Enterobacter cloacae MF01 and also to explore the generation of nanostructures to be used in future biotechnological processes. Resistance of E. cloacae MF01 to gold and tellurium salts as well as the putative mechanisms involved -both in aerobic and anaerobic growth conditions- was evaluated. These metal(loid)s were selected because of their potential application in biotechnology. Resistance to auric tetrachloride acid (HAuCl4) and potassium tellurite (K2TeO3) was assessed by determining areas of growth inhibition, minimum inhibitory concentrations, and growth curves as well as by viability tests. E. cloacae MF01 exhibited higher resistance to HAuCl4 and K2TeO3 under aerobic and anaerobic conditions, respectively. In general, their toxicity is mediated by the generation of reactive oxygen species and by a decrease of intracellular reduced thiols (RSH). To assess if resistance implies toxicant reduction, intra- and extra-cellular toxicant-reducing activities were evaluated. While E. cloacae MF01 exhibited intra- and extra-cellular HAuCl4-reducing activity, tellurite reduction was observed only intracellularly. Then, Au- and Te-containing nanostructures  were synthesized using crude extracts from E. cloacae MF01 and their size, morphology, and chemical composition was evaluated.Microorganism survival in the presence of toxic substances such as metal(loid)s lies chiefly on their ability to resist (or tolerate) such elements through specific resistance mechanisms. Among them, toxicant reduction has attracted the attention of researchers because metal(loid)-reducing bacteria are being used to recover and/or decontaminate polluted sites. Particularly, our interest is to analyze the toxicity of gold and tellurium compounds for the environmental microorganism  The refin vitro using crude cell extracts or purified proteins. In this frame, a number of enzymes involved in metal(loid) tolerance have been identified which, apart from their normal metabolic functions can help in the process of metal reduction. In particular, some of them belong to the flavoprotein family  reduction has also been observed n family  includinrganisms , NDH-II  could also participate in metal(loid) reduction . EPS canE. coli and Geobacillus sp., respectively, have generated great interest due to its potential application in the field of biotechnology. Nanostructure production by biological methods is considered a safe, economical and environmentally friendly process, in contrast to chemical procedures that require high temperatures, anaerobic conditions and/or the presence of a number of toxic components s because of their ability to change the compound’s oxidation state, influencing their solubility that results in the formation of less toxic and often insoluble derivatives . In somemponents .The specific, mechanical, optical and fluorescence properties exhibited by metal(loid)-containing nanostructures makes them excellent candidates to be used in the field of physics, chemistry, electronics, materials science, biomedicine, electronics and agriculture, among others . ParticuEnterobacter cloacae MF01 which could represent interesting candidates for biotechnological processes.Summarizing, it is of great interest to unveil the mechanism by which microorganisms are able to tolerate metal(loid) since it could involve the reduction of these elements to less toxic forms. In this line, this work describes the generation of Au- and Te-containing NS by Enterobacter cloacae MF01 was isolated from environmental water samples from central Chile . Growth in solid media was carried out in 2% (w/v) LB agar plates.4) or 4 mM potassium tellurite (K2TeO3). The zone of growth inhibition (cm2) was determined after incubating the plates at 37°C for 24 h.One hundred μL of cells grown to stationary growth phase were evenly spread on LB agar plates. After drying briefly in air, three sterile filter paper disks (0.6 cm diameter) were placed on each plate, to which 10 μL of the toxican to be tested were added: 50 mM auric tetrachloride acid  or 0.4 (anaerobic conditions) were added to each well and incubation proceeded with constant shaking at 37°C. Minimal inhibitory concentrations (MICs) were determined by monitoring turbidity at 600 nm after 24 h.Serial dilutions (1:2) were made from sterile solutions of HAuCl600 ∼0.6 (aerobiosis) or 0.4 (anaerobiosis). Then, 10 μL were added to 1 mL of fresh LB medium containing sublethal concentrations of each metal(loid) in 48-well plates. Growth was monitored at 600 nm every 30 min at 37°C for 15 h in a TECAN Infinite® M200 plate reader.Overnight cultures were diluted 1:100 with fresh LB medium and incubated at 37°C to OD600 (%), lag phase duration (h), growth rate (μ) and generation time (h) were determined. The maximum OD600 reached by the culture was referred to that of the control (100%). The duration of the lag phase was determined as the end point of the latency phase and beginning of the exponential phase of bacterial growth. The growth rate was estimated as the change in the OD600 per h during the exponential growth phase. Finally, the generation time was calculated from the equation g = 0.693/μ.Data were plotted and growth parameters corresponding to the maximum ODE. cloacae MF01 were diluted 1:100 with fresh LB medium and incubated at 37°C and 150 rpm to OD600 0.6 (aerobic) or 0.4 (anaerobic). Subsequently, 180 μL aliquots of the cultures were added to 20 μL of fresh LB medium containing different concentrations of the toxicants in 96 well plates . After 15 min, aliquots of 20 μL were taken and added to a 96-well plate containing 180 μL of 0.9% NaCl. Serial dilutions (1:10) were made in the 96-well plate, from which 4 μL were seeded in LB agar plates (drop). The number of colony forming units (CFUs) was determined after incubating at 37°C for 24 h.Overnight cultures of 4 or K2TeO3 were determined using 2′,7′-dichlorodihydrofluorescein diacetate as probe . Overnight E. cloacae MF01 cultures were diluted 1:100 with fresh LB medium and incubated at 37°C with shaking to OD600 ∼0.6 (aerobic) or ∼0.4 (anaerobic). Then, cells were treated with the toxicants (0.5x of the corresponding MIC) and with ascorbic acid (10 mM). One mL aliquots were taken at different times  and sedimented at 12,000 ×g for 5 min. Pellets were washed three times with 50 mM Tris-HCl buffer pH 7.4 and subsequently, 50 μL were added to 930 μL of 50 mM Tris-HCl buffer pH 7.4, treated with H2DCFDA  and incubated in the dark for 30 min. Cells were centrifuged as above for 5 min and washed with 50 mM Tris-HCl buffer pH 7.4. Finally, 200 μL of the cell suspension were used to determine fluorescence intensity in a Tecan Infinite® M200 Pro plate multi lector . Fluorescence intensity was normalized by OD600. Per cent of fluorescence intensity regarding that of the control condition (100%) was calculated.Intracellular total ROS levels in cells exposed to HAuClE. cloacae MF01 were diluted 1:100 with fresh LB medium and incubated at 37°C to ∼OD600 0.6 (aerobic) or 0.4 (anaerobic). Cultures were treated with the toxicants as above and then aliquots of 500 μL were taken at different times  and sedimented at 12,000 ×g for 2 min. Sediments were suspended in 1 mL of 5 mM EDTA, 0.1% SDS, 0.1 mM DTNB and 50 mM Tris-HCl buffer pH 8.0. The suspension was incubated for 30 min at 37°C and subsequently, vigorously shaken and sedimented at 12,000 ×g for 10 min. Finally, the supernatant was rescued and the absorbance at 412 nm measured in a Tecan Infinite® M200 Pro plate multireader. The RSH content was calculated through a calibration curve constructed with GSH standards (0–200 μM). RSH concentration was normalized by protein concentration. Per cent of RSH was calculated with respect to the respective control condition (100%).Overnight cultures of Protein concentration was determined by the method described by Enterobacter cloacae was grown to OD600 ∼0.6 (aerobic) or 0.4 (anaerobic), treated with the toxicants as above for 15 min and centrifuged at 12,000 ×g for 4 min at 4°C. After storing the supernatant, the sediment was suspended in 50 mM Tris-HCl pH 7.4 buffer supplemented with 0.1 mM phenylmethylsulfonyl fluoride . After sonicating on ice (four pulses of 20 s each), the cell debris was removed by centrifugation  for 10 min at 4°C. The supernatant was ultracentrifuged at 120,000 ×g for 1 h in a Beckman optima LE-80K centrifuge using a 70.1 Ti rotor. This new supernatant was considered the crude extract while the sediment represented the membrane fraction.3 (up to 2%), filtered  and used for determining gold and tellurium in a PerkinElmer Optima 2000 DV ICP-OES device. Calibration curves were made with gold and tellurium commercial standards. Per cent of gold and tellurium was calculated regarding the respective controls .Supernatants, intracellular and membrane fractions from cells exposed to the toxicants were diluted with HNO4 and K2TeO3 reducing activities were assessed at 37°C using 20 mM potassium phosphate (pH 6.0–7.0) and 50 mM Tris-HCl (pH 8.0–9.0) buffers, respectively. Generation of metallic gold and tellurium was monitored in a TECAN Infinite M200 Pro multimode plate reader at 500 nm  was defined as the amount of enzyme that increased the absorbance by 0.001 units in 1 min under the assay conditions. The enzymatic activity was normalized by the protein concentration.HAuCl0 nm Te,  and 540 0 nm Te, . ReactioE. cloacae MF01 were grown to stationary phase and centrifuged at 4°C. Supernatants were filtered under vacuum using a 0.45 μm nitrocellulose filter (Merck Millipore). EPS were precipitated by adding three volumes of cold ethanol to the filtrate. After incubating at 4°C, the sample was sedimented at 12,000 ×g for 10 min at 4°C and washed three times with 70% ethanol. EPS were dissolved in 2 mL of miliQ water and dialyzed for 24 h at 4°C against nanopure water .Purification of bacterial EPS was accomplished by using the modified cold ethanol precipitation method described by 2SO4. The mixture was cooled on ice for 2 min and the production of furfural derivatives was monitored at 315 nm. A D-glucose calibration curve was used as standard. Protein content was determined as described in Section “Materials and Methods.”Total sugar content in EPS was estimated using the modified UV-sulfuric acid method of 4 or K2TeO3, 5 mg/mL BPN’ subtilisin (Sigma) or 1 U/μL benzonase nuclease (Novagen) and 10 μL of the purified EPS. Controls to rule out abiotic reduction were run in each case. Reducing activities of HAuCl4 or K2TeO3 were defined as the difference in absorbance after incubation at 37°C for 24 h at 540 or 500 nm, respectively.Extracellular polymeric substance-mediated metal(loid) reducing activity was assessed as specified in Section “Metal(loid)-Reducing Activity by Crude Extracts.” The reaction was carried out in a final volume of 200 μL that contained the appropriate buffer, 1 mM HAuClE. cloacae MF01 were treated with 8 μM K2TeO3 (0.5x the MIC in