The structural and optical properties of the prepared materials were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), and UV-vis adsorption. The specific surface area was also analyzed by Brunauer–Emmett–Teller (BET) method. The anatase/rutile mixed-phase TiO2 nanocomposites containing of rod-like, cuboid, and some irregularly shaped anatase nanoparticles (exposed {101} facets) with sizes ranging from tens to more than 100 nanometers, and rod-like rutile nanoparticles (exposed {110} facets) with sizes ranging from tens to more than 100 nanometers. The photocatalytic activities of the obtained anatase/rutile mixed-phase TiO2 nanoparticles were investigated and compared by evaluating the degradation of hazardous dye methylene blue (MB) under ultraviolet light illumination. Compared to the commercial Degussa P25-TiO2, the mixed-phase TiO2 nanocomposites show better photocatalytic activity, which can be attributed to the optimal anatase to rutile ratio and the specific exposed crystal surface on the surface. The anatase/rutile TiO2 nanocomposites obtained at pH 1.0 (pH1.0-TiO2) show the best photocatalytic activity, which can be attributed to the optimal heterojunction structure, the smaller average particle size, and the presence of a specific exposed crystal surface. The enhanced photocatalytic activity makes the prepared anatase/rutile TiO2 photocatalysts a potential candidate in the removal of the organic dyes from colored wastewater.Anatase/rutile mixed-phase TiO The clow cost ,4. TiO2 ficiency . Howevernd so on . It is ractivity ,6,7. Amoactivity . Kawaharactivity . Ohno eteratures . Among tss, etc. ,13. Cao articles . Peng etene blue . Atitar P25-TiO2 .2 with tailored facets for photocatalysis also has attracted considerable attention in recent years. In 2007, Wen et al. successfully obtained anatase TiO2 nanocrystals with exposed {010} facets and controllable morphologies by using the exfoliated layered titanate nanosheets as the precursor under hydrothermal conditions [2 nanorods were prepared under hydrothermal conditions, which exhibited efficient solar water-splitting performance [2 nanosheets with dominant {001} facets were synthesized in the absence of any fluorine precursor or hazardous materialat room temperature [2 dominated by {100} facets were synthesized by using titanate nanofibers derived from alkali treatment as precursor, which exhibited smart electrorheological behavior underexternal electric field [2 nanocrystals with exposed special high energy facets [Besides heterojunction structure, TiOnditions . After tnditions . Lee et nditions . Recentlperature . The tetic field . We alsoy facets ,24,25.2 with exposed {101} facets and {110} facets was synthesized through a simple sol-gel route with further calcination. This approach uses rich analytical ethanol and deionized water as the solvent, inexpensive titanium tetrachloride as the titanium source, and low-cost ammonium fluoride as the capping agent, which effectively decreases the production cost. The anatase/rutile mixed-phase TiO2 nanocomposites have a smaller nanocrystal size and a narrower grain distribution. The photocatalytic activity of the obtained anatase/rutile nanocomposites was evaluated using the degradation tests of methylene blue (MB) under UV light illumination. The obtained anatase/rutile mixed-phase TiO2 exhibited excellent photocatalytic activity because of the synergistic effect of the optimum anatase to rutile radio, and the specific exposed crystal surface, compared to the commercial Degussa P25-TiO2. The improvement in photocatalytic activity makes the prepared anatase/rutile nanocomposite a potential candidate in wastewater purification.In this work, anatase/rutile mixed-phase TiO2 composites synthesized by the sol-gel method using titanium tetrachloride as the titanium source. It is worth noting that both the XRD patterns of pH0.5-TiO2 and pH1.0-TiO2 show the same diffraction peaks, which can be ascribed to the tetragonal anatase phase  and the tetragonal rutile phase , and rutile TiO2 is the major phase because it is the most stable among the three polymorphs  under strong acidic conditions [2; while the peaks located at 2θ = 27.74°, 36.42°, 41.58°, 44.40°, 54.64°, 56.56°, 63.09°, 64.20°, 69.26°, and 69.83° are indexed to the (110), (101), (111), (210), (211), (220), (002), (310), (301), and (112) diffraction peaks of rutile TiO2.The proportion of anatase/rutile TiO2 composite can be calculated by the following formulas (1) and (2) [WA and WR represent the mass fraction of anatase TiO2 and rutile TiO2, respectively. IA and IR represent the integral intensity of diffraction peaks on crystal surface of anatase TiO2 (101) and rutile TiO2 (110), respectively. As shown in 2 (101) crystal surface and rutile TiO2 (110) crystal surface in the composite is 47.6% (IA) and 100.0% (IR), respectively. Therefore, the proportion of anatase TiO2 and rutile TiO2 in the composite is 29.66% and 70.34%, respectively. As shown in 2 (101) crystal surface and rutile TiO2 (110) crystal surface in the composite is 50.3% (IA) and 100.0% (IR), respectively. Therefore, the proportion of anatase TiO2 and rutile TiO2 in the composite is 30.83% and 69.17%, respectively. The average crystallite size of pH0.5-TiO2 and pH1.0-TiO2 are readily calculated from XRD patterns to be 27.2 nm and 25.7 nm, respectively. The patterns exhibited that with increasing pH, the intense anatase and rutile peaks become weak, which is due to the decrease in crystalline size, implying that increasing pH inhibits the formation of rutile TiO2 and enhances the formation of anatase TiO2 during the hydrothermal reaction process at 180 °C for 24 h.nditions . As show2 nanoparticles was characterized by TEM and HRTEM, as shown in 2 is a mixture of anatase and rutile phase (see d = 3.26 Å) on the lateral surface were also observed  atomic planes with a lattice distance of 3.52 Å can be clearly seen, indicating that the two square surfaces of top and bottom are the {101} facets.The morphology of the TiOhase see a and hashase see a,b. The hase see c. In addobserved c. The laobserved d. Figure2 is also amixture of anatase and rutile phase (see d = 3.46~3.52 Å) on the lateral surface were also observed, indicating that it exposed the {101} facets on the lateral surfaces  in aqueous solution under low-pressure mercury lamp. The photocatalytic MB is thought to be degraded by the following reactions [The photocatalytic activity of the pH0.5-TiOeactions ,29:2/rutile TiO2 + hv→ anatase TiO2/rutile TiO2(eˉ + h+)anatase TiO+ + H2O → ·OH + H+h+ + OHˉ→ ·OH h2→ O2ˉeˉ + O2ˉ + H2O→ ·OOH + OHˉO2O→H2O2 + ·OH·OOH + H+ →H2O2eˉ + ·OOH + H2O2 →2·OHH·OH + MB → peroxides or hydroxylatedintermediates →→ degraded or mineralized products.2, pH1.0-TiO2, P25-TiO2, and absence of any other catalysts. The corresponding time-dependent photodegradation profiles of MB are shown in 2 shows the highest photocatalytic activity and pH0.5-TiO2 shows slightly lower photocatalytic activity than pH1.0-TiO2, while P25-TiO2 shows much lower photocatalytic activity than pH1.0-TiO2. Totals of 85.8%, 82.6%, and 79.4% MB were photodegraded for pH1.0-TiO2, pH0.5-TiO2, and P25-TiO2, respectively, under UV irradiation for 90 min. It also can be seen that the blank sample shows the lowest photocatalytic activity, displaying only 8.9% photodegradation of MB under UV irradiation for 90 min.The above reactions are chosen because both the electrons and holes eventually participate in the formation of ·OH radicals, which degrade the MB. 2, pH1.0-TiO2, and P25-TiO2, respectively. Due to the quantum size effect, smaller average crystal size provides more powerful redox ability, resulting in a reduction in the net electron–hole recombination, thereby increasing the photocatalytic activity [2, pH1.0-TiO2, and P25-TiO2 anatase/rutile TiO2 samples is 47.5, 53.2, and 52.5 m2/g [2/g (≤3.2 m2/g difference), and the experimental error for BET analysis is around ±10%, indicating that the specific surface area is not an important factor affecting the photocatalytic activity in the above catalytic reactions. Thus, the improvement in photocatalytic activity of pH0.5-TiO2 and pH1.0-TiO2 nanoparticles should be attributed to factors other than the specific surface area. It is well known that the photocatalysis of a crystal also depends on the surface structure and surface activity of the crystal facets [2 and pH1.0-TiO2 are both mixtures of anatase and rutile phases, similar to P25-TiO2. The heterojunction structure of the mixed anatase/rutile