (ETR) dropped. Decreased nonphotochemical quenching (NPQ) paralleled a drop in xanthophyll cycle de‐epoxidation under UVB. Reactive oxygen species (ROS) and D1 protein accumulation were inversely correlated. PAB exhibited elevated ROS production at earlier times. Once ROS decayed, D1 protein recovered two‐fold compared with P and PA at later stages. Therefore, PsbA gene was still transcribed, suggesting ROS involvement in D1 recovery by its direct effect on mRNA‐translation. We add evidence of an UVB‐induced positive effect on the cells when P is present, providing photoprotection and resilience, by means of D1 repair. This allowed cells to survive. The photoprotective mechanisms described here (which are counterintuitive in principle) conform to an important ecophysiological response regarding light stress acclimation.     

Impact of Peels Extracts from an Italian Ancient Tomato Variety Grown under Drought Stress Conditions on Vascular Related Dysfunction

Background: Tomato by-products contain a great variety of biologically active substances and represent a significant source of natural antioxidant supplements of the human diet. The aim of the work was to compare the antioxidant properties of a by-product from an ancient Tuscan tomato variety, Rosso di Pitigliano (RED), obtained by growing plants in normal conditions (-Ctr) or in drought stress conditions (-Ds) for their beneficial effects on vascular related dysfunction. Methods: The antioxidant activity and total polyphenol content (TPC) were measured. The identification of bioactive compounds of tomato peel was performed by HPLC. HUVEC were pre-treated with different TPC of RED-Ctr or RED-Ds, then stressed with H₂O₂. Cell viability, ROS production and CAT, SOD and GPx activities were evaluated. Permeation of antioxidant molecules contained in RED across excised rat intestine was also studied. Results: RED-Ds tomato peel extract possessed higher TPC than compared to RED-Ctr (361.32 ± 7.204 mg vs. 152.46 ± 1.568 mg GAE/100 g fresh weight). All extracts were non-cytotoxic. Two hour pre-treatment with 5 µg GAE/mL from RED-Ctr or RED-Ds showed protection from H₂O₂-induced oxidative stress and significantly reduced ROS production raising SOD and CAT activity (* p < 0.05 and ** p < 0.005 vs. H₂O₂, respectively). The permeation of antioxidant molecules contained in RED-Ctr or RED-Ds across excised rat intestine was high with non-significant difference between the two RED types (41.9 ± 9.6% vs. 26.6 ± 7.8%). Conclusions: RED-Ds tomato peel extract represents a good source of bioactive molecules, which protects HUVECs from oxidative stress at low concentration.     

Microwave spectrum,barrier to internal rotation and dipole moment in 5-methyl-pyrimidine

The microwave rotational spectrum of 5-methyl-pyrimidine has been investigated in the region from 8 to 27 GHz, the three types of lines to be expected for a molecule of this symmetry and with a very low sixfold barrier hindering internal rotation of the methyl top have been found: m = 0; |m| ≠ 0,3; |m| = 3n. From the m = 0 (a-type transitions) the rotational constants A′ (less methyl top) = 6108.41, B = 2642.198, C = 1844.196 MHz and the dipole moment μ = 2.881D have been determined. From the wide splitting of the lines |m| = 3, |k| = 1 the potential barrier has been derived as V₆ = 11.73 cal/mole.     

Recent advances in fluorescent probes for the detection of reactive oxygen species

Reactive oxygen species (ROS) have captured the interest of many researchers in the chemical, biological, and medical fields since they are thought to be associated with various pathological conditions. Fluorescent probes for the detection of ROS are promising tools with which to enhance our understanding of the physiological roles of ROS, because they provide spatial and temporal information about target biomolecules in in vivo cellular systems. ROS probes, designed to detect specific ROS with a high selectivity, would be desirable, since it is now becoming clear that each ROS has its own unique physiological activity. However, dihydro-compounds such as 2′,7′-dichlorodihydrofluorescein (DCFH), which have traditionally been used for detecting ROS, tend to react with a wide variety of ROS and are not completely photostable. Some attractive fluorescent probes that exhibit a high degree of selectivity toward specific ROS have recently been reported, and these selective probes are expected to have great potential for elucidating unknown physiological mechanisms associated with their target ROS. This review focuses on the design, detection mechanism, and performance of fluorescent probes for the detection of singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), hydroxyl radicals (^.OH), or superoxide anion (O₂ ^−.), a field in which remarkable progress has been achieved in the last few years.     