aerobic conditions) and incubated at 37°C for 24 h. After plating on LB agar plates, incubation was continued under the same conditions for 24 h. Samples were fixed by mild heating in glass covers and then dried for later visualization. Prior to imaging, samples were stained, mounted on conductive adhesive and sputter coated with gold film. Electron microscopy analysis was performed using a SEM Zeiss EVO MA-10 equipment and imaged at an accelerating voltage of 20 kV and 8 mm of working distance. This study was conducted at the Center for the Development of Nanoscience and Nanotechnology – CEDENNA, Universidad de Santiago de Chile (USACH).To confirm the formation of exopolysaccharide material after toxicant exposure, cultures of E. cloacae MF01 (200 μg/mL protein) were used to produce nanostructures by incubation with 1 mM HAuCl4 or K2TeO3 for 24 h at the optimal conditions of temperature, pH, cofactor, and growth conditions (as determined from reduction assays) in a final volume of 1 mL. Controls to rule out abiotic synthesis were run in each case. Concentrations of these nanostructures were calculated by dry weight, which was expressed as μg/ml.Crude extracts from The morphology of the synthesized nanostructures was analyzed by TEM using a Hitachi Transmission Electron Microscope HT7700 . To do this, a drop of the synthesized nanostructures was placed on a copper grid and analyzed briefly after.Energy dispersive X-ray spectroscopy (EDS) analysis was carried out to determine the chemical composition of the synthesized nanostructures. Samples were analyzed by scanning electron microscopy (SEM) using a Zeiss EVO MA-10 microscope with a tungsten filament gun and by EDX spectra which were collected using an Oxford Instruments X-act system attached to a microscope equipped with a Penta FET Precision detector . Prior to imaging, samples were supported on glass slides and stained with Gram-Hucker kit, fixed on conductive adhesive over pin stub mount and sputter coated with gold film to protect the surface from damage and calcinations and to minimize charge related artifacts. The samples were imaged at an accelerating voltage of 20 kV and 8 mm of working distance.t-test were used considering p < 0.05. The statistical significance was indicated as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant.Plots and statistical analyses were carried out using the GraphPad Prism 7.0 . Analysis of variance (ANOVA) and 4 and K2TeO3 for E. cloacae MF01 both under aerobic and anaerobic conditions were determined. Under aerobic conditions, the MIC of HAuCl4 was twice that in the absence of oxygen (0.50 vs. 0.25 mM), thus suggesting that the anaerobic condition makes the isolate more sensitive to the toxicant. Conversely, TeO32- was more toxic for E. cloacae MF01 in aerobic growth conditions (0.016 vs. 0. 031 mM).Minimal inhibitory concentrations of HAuClE. cloacae MF01 exposed to HAuCl4 or K2TeO3 under aerobic and anaerobic growth conditions. While the bacterium grew better in the presence of HAuCl4 in aerobiosis, it turned to be more resistant to tellurite in the anaerobic condition.Supplementary Figure 4 and K2TeO3 on E. cloacae MF01 growth was evaluated in aerobiosis and anaerobiosis .The chemical composition of purified EPS from in vitro nanostructure synthesis by crude extracts of this bacterium lasted about 16 h. NS were generated at different pH values, electron donors and in the presence or absence of oxygen. Gold-containing nanostructures (AuNS) were synthesized in the following conditions: (i) O2 + NADH + pH 9.0, (ii) O2 + NADPH + pH 8.0, and (iii) O2 + NADPH + pH 9.0, (iv) -O2 + NADH + pH 8.0, (v) -O2 + NADH + pH 9.0, and (vi) -O2 + NADPH + pH 9.0. In turn, tellurium-containing nanostructures (TeNS) were synthesized aerobically and anaerobically in the following conditions: (i) NADH + pH 8.0, (ii) NADH + pH 9.0, (iii) NADPH + pH 8.0, and (iv) NADPH + pH 9.0. Supplementary Table In general, Nanostructure and chemical composition of AuNS and TeNS were determined by transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS), respectively. Figures Finally and independently of the presence of oxygen, TeNS exhibited a uniform, nanostick-like morphology of ∼50 nm. As with AuNS, in some cases aggregates of ∼200 nm were observed Figures , 8D. TelEnvironmental bacteria thrive in places where growth conditions are very variable, often hostile, such as high or low temperatures, nutrient deprivation, and presence of toxic agents, among others . In geneE. cloacae MF01 is highly resistant to HAuCl4 under aerobic growth conditions, toxicity tests showed that precisely in this condition it contains enhanced ROS levels as well as a decreased RSH concentration. ROS resistance may be related to the antioxidant defense system of this bacterium, i.e., catalase, peroxidase, and superoxide dismutase which eliminate toxic forms of oxygen would protect cell components that are prone to damage such as proteins, cell membranes, and the genetic material  of the metal(loid) remains in the extracellular fraction. While tellurite entrance is mediated mainly by the phosphate transporter PitA and/or by the acetate transporter ActP  glutathione reductase, flavorubredoxin reductase, dihydrolipoamide dehydrogenase and a putative oxidoreductase from E. coli  tet manner , (iii) ct manner , (iv) is glup. BNF22 , (vi) dip. BNF22 , thiored E. coli . In addiactivity .4- and K2TeO3-reducing activities were also evaluated in the pH range 6.0–9.0. To determine if any protein and/or nucleic acid contributed to the observed activity, EPS preparations were treated with proteases or nucleases prior the reducing assay. A similar mechanism has been described in E. cloacae SUKCr1D, where the EPS from this microorganism can reduce Cr(VI) to Cr(III)  .E. cloacae SUKCr1D is mainly composed of polysaccharides (22 mg/g), proteins (130 mg/g), nucleic- (28 mg/g), and uronic acids (56 mg/g). Our results of EPS-mediated gold-reducing activity support the importance of proteins in the process, since EPS treatment with proteases decreased gold-reducing activity almost to half of that exhibited by untreated EPS irrespective of the presence of oxygen.Extracellular polymeric substance dependent gold-reducing activity was observed only at pH 6.0 and 7.0, in contrast to the results obtained with crude extracts, which revealed gold-reducing activity mainly at pH 8.0 and 9.0. An obvious explanation is that extracellular gold reduction is being carried out by different agents from those forming part of the EPS . Since E4, no EPS-mediated tellurite reductase activity was found under the tested conditions. Despite this, a white precipitate was always formed. Probably tellurite interacts with positively charged groups that are present in EPS components , which could chelate the toxicant -containing NS at the same conditions of pH, enzyme cofactor, and growth conditions that were utilized in metal(loid)-reducing activity tests. In general, reducing activity and NS generation correlated well, excepting for HAuCl4-reducing activity from anaerobic cultures at pH 6.0 in the presence of NADH.2 and pH 9.0/NADPH/-O2 showed different morphology and smaller size than their counterparts synthesized under the other conditions tested. TeNS appeared as aggregates, which is probably due to size reduction and increased attractive forces that allow NS to interact with each other  content since metal(loid) oxidation is avoided thus facilitating NS formation .E. cloacae MF01 showed higher resistance to HAuCl4 and K2TeO3 under aerobic and anaerobic growth conditions, respectively. Toxicant’s toxicity was due to ROS generation and RSH depletion. E. cloacae MF01 displayed intra- and extra-cellular HAuCl4 reduction and only intracellular K2TeO3-reducing activity. Since in most cases the highest reducing activity did not correlate with higher toxicant resistance, it could be inferred that another resistance mechanism, still unknown, may be involved in alleviating the effects of these toxicants. Finally, the biological synthesis of Au- and Te- containing NS by crude extracts from E. cloacae MF01 could represent a start point to support future interesting biotechnological applications.The environmental isolate FC, CM-V, CV, and FA conceived and designed the experiments and analyzed the data. FC, EV, KJ, MF, and CM-V performed the experiments. CV and FA contributed reagents, materials, analysis tools, and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ginkgo biloba L. leaf extracts and herbal infusions are used worldwide due to the health benefits that are attributed to its use, including anti-neoplastic, anti-aging, neuro-protection, antioxidant and others. The aim of this study was to evaluate the effect of an aqueous Ginkgo biloba extract on HepG2 cell viability, genotoxicity and DNA protection against paraquat-induced oxidative damage. Exposure to paraquat (PQ), over 24 h incubation at 1.0 and 1.5 µM, did not significantly reduce cell viability but induced concentration and time-dependent oxidative DNA damage. Ginkgo biloba leaf extract produced dose-dependent cytotoxicity (IC50 = 540.8 ± 40.5 µg/mL at 24 h exposure), and short incubations (1 h) produced basal and oxidative DNA damage . However, lower concentrations  of Ginkgo biloba leaf extract were not cytotoxic and reduced basal DNA damage, indicating a protective effect at incubations up to 4 h. On the other hand, longer incubations (24 h) induced oxidative DNA damage. Co-incubation of HepG2 cells for 4 h, with G. biloba leaf extract (75 µg/mL) and PQ (1.0 or 1.5 µM) significantly reduced PQ-induced oxidative DNA damage. In conclusion, the consumption of Ginkgo biloba leaf extract for long periods at high doses/concentrations is potentially toxic; however, low doses protect the cells against basal oxidative damage and against environmentally derived toxicants that induce oxidative DNA damage. Ginkgo biloba L., the oldest living tree, has a long history of use in traditional Chinese medicine; nowadays the aerial parts (used as an infusion) and the standardized leaf extract (EGb761)- of G. biloba are among the most widely sold phytomedicines or dietary supplements in Europe and the USA [Plants and herbs have been used since ancient times and today many people still rely on medicinal plants for their primary health care, others use them as a natural source of bioactive molecules. act EGb76- of G. b the USA . Severalact EGb76- of G. b the USA ,3,4,5, a the USA , antagon the USA ,5, inhibact EGb76- of G. b the USA  and anti the USA ,11,12.Ginkgo biloba leaf extract are inconsistent; some authors highlight its ability as an antioxidant with the ability to scavenge hydrogen peroxide [G. biloba extract is pro-oxidant [The bioactivity of natural compounds such as antioxidant, anticancer, and antiaging activity, has gained importance in the phytopharmaceutical industry due to the growing interest in natural compounds for application in preventive