TiO2 can mediate the migration of photogenerated holes across the interface into rutile TiO2 and reduce the recombination of the photogenerated electrons and holes [2, 31% and 69% for pH1.0-TiO2, and 87% and 13% for P25-TiO2 [2 has a greater anatase/rutile ratio (87%/13% = 6.39/1), the photodegradation efficiency is less than those of the pH0.5-TiO2 (30%/70% = 0.43/1) and pH1.0-TiO2 (31%/69% = 0.45/1) samples, indicating that the presence of an optimum anatase to rutile radio in the biphasic system is beneficial to increase the photocatalytic efficiency [2 nanocomposites first increases with increasing rutile nanocrystals content (P25-TiO2(13%) ˂ pH1.0-TiO2(69%)), and then decreases (pH0.5-TiO2(70%) ˂ pH1.0-TiO2 (69%)). As the rutile nanoparticle increases, photogenerated electron–hole pairs in the anatase phase can be separated by transferring the electrons to the rutile nanoparticle, and the holes remain in the anatase nanoparticle, which reduces the recombination rate of anatase, leading to the improvement of the photocatalytic efficiency. Since both electrons and holes participate in the degradation reactions, both anatase and rutile nanoparticles must have access to the MB solution [2 and pH1.0-TiO2, the content of anatase and rutile, the particle morphology , and the exposed facets ({110}/{101}) are similar, so the difference in photocatalytic activity can be attributed to the difference of the specific surface area, anatase to rutile radio, and the particle size. Furthermore, as photocatalysis is a surface phenomenon, it is closely related to the exposure of the crystal surface [− and h+ on the external anatase crystal structure. For rutile particles, it is suggested that the oxidation site is mainly on the {011} facets with higher electronic energy levels and the reduction site is mainly on the {110} facets with lower electronic energy levels; the difference in the energy levels drives the electrons and holes to different crystal facets. The synergistic effect between the {011} and {110} facets accelerates the separation of electrons and holes and suppresses the recombination of photogenerated electrons and holes, resulting in improvement of the photocatalytic activity [2 surface [2) < {100} facets (0.824 J/m2) < {001} facets (1.398 J/m2) [2 surface, resulting in the enhancement of the photocatalytic activity [2 and pH1.0-TiO2 samples with dominant exposed {110} and {101} facets exhibited the higher photocatalytic activity, compared with that of P25-TiO2 without an exposed specific surface. P25-TiO2 shows the lower photocatalytic activity, which can be attributed to the absence of optimal anatase to rutile ratio and specific exposed crystal surface [It is well known that the photocatalytic activity of a catalyst is determined by many factors, such as crystalline phase, degree of crystallinity, crystal size, specific surface area, crystal morphology, dopant, doping amount, and heterojunction structure ,32,33,342.5 m2/g , respect2.5 m2/g . Howeverl facets . Based ond holes . HoweverP25-TiO2 , respectsolution . With th surface . Therefoactivity . On the 98 J/m2) . It has  surface .In synthesis of the nanoparticles, 50 mL titaniumtetrachloride  was added dropwise into 150 mL analytical ethanol  under vigorous stirring. After 30 min, the above solution was added dropwise into 440 mL deionized water to obtain a colloidal solution under continuous stirring. After 24 h, 50 mL of the above colloidal solution was transferred into two Teflon autoclaves with a capacity of 80 mL and the pH was adjusted to 0.5 and 1.0, respectively. Then, 0.20 g of ammonium fluoride solid was added into the above Teflon autoclaves under vigorous stirring. After that, the temperature of the above autoclaves was raised to 180 °C, maintained for 24 h, and then cooled to room temperature. Two white colloidal precipitates were obtained at the bottom of the autoclaves. The white colloidal precipitates were isolated from the solution by filtering, and were washed with deionized water 4 or 5 times. After the white colloidal precipitates were dried overnight at 80 °C, two white gels were obtained. The white gels were grounded carefully and then annealing at 450 °C for 1 h in a high temperature box furnace.−1 in the 2θ range of 5°–80° at room temperature. Transmissionelectron microscopy (TEM) and high-resolution transmissionelectron microscopy (HRTEM) were carried out on a JEOL Model JEM-2100-F microscope at an accelerating voltage of 200 kV. Nitrogen adsorption/desorption isotherms were measured at −196°C on a TriStar II 3020 volumetric adsorption analyzer. Prior to the measurement, the obtained samples were degassed under high vacuum at 120 °C for 5 h. The surface area was calculated from the adsorption isotherm plot in the range of relative pressure from 0.05 to 0.30 by BET method. UV-Vis adsorption spectra were analyzed by the TU-1901 spectrophotometer .The structures of crystalline samples were investigated by using a powder X-ray diffractometer  equipped with monochromated Cu Ka (λ = 0.15406 nm) radiation at a scanning speed of 8°·min2 and pH1.0-TiO2 were dispersed in 150 mL of methylene blue  solution (2.92 × 10−5 mol·L−1) under dark and stirring conditions, respectively. The resulted suspension was stirred for 2 h in the darkness to reach adsorption–desorption equilibrium. The irradiation was carried out under stirring with a 175 W ultraviolet lamp  located 40 cm horizontally from the sample. The concentration change of MB in the suspension solution with irradiation time was determined by using a TU-1901 spectrophotometer. For the comparison, the commercially available Degussa P25-TiO2 sample  with an average particle size of 27.8 nm and a composition of ~87% anatase, ~13% rutile phase, and BET surface area of 52.5 cm2/g was used as the standard sample for the photocatalytic measurement.Seventy-five milligrams of pH0.5-TiO2 nanocomposites have been successfully synthesized through a simple sol-gel route with further calcinations by using titanium tetrachloride as the titanium source, analytical ethanol and deionized water as the solvent, and ammonium fluoride as the capping agent. The relative amount of anatase and rutile can be slightly changed by adjusting the pH value of the suspension. The anatase/rutile mixed-phase TiO2 nanocomposites contained rod-like, cuboid, and some irregularly shaped anatase nanoparticles (exposed {101} facets) with sizes range from tens to more than 100 nanometers, and rod-like rutile nanoparticles (exposed {110} facets) with sizes range from tens to more than 100 nanometers. The degradation experiments demonstrate that the mixed-phase TiO2 nanocomposites show better photocatalytic activity than commercial Degussa P25-TiO2 under UV light irradiation because of the synergistic effect of the optimum anatase to rutile ratio and the specific exposed crystal surface. The anatase/rutile TiO2 nanocomposites obtained at pH = 1.0 (pH1.0-TiO2) show the best photocatalytic activity, which can be attributed to the optimal heterojunction structure, the smaller average particle size, and presence of a specific exposed crystal surface. This work presents a simple and economical method to prepare efficient anatase/rutile TiO2 photocatalysts for wide applications in the removal of the organic dyes from colored wastewater.Anatase/rutile mixed-phase TiO