Near-Infrared Light-Triggered Chlorine Radical (.Cl) Stress for Cancer Therapy

Free radicals with reactive chemical properties can fight tumors without causing drug resistance. Reactive oxygen species (ROS) has been widely used for cancer treatment, but regrettably, the common O₂ and H₂O₂ deficiency in tumors sets a severe barrier for sufficient ROS production, leading to unsatisfactory anticancer outcomes. Here, we construct a chlorine radical (^.Cl) nano-generator with SiO₂-coated upconversion nanoparticles (UCNPs) on the inside and Ag⁰/AgCl hetero-dots on the outside. Upon near-infrared (NIR) light irradiation, the short-wavelength emission UCNP catalyzes ^.Cl generation from Ag⁰/AgCl with no dependence on O₂/H₂O₂. ^.Cl with strong oxidizing capacity and nucleophilicity can attack biomolecules in cancer cells more effectively than ROS. This ^.Cl stress treatment will no doubt broaden the family of oxidative stress-induced antitumor strategies by using non-oxygen free radicals, which is significant in the development of new anticancer agents.     

Development of environmentally friendly biological algicide and biochemical analysis of inhibitory effect of diatom Skeletonema costatum

Skeletonema costatum is a diatom widely distributed in red tide microalgae blooms and as one of the main algae causing harmful algal blooms, because of their rapid reproduction and production of toxic and harmful substances, often play a negative role in aquatic ecosystems, and human health and wellbeing. Bacillomycin D is a nonribosomal cyclic antifungal lipopeptide in the iturins family. In this study,Bacillomycin D was tested for its ability to inhibit the growth of S. costatum. The EC_50...     

Artificial cell-microencapsulated phenylalanine ammonia-lyase

Phenylalanine ammonia-lyase (PAL) is immobilized in collodion artificial cells. Once technical problems associated with the encepsulation of this enzyme were solved, the enzyme kinetics were compared to PAL in free solution. Microencapsulated PAL has an apparent enzyme activity that is 20% of the activity of enzyme in free solution. TheK _m for both free and immobilized PAL is 475 μM. TheV _m for the microen-capsulated PAL is 9 μM/min, whereas that of PAL in free solution is 55 μM/min.     

Normalized potential sweep voltammetry: Part III. Elimination of the effect of uncompensated Resistance and the application to heterogeneous kinetics of rapid charge-transfer processes

Uncompensated resistance (R_u) has a distoring effect on normalized potential sweep voltammetry (NPSV) slopes. This provides a simple and effective method to determine R_f^o, the value of the potentiostat feedback resistance necessary for full compensation. If the NPSV range is divided into overlapping segment, 1 and 2, corresponding to I_N of 0.20–0.50 and 0.50–0.80 respectively, the slopes m₁ and m₂ differ significantly when R_f differs form R_f^o. The difference, m₁-m₂, is negative for R_f<R_f^o and positive for R_f>R_f^o. Fine tuning of the potentiostat R_f setting so that the average value of the difference is the theoretical value can be accomplished in a minimum of time. Under these conditions, m₁ and m₂, as well as m_T, the slope of the entire correlation have very nearly the same values. Linear equations were derived from theoretical data which allow heterogeneous rate constants to be obtained directly from NPSV slopes. The precision in the NPSV slopes was observed to be of the order of ±0.002 which implies that the method should give reliable rate constants as great as 10cm s^? at a voltage sweep rate of 100V s^?1. The method is demonstrated using the reduction of benzonitrile and perylene in N,N-dimethylformamide and acetonitrile as examples.     