medicine. Data on the antioxidant activity of peroxide ,14, as aperoxide , and itsperoxide ; others -oxidant ,17 or ev-oxidant ,18,19, wSaccharomyces cerevisiae following an oxidative shock, when pre-exposing or simultaneously exposing S. cerevisiae with G. biloba water extract in the mg/mL range of concentration. In an in vivo study, a commercially available G. biloba extract (EGb) was shown to reduce comet tail intensity of rat hepatocytes in a hepatocellular carcinoma model using N-nitrosodiethylamine as a tumor inductor [Concerning DNA repair, Marques and collaborators  showed ainductor .G. biloba leaf extract in DNA and its potential protective effect against paraquat-induced DNA oxidative damage in HepG2 cells. We used HepG2 cells that retain the enzymatic activity of normal human hepatocytes and mimic in vivo responses [Paraquat  is a non-selective herbicide, which is still used even though its use is restricted, or even forbidden in some countries, and exposure to it may increase the risk of cancer  as it inesponses , and theesponses .In this work, we aimed to use PQ as an oxidative stress inducer that causes significant DNA damage without producing a reduction in cell viability. Exposure of HepG2 cells to PQ (1.0–8.0 µM) for 24 h did not alter cell viability, as measured by the cell viability indicator Alamar Blue A. These G. biloba extract on cell viability, cells were exposed for 24 h . P > 0.05). But, although the level of oxidized purines remained similar to control (P > 0.05) at 1 h treatment, it peaked at 4 h of treatment , with this value being 4.5-fold higher on average, than control (P < 0.05). The % DNA in the tail at 24 h  is slightly higher than at 4 h, but the differences are statistically significant , which may be converted into hydrogen peroxide (H2O2) by superoxide dismutase (SOD) and then eliminated by catalase (CAT). The activity of these enzymes determines the production of oxygen reactive species (ROS) that contribute to DNA damage [Paraquat is an oxidizing molecule that has been shown to induce DNA damage in some studies . Concentrations of 750 and 1500 µg/mL significantly increased strand breaks (light grey bars) compared to control, and oxidized purines (green bars) attain a higher percentage for 1500 µg/mL of extract . However, at 75 µg/mL the extract did not induce DNA damage for 1 h exposure , and its effect on oxidative damage (with FPG) only shows a significant increase in oxidized purines at 24 h incubation (~2-fold higher than control). These results are in line with the results of cell viability  at concentrations up to 2 mg/mL. These authors, pointed out that cell toxicity is a result of oxidative stress [Ginkgo biloba aqueous extract at higher concentrations induces oxidative DNA damage , which is indicative of loss of membrane integrity. Other studies have suggested that the pro-oxidant activity of G. biloba extract causes cell toxicity [G. biloba extract.Hecker and co-workers  reportedtoxicity ,39,43. FG. biloba extract and 4 h incubation, as this concentration and period of incubation do not cause cell toxicity and DNA damage .From the above experiment, we chose to use 75 µg/mL G. biloba extract and 4 h incubation, because this concentration and period of incubation do not cause cell toxicity or DNA damage with significant differences. HepG2 cells were concomitantly exposed to 75 µg/mL G. biloba and to 1.0 or 1.5 µM PQ . From the above experiment, we chose to use 75 µg/mL .5 µM PQ  for 4 h.e damage , as obseSaccharomyces cerevisiae with G. biloba water extract plus hydrogen peroxide, where G. biloba showed a DNA protective effect [Our findings corroborate the results of other experimental models, such as in the case of simultaneous incubation of e effect . G. biloba extract at high concentrations, which may induce mutations in the DNA sequence that will join to naturally occurring mutations, is of interest due to concerns about the potential health risks for the general population who are exposed to diet supplements. Over time, cell antioxidant defenses weaken or become inadequate, increasing the risk of developing genetically-based diseases, such as cancer. A study in Swiss albino mice showed that long-term treatment with G. biloba extract can cause aneuploids, decreased fertility and hampers the pre-implantation of embryos due to embriotoxic and neurotoxic effects. This also warrants its careful use as a remedy for male impotence and/or erectile dysfunction [G. biloba for long periods of time [G. biloba extract are cytotoxic . Taking into consideration that most of the compounds in the infusion will suffer pH degradation (in the stomach and intestine) and/or oxidation, and that absorption will not be complete as some compounds will not be absorbed because of the presence of fibers in the gut, we may speculate that the amount of extract reaching the whole body is in the antioxidant range. However, caution must be taken if several Ginkgo biloba leaf at low concentrations (up to 200 µg/mL), did not produce cell toxicity or induce DNA damage for short exposures, but instead resulted in a reduction in oxidative DNA damage, indicating an antioxidant effect. However, at higher concentrations, cell toxicity and oxidative DNA damage were observed in a concentration and time-dependent manner. Most importantly, at low concentrations, i.e., as found in the regular usage of a tea-bag per day, a protective effect against PQ-induced DNA damage was observed. Given these results, an antioxidant effect for low concentrations and oxidant properties of Ginkgo biloba extract at high concentrations were observed, thus care should be taken when using frequent infusions or extracts directly from leaves for long periods and at high dosages. The moderate consumption of G. biloba infusions might contribute flavonoids and other phenolic compounds that act as free radical scavengers to reduce cell oxidative stress. However, high doses, in addition to providing beneficial compounds, also appear to contribute compounds that generate cellular stress, which can be translated into DNA damage. PQ was used as a model for oxidative DNA damage, further studies could examine the protective effect of Ginkgo biloba extract (at low doses) against other environmental stressors, such as food containing contaminants .Paraquat may be used experimentally as oxidative stress inducer. The aqueous extract of Ginkgo biloba leaves were collected at Vila Real, Portugal from a tree sample in the University Botanical Garden . Aqueous extraction was performed as described by Ding and co-workers in [g for 15 min. The supernatant was collected, filtrated, the pH adjusted to 6.5 and it was then frozen at −20 °C before its lyophilization. After lyophilization, a yield of 15.3%  × 100%) was obtained.rkers in . Briefly2 in air at 37 °C, as described previously [The human hepatoma cell line  was maintained in culture in DMEM  with 25 mM glucose, supplemented with 10% fetal bovine serum , 2 mM L-glutamine  and antibiotics  in an atmosphere of 5% COeviously ,46.4 cells/mL (100 µL/well) for 24 h. After seeding, the culture media was replaced by FBS-free culture media supplemented with PQ (1–8 µM) or with G. biloba extract at the desired concentration (from 50 to 1500 µg/mL). To prepare these solutions, a 20 mg/mL stock solution of G. biloba extract was prepared in FBS-free culture medium, and from this, subsequent dilutions were made in FBS-free culture medium. Cell viability was evaluated with Alamar blue assay . Thus, 24 h after exposure of the cells, FBS-free culture media supplemented with 10% (v/v) of Alamar blue was added (100 µL to each well), and absorbance at 570 nm  and 620 nm  was read ~4 h later. Data were analyzed by calculating the percentage of Alamar blue reduction (according to the manufacturer’s recommendation) and expressed as a percentage of control (untreated cells), as reported in [HepG2 cells were handled as described in ,46. Brieorted in .5 cells/mL (1 mL/well). After seeding for 24 h, the culture medium was discarded and replaced by Ginkgo biloba extract and/or paraquat  diluted in FBS-free culture medium. Negative controls were made throughout using FBS-free culture medium. HepG2 cells were seeded onto 12-well culture plates at a density of 1 × 10G. biloba extract on DNA damage, cells were exposed to 75, 750 and 1500 µg/mL of G. biloba extract (stock solution diluted in FBS-free culture media) for 1 h, and for 75 µg/mL of G. biloba extract, comet assay was performed at 1, 4 and 24 h after cell exposure. (iii) Finally, a set of cells were submitted to G. biloba extract (75 µg/mL) incubation for 4 h and simultaneously incubated (co-incubation) with 1.0 or 1.5 µM PQ.Treatments were performed in three distinct conditions: (i) For optimization of the binomial concentration-time to maximize PQ-induced oxidative stress, cells were incubated with PQ at final concentrations of 1.0, 1.5, 2.0 and 5.0 µM for 30 min or 1 h, and for lower PQ concentrations (1.0 and 1.5 µM), incubations of 0.5, 1, 4 and 24 h were used. (ii) To study the effect of w/v) low-melting agarose  in PBS, heated at 37 °C, and embedded in gels, which were placed onto glass microscope slides pre-coated with 1% (w/v) normal melting point agarose  in water. Each gel (70 µL) contained approximately 2 × 104 cells [Comet assay was performed as described previously ,34,48,49The two gels, placed on each side of a microscope slide, served as duplicates. Slides with cells were immersed into cold lysis solution  for 1 h at 4 °C. Then, slides were placed in electrophoresis solution  for 40 min at 4 °C in the dark . Subsequently, gels underwent electrophoresis at 25 V and 300 mA, for 30 min at 4 °C in the dark. After electrophoresis, gels were washed twice with cold (4 °C) distilled water. Strand breaks allow DNA to extend from the nucleoids towards the anode, forming a “comet tail”.w/v) bovine serum albumin, pH adjusted to 8 with KOH) at 4 °C. After washing, 50 µL of FPG was dropped onto each gel, covered with glass and incubated at 37 °C for 30 min in a humid chamber. Alkaline treatment and electrophoresis then followed as described above. The additional breaks formed at the oxidized purines lead to an increase in the amount of DNA in the tail. FPG (a lesion specific enzyme) was used to measure oxidized purines, including 8-oxoguanine. After lysis the slides were washed three times with enzyme reaction buffer  after staining with ethidium bromide , and computerized image analysis was applied using Comet Assay IV software. The percentage of DNA in the tail  was taken as a measure of DNA damage.50  was calculated as reported by Silva et al [P-value < 0.05). The Tukey test was applied for multiple comparisons.Data from different experiments are presented as mean ± standard deviation (S.D.) or as mean ± S.E.M. (as mentioned). The value of ICva et al . Statist