A RSNMC-modified glassy carbon (GC) electrode was employed for the determination of ascorbic acid (AA) and exhibited a linear response in the concentration range of 0.005–6.0 mM with a detection limit of 0.003 mM. These results demonstrate that RSNMC has potential as an advanced and cheap electrode material for electrochemical sensing and other electrocatalytic applications.Rice starch (RS-)based nano/mesoporous carbon (RSNMC) was prepared via a hard-templating route using cheap rice starch as a carbon source. XRD and TEM characterization indicated the formation of organized nanoporous RSNMC. Nitrogen absorption–desorption studies revealed a high surface area of up to 488 m Nano/mesoporous carbon materials (NMC) are of interest because of their high surface area, uniform pore structure, mechanical strength, chemical stability, and relative lack of reactivity ,4,5,6,7.In this context, sustainable nanoporous carbon materials from low-cost biomass have become an important area of investigation ,6. RecenIn this study, we describe a sustainable and cost-effective one-pot synthesis of rice starch-derived nano/mesoporous carbon material (RSNMC) and its application as an electrode material for the detection of ascorbic acid (AA). Ascorbic acid (vitamin C) is an essential nutrient for living cells and plays an important role in metabolic and cell development processes ,11,12,13Surfactant Pluronic P123 (2.0 g)  was added to 60 mL of 2 M HCl at 38 °C with vigorous stirring for 2 h. Tetraethylorthosilicate (TEOS) (4.2 g) (Sigma-Aldrich) was added dropwise to the surfactant-containing solution with stirring for another 8 min and left to stand for 24 h at 38 °C. This solution was then autoclaved at 100 °C for 24 h, filtered, and dried at room temperature. Finally, calcination was performed at 550 °C for 6 h in air ,33,34,35Rice starch (2.0 g) (Sigma-Aldrich) in 20 mL of deionized water was stirred in a beaker at 85–90 °C. SBA15 mesoporous silica template (2.3 g) a was addThe RSNMC was predried at 60 °C for 1 h in a conventional oven. RSNMC (5 mg) was added to 20 mL of ethanol and sonicated for 30 min to obtain a well-dispersed, homogeneous suspension. The bare glassy carbon (GC) electrode was polished with 0.50 and 0.05 μm alumina slurry and rinsed with water. It was then sonicated in deionized water for 5 min and dried under a high-purity nitrogen stream. The RSNMC ethanol suspension (4 µL) was drop-cast onto the cleaned GC electrode and dried under a nitrogen atmosphere at room temperature to obtain the RSNMC/GC electrode.3Fe(CN)6, and AA. Electrochemical measurements were performed under a Nitrogen atmosphere using solutions deoxygenated by purging with N2 for about 15 min. All measurements were carried out at room temperature (25 °C).Electrochemical experiments were performed on a Bioanalytical Systems BAS 100B/W electrochemical workstation with three electrodes including the RSNMC-modified GC working electrode, an Ag/AgCl reference electrode, and a platinum wire counter electrode. A 0.1 M PBS, pH 7.0 solution was used as the supporting electrolyte for the potential cycling of potassium ferricyanide, K29Si and 75.468 MHz for 13C. Powdered material was placed in the 4 mm zirconium rotor and rotated at the magic angle at 7 kHz. Spectra were acquired using a 42 ms acquisition time, sweep width of 50 kHz, 2 K data points, and high-power 1H TPPM decoupling. Recycle times ranged from 30 s to 100 s and were verified to be sufficient for relaxation without signal saturation. 29Si NMR spectra were recorded after a single pulse to allow for quantification of the Si species. The 90° pulse was followed by high-power 1H TPPM decoupling at 87 kHz. X-ray photoelectron spectroscopy (XPS) was performed using a Kratos Axis ULTRA X-ray photoelectron spectrometer equipped with a 165 mm hemispherical electron energy analyzer using a monochromatic Al Kα (1486.6 eV) X-ray source at 150 W . A survey wide scan was collected at an analyzer pass energy of 160 eV and multiplex (narrow) high-resolution scans of 20 eV. The base chamber pressure was 1.0 × 10–9 Torr, increasing to 1.0 × 10–8 Torr during sample analysis. The binding energies were referenced to the C 1 s peak of adventitious carbon at 284.6 eV to account for charging effects.X-ray diffraction (XRD) patterns were recorded using a Bruker radiation D8 Advanced Diffractometer with Cu Kα radiation for angles 1 to 10 2θ (degrees). A Rigaku Miniflex diffractometer (Japan) with Cu Kα radiation was used for the 2θ range from 10 to 80° under identical conditions at a scanning rate of 2 degrees. An automated adsorption analyzer  was used to study the Brunauer–Emmett–Teller (BET) surface areas, pore sizes, and pore volumes. All samples were outgassed at 180 °C for 12 h under vacuum prior to analysis using the degas port of the adsorption analyzer. Pore size distribution (PSD) was obtained using the Barrett–Joyner–Halenda (BJH) model from the desorption branch of the isotherm ,37. The 6mm)  prepared in PBS, pH 7.0). The cyclic voltammogram (CV) curves obtained by using a RSNMC-modified GC electrode and a bare GC electrode at a potential sweep rate of 20 mV/s are shown in 2) of the electrode, D is the diffusion coefficient of [Fe(CN)6]3− (taken to be 7.60 × 10−6 cm2∙s−1 in aqueous medium), c* corresponds to the bulk concentration of the redox probe (mol∙cm−3), and v is the scan rate (V/s). The calculation indicates an electroactive surface area for RSNMC/GC electrode of 0.052 cm2, which is 1.7 times higher than that of a GC electrode (0.03 cm2). In addition, the peak potential separation (ΔEp) between the anodic and cathodic peaks is 57 mV for the RSNMC/GC electrode and 103 mV for the GC electrode. The decrease in ΔEp indicates that the RSNMC/GC electrode not only possesses a high surface area, but also accelerates the electron transfer rate of ferricyanide [The electrochemical performance of RSNMC as a novel electrode material was evaluated using potassium ferricyanide , large pore volume (1.14 cm3∙g−1), and narrow pore size distribution (3.92 nm) without requiring chemical activation. A RSNMC-modified GC electrode displayed enhanced electrocatalytic activity towards the electrochemical oxidation of ascorbic acid as indicated by current enhancement and an associated peak shift compared with an unmodified GC electrode. The constructed porous carbon interface displayed strong signal amplification. The RSNMC-modified GC electrode demonstrated a reproducible, linear relationship for ascorbic acid detection over the range 0.005–6.0 mM and a detection limit of 0.003 mM. The preparation of RSNMC is very straightforward and our results indicate that it has superior performance as an electrode material.In summary, we have successfully demonstrated that rice starch can be used as an inexpensive biomass precursor source for nano/mesoporous carbon (RSNMC). Carbonaceous nanostructures were prepared using a hard, mesoporous silica template and exhibited a high surface area (488 m


Lariophagus distinguendus  complex, which includes cosmopolitan parasitoids of coleopteran stored-product pests, were studied using glass-needle based microdissection, reverse and cross-species fluorescence in situ hybridisation (FISH). This experiment strongly indicates that the largest metacentric chromosome in the karyotype with n = 5 originated from a particular fusion between the only acrocentric and a smaller metacentric chromosome of the set with n = 6, therefore confirming our previous hypothesis based on the karyotypic analysis using chromosome morphometrics. This study represents the first successful application of both microdissection and whole chromosome painting for the reconstruction of karyotypic rearrangements in closely related species of parasitoids, as well as in the order Hymenoptera in general.Karyotypes of two cryptic species of parasitoid Hymenoptera with n = 5 and 6 belonging to the  Parasitoid Hymenoptera are one of the most species-rich, taxonomically complicated and economically important groups of insects . SpecifiGlass-needle based chromosome microdissection (midi) is a powerful tool for characterization of karyotypic transformations, both intra- and interspecific ones –8. For eNasonia vitripennis  , was analysed using the aforementioned approach, i.e. midi and reverse FISH  was added, and the whole cycle except for the first step was repeated another seven times. After that, 45 μL of the PCR mix for high-temperature cycles (HTC) containing 28.0 μL water, 5.0 μL 10× AmpliTaq Gold DNA Polymerase Buffer, 0.222 mM dNTP, 2.778 mM MgCl2, 0.622 μM DOP primer, 0.6 μL 5 U μL-1 AmpliTaq Gold DNA Polymerase was added and HTC-PCR was performed. The latter procedure  was repeated 33 times, followed by another 2 min at 72°C and stopped by keeping the mixture at 4°C.The probes obtained from rding to . To ampl2, 0.1 mM labelled dUTP, 0.1 μL 5 U μL-1 AmpliTaq Gold DNA Polymerase. The PCR procedure included 3 min at 95°C, followed by 20 amplification cycles , another 5 min at 72°C and stopped by keeping the mixture at 4°C. The PCR product was then precipitated in ethanol, centrifugated at 13,000 rpm for 20 min, and resuspended in 20 μL hybridisation buffer (see below).The probes LD5-1 and LD6-A were then respectively labelled with Spectrum Orange-dUTP and Spectrum Green-dUTP  in a secondary DOP-PCR using the procedure described in . SpecifiL. distinguendus (n = 5 and 6) were used for the bidirectional reverse FISH and ZOO-FISH experiments, that were carried out following [-1) under 24 × 60 mm coverslips for 1 h at 37°C in a moisture chamber. After that, the slides were treated with 1× phosphate buffered saline  for 5 min on a shaker at RT, and this procedure was also repeated after each