Cloning,Expression, Purification,and Characterization of β-Galactosidase from Bifidobacterium longum and Bifidobacterium pseudocatenulatum

Expression and purification of β-galactosidases derived from Bifidobacterium provide a new resource for efficient lactose hydrolysis and lactose intolerance alleviation. Here, we cloned and expressed two β-galactosidases derived from Bifidobacterium. The optimal pH for BLGLB1 was 5.5, and the optimal temperature was 45 °C, at which the enzyme activity of BLGLB1 was higher than that of commercial enzyme E (300 ± 3.6 U/mg) under its optimal conditions, reaching 2200 ± 15 U/mg. The optimal pH and temperature for BPGLB1 were 6.0 and 45 °C, respectively, and the enzyme activity (0.58 ± 0.03 U/mg) under optimum conditions was significantly lower than that of BLGLB1. The structures of the two β-galactosidase were similar, with all known key sites conserved. When o-nitrophenyl-β-D-galactoside (oNPG) was used as an enzyme reaction substrate, the maximum reaction velocity (V_max) for BLGLB1 and BPGLB1 was 3700 ± 100 U/mg and 1.1 ± 0.1 U/mg, respectively. The kinetic constant (K_m) of BLGLB1 and BPGLB1 was 1.9 ± 0.1 and 1.3 ± 0.3 mmol/L, respectively. The respective catalytic constant (k_cat) of BLGLB1 and BPGLB1 was 1700 ± 40 s⁻¹ and 0.5 ± 0.02 s⁻¹, respectively; the respective k_cat/K_m value of BLGLB1 and BPGLB1 was 870 L/(mmol∙s) and 0.36 L/(mmol∙s), respectively. The K_m, k_cat and V_max values of BLGLB1 were superior to those of earlier reported β-galactosidase derived from Bifidobacterium. Overall, BLGLB1 has potential application in the food industry.     

Reactions of O with CO van der Waals molecules and clusters

The reaction of O(³ P) with COR _m clusters to produce electronically excited CO₂ was studied under molecular beam conditions. It was found that the spectrum of the chemiluminescence produced extended from the blue all the way to the near infrared. The dependence of the total emission intensity on stagnation pressure was investigated for (CO) _m as well as for COR _m , R=He, Ne, Ar, N₂, CO₂ and SO₂. The low pressure data indicate that small (CO) _m polymers are more efficient than clusters of CO with other species in inducing the chemiluminescent reaction. The larger CO-rare gas clusters, however, exhibited larger reaction cross-sections than those of the CO polymers. Rare gas clusters ofm≧5, on the other hand have successively smaller cross sections for reaction. The reactivity of the CO₂ and SO₂ clusters seems to peak at aboutm=1 and then decreases for larger species. An equilibrium model for cluster formation was proposed and it was found capable of explaining and simulating the experimental observations. Contrary to what was reported from afterglow experiments, no barrier for the reaction was detected.     

Antioxidant and Anticholinesterase Activities of Extracts and Phytochemicals of Syzygium antisepticum Leaves

Bioassay-guided separation of young leaves extracts of Syzygium antisepticum (Blume) Merr. & L.M. Perry led to the isolation of four triterpenoids (betulinic acid, ursolic acid, jacoumaric acid, corosolic acid) and one sterol glucoside (daucosterol) from the ethyl acetate extract, and three polyphenols (gallic acid, myricitrin, and quercitrin) from the methanol (MeOH) extract. The MeOH extract of S. antisepticum and some isolated compounds, ursolic acid and gallic acid potentially exhibited acetylcholinesterase activity evaluated by Ellman’s method. The MeOH extract and its isolated compounds, gallic acid, myricitrin, and quercitrin, also strongly elicited DPPH radical scavenging activity. In HEK-293 cells, the MeOH extract possessed cellular antioxidant effects by attenuating hydrogen peroxide (H₂O₂)-induced ROS production and increasing catalase, glutathione peroxidase-1 (GPx-1), and glutathione reductase (GRe). Furthermore, myricitrin and quercitrin also suppressed ROS production induced by H₂O₂ and induced GPx-1 and catalase production in HEK-293 cells. These results indicated that the young leaves of S. antisepticum are the potential sources of antioxidant and anticholinesterase agents. Consequently, S. antisepticum leaves are one of indigenous vegetables which advantage to promote the health and prevent diseases related to oxidative stress.