Thyroid hormone (TH) is essential for normal brain development. TH insufficiency during early stages of development may increase the risk for attention deficit/hyperactivity disorder, in which malfunction of brain monoaminergic systems is likely involved. However, little is known about the effects of perinatal hypothyroidism on behaviors and brain monoaminergic systems in offspring mice. The present study examined in mice (1) whether perinatal hypothyroidism causes hyperactive behavioral phenotypes, (2) how perinatal hypothyroidism influences brain monoaminergic systems, and (3) whether hyperactive behavioral phenotypes are associated with the state of brain monoaminergic systems. When dams were exposed to propylthiouracil, offspring mice developed hypothyroidism during the perinatal period. Offspring mice with perinatal hypothyroidism exhibited hyperactive behavioral phenotypes such as hyper-ambulatory activity and an increased response rate in the two-way active avoidance test in a male-specific manner. Significant decreases in dopamine (DA) and serotonin turnover were observed in the striatum (ST), nucleus accumbens, hypothalamus, and hippocampus in male mice with perinatal hypothyroidism. A significant correlation between ambulatory activity and DA turnover in the ST and an augmented ambulatory response to the DA reuptake inhibitor bupropion suggested that DA in the ST was involved in the hyper-ambulatory activity in mice with perinatal hypothyroidism. Many arRisk factors for ADHD in humans produce ADHD-like behavioral phenotypes in mice, suggesting that mice are a useful animal species to test the potential of putative factors that induce ADHD-like neurodevelopmental disorders. Manipulation of the candidate gene for ADHD identified in humans ,14,22,41Malfunction of brain monoaminergic systems may be involved in the etiology of ADHD . Human gIn the present study, in mice, we focused on (1) whether perinatal hypothyroidism causes hyperactive behavioral phenotypes, 2) how perinatal hypothyroidism influences brain monoaminergic systems, and (3) whether behavioral phenotypes are associated with the state of brain monoaminergic systems. The TH synthesis inhibitor propylthiouracil (PTU) was used to induce hypothyroidism. In mice, midbrain DA neurons start to extend axons at GD11.5, and 5-HT neurons start extending axons at approximately GD11, followed by increase in axonal projection density until PND60 . Althoug how peri22.1ad libitum. The cages were placed in a room artificially illuminated by fluorescent lamps on a 12L:12D schedule (light period: 07:00-19:00) at a room temperature of 25 ± 1 °C.ICR (or CD-1) strain mice  were used. Mice were housed in aluminum cages with stainless-steel mesh tops and paper bedding. Commercial solid food (Clea Japan) and tap water were provided Total number of animals used for each treatment group were; tap water group; dams; 22, male pups; 134, female pups; 70, 125 ppm PTU group; dams; 20, male pups; 110, female pups; 53, 250 ppm PTU group; dams; 22, male pups; 115, female pups; 55, 500 ppm PTU group; dams; 20, male pups; 104, female pups; 51.All experiments were performed following approval by the Committee for Experimental Animals of the National Institute for Environmental Studies, Japan.2.2ad libitum as drinking water to pregnant females. BUP was prepared in 0.9% NaCl  solution  and given to offspring mice. The administration volume was 1 mL/100 g body weight regardless of the dosage.Propylthiouracil -pyrimidinone; PTU) and bupropion HCl (BUP) were purchased from Sigma-Aldrich . PTU was dissolved in tap water at concentrations of 125, 250, or 500 ppm, and the solution was available 2.3Serum T4 levels in collected blood samples from dams and male and female offspring mice were measured by radioimmunoassay using DPC・total T4 kits , Tokyo, Japan) according to the company’s instructions. The limit of detection was 1.0 μg/dL.2.4Ambulatory activity of the mice was measured using a tilt-type ambulometer . The apparatus is explained in detail elsewhere .2.52.5.1via an interface , and computer software (O’Hara & Co.) was used to control the avoidance schedule and record data.Two-way active avoidance test was conducted using the apparatus previously reported ,35. Brie2.5.2Experimental conditions of two-way active avoidance test was same to those in the previous studies ,35. Brie2.6g for 15 min at 4 °C. The supernatant was frozen in liquid nitrogen and stored at −135 °C until the assay. DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), 5-HT, and 5-hydroxyindoleacetic acid (5-HIAA) levels were measured using high performance liquid chromatography with electrochemical detection (HPLC/ECD). The mobile phase  was added at 1.0 mL/min by the HPLC/ECD system  to a 4.6 mm × 250 mm ODS-C18 column  maintained at 19 °C. Monoamines were detected with an electrochemical detector  using a glassy carbon electrode maintained at +750 mV relative to an Ag/AgCl reference electrode. Protein concentration was determined using a BCA protein assay kit .Mice were sacrificed by decapitation, and the brain was removed and placed on ice. The frontal cortex, hypothalamus (HYPO), striatum (ST), hippocampi (HIPP), and nucleus accumbens (NAcb) were bilaterally dissected out, frozen in liquid nitrogen, and stored at −135 °C. Tissue was sonicated in 0.4 N perchloric acid and centrifuged at 17,760 × 2.72.7.1The day when a plug was observed in the dam was defined as GD0. Each dam was individually kept in a cage. Administration of 125, 250, or 500 ppm PTU solution was started on GD15. Tap water was given to control dams. The dams were observed every day, and the day when delivery was observed was defined as PND0. On PND0, the litter size and body weight of offspring were measured. Every 3–4 days after delivery, the body weight and eye opening of offspring were checked. According to our preliminary study, PND25 was set as the day for weaning in this study, as apparent growth retardation was observed in PTU-exposed offspring. On PND25, male and female offspring were separated, and their body weight was measured. Exposure to PTU was terminated on PND25, and tap water was given to all offspring mice thereafter. After weaning, the body weight of offspring was measured every 3–4 days.2.7.2On PND25, dams and one or two male and female offspring randomly selected from each litter were deeply anesthetized, and blood was transcardially collected. The serum samples of the collected blood were stored and subjected to T4 measurement at a later time.2.7.32.7.3.1Ambulatory activity of male and female offspring mice was measured 2–4 months after birth. Mice were individually placed in the activity cages of the ambulometer, and the ambulatory activity was continuously measured for 30 min.In the test to evaluate the response to the ambulation-promoting effect of BUP, individual mice were placed in the activity cages, and 30 min later, saline or 1.25, 2.5, 5, or 10 mg/kg BUP was subcutaneously administered, followed by measurement of ambulatory activity for 60 min. The test was repeated in the same mice starting with saline, followed by BUP challenges from a low dose to a high dose at 3- to 4-day intervals.2.7.3.2The two-way active avoidance test was performed 6–8 months after birth in male offspring mice and 8–10 months after birth in female offspring mice. Mice were individually placed in the experimental chambers and underwent an avoidance session for 40 min  in 1 day. The avoidance session was repeated in the same mice for 5 consecutive days.2.7.4The brain monoamine content assay was conducted on male offspring mice. Ambulatory activity was measured on PND46-48. Mice were individually placed in the activity cages of the ambulometer, and the ambulatory activity was continuously measured for 30 min. After measurement of the ambulatory activity, the mice were returned to their home cages. The mice were sacrificed by decapitation on PND60, and the brains were collected. The samples were kept at −135 °C and later subjected to the monoamine content assay using the HPLC/ECD system.2.8P < 0.05 was considered statistically significant in all statistical analyses.Changes in body weight were analyzed using repeated measures analysis of variance (ANOVA), followed by one-way ANOVA for each PND. The difference between tap water-treated controls and each PTU-exposed experimental group was tested using Dunnett’s test. The day of eye opening was analyzed using the Wilcoxon test.2).As data were not distributed normally, ambulatory activity was analyzed using the Kruskal-Wallis or Wilcoxon tests. Similarly, monoaminergic neurotransmitter contents, metabolite contents, and turnover in the five brain regions were also analyzed using the Wilcoxon test. Relationships between ambulatory activity and each monoaminergic neurotransmitter content, metabolite content, and turnover in the five brain regions were examined with regression analysis after square root transformation of the ambulatory activity and the monoaminergic indices. The best-fit regression equation was obtained with the least-squares method. The significance of the best-fit regression equation was evaluated with ANOVA. Correlations between the ambulatory activity and each monoaminergic index were also evaluated using the coefficient of determination . Serum T4 levels were measurable in one dam, but were below the detectable level (1.0 μg/dL) in other dams in the 125 ppm PTU-administered group. Serum T4 was not detected in any male or female offspring of the 125 ppm PTU group. Serum T4 was not detected in any dams or male or female offspring in the 250 or 500 ppm PTU-administered groups .A delay in body weight gain is a typical physical sign of hypothyroidism during development. Perinatal PTU exposure retarded body weight gain in male and female offspring after birth in a concentration-dependent manner  = 9.0736, P = 0.0044; Postnatal day F = 2063.7214, P < 0.0001; Interaction F = 3.4485, P < 0.0001; female offspring; PTU dose F = 3.0844, P = 0.0769; Postnatal day F = 1655.5591, P < 0.0001; Interaction F = 2.2202, P < 0.0001) , (b)). Eye opening is a milestone in the development of pups. The day of eye opening was significantly delayed in PTU-exposed offspring in a concentration-dependent manner ).3.23.3The response rate in the two-way active avoidance test is the average number of interruptions (during 40 min) of two sets of photo beam sensors horizontally placed in the experimental chamber. Thus, the response rate represents the degree of horizontal movement of the mouse in the experimental chamber.Male offspring mice that were perinatally exposed to 500 ppm PTU exhibited a significantly increased response rate  = 3.2559, P = 0.0238; Session, F(4. 