of the two following steps, i.e. treatment with 0.005% pepsin solution under a coverslip for 10 min at 37°C, and post-fixation with 1% paraformaldehyde solution  for 10 min at RT. The preparations were then dehydrated in ethanol (see above) and air-dried.Chromosomal preparations of ollowing . The prePrior to in situ hybridisation, the preparations were incubated in a formamide solution  buffer (Merck), 50% deionized formamide, pH 7.0) for 3 min at 75°C, then dehydrated in cold ethanol  and air-dried. The hybridisation mix containing 20 μL of the hybridisation buffer (10 mg formamide and 4 mg dextran sodium sulphate in 2× SSC and 0.1 mM phosphate buffer) and 100 ng of the labelled probe was then denatured in a thermocycler for 10 min at 85°C, applied to the preparation, covered with a coverslip and incubated for 14 h at 37°C in a dark moisture chamber. After removing the coverslip, the slide was rinsed in 1× SSC at 65°C. The slide was then rinsed in a washing buffer (4× SSC containing 0.05% Tween 20) for 5 min at RT on a shaker, dehydrated in ethanol (see above), air-dried and mounted in Vectashield Mounting Medium with DAPI .0t-1 DNA was applied to block these sequences. To prepare C0t-1 DNA, genomic DNA was first extracted from ten unsexed pupae of each species according to [2, 0.025 M KCl). After slowly adding 10% solution of Triton X-100 in buffer A to the final concentration of 1%, the mix was centrifugated three times at 800× g and 4°C for 5 min, with the supernatant discarded and the pellet resuspended in 1 mL of ice-cold buffer A every time. After final resuspension, 4 mL of buffer B  and RNase A  at RT was added. After this step, equal amounts (2.5 mL) of 0.4% sodium dodecyl sulphate (SDS) and 0.1 mg mL-1 of proteinase K (Thermo Fisher Scientific) in buffer B were added, and the mixture was stirred. It was then incubated for 1 h at 60°C and vortexed every 20 min.Since initial experiments demonstrated substantial cross-hybridisation of both probes LD5-1 and LD6-A to non-target chromosomes due to the presence of high-copy repeat sequences, Crding to  with a f0t-1 DNA was isolated following the procedure described in [-1 using 5 M NaCl and double-distilled H2O to a final concentration of 0.3 M NaCl. Then the DNA was sheared by autoclaving it several times on a liquid cycle (5 min each), until the necessary fragment size of 100 to 1,000 bp (controlled by electrophoresis) was reached. After denaturing in a water bath at 95°C for 10 min and cooling in ice water for 10 s, DNA was reannealed by placing it again in a water bath at 65°C. To digest the remaining single-stranded DNA, calculated amounts of l0× S1 nuclease buffer  (Merck) and S1 nuclease (Thermo Fisher Scientific) were then added to achieve the concentration of 1 U S1 nuclease μg-1 DNA, and the mix was immediately put in a water bath at 37°C for 8 min. The nuclease digestion was then stopped by DNA extraction with phenol equilibrated with Tris-EDTA (TE) buffer, and the supernatant was further extracted with an equal volume of phenol-chloroform followed by an equal volume of chloroform . C0t-1 DNA was then precipitated overnight using 2.5 volumes of 100% ethanol, dried, resuspended in TE buffer and stored at -20°C until needed. Different amounts of C0t-1 DNA, i.e. 20 and 70 μg, were added to the hybridisation mix, with the latter amount appeared to yield the best results.In turn, Cribed in . SpecifiChromosomes were visualized using an epifluorescence microscope Zeiss Axioplan (Carl Zeiss AG) fitted with a digital CCD camera Olympus DP70 . Additional images of Giemsa-stained chromosomes were obtained using an optic microscope Zeiss Axioskop 40 FL fitted with a digital CCD camera Zeiss Axiocam MRc (Carl Zeiss AG). Chromosomes were classified as M or A according to their arm ratios . Images L. distinguendus (n = 6) demonstrated that LD6-A probe hybridised with the only A pair (L. distinguendus (n = 5) showed that LD5-1 probe strongly painted the largest M pair  resulted from a chromosomal fusion of the unique pair of acrocentrics and a smaller M chromosome pair in the karyotype of L. distinguendus (n = 6), thus confirming our previous assumptions on the type and direction of karyotype transformation in this complex respectively made on the basis of chromosome morphometrics and cladistic analysis of different populations [Nasonia vitripennis [Encarsia Förster, 1878 (Aphelinidae) [The results obtained clearly show that the largest M pair in the chromosome set of ulations . It therndus n =  resultedulations  used revripennis –20 or a linidae) –34, althlinidae) . We therlinidae) . We also


In view of wide range of biological activities of oxazole, a new series of oxazole analogues was synthesized and its chemical structures were confirmed by spectral data . The synthesized oxazole derivatives were screened for their antimicrobial and antiproliferative activities.The antimicrobial activity was performed against selected fungal and bacterial strains using tube dilution method. The antiproliferative potential was evaluated against human colorectal carcinoma (HCT116) and oestrogen- positive human breast carcinoma (MCF7) cancer cell lines using Sulforhodamine B assay and, results were compared to standard drugs, 5-fluorouracil and tamoxifen, respectively.3, 5, 6, 8 and 14 showed promising activity against selected microbial species. Antiproliferative screening found compound 14 to be the most potent compound against HCT116 (IC50 = 71.8 µM), whereas Compound 6 was the most potent against MCF7 (IC50 = 74.1 µM). Further, the molecular docking study has been carried to find out the interaction between active oxazole compounds with CDK8 (HCT116) and ER-α (MCF7) proteins indicated that compound 14 and 6 showed good dock score with better potency within the ATP binding pocket and may be used as a lead for rational drug designing of the anticancer molecule. The performed antimicrobial activity indicated that compounds  Multidrug resistance and emergence of new infectious diseases are amongst the major challenges in the treating of microbial infections which necessitates the discovery of newer antimicrobial agents . Cancer Molecular docking studies provide the most detailed possible view of drug-receptor interaction and have created a new rational approach to drug design. The CDKs (cyclin dependent kinase) is an enzyme family that plays an important role in the regulation of the cell cycle and thus is an especially advantageous target for the development of small inhibitory molecules. Selective inhibitors of the CDKs can be used for treating cancer or other diseases that cause disruptions of cell proliferation . EstrogeOn the basis of the information obtained from literature survey Fig. , in the 1–15) were accomplished using the synthetic procedure depicted in Scheme H-chromen-2-one (I) was prepared by the reaction of salicylaldehyde and ethyl acetoacetate in the presence of piperidine. Further, the reaction of I with bromine resulted in the formation of 3-(2-bromoacetyl)-2H-chromen-2-one (II). The later was refluxed with urea to synthesize 3-(2-aminooxazol-5-yl)-2H-chromen-2-one (III). The reaction of 3-(2-aminooxazol-5-yl)-2H-chromen-2-one (III) with substituted aldehydes yielded the title compounds 3-(2-(substituted benzylideneamino)oxazol-5-yl)-2H-chromen-2-one derivatives (1–15). The physicochemical and spectral characteristics of the synthesized oxazole derivatives are given in Table −1), 1H/13C–NMR  and Mass spectral) studies helped in determining the molecular structures of the synthesized derivatives (1–15). The IR spectrum indicated that the appearance of bands at 3398–2924 cm−1, 1456–1415 cm−1, 1680–1595 cm−1, 1382–1236 cm−1 and 1724–1693 cm−1 displayed the presence of C–H, C=C, C=N, C–N and C=O groups, respectively in the synthesized compound. The absorption bands around 1292–1130 cm−1 corresponded to C–O–C stretching of oxazole compounds. In case of 1H-NMR spectra the presence of multiplet signals between 6.88 and 8.69 δ ppm reflected the presence of aromatic protons in synthesized derivatives. The compound 14 showed singlet (s) at 6.76 δ ppm because of the presence of OH of Ar–OH. The appearance of singlet (s) at 7.51–8.4 δ ppm and 6.9–7.37 δ ppm is due to the existence of N=CH and C–H of oxazole, respectively. Compound 8 showed multiplet and doublet signals at 3.11 δ ppm and 1.29 δ ppm due to existence of –CH and (CH3)2 groups of –CH(CH3)2 at the para-position. The compounds, 1, 2 and 14 showed singlet at 3.73–3.89 δ ppm due to the existence of OCH3 of