127) = 12.3038, P < 0.0001; Interaction F = 0.7636, P = 0.6883: One-way ANOVA; 1 st session: F = 4.6606, P = 0.004; 2nd session: F = 3.4531, P = 0.0185; 3rd session: F = 2.337, P = 0.0767; 4th session: F = 1.8067, P = 0.1491; 5th session: F = 2.376, P = 0.073) . On the A change in the avoidance rate according to the number of trials and/or sessions indicates that mice have learned the avoidance response. No significant effect of PTU exposure was observed on the acquisition process in the male mice  = 2.6247, P = 0.0533; Session, F = 150.2645, P < 0.0001; Interaction, F = 1.2102, P = 0.2723). Thus, perinatal exposure to PTU produced enhanced horizontal motor activity in the avoidance test in offspring mice in a male-specific manner.3.4Different offspring mice given tap water or 125–500 ppm PTU solution from GD15 to PND25 were prepared, and were used in the following experiment.The ambulatory activity was measured in the male offspring mice. Male offspring that were exposed to PTU showed significantly enhanced ambulatory activity /DA, and 5-HIAA/5-HT in the NAcb , the DOPAC content, HVA/DA, and 5-HIAA/5-HT in the HYPO , and the 5-HT content and 5-HIAA/5-HT in the HIPP  of the male offspring mice. No effects of perinatal PTU exposure on the monoaminergic state were observed in the frontal cortex (data not shown).2) were:Correlations between the ambulatory activity and each index for the monoaminergic state in the five brain regions were examined using regression analysis. A significant correlation was found between the ambulatory activity and (HVA+3-MT)/DA in the ST . The regThus, the hyper-ambulatory activity was accompanied by changes in the brain monoaminergic state, and alteration of DA turnover in the ST was negatively correlated with the hyper-ambulatory activity.3.5Hyper-ambulatory activity was accompanied by changes in the brain monoaminergic state. In particular, changes in DA turnover in the ST may be involved in the hyper-ambulatory activity. BUP inhibits the DA transporter, increases extracellular DA levels in the ST, and thus, promotes ambulation in mice . Therefo24 = 33.8394, P < 0.0001, 125 ppm; χ24 = 37.8806, P < 0.0001, 250 ppm; χ24 = 57.5084, P < 0.0001, 500 ppm; χ24 = 38.2188, P < 0.0001). Obtained dose-response relationships for the ambulation-promoting effect of BUP indicated that mice given 250–500 ppm PTU exhibited a significantly augmented ambulatory response to 5–10 mg/kg BUP (Different male offspring mice given tap water or 125–500 ppm PTU solution from GD15 to PND25 were prepared, and were used. After 30 min adaptation period, saline or 1.25, 2.5, 5, or 10 mg/kg BUP was subcutaneously administered, followed by measurement of ambulatory activity for 60 min. The test was repeated in the same mice starting with saline, followed by BUP challenges from a low dose to a high dose at 3- to 4-day intervals. BUP produced significant ambulation-promoting effects in all treatment groups (saline; χg/kg BUP . Thus, p4T4 was below the detectable limit in offspring mice at the end of perinatal exposure to 125–500 ppm PTU. In addition, a delay in eye opening and retardation of body weight gain were also observed in offspring mice. These results indicate that offspring mice experienced hypothyroidism following exposure of their dams to 125–500 ppm PTU from GD15 to PND25. The delay in eye opening and retardation of body weight gain were concentration dependent. On the other hand, the behavioral effects were concentration-dependent in some degree, and concentration dependency was not apparent for the PTU effects on brain monoamine contents and turnover. Concentration-dependent retardation of body weight gain was observed, although growth curves for the PTU-exposed mice were almost parallel to that for control mice after termination of PTU exposure. This could be due to rapid elimination of PTU and recovery of TH levels after termination of PTU exposure . Such raAmbulatory activity was measured using the tilting cage method, and therefore, ambulatory activity represents motor activity of mice in the cage. The tilting cage method is more sensitive to horizontal movement such as locomotion of the mouse in the activity cage than to vertical movement . The resIn the present study, we observed hyper-ambulatory activity and an increased response rate in the avoidance test in offspring mice with perinatal hypothyroidism. The hyperactive characteristics were evident in male offspring, but not in female offspring. TH produces its physiological effects through TRα, TRβ, and indirect molecular pathways, and the roles of TRα and TRβ for locomotor activity are likely different in mice. Mice expressing human mutant TRβ1 and TRβ-KO mice exhibit hyper-locomotor activity ,33, wherDA and 5-HT systems in mouse brain considerably develop during the perinatal period . TRs areAmbulatory and/or locomotor activity is related to DA tone in the ST ,37. We oThe hyperactive behavioral phenotypes of mice with perinatal hypothyroidism were male specific. This phenomenon is of interest, as the prevalence of ADHD is higher in boys than in girls in the ratio of 3–4:1 . TestostA significant decrease in 5-HT turnover was also observed in the ST in mice with perinatal hypothyroidism. As mouse locomotion involves interactions between DA and 5-HT ,13,30,38In conclusion, the present study revealed the hyperactive behavioral phenotypes and the altered monoaminergic state in various brain regions in male mice with perinatal hypothyroidism. Alteration in DA tone in the ST was likely involved in the hyperactive behavioral phenotypes.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The central oscillator generating cyanobacterial circadian rhythms comprises KaiA, KaiB, and KaiC proteins. Their interactions cause KaiC phosphorylation and dephosphorylation cycles over approximately 24 h. KaiB interacts with phosphorylated KaiC in competition with SasA, an output protein harboring a KaiB-homologous domain. Structural data have identified KaiB–KaiC interaction sites; however, KaiB mutations distal from the binding surfaces can impair KaiB–KaiC interaction and the circadian rhythm. Reportedly, KaiB and KaiC exclusively form a complex in a 6:6 stoichiometry, indicating that KaiB–KaiC hexamer binding shows strong positive cooperativity. Here, mutational analysis was used to investigate the functional significance of this cooperative interaction. Results demonstrate that electrostatic complementarity between KaiB protomers promotes their cooperative assembly, which is indispensable for accurate rhythm generation. SasA does not exhibit such electrostatic complementarity and noncooperatively binds to KaiC. Thus, the findings explain KaiB distal mutation effects, providing mechanistic insights into clock protein interplay. Circadian rhythms enable organisms, including mammals and plants, to coordinate their biochemistry and physiology with daily environmental alterations that are caused by Earth’s light and dark cycles. Cyanobacteria are photoautotrophic organisms that use circadian rhythms to regulate their photosynthetic activity. A similar regulatory mechanism has been observed in some eukaryotic algae and plants; accordingly, cyanobacteria are considered to be one of the most useful model organisms to study circadian biology . CyanobaKaiC forms a homohexamer with a double-ring structure composed of the CI and CII rings a 4,5]. ,5. 4,5].Thermosynechococcus elongatus SasA). The phosphorylated SasA is released from KaiC, and it subsequently interacts with and transfers the phosphate group to its cognate response regulator, RpaA . I. I20]. I protein ,22. Ther of KaiC . Further10–108 and TeKaiCDD formed a complex exclusively in a 6:6 stoichiometry , which was confirmed to be monomeric . Furthermore, the mutation caused considerable reduction in the amplitude of the circadian oscillation. These results indicate that the electrostatic complementarity at the KaiB–KaiB interaction surface is a determining factor for an accurate circadian clock oscillation. Previous studies have reported that a KaiB R22A mutation decreased its affinity for the KaiC hexamer and a KaiB D90G mutation severely impaired the S. elongatus PCC 7942 circadian rhythm [As shown in n rhythm . BecauseThe KaiB hexameric ring formed on the KaiC hexamer can bind KaiA molecules ,26, resuThe findings suggest that the cooperative binding of the KaiB protomers to the KaiC hexamer enables sharp phase switching of the circadian oscillator complex through the synergistic release of the SasA clock-output protein and the coincidental sequestration of the KaiA phosphorylation enhancer. These mechanisms contribute to the synchronous nature of the cyanobacterial circadian system.Synechococcus elongatus PCC 7942 and T. elongatus were expressed in Escherichia coli. SyKaiA and SyKaiC were expressed and purified as Strep-tagged recombinant proteins [10–108, TeKaiB10–108/DD, SyKaiBDD, SyKaiCDT, TeKaiCDD, and SasAN were expressed and purified by the same method used for their wild-type counterparts [10–108 and the corresponding Arg22 and Lys25 in SyKaiB were substituted with aspartate residues.KaiA, KaiB, KaiC, and SasA from proteins ,28. TeKaproteins ,21. The terparts ,22,29,30DD was performed with a Superdex 200 increase column  equilibrated with 20 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA and 1 mM DTT at 0.75 min/mL flow rate. The elution profiles of proteins were monitored by absorbance at 280 nm.Size exclusion chromatography analysis of TeKaiB or TeKaiB10–108 or TeKaiB10–108/DD (0.2 mg/mL) was dissolved in 20 mM phosphate buffer (pH 7.8) containing 150 mM NaCl. Measurements of circular dichroism spectra were performed in a 1-mm quartz cuvette at a room temperature using a spectropolarimeter . After subtraction of the spectrum of the buffer alone, data were represented as mean residue ellipticities.TeKaiBDD (20 µM) was titrated with either KaiB proteins (TeKaiB10–108 and TeKaiB10–108/DD) or SasA proteins (TeSasAWT and TeSasAN). The mixed protein solutions were incubated at 37 °C for 6 h, and were subsequently buffer-exchanged into 150 mM ammonium acetate, pH 6.8, using a Bio-Spin 6 column  according to the manufacturer’s instructions. In analyses of the Kai proteins from Synechococcus elongatus PCC 7942, the purified SyKaiCDT (20 µM) was mixed with SyKaiB or SyKaiBDD (30 µM), followed by incubation at 30 °C for 6 h. Approximately 2–5 µL of the buffer-exchanged protein solutions were immediately analyzed by nanoflow electrospray ionization MS using gold-coated glass capillaries prepared inhouse. Spectra were recorded on a SYNAPT G2-Si HDMS  in the positive ionization mode at 1.33 kV with a 150 V sampling cone voltage and source offset voltage, 0-V trap and transfer collision energy, and 5-mL/min trap gas flow. The spectra were calibrated using 1 mg/mL cesium iodide and analyzed using MassLynx software (Waters). Spectral measurements were performed at least in triplicate.The purified TeKaiCWT, or SyKaiBDD at 30 °C, and 3 μL aliquots of the reaction mixtures were removed to stop the reaction at specific time intervals, as described previously [Reaction mixtures of SyKaiA (1.5 µM) and SyKaiC (3.4 µM) were incubated in the presence of 4.2 µM SyKaiBeviously . The alieviously . The releviously .


The aim of the study was to evaluate power performance and muscle morphology adaptations in response to 5 weeks of fast-eccentric squat training (FEST) performed twice per week, with three different training volumes. Twenty-five moderately trained females were assigned into three groups performing eight repetitions of FEST of either four sets , 6 sets  or eight sets . Before and after the intervention, countermovement jumping height (CMJh) and power (CMJp), half squat maximal strength (1-RM), quadriceps cross-sectional area (QCSA) and vastus lateralis (VL) architecture and fiber type composition were evaluated. Significant increases (p < 0.05) were found for all groups, with no differences among them in 1-RM , CMJh , CMJp  and QCSA . Muscle fiber type distribution remained unaltered after training in all groups. VL fascicle length increased and fascicle angle decreased only in 6 × 8 and 8 × 8 groups. In conclusion, four sets of eight fast-eccentric squats/week increase lower body power and strength performance and maintain type IIX muscle fibers after 5 weeks, at least in moderately trained females. Muscle power determines the ability to perform explosive actions both in sports and in everyday life. Resistance training performed with maximum concentric velocity is an effective intervention to enhance muscle power ,2. InterPower resistance training involves primarily high-velocity movements with body weight or with additional external loads, e.g., jumps , ballistAmong other resistance training regimens, eccentric training has stimulated the interest of sports scientists towards increasing explosive performance. This type of training exploits the ability of the muscle to produce greater maximum forces during the eccentric phase of action , and it Three groups of young, female, physical education students, with minimal resistance training experience, were trained for 5 weeks with either four, six, or eight sets, of eight repetitions of fast eccentric-only half squats. Before and after the training period, lower body strength and power performance as well as vastus lateralis muscle morphology and architecture were evaluated. After recruitment of the participants, before any other testing, anthropometric characteristics and lower extremity dominance  were assessed. Counter movement jumping performance (CMJ) and half-squat maximum strength (1-RM) in a Smith machine  were tested in the same day, one week later. Muscle architecture was assessed with ultrasonography and muscle biopsies were obtained from the same area of non-dominant vastus lateralis, three days later. After concluding these evaluations, each participant performed two training familiarization sessions, with 1-day rest between them. The training program included fast eccentric-only half squats in a Smith machine (see “Training” section). Participants completed a 5–week intervention, twice a week with at least 72 h rest between the training sessions. Finally, all the initial evaluations (T1) were repeated with the same order at least 1 week after the end of the last training session (T2).Participants were recruited via advertisements at the local university. Responders visited the laboratory and completed a weekly recall self-reported physical activity questionnaire. The inclusion criteria for participants were: 1) female gender; 2) absence of systematic exercise training at least during the previous 12 months; 3) weight stability (±2 kg) for three months prior to entry; 4) absence of restraining orthopaedic/neuromuscular maladies; 5) age range between 18 to 27 years; and 6) absence of drug abuse or medications which are known to affect the neuromuscular system. Finally, twenty-five healthy female physical education students, gave their written consent to participate in the study, after being informed of the experimental procedures. They were assigned into three groups matched for non-dominant lower extremity vastus lateralis’ fascicle length and half-squat 1-RM . Basic anthropometric characteristics for the three groups are presented in All participants trained twice per week for 5 weeks, with at least 72 hours between sessions. Each group performed 8 repetitions of eccentric-only half squats in each set, but different number of sets in accordance to the respective training group: 4 × 8, 6 × 8, or 8 × 8 (sets × repetitions) with 3 minutes rest between sets. Each training session was initiated with a 5-minute warm-up on a stationary bicycle at 50–75 Watts and static stretching for the muscles of the lower extremities. Each participant performed the downward movement only (eccentric contraction) of the half-squat with instructions to lower the load as fast as possible, in a controlled movement and stop when knees reached an angle of 90°, with a rapid isometric contraction ,12. Then2 × 2−1) × 9.81 and Maximum Power (W) = (body weight + Fmax) × 9.81 × flight time. The signal was filtered using a fourth order low pass Butterworth filter with a cutoff frequency of 10 Hz. The best performance in jump height was used for further analysis. The ICCs for jump height and power were 0.87,  and 0.91 , respectively, n = 13. Participants warmed-up for 5 minutes on a stationary bicycle at 50 Watt before performing three CMJs with submaximal intensity. Subsequently, they performed 3threemaximal CMJs with arms akimbo, with 2 min rest between each jump, on a force platform  as previously described ,17,20). Maximal half-squat strength was assessed in a Smith machine. Briefly, 10 minutes after the CMJ testing, participants performed 2–3 half-squat sets of 6–8 repetitions with increasing loads. Subsequently, they performed single repetition sets with incremental loads, with 3-minute rest between sets, until they were unable to lift a heavier load. Knee bending was not allowed beyond 90°. An adjustable iron rack was placed in the Smith machine to restrict knee bending under 90°. In all cases, two of the authors were present and vocally encouraged the participants. The ICC for 1-RM testing has been previously determined in our laboratory  ,8.Ultrasound images, both before and after the training intervention were obtained during the morning hours. B-mode axial-plane ultrasound measurement  was performed with a 10 MHz linear-array probe (38-mm width). All ultrasound images were analyzed with image analysis software . Ultrasound images were obtained from the non-dominant lower extremity, performing the Extended-Field-Of-View technique on vastus lateralis  and on quadriceps , as previously described ,25,40. BSecondly, panoramic ultrasound images were obtained transversely on the femur, at the 40% of the distance  from the center of the patella to the medial aspect of the anterior superior iliac spine . Each paMuscle samples were obtained from vastus lateralis of the non-dominant lower extremity, with Bergström needles under local anesthesia. Initial (T1) samples were obtained 20 cm from mid patella while the final (T2) samples were obtained 5 cm proximally to the initial site. Samples were aligned, placed in embedding compound and frozen in isopentane pre-cooled to its freezing point, and subsequently stored in liquid nitrogen until analysis. Serial cross sections of 10 μm thickness were cut at −20 °C and stained for myofibrillar ATPase after pre incubation at pH 4.3, 4.6, and 10.3 ,19,42,43A post hoc power analysis , was performed according to the study design, the number of the participants and the lower Partial Eta Squared of the significant contrasts that were found, which revealed an actual power of 0.897 for the results of the present study. The assumption of normality was tested prior to any further statistical analysis, with the Shapiro–Wilk test (p > 0.05). All data are presented as mean and standard deviation (±SD). Two-way repeated measures analyses of variance . When significant differences were found, Bonferroni Post-Hoc tests were used for the investigation of the differences of the absolute values among the three groups, before and after training. One-way ANOVAs followed by Bonferroni Post-Hoc tests were used to compare the percentage changes  among the three groups. Partial eta squared was also evaluated, while the magnitude of effect size (ES) for pairwise comparisons, was determined by Hedges’ g . Pearson’s product moment correlation coefficient was used to explore correlations between the changes in performance and biological parameters evaluated in the present study. Statistical analyses were performed with SPSS Statistics . P ≤ 0.05 was used as a 2-tailed level of significance.2: 0.870). Specifically, it was increased 14.8 ± 8.2%, 13.1 ± 9.2% and 21.6 ± 7.0% for the 4 × 8 group , the 6 × 8 group  and the 8 × 8 group . No significant differences were found for 1RM increases among the groups , although the percentage increase in the 8 × 8 group was higher (2: 0.669) in all groups, with no significant differences among groups . Specifically, CMJ height increased by 12.5 ± 8.5%, 11.3 ± 9.3% and 7.0 ± 6.2% for the 4 × 8 group , 6 × 8 group  and 8 × 8 group, respectively , by 9.1 ± 6.0%, 7.1 ± 5.2% and 5.0 ± 3.9% for the 4 × 8 group , 6 × 8 group , and 8 × 8 group , respectively, with no significant differences among groups .No significant differences were found in the initial evaluations among the groups (p > 0.05). Half-squat 1RM increased significantly after the training intervention in all groups (Factor Time: p < 0.001; ηs higher . CMJ hei2: 0.226–0.567; 2: 0.606; Vastus lateralis fascicle length and pennation angle were altered significantly (Factor Time: p < 0.001; η2: 0.185–0.808; Vastus lateralis muscle fiber composition was not altered after the training in any of the groups (Factor Time: p > 0.05; The main finding of the present study was that four sets with eight repetitions of fast eccentric half-squats performed twice per week are adequate to increase lower body muscle strength and power during the initial 5 weeks of training in moderately trained females. It seems that two to three times larger training volumes than 4 × 8 twice a week, of this exercise, may not result in larger increases in explosive performance, during the initial 5 weeks of training, however, performing increased number of repetitions per training session result to higher increases of muscle mass. Interestingly, the changes of VL muscle fiber composition, thickness, fascicle length, pennation angle and the quadriceps cross sectional area could not explain the changes in power performance induced by either of the training volumes. Therefore, these physiological variables may not provide a direct explanation of the performance increase during the initial phase of fast eccentric training in these previously untrained females. Previous studies report that resistance training-induced increases in muscle mass, have been linked with increases in explosive performance ,27,29. HInterestingly, neither of the training volumes used here, induced a significant reduction in vastus lateralis type IIX muscle fiber percentage. This is in accordance with previous studies reporting that power training may preserve the proportion of type IIX muscle fibers, at least in sedentary or moderate trained participants during the initial weeks of explosive training ,24,25,32Vastus lateralis fascicle length increased only after 6 × 8 and 8 × 8 of fast eccentric-only squats. Fascicle length is thought to reflect the number of sarcomeres in series in a muscle fascicle . An incrResistance training elicits significant neural adaptations, especially during the first training weeks . AccordiIn conclusion, the current data suggest that 64 repetitions per week of fast eccentric half-squat training are sufficient to increase lower body muscle strength and power, after 5 weeks of training, in moderately trained females. This training regimen induces significant neuromuscular adaptations including increases in muscle fiber cross sectional area, muscle architectural changes, and maintenance of type IIX muscle fibers. Although not measured here, neural adaptations may have also contributed significantly to the increase in explosive performance.