Ar–OCH3. The compounds, 3 and 5 showed singlet at 5.08 δ ppm due to the existence of –CH2–O group of (benzyloxy)benzene. The compound 10 displayed doublet signal at 5.59–6.95 δ ppm due to the existence of –CH=CH group of -prop-1-en-1-ylbenzene. The 13C–NMR spectrum indicated that the carbon signals around at 161.1, 128.5 (coumarin), 151.9 (N=CH), 136.1 (oxazole) of the synthesized compounds. Mass of synthesized compounds showed in (M++1).The synthesis of oxazole derivatives –15 were 3 was moderately potent against S. aureus with MICsa value of 14.8 µM and compound 8 was moderately active against B. subtilis with MICbs value of 17.5 µM. Compound 3 (MICec = 14.8 µM) was found to be effective against E. coli. Compound 14 (MICpa = 17.3 µM) and compound 6 (MICse= 17.8 µM) exhibited promising activity against P. aeruginosa and S. enterica, respectively. The antifungal activity results indicated that compound 6 (MICan = 17.8 µM) displayed most potent activity against A. niger and compounds 3 and 5 (MICca= 29.6 µM) were found to be moderately potent against C. albicans. The antibacterial screening results are comparable to the standard drug (cefadroxil), whereas antifungal results of compound 6 showed less activity against A. niger and compound 5 showed more against C. albicans than the standard drug (fluconazole) and these compounds may be used as a lead compound to discover novel antimicrobial agents.The in vitro antimicrobial potential of the prepared oxazole derivatives was determined by tube dilution technique Table , 4 and 514 exhibited good activity with IC50 = 71.8 µM. In the case of MCF7, compound 6 exhibited good activity with IC50 = 74.1 µM. Reference drugs used in the study were 5-flourouracil (for HCT116) and tamoxifen (MCF7). They had yielded IC50 values of 12.7 µM and 4.3 µM, respectively and these compounds may be used as a lead compound to discover novel anticancer agents. Results are displayed in Table The synthesized derivatives were also screened for their cytotoxic effect using Sulforhodamine B (SRB) assay  against The mammalian cyclin-dependent kinase 8 (cdk8) protein which is a component of the RNA polymerase has been one of the proteins responsible for acute lymphoblastic leukaemias. CDK-8 is a heterodimeric kinase protein responsible for regulation of cell cycle progression, transcription and other functions. CDK-8 phosphorylates the carboxyterminal domain of the largest subunit of RNA polymerase II like protein kinases. Therefore, the inhibition of CDK-8 protein may be crucial for controlling cancer . Since c14 and compound 6 against human colorectal carcinoma HCT116 and oestrogen- positive human breast carcinoma MCF7 cancer cell lines respectively. The molecular docking study was carried out on GLIDE docking program. The compound 14 was docked in the active site of the cyclin dependent kinase cdk8 (PDB: 5FGK) co-crystallized wit 5XG ligand. The results were analysed based on the docking score obtained from GLIDE. Ligand interaction diagram and displayed the binding mode of compound 14 in the active site of cdk8 having co cystallised ligand 5XG and 5-fluorouracil (the standard inhibitor of cancer) is having a different binding mode to that of active compound  co-crystallized wit OHT (Tamoxifen) ligand. The results were analysed based on the docking score obtained from GLIDE. Ligand interaction diagram and show the binding mode of compound 6 in the active site of ER apha having co cystallised ligand OHT and Tamoxifen (the standard inhibitor of cancer) is having a different binding mode to that of active compound  with better potency (71.8 µM) within the ATP binding pocket (Table 6 showed the best dock score (− 6.462) with better potency (74.1 µM) within the ATP binding pocket 2) at para position of the substitution part of the synthesized compound 8, increased the antibacterial activity against B. subtilis. Presence of para-(phenoxy-methyl)benzene group (compound 3), enhanced the antibacterial activity against E. coli and S. aureus as well antifungal activity against C. albicans whereas (Compound 5) also improved the antifungal activity against C. albicans.The different substitution of aldehydes were used to synthesized the final derivatives of 3-(2-aminooxazol-5-yl)-23) at meta and para position of the substitution portion of the synthesized compound 14, increased the antibacterial activity against P. aeruginosa and also increased anticancer activity against HCT116 cancer cell line whereas electron withdrawing groups (–Cl) at para-position of the synthesized compound 6, improved the antimicrobial activity against S. enterica and A. niger as well as anticancer activity against MCF7 cancer cell line. These compounds may be used as a lead compound to discover novel antimicrobial and anticancer agents.Presence of electron releasing group  downfield from tetramethylsilane . Proton NMR spectra are given as multiplicity  and number of protons. Infrared  spectra were recorded as KBr pellets on Shimadzu FTIR 8400 spectrometer. Waters Micromass Q-ToF Micro instrument was used for obtaining the Mass spectra.The chemicals procured were of analytical grade and were further used without any purification. Melting point (m.p.) was determined in open glass capillaries on a Stuart scientific SMP3 apparatus. Reaction progress of every synthetic step was confirmed by TLC plates on silica gel sheets. Step 1: Synthesis of 3-acetyl-2H-chromen-2-one (I) To a solution of salicylaldehyde (0.025 mol) and ethyl acetoacetate (0.025 mol) in methanol (15 mL), 2–3 drops of piperidine was added, shaken with stirring and allowed to stand at room temperature for 30 min. Needle shaped crystals of 3-acetyl-2H-chromen-2-one (I) were obtained which were filtered dried and recrystallized from methanol  and oestrogen- positive human breast carcinoma (MCF7) using Sulforhodamine-B assay. HCT116 was seeded at 2500 cells/well (96 well plate) whereas MCF7 was seeded at 3000 cells/well (96 well plate). The cells were allowed to attach overnight before being exposed to the respective compounds for 72 h. The highest concentration of each compound tested (100 µg/mL) contained only 0.1% DMSO (non-cytotoxic). Sulforhodamine B (SRB) assay  was thenhttp://www.rcsb.org/pdb/home/home.do) for docking of potent synthesized oxazole compounds. Docking score obtained from GLIDE and ATP binding site was targeted and the grid was created. The active site grid covered the important amino acids interacting with ATP [The protein target for oxazole derivatives was identified through the literature. Since the oxazole nucleus has vast medicinal properties, so the targets enzymes/receptors were found targeted with anticancer effect of oxazole compounds were collected for selection . Cyclin-with ATP .3, 5, 6, 8 and 14 had the best antimicrobial activity and had MIC values comparable to the standard drugs whereas in the case of anticancer activity, compound 14 was found to be moderate activity against HCT116 while compounds 6 was moderate activity against MCF7. Further molecular docking study indicated that compound 14 showed the best dock score (− 7.491) with better potency (71.8 µM) within the ATP binding pocket. Compound 6 showed the best dock score (− 6.462) with better potency (74.1 µM) within the ATP binding pocket. Hence these compounds may be taken as lead compound for further development of novel antimicrobial and anticancer agents.A series of oxazole derivatives was designed, synthesized and evaluated for its antimicrobial and antiproliferative activities. The biological screening results indicated that the compounds