After that, CuFe2O4@MC was characterized with FESEM, EDS, FT-IR, XRD, TGA, and VSM techniques. To measure CuFe2O4@MC photocatalytic activity, ciprofloxacin (CIP) removal ability of CuFe2O4@MC was investigated under the conditions such as initial CIP concentrations , pHs , photocatalyst loadings , and irradiation time . Kinetic process was evaluated with the pseudo-first order and the Langmuir-Hinshelwood models. CIP concentration was measured with high performance liquid chromatography (HPLC). The maximum CIP removal efficiency in the optimal conditions which contained pH = 7, CIP initial concentration of 3 mg/L, photocatalyst loading of 0.2 g, and at irradiation time 90 min was achieved 72.87 % and 80.74 % from real and synthetic samples, respectively. Also, COD removal efficiency in the optimal conditions was achieved 68.26 %. Furthermore, the CuFe2O4@MC reusability and chemical stability were examined and 73.78 % of CIP was degraded after the fourth cycle.Herein, magnetically separable CuFeAdvantages of this technique were as follows:•2O4@MC as a new nanobiomagnetic photocatalyst was synthesized with a facile, fast, and green method and were characterized with FESEM, EDS, FT-IR, XRD, TGA, and VSM techniques.CuFe•2O4@MC were confirmed and significant photocatalytic activity of CuFe2O4@MC was observed.Ferromagnetic property and pure-phase spinel ferrites of CuFe•Easily gathering, reusability and good chemical stability were interests of this nanobiomagnetic photocatalyst. Specification Table+) pairs production, and hydroxyl radical (OH•) or other oxidizing radicals were generated for oxidation of hazardous materials [•) production, photocatalysis efficiency, response to light, and photocatalytic activity of photocatalysts increase using nanophotocatalysts, due to the smaller size, greater surface area, the higher diffusion power, and superior behavior of nanomaterials [Pharmaceutical pollutants could enter to the aquatic environment from residual part of pharmaceutical industry, hospital wastewater, and human waste disposal. Antibiotics are one of these pollutants, which metabolize imperfectly and enter to various sources of water . Ciproflaterials . Moreoveaterials .2O4, ZnFe2O4, and CuFe2O4 as photocatalyst is one of the effective methods for antibiotic removal from aqueous solutions [2O2 has been used [2O4@methylcellulose as a magnetic nanobiocomposite photocatalyst from aqueous solutions. CuFe2O4@methylcellulose is a new class of biocomposites, which built from organic linkers and inorganic metal nodes through coordination bonds [2O4@methyl cellulose (MC) photocatalyst was designed with a facile, fast, and green microwave-assisted method.Photocatalytic degradation by using metal oxides and metal spinel ferrites such as CoFeolutions . The phoeen used ,23. To peen used . These peen used ,26. Due on bonds . Therefo2O4@MC was prepared with a new microwave-assisted method and characterized. After that, the photolysis, adsorption, and photocatalytic processes were compared with each other. Then the operational parameters effects on the CIP removal efficiency were evaluated and optimized. In the next step, comparison of the photocatalytic performance of CuFe2O4@MC and CuFe2O4 was done. Also, in the optimal conditions, the CIP degradation from hospital wastewater and the COD removal were investigated. Finally, the CIP photocatalytic removal kinetics were evaluated and CuFe2O4@MC reusability and chemical stability were obtained. The study stages have been shown in the The research steps were as follows: at first, CuFe3.6H2O, CuCl2.2H2O, NaOH, and methyl cellulose (MC) were purchased from Merck Company (Germany) and used without more purification. Also, CIP (99 % purification) was purchased from Tamad Pharmaceutical Company . All the materials were in analytical grade and used without further purification. Deionized water was used during the tests.FeCl3.6H2O , plus CuCl2.2H2O , were dissolved in the 50 mL deionized water (DW). Then, MC (1 g) was added to the gained solution, and the mixture was stirred at the room temperature (25 °C). After that, sodium hydroxide was added to the suspension during 1 h. Dark brown solution was transferred to microwave oven (3 × 5 min at 450 W) . Afterwards, CuFe2O4@MC as a lightweight sediment powder was observed. In the next step, the obtained nanobiomagnetics were separated by using an external magnet and was washed several times with DW. The final product was dried in an oven vacuum at 100 °C for 24 h [2O4@MC was reported in the diffraction angle range of 2θ = 15○–70○ by an X'Pert PRO MPD PA nalytical using Ni-FILTERED Cu Kα radiation. Thermal gravimetric analysis (TGA) was carried out using an STA (PC Luxx 409-NETZSCH) instrument at the rate of 10 °C min−1 in air. The CuFe2O4@MC magnetic properties were characterized with a vibrating sample magnetometer (VSM) (LakeShore Cryotronics-7404) at the room temperature (25 °C). The CuFe2O4@MC chemical composition, microstructure, and morphology were evaluated with field emission scanning electron microscope-energy dispersive spectroscopy (FESEM-EDS) (MIRA3TESCANXMU)  .Fig. 2Sy8; 259 × 4.6 × 5 mm with UV detector at wavelength of 272 nm with a hybrid mobile phase 10 micro molar HCl and acetonitrile (80:20 v/v), and input flow rate of 1 mL/min  at the wavelength of 248.30 nm and 324.70 nm, respectively. The CIP concentration was specified with HPLC device  using column with the properties of C1 mL/min . Chemica2O4@MC = 0.2 g, and CIP concentration =3 mg/L [2O4@MC, and the photocatalytic process was obtained 10.11 %, 17.6 % and 80.77 %, respectively. It means that the higher CIP removal efficiency is depends on the photocatalytic mechanism against photolysis and adsorption processes.In the researches which are about photocatalysis processe, comparison of the adsorption, photolysis results, and the photocatalytic mechanisms are valuable so, the mentioned processes were compered at irradiation time = 90 min, at pH = 7, CuFe =3 mg/L . The res2O4@MC was separated with an external magnet. After that, the degradation efficiency (η) was obtained by Eq. t and C0 show the obtained CIP concentration at different periods of irradiation time (t) and at nil min, respectively, indicated by HPLC.Effects of initial CIP concentrations , pHs , nanocatalyst loadings , and UV-C irradiation time  were optimized in a batch photoreactor. A plexiglas rectangular cubic shape photoreactor with internal dimensions of 25 cm (length), 10 cm (width), and 5 cm (height), an applicable volume of 300 mL was used in this study which had three UV-C lamps . The lamps were installed on the top of the reactor. To generate more hydroxyl radicals, a reactor was designed which could supply the minimum distance between the light supplier and the catalyst. To mix the reactor contents a peristaltic pump with a flow of 1 mL/s was used. The designed photoreactor is shown in 2O4@MC and CuFe2O4 photocatalytic performance in the CIP degradation was compared in the optimal conditions such as pH of 7, irradiation time of 90 min, CIP concentration of 3 mg/L, and photocatalyst loading of 0.2 g. The CIP degradation efficiency with these photocatalysts was obtained 80.77 % and 51.03 %, respectively.The CuFepseudo-first order (Eq. 2) and the Langmuir-Hinshelwood (Eq. 3) kinetic models  for concentrations of 3, 5, 7, and 9 mg/L was 0.902, 0.923, 0.922, and 0.929, respectively. The Langmuir-Hinshelwood equilibrium adsorption coefficient and the superficial reaction rate constant were gained 0.202 L/mg and 0.141 mg/L min, respectively. The high correlation coefficient (R2 = 0.930) indicated that the CIP photocatalytic degradation followed the Langmuir-Hinshelwood kinetic model.The kinetic linear models indicate that the CIP degradation, following the 2O4@MC photoactivity was examined. At first, The CuFe2O4@MC was separated with a magnet, and after that, was washed with ethanol/water and dried at 100 ○C for 2 h. The recycled photocatalyst in each run was added to a fresh solution of CIP the under UV-irradiation condition. In the first cycle, the CIP removal efficiency was gained 80.77 %. The results demonstrated that the CuFe2O4@MC photocatalytic activity had significant reduction in the second cycle (75.25 %) and maintained relative stability. Adsorption of intermediate products on the photocatalytic active sites can reduce the percentage of the CIP degradation. However, the CIP removal efficiency was obtained 73.78 % in the fourth cycle. Moreover, the Fe and Cu metal ions leaching in the photocatalytic process and loss of them could cause the CIP degradation efficiency reduction. Due to this reason, after the fourth cycle, copper and iron ion concentrations were determined in the solution. However, the mentioned ions were not detected in the solution. The CuFe2O4@MC chemical stability was determined with the XRD analysis, which showed the XRD peaks intensity after the fourth cycle, did not have an obvious change. This result cause to understand that this photocatalyst has good chemical stability and could be recycled.Considering the importance of the photocatalyst reusability in practical applications, the recycled CuFe2O4@MC. The maximum CIP removal efficiency occured in the optimum conditions at pH 7, CIP concentration 3 mg/L, photocatalyst loading 0.2 g, and irradiation time 90 min. Then, after being used in the fourth cycle CuFe2O4@MC was separated with a magnet and recycled without loss considerable photocatalytic activity. CuFe2O4@MC has a lot of advantages such as high reusability, stability, environmentally-friendly and great photocatalyst activity. As well as, it can be applied for the contaminated water treatment.Briefly, a strong, magnetically separable photocatalyst was prepared by a simple, fast, green, and new microwave-assisted method and by using iron and copper salts on MC in an alkali medium. The magnetic nanobiocomposite characterization showed pure phase spinel ferrites, spherical particle morphology with smaller agglomeration, and the ferromagnetic nature of CuFe


Extra treatment, i.e., heating or prolonged storage time, facilitated starch/DES plasticizing. Starch with selected DES was also extruded and the influence of preconditioning and extrusion rotational speed were subsequently studied on thermocompressed films. Extrusion at 100 rpm led to films with TS up to ca. 10 MPa and ε up to 52%. Some differences in film samples morphology obtained via two processing methods were observed. X-ray diffractograms revealed that extruded samples exhibited a V-type peak at 18.2°, with intensity depending on plasticizer total molecular size. Applied techniques , FTIR-Attenuated Total Reflection (ATR), and moisture sorption) indicated that S-based DES forms stronger interactions with starch than glycerol (G) only used as conventional plasticizer, thus leading to better mechanical properties and inhibited tendency to starch recrystallization (studied up to one year).The aim of this work was to prepare sugar alcohol-based deep eutectic solvents (DES) and test them as starch plasticizers. Thermoplastic starch (TPS) films were obtained via a simple and convenient thermocompression method. Influence of starch/DES premixtures conditioning  on TPS properties was investigated. TPS/sorbitol (S)-based DES exhibited similar tensile strength (TS) (8.6 MPa) but twice higher elongation at the break ( Amylose is almost completely linear chains mainly based on α(1–4) bonds with molar mass of 105–106 g/mol. Amylopectin is highly branched polymer linked with α(1–4) and α(1–6) bonds with molar mass 107–109 g/mol. Amylose/amylopectin content in starch can be varied from <1% to a little above 80%, depending on the starch source [Tg) of unmodified starch near its degradation hinder its processing as conventional thermoplastic materials. For that reason, it requires plasticizer to lower the Tg. Starch plasticizers are polar media that disrupt external OH hydrogen bondings between chains forming themselves new bonds with the polysaccharide [Starch is one of the most abundant natural polymers containing two fractions of α-ased on α–4 bonds h source ,3, but occharide ,12 amideccharide , or ioniccharide ,15, are ccharide . They arccharide ,17. DES ccharide ,23,24,25Starch plasticization can be performed via: (i) casting from aqueous system after starch gelatinization  or melt processable techniques, i.e., (ii) thermocompression (with high temperature and pressure) directly from starch/plasticizer premixture, (iii) extrusion, i.e., mixing and shear forces action at high temperature. Afterwards, extrudate can be processed via thermocompression, melt blowing, or injection molding. The last methods are especially convenient to processing a large amount of material, e.g., in industrial scale (with no requirement of solvent application).Sugar alcohols, especially sorbitol (S), were used as starch plasticization, but mainly in the casting method ,10,11,12Films were conditioned and characterized . Influence of preconditioning parameters (storage time and temperature) on the properties of final products was also studied. Mechanical properties, morphology , electrical resistivity and moisture content/sorption of the films obtained via two processing methods were investigated and compared. Moreover, for extrudates, the impact of preconditioning and different processing parameters, such as rotational speed, on final films properties was investigated. Flow chart of this study is presented in Tm 198 °C), maltitol—M , and xylitol—X  were purchased from Alfa Aesar . Glycerol—G  curve) was purchased from Eurochem , sorbitol—S  from Acros Organics.Native potato starch—PS  was supplied by Zetpezet S.A. . Betaine—B , choline chloride—CC  to remove moisture.In the first step, starch/DES premixtures were prepared. DES and starch (35 wt parts of DES per 100 wt parts of dry starch) were placed in a mortar and ground to obtain a homogenous paste, then the composition was placed into PP vial, sealed, and stored for 24 h at ambient conditions. Some sealed samples were additionally treated, i.e., heated at higher temperature for certain time (standard heating procedure: 1 h at 80 °C—“ht” abbreviation with the sample name). Comparison films with pure polyols and CC were also prepared.The premixtures were placed in steel frame between PET sheets and thermocompressed  at 150 °C, 12 tons (153 bars) for 5 min, cooled down to 85 °C under pressure, and then stored in a climate chamber for 7 days before testing . L/D ratio 40:1, screw diameter 16 mm . Temperature profile from the feed throat to the nozzle was 80/100/130/135/140/140/140/140/135/120 °C and screw speed was maintained at 50 or 100 rpm. After processing, extrudates were granulated (granulator Prism Varicut) obtaining ca. 3 mm pellets. After 14 days of storage at ambient conditions in sealed PE bags, pellets were thermocompressed at 150 °C, 12 tons for 2 min, cooled down under pressure to ca. 85–90 °C and stored in a climate chamber  for 48 h before testing.Preparation of TPS/DES extrudates was as follows: Premixture of starch and selected DES was processed (i) after 1 day storage (1d); (ii) 4 days storage (4d); (iii) as fresh (f) composition: 30 min after starch with DES mixing; and (iv) preheating (ht) (heating at 80 °C for 1 h and stored for 24 h before extrusion). The systems were extruded with laboratory twin-screw co-rotational extruder with Phase transitions, i.e., starch plasticization, as well as influence of pretreatment  and storage time on transition temperature, were investigated using DSC technique . DES and PS/DES premixtures (before thermocompression) after 24 h storage were analyzed in aluminum hermetic pans with nitrogen as the cooling agent with heating rate 5 °C/min. Standard runs at the temperature range from −90 to 250 °C for DES and from 20 to 200 °C for PS/DES premixtures were applied.Viscosity of DES was measured with a Brookfield RV rheometer  equipped with cone/plate at 25 °C and with 20 and 50 rpm. Average viscosity values are placed in ε), tensile strength (TS), and Young’s modulus (YM) with standard deviations were calculated with Bluehill 3 software.Mechanical tests for TPS/DES films were performed using Instron 5982 (load cell 1 kN). The films (thickness 0.50–0.65 mm) were cut into 10 mm wide, 100 mm long stripes. The initial grip separation was 50 mm and the cross head speed was 10 mm/min. At least eight replicated samples for each system were tested and the mechanical parameters: elongation at break  wavelength 1.54 Å). The XRD analysis of the samples processed via two investigated methods was repeated after 12 months of storage in ambient conditions. Starch-based materials were analyzed in a form of film or as a powder directly obtained from extrudate to examine if an additional thermocompression affects TPS crystalline structure.Dynamic mechanical thermal analysis  was used to measure and compare transition temperatures and storage modulus curves of TPS/G and TPS/DES films obtained from premixture and from extrudate. The measurements were carried out in a dual cantilever mode at frequency of 1 Hz, heating rate 3 °C/min, and temperature range from −80 to 140 °C.−1.FTIR analysis was performed using the Nexus  technique equipped with ATR. For each sample, 32 scans were taken from 4000–400 cmElectrical surface and bulk resistivities of the TPS/S:CC 1:2 films obtained via thermocompression, as well as extrusion and thermocompression, were measured and compared using an electrometer 6517A with electrode set Keithley 8009 .Moisture content in TPS films was measured using moisture analyzer . For moisture sorption and swelling tests, TPS/G and TPS/DES composite samples 20 × 20 mm were prepared. Samples were placed in a vacuum dryer (250 milibar) at 65 °C/24 h. Mass of dried samples, as well as stored in a climate chamber and kept at 25 °C/50% and 75% RH  or immersed in distilled water for 24h (for swelling behavior investigation), were determined.Statistical analysis of the mechanical test data for extruded samples was subjected to a one way analysis of variance and the significant difference was determined by the significance difference test (t-Student’s test).DES analysis results  is presented in Tg value, ca. −69 °C, was found for X:G 1:2. The higher G content in polyol-based eutectic mixture the less viscous DES. Heating of DES causes rapid drop of viscosity, therefore, preheating of some DES before addition into starch is advised to better mutual mixing. When X and S-based DES are compared, mixtures with S exhibit higher viscosity. It can be caused by a higher number of OH groups onto longer polyol chains are able to form more H-bonds between molecules, in cases of the latter. Comparing onset degradation temperature values, the highest thermal stability exhibited S:CC 2:1 (ca. 268 °C), whereas the lowest was S:G 1:2 (156 °C). According to Francisco et al. [Tg instead of Tm are called “low transition temperature mixtures”; however, most related literature called CC mixtures with polyols DES [All mixtures exhibited glass transitions at low temperatures except for M:CC 1:4, where a melting peak with minimum at ca. 80 °C was observed. The lowest o et al. , mixtureyols DES .H) are presented in Tmin and ∆H. This phenomenon is related with facilitation of thermocompression efficiency (see point 3.3.1).An influence of additional pretreatment of compositions  on transition occurred in starch (+moisture)/DES systems was investigated using the DSC technique. Relevant temperatures and enthalpy values . Moisture evaporation  with high YM (451 MPa), indicating an antiplasticizing effect. As our data show, CC alone is also not efficient plasticizer, even after prolonged conditioning time. When S:CC 2:1 was used as plasticizer (without preheating), TPS on its base exhibited almost the same high TS value (8.6 MPa) and insignificantly lower YM (428 MPa), but ε was twice higher than for TPS/S. Slightly lower TS near 7 MPa and high ε value above 69%, almost twice higher than for TPS/G, was observed for TPS/S:G 1:2. In other works related to starch processing with multicomponent plasticizers [ε values of TPS materials with DES presented in this work are higher than for materials with individual components. Introduction of the mentioned polyols in eutectic liquid form, considered as “loose” plasticizers with greater total size, eases the polysaccharide chain’s mobility. Despite that plasticizer still contains molecules with a high amount of OH groups that interact with starch, there is no decrease of TS. In the literature, very often the mixed polyols were introduced in their solid form, so the mechanical properties values are between those for TPS with individual components. In Mikus et al.’s work [ε values for wheat TPS films plasticized with G and S mixture (12.5%/12.5%), in comparison with TPS plasticized with one type of polyol, was registered; however, TS (6.5 MPa) was much lower than for TPS/S (14.6 MPa).Thermocompression tests were performed for starch not only with DES but also with individual polyols , as well as with CC, for comparison their plasticizing ability . The mosticizers , mechani.’s work , an incrε values, and when it was increased from 80 to 100 °C, ε decreased , except TS values for TPS/S:CC 1 d. It is noteworthy that YM values for TPS plasticized with S:CC are as a rule 2–3 times higher in comparison to the respective parameter values for films with G, and the differences are statistically significant (P < 0.05). Moreover, standard deviation values are, respectively, lower in the case TPS/S:CC samples extruded with 100 rpm than 50 rpm, suggesting better mixing during processing and homogeneity of the final material , TS of TPS/G is two times lower than for TPS/S:CC 2:1. The highest TS (10.1 MPa) and ε (51.6%) were achieved for the TPS/S:CC 2:1 sample stored for 1 day before processing at 100 rpm. As data from Comparing samples obtained with two rotational speeds of extrusion, it can be found that the ones prepared with 100 rpm exhibited slightly higher TS and YM, as well as material . TPS/G iThe influence of starch/S:CC 2:1 pretreatment, processing method, and storage time on starch crystallinity structure was investigated by XRD technique. ε values, especially when we compare it with results for only thermocompressed TPS where the peak did not occur. Similar results were obtained in Li and Huneault’s work [An intensity of peak at 18.2°, which is assigned to single helical structure and is characteristic for extruded TPS materials processed at higher temperatures with low water content , increast’s work , where tt’s work ,38, whicThere are no significant differences between XRD patterns for films prepared using various methods of the premixture treatment before extrusion. However, for preheated and freshly extruded samples, some visible peak at 17.1° and for TPS/S:CC ht barely visible at 20.7° can be observed C. DuringThere are no differences for the same sample analyzed after film preparation and after one year, which indicated lack of starch further recrystallization . The samComparing rotational speeds (50 and 100 rpm) for TPS/S:CC films, there were no significant differences on XRD pattern, similar to that between the powder and film form of TPS/S:CC samples (data not shown). It evidenced that subsequent thermocompression of the extrudate do not affect morphological structure .The last issue is the differences between crystallinity for the same compositions processed via two different methods. Shear forces during extrusion disrupt granules and pull out amylose, as it happens during starch gelatinization in water. Moreover, amylopectin is more sensitive to these forces and can degrade during processing in the extruder ,40,41, uδ curves are shown. The samples revealed two main relaxation regions of separated phases; the first with a maximum between −21 °C and −52 °C  related to secondary relaxation of the domain rich in plasticizer small molecules, and the second was above 14 °C  of starch-rich domain. Much higher temperatures of both relaxations exhibit films with S-based DES, rather than with G. This indicates a stronger interaction between starch with S:CC 2:1 than with G. Curves for TPS/DES films obtained by both processing methods (samples stored for 1 day before processing) are much different from each other. Tβ are actually placed in the same area, but Tα of thermocompressed TPS/DES film (33.7 °C) is lower than for film from the extrudate (42.1 °C). Higher intensity of the α-relaxation peak evidences higher mobility of polymeric chains, caused by highly amorphous state of amylopectin and their partial mechanical degradation  values . A small peak in this wavenumber range has been observed in Vu and Lumdubwong’s work [S and their eutectic mixtures exhibited some peak at about 886 cmg’s work  for star− mobility; however, mixing with stress forces during extrusion and formation of amorphous structure resulted in disrupting these bonds.Results for surface and bulk electrical resistivity measurements for selected TPS films plasticized with S:CC 2:1 are presented in All analyzed TPS films were kept in a climate chamber for 7 days before testing. Moisture content values in these samples are presented in Similar results, i.e., an influence of molecular size of plasticizer on TPS sorption properties, were observed in other work . Resultsε value. Films with S:CC 2:1 prepared from extrudate have better mechanical properties than those only thermocompressed. Maximum values of TS, ε, and YM for films based on extrudates attained ca. 10 MPa, 52%, and 616 MPa, respectively. XRD results pointed out amorphous morphology, but V-complex of amylose with DES after extrusion is formed. Intensity of this peak was dependent on plasticizer total molecular/structure size. All applied techniques , indicated that S-based DES forms stronger interactions with carbohydrate polymer chains than G, leading to better mechanical properties  and inhibited recrystallization of B-type semicrystallinity of PS (studied up to one year).In the presented work, for the first time, sugar alcohol-based DES were used for PS plasticization via hot melting methods: thermocompression and extrusion with subsequent thermocompression. Influence of starch/DES premixtures conditioning (preheating temperature and storage time) before processing on final films properties was also investigated. Additional preconditioning of starch/DES mixtures facilitated starch plasticization and influenced mechanical properties, especially on elongation at the break of TPS/DES films—Samples modified with S-based DES exhibited high TS comparable to TPS with S only but with twice higher Generally, using sugar alcohol-based DES allows the formation of TPS materials with better mechanical properties and lower hygroscopicity features than TPS plasticized only with polyols. Due to the non-toxic, cheap character of DES components, such materials can be applied in food packaging, or even as edible films.