However, proper pre-deformation is an essential part of repeatedly obtaining large and stable shape change. In this paper, one-time uniaxial tensile pre-deformation was carried out at the temperature range −70–−80 °C. Then, the two-way shape memory effect (TWSME) of the alloy was evaluated by the martensitic transformation strain (εM) which was measured by a thermal expansion test to investigate the relationship between the pre-deformation strain (εT) and the TWSME. The results showed that εM of the columnar-grained Cu71.7Al18.1Mn10.2 alloy increased at first and then decreased with the increase of εT. The maximum value 2.91% of the εM could be reached when εT was 6%. The effects of the εT on transformation temperatures were also measured by differential scanning calorimetry. Based on the variations of transformation temperatures, the relationship between the internal stress induced by the pre-deformation process and the εM, and the influence mechanism of the pre-deformation strain on the TWSME in columnar-grained Cu71.7Al18.1Mn10.2 alloy, were discussed. The results obtained from this work may provide reference for potential applications of Cu-based SMAs, such as self-control components, fasteners, etc.Columnar-grained Cu Mf) which is often called cryogenic pre-deformation, and then restoring the original shape by subsequently increasing the temperature to a high temperature  without any external stress [εT). If the uniaxial tensile loading is chosen for the cryogenic pre-deformation, the TWSME can be evaluated by the elongation induced by the martensite transformation strain (εM) upon cooling or the contraction induced by the reverse/austenite transformation strain (εA) upon heating. For a fully recovered deformation, the transformation strain for the thermal cycle (εTW) can be used as εTW = εM = εA.The two-way shape memory effect (TWSME) associated with the forward and reverse transformations of martensite is a phenomenon in which the reversible and spontaneous shape change during heating and cooling occur without any external stress . The TWSl stress . In thisεT applied on the sample is usually 2–5 times as large as the εTW finally obtained [εTW of 4.1% and 2% were obtained in Ni–Ti alloy and Cu–Zn–Al alloy after pre-deformation with εT of 13.2% and 7%, respectively [εTW in SMAs, which could guarantee a sufficient εT to apply onto the SMAs without cracking or fracturing. For Cu-based SMAs, single crystal alloys exhibit good ductility [εTW due to their low ductility. The development of ductile polycrystalline Cu-based SMAs becomes a key to realizing wide industrial application. In the 1990s, Kainuma et al. developed ductile Cu–Al–Mn alloys with a high Mn content of about 10 at.% and a low Al content of 16–18.5 at.% due to an appropriate decrease of the degree of order in the L21 austenite phase, in which the martensite phase has the 18R structure [εTW = 3.2% was obtained after ~12.5% pre-deformation strain [During thermomechanical treatment of SMAs, much research indicated that the obtained ,3,4,5. Fuctility ,7. Neveructility . Meanwhiuctility , while ouctility ) and laructility  which letructure ,12. Omorn strain ,15 prepaεTW/εT) is another way to obtain higher εTW under a certain pre-deformation strain. Considering the energy dissipation, minimization of the energy dissipation induced by microstructure defects, grain boundaries, etc. during thermomechanical pre-deformation of SMAs, could increase the pre-deformation efficiency [71Al18Mn11 alloys at room temperature [εTW in columnar-grained Cu–Al–Mn alloys was experimentally investigated. The internal stresses induced by tensile pre-deformation were also analyzed by transformation temperature measurement to discuss the effect mechanism of pre-deformation strain on the TWSME of columnar-grained Cu–Al–Mn alloys.On the other hand, improving pre-deformation efficiency , electrolytic aluminum (99.99 wt.% purity) and electrolytic manganese (99.9 wt.% purity), which were cast into precast ingots by vacuum induction melting. Subsequently, the precast ingots were re-melted and directionally solidified to obtain columnar-grained ingots with a dimension of Φ50 × 150 mm . The ing3 hydrochloric solution. The phase and microstructure were analyzed by Rigaku D/MAX-RB12KW X-ray diffraction  with Cu Kα radiation and a Tecnai G2 F20 field emission transmission electron microscope . The orientation of the grains was analyzed by electron backscatter diffraction (EBSD).The metallographic samples were cut from the middle part of the ingot by electrical-discharge machining, which was observed by Nikon Eclipse LV150 optical microscope  after being mechanically polished and etched by a FeCl2. During deformation a local strain was measured using an Instron extensometer with a gauge length of 25 mm. The longitudinal direction of the samples was parallel to the solidification direction (SD). Superelasticity was examined by a cycle tensile test with a strain rate of 5 × 10−4 s−1 at room temperature in an austenite phase state [The SE test and cryogenic pre-deformation experiments were conducted using dog-bone-shaped samples with a gauge length of 30 mm and a section area of 8 × 3 mmse state , that is−4 s−1 in a nearly isothermal condition, held for 300 s, and then unloaded at a strain rate of 1 × 10−4 s−1. Subsequently, the cryogenic pre-deformation samples were taken into ambient temperature and L0 = 20 mm rectangular plate samples for the thermal expansion test were cut from the middle of the cryogenic pre-deformation samples with a 25 mm gauge length and examined by a laser thermal expansion instrument  with an accuracy of 1 × 10−3 mm at a heating and cooling rate of 2 °C/min. It should be noted that during the thermal expansion measurement, a very small pressure of about 0.1–1 N was applied to the samples.The cryogenic pre-deformation experiment was conducted by Instron universal mechanical test systems with a thermostatic chamber, and nine specimens were used to load to 2%, 3%, 4%, 5%, 6%, 8%, 10%, 12%, 14%, respectively. All cryogenic pre-deformation specimens were cut from the same ingot to avoid the effects of deviation from the composition and casting processes on the comparability of test results. The detailed procedure of cryogenic pre-deformation experiment was as follows. First, the cryogenic pre-deformation samples were cooled to −70–−80 °C into martensite phase state for 1 h using liquid nitrogen . The cryogenic pre-deformation samples in the martensite phase state were loaded to a specific strain with a strain rate of 5 × 1071.7Al18.1Mn10.2 sample are shown in Ms = −10.3 °C, the martensite transformation finish temperature Mf = −29.3 °C, the austenite transformation starting temperature As = −3.9 °C, and the austenite transformation finish temperature Af = 7.6 °C. The transformation heat was also measured based on the DSC curves as 5.7 J/g for the martensitic transformation on cooling and 6.5 J/g for the reverse transformation on heating.The DSC curves of the Cu71.7Al18.1Mn10.2 alloy sample was composed of a single β1 phase (austenite phase) at room temperature as proved by XRD  of 18%. With the increase of cycles and total strain, the SE strain (εSE) increased at first and then decreased, where εSE= εt − εr − εe, εr was unrecovered strain and εe was elastic strain, as shown in εt was up to 10%, the maximum εSE was 8.18% (σM) at the first cyclic strain was about 60 MPa and decreased with the increase in the number of cycles. The stress plateau slope dσSE/dε of the stress induced martensitic transformation stage was 7.3 MPa. The cyclic stress-strain curves showed that the columnar-grained Cu71.7Al18.1Mn10.2 alloy exhibited excellent superelasticity along the SD. The normal tensile test was also conducted and showed that the elongation reached more than 40%, which indicated a high ductility of the columnar-grained Cu71.7Al18.1Mn10.2 alloy sample.The SE cyclic stress-strain curve tested at room temperature was plotted in as 8.18% a. The cr71.7Al18.1Mn10.2 alloy samples extended at −70–−80 °C (<Ms −50 °C) are shown in σMR) [σ) could be described as Δσ = σs − σMR, where σs was the maximum loading stress for each stress-strain curve in The cryogenic pre-deformation tensile curves of 2%, 3%, 4%, 5%, 6%, 8%, 10%, 12%, 14% strains respectively for nine columnar-grained Cu in σMR) . FurtherεTW is calculated by L is the average absolute value of expansion during the cooling process and of contraction during the heating process along the longitudinal direction of the samples. εTW are shown in εTW increased with increasing εT to a maximum of 2.91% when εT = 6% and then decreased as shown by black dots in εT reached 12%, the εTW decreased to nearly zero, which indicated TWSME disappeared when undergoing a pre-deformation strain that was equal to or higher than 12%. The polynomial fitting  done by pre-deformation corresponding to the WT than their polycrystalline counterparts. The value of 71.7Al18.1Mn10.2 alloy was ~55.0% corresponding to ~3% εTW. Comparatively, the εTW obtained by bending pre-deformation in ordinary polycrystalline Cu–Al–Mn alloys [WT for columnar-grained Cu71.7Al18.1Mn10.2 alloy was only ~3 MJ/m3, while for the Ti–Ni alloy, it was ~15 MJ/m3. 71.7Al18.1Mn10.2 alloy were better than ordinary polycrystalline Cu-based SMAs and reached a single crystal level. In terature ,27,28,29εTW was much larger than the thermal expansion. 71.7Al18.1Mn10.2 alloy sample without deformation pre-deformation. The thermal expansion coefficient was measured to be ~12.9 × 10−6/°C for both the austenite and martensite. It was seen that a near-linear dimensional expansion of ~0.2% was involved in transformation from −80 to 80 °C without deformation pre-deformation. Hence, when compared to the εTW mentioned above, the inherent thermal expansion of columnar-grained Cu71.7Al18.1Mn10.2 alloy could be ignored.For SMAs, the 71.7Al18.1Mn10.2 alloy samples. The cryogenic pre-deformation was attributed to the variants of self-accommodation and reorientation with an internal elastic energy  ΔG=ΔIn this equation, the superscript “A–M” denotes the reversible transformation between the austensite and the martensite. The critical temperature for the reversible transformation can be determined at T0 was directly influenced by It was seen that The 71.7Al18.1Mn10.2 alloy samples with the increase of pre-deformation strain could be obtained and plotted in 71.7Al18.1Mn10.2 alloy samples decreased at first and then increased with the increase of the pre-deformation strain. The change of transformation temperatures could be attributed to the internal stress created during cryogenic pre-deformation. When the sample was loaded with small strains during cryogenic pre-deformation, the reorientation of self-accommodation martensite variants became the dominative deformation mechanism. The internal stress of the sample decreased with the completion of reorientation. When the reorientation completely finished, the plastic deformation was induced by a dislocation slip mechanism, which would increase the internal stress by interaction of dislocations and other microstructure defects such as grain boundary and phase interface. 71.7Al18.1Mn10.2 samples after cryogenic pre-deformation at room temperature, which illustrated that martensites formed preferentially in some grains with little resistance during transformation. The martensite plates were thin when pre-deformation strain was ≤6% as shown in As shown in 71.7Al18.1Mn10.2 alloy. With the raising of pre-deformation strain, TWSME increased at first and then decreased, and the maximum transformation strain of 2.91% was measured under 6% pre-deformation strain. Meanwhile, the high pre-deformation efficiency of 57% and low applied pre-deformation work of ~3 MJ/m3 were found in the columnar-grained Cu71.7Al18.1Mn10.2 alloy, which indicated that the columnar-grained Cu-based SMAs show a better shape memory property than ordinary polycrystalline SMAs.In this paper, we have investigated the relationship between pre-deformation strain and TWSME in the columnar-grained Cu


The protein absent in melanoma 1 (AIM1) is a member of the βγ-crystal lens superfamily that is associated with the development of multiple cancers. The binding of AIM1 to β-actin affects the migration and invasion of prostate cancer epithelial cells. The C-terminus of AIM1 is required for the β-actin interaction. However, the characteristics of AIM1 in vitro and the interaction mode between AIM1 and β-actin remain unknown. We describe novel methods to prepare pure recombinant AIM1 and identify possible binding modes between AIM1 and β-actin; we also obtain the crystal of the first two βγ-crystallin domains of AIM1 (g1g2) for future structural biology research. We first express and purify AIM1 after cloning the sequence into a modified pET-28a_psp expression vector. Next, we define the minimum unit formed by the βγ-crystallin domain repeats that bound to β-actin and perform its physiological function. Finally, we made the structural model of the AIM1 g1g2 that can be used to guide future biomedical investigations and prostate cancer research. More than 90% of cancers lead to deaths associated with metastasis. Cancer cells must have the ability to migrate into and invade surrounding tissues, then enter the lymphatic and blood microvasculature and eventually transfer to distant tissues and adapt to the environment to form a tumor . Human cA correlation between cytoskeletal dynamics and tumor development has been identified ,3,4,5. PAIM1 in adult skin, lung, heart, liver and kidney, and it plays a transient regulatory role in embryonic tissues [AIM1 gene and its major transcripts reveal that AIM1 is a member of the βγ-crystallin superfamily [The absent in melanoma 1 (AIM1) gene is a novel gene whose expression is correlated with tumor suppression in the human melanoma model ,12,13. H tissues . The chaerfamily . Crystalerfamily . The funerfamily . AIM1 coerfamily . The firerfamily ,20. The Previous studies show that AIM1 is a β-actin-binding protein that suppresses the invasion of prostate cancer cells. Depletion of AIM1 in prostate epithelial cells increases cytoskeletal remodeling, intracellular traction forces, cell migration and invasion, and cancer growth . AIM1 tiWe developed novel methods to prepare pure recombinant proteins and observe the AIM1 interaction with β-actin. For protein purification, we first performed preliminary purification with Ni-NTA, then removed the 8x His-tag and obtained the purified, unlabeled protein with a 1 mL HisTrap HP column. After separation on a 1 mL HiTrap QHP column, the pure protein was obtained, and finally, Superdex 200 increase 10/300GL resin was used to detect the aggregation state of the protein. g1, g1g2, g1g2g3 are dimers in solution. Dot blots revealed that g1g2 was the minimum unit that interacted with β-actin. In addition, we constructed an artificial recombinant protein containing two g1 repeats (named g1g1) and found that it also bound β-actin, further confirming that the first Greek motif g1 is required for the main physiological functions of AIM1.We converted the commercially available pET-28a vector with a 6x His-tag to a pET-28a_psp vector with an 8x His-tag A,B. In oEscherichia coli and used for overexpression. The plasmids expressing g1, g1g2, g1g2g3 and g1g1 were constructed using the same method. Escherichia coli BL21 (DE3) cells harboring recombinant plasmids were cultured in LB medium. When the OD600 ranged between 0.6 and 0.8, 0.2 mM β-d-1-thiogalactopyranoside (IPTG) was added and cultured at 25 °C overnight. On SDS-PAGE gels, an overexpressed band was observed at the size of the target band.The coding sequences of these genes showed no variations or mutations from the T7 promotor to the stop codon. Plasmids containing the correct sequences were transformed into Escherichia coli culture and concentrate the protein to 3.5 mg/mL.This section describes g1g2 as an example. Resuspended cells were passed through a cryogenic overpressure cell breaker at 1200 psi. Both the supernatant and the precipitate were collected using high-pressure crushing followed by centrifugation. The recombinant soluble protein was purified on a Ni-NTA column (2 L of cells produce 15 mg of protein). Samples were analyzed by 12% SDS-PAGE and stained by Coomassie Blue a. Then, The g1, g1g2, g1g2g3 and g1g1 proteins were applied to Superdex 200 increase 10/300GL resin to detect the aggregation in aqueous solution, and 1 mg of sample was assayed. Proteins were analyzed in the following Buffer: 150 mM NaCl, 20 mM Tris-HCl and 1 mM DTT. The chromatograms of the four proteins and the marker overlapped a. The g1Dot blot assays were used to detect interactions between proteins. The control group and the experimental group included these four proteins incubated with β-actin. The proteins were loaded at concentrations of 0.4, 0.2 and 0.1 mM. The g1 did not bind to β-actin, while 0.4 mM g1g2 showed strong binding to β-actin. Interestingly, g1g1 also interacted with β-actin b. These Based on the experimental results described above, we have modeled the interaction between g1g2 and G-actin . As showThe production of soluble and active recombinant proteins is vital for structure-function analysis and therapeutic applications. Development of cancer drugs requires the ability to express and purify recombinant proteins, which are required in biological research to investigate enzyme activity, ligand binding, protein interactions, or other functions in vitro. The correlation between AIM1 and cancer has been reported in many studies ,23,24, bThe first domain of AIM1 (which contains one motif β and γ) is named g1 in this paper and is very important for the stability and function of AIM1, according to the previous studies. In the present study, the first two domains of AIM1, g1g2, are the minimum unit required to bind β-actin. We designed a possible model for AIM1 g1g2 assembly with G-actin, as shown in Although AIM1 is a member of the βγ-crystallin family, its function is quite different, which is a hindrance to our further studies of its function. Currently, we only resolve the structure of the C-terminal g1 domain of this protein. The three-dimensional structure of the full-length protein need to be obtained to better understand its physiological functions; the crystallization of the protein is currently ongoing, and some crystallization procedures are being further optimized (data not shown). Based on our findings, at least two motifs are required for the physiological function of AIM1. This domain is only a very small part of the protein, and we are planning to perform further experiments.Escherichia coli strains DH5α and BL21 (DE3) were the host strains for amplification and protein expression, respectively. pET-28a (+) was purchased from Novagen company. Subcloning of artificial genes encoding g1, g1g2, g1g2g3 and g1g1 were inserted into pET-28a_psp vectors and oligonucleotide primers were synthesized. The BamH1 and Sal1 restriction enzymes, T4 ligase and prestained protein ladder were purchased from Thermo Scientific. The Ni-NTA column, 1 mL HisTrap HP column, 1 mL HiTrap QHP column and Superdex 200 increase 10/300GL were purchased from GE Healthcare. AKTA pure 25 was purchased from GE Healthcare for protein purification. For dot blot assays, Super Enhanced ECL-chemiluminescence was purchased from sbjbio life science. All other Technologies from GBCBIOO chemicals used in the present study were of biochemical research grade.Escherichia coli DH5α strain, colonies were selected for sequencing and the correct sequences were confirmed. The final modified vectors contained an N-terminal 8x His-tag and with the Prescission Protease cleavage site (MGSSHHHHHHHHLEVLFQGP).We have modified the commercial pET-28a (+) vector to facilitate the removal of the 8x His-tag. According to the base sequence of pET-28a (+), primers for pET-28a_psp were designed. The target fragment was obtained by PCR, purified, and treated with PNK enzyme to induce phosphorylation. After a second purification step, 50 ng of the system was brought to 50 mL and connected with T4 DNA ligase. The plasmid was transformed into the The g1, g1g2, and g1g2g3 proteins are AIM1 fragments of different lengths, and g1g1 is a repeating amino acid sequence of g1. The g1 fragment representing amino acids 1-97, g1g2 fragment representing amino acids 1-186 and g1g2g3 fragment representing amino acids 1-293 were artificially synthesized. The genes encoding g1, g1g2, g1g2g3 and g1g1 were amplified by PCR using five pairs of primers .Escherichia coli BL21 (DE3) cells and spread on to an LB agar plate containing 30 μg/mL kanamycin. Colonies were evaluated using colony PCR.These forward and reverse primers included the BamH1 and Sal1 restriction sites in pET-28a_psp. The pET-28a_psp plasmid was linearized by double digestion with the BamHI and SalI restriction enzymes and purified using a DNA Gel Extraction kit 50-Prep. The amplified PCR products and the linearized pET-28a_psp vector were ligated using DNA ligase. Finally, the recombinant plasmids were transformed into 600 reached 0.6–0.8, and then protein expression was induced with 0.2 mM β-d-1-thiogalactopyranoside (IPTG) at 25 °C for 16 h. Cells were harvested by centrifugation (4000 rpm for 15 min at 4 °C) and stored at −80 °C until further use.The colonies containing correct sequences of the g1, g1g2, g1g2g3 and g1g1 cDNAs were grown in large-scale cultures in LB medium containing 30 μg/mL kanamycin at 37 °C. The cells were grown at 37 °C with shaking at 180 rpm until ODw/v). Resuspended cells were passed through cryogenic overpressure cell breaker at 1200 psi. The resulting lysate was centrifuged for 60 min at 12,000 rpm to obtain the supernatant. Firstly, the target protein was purified using a combination of affinity chromatography steps with Ni2+-nitrilotriacetic (Ni-NTA) agarose resin , and Buffer A  was used to remove hybrid proteins, followed by Buffer B  to elute the target protein. Secondly, the purification was performed by fast protein liquid chromatography (FPLC) on 1 mL HisTrap HP column  to obtain the target protein without 8x His-tag. The protein solution was injected into the equilibrated column at 0.5 mL/min flow rate, eluted using a linear imidazole gradient from 10 to 250 mM. Every 2 mL fraction was collected over the full gradient length. Thirdly, anion exchange chromatography was performed on a 1 mL QHP column  with NaCl gradient. Finally, the pure protein was separated on Superdex 200 increase 10/300GL  to obtain the homogeneous protein in solution. The column was first equilibrated with Buffer , and the target proteins were concentrated. The aliquoted protein was flash-frozen and stored at −80 °C until further use.The previously collected strains were thawed and resuspended at in 40 mM Tris-HCl, pH 8.0, 250 mM sodium chloride, 1 mM β-mercaptoethanol and 1 mM PMSF  at a 1:4 ratio of 1:4 for two hours on ice. The mixture was incubated with the appropriate amount of Ni-NTA for two hours at low temperature on a 3D shaker, and finally a Buffer containing imidazole was used to elute the desired protein. The proteins were quantified, spotted on PVDF membranes (unactivated), naturally air dried and blocked with 5% skim milk powder in TBST for 1 h. Membranes were incubated with a β-actin antibody overnight, washed, incubated with a secondary antibody, and exposed to ECL for detection.We developed an effective procedure for the expression and isolation of milligram quantities of the highly purified six C-terminal Greek motifs of the AIM protein. The purified proteins were stable and formed a dimer in solution. The purified AIM1 g1g2 protein is the minimum unit required to bind β-actin. This protocol will be useful for further biochemical and structural biological studies.


Single-phase solid solutions were obtained in the case of calcium content x = 0.03 and 0.05, whereas a calcium-enriched impurity phase was found at x = 0.10. Water uptake and release were studied by means of thermogravimetric analysis, thermodesorption spectroscopy and dilatometry. It was shown that lower calcium content in the main phase leads to a decrease in the water uptake. Conductivity was measured by four-probe direct current (DC) and two-probe ascension current (AC) methods at different temperatures, pO2 and pH2O. The effects of phase composition, microstructure and defect structure on electrical conductivity, as well as correlation between conductivity and water uptake experiments, were discussed. The contribution of ionic conductivity of La1−xCaxScO3−α rises with decreasing temperature and increasing humidity. The domination of proton conductivity at temperatures below 500 °C under oxidizing and reducing atmospheres is exhibited. Water uptake and release as well as transport properties of La1−xCaxScO3−α are compared with the properties of similar proton electrolytes, La1−xSrxScO3−α, and the possible reasons for their differences were discussed.In this study, oxide materials La Oxide materials with proton conductivity have been attracting attention as key materials for highly efficient and environment friendly protonic ceramic fuel cells (PCFC). The use of proton-conducting electrolytes in comparison with oxygen-ion conductors has several advantages. For example, the proton transfer process has significantly lower activation energy than oxygen-ions, which leads to a reduction in the operating temperature of devices without any loss of their power characteristics ,2,3. 2+B4+O3-type perovskites  because of their high proton conductivity. However, some of these materials can decompose in atmospheres containing water vapor and carbon dioxide due to the high content of alkaline earth-metal cations [Traditionally, researchers have focused on the study of solid oxides with A cations ,6.3+B3+O3 structure have a higher chemical stability and mechanical durability. Studies of the physicochemical properties of acceptor-doped La1−xMxBO3−α  solid solutions demonstrate a tendency to increase the total conductivity and proton mobility with the decrease in ionic radii of the B site cations [3 can be considered as promising materials for solid electrolytes of PCFC [Perovskites with A cations ,8,9. Thu of PCFC ,6,7,8,9.0.9Sr0.1Sc0.9Mg0.1O3−α [1−xMgxO3−α [1−xSrxScO3−α [1−xCaxScO3−α. However, the influence of Sr–Ca substitution on water vapor solubility, conductivity and thermal expansion of La1−xCaxScO3−α, depending on temperature, partial pressures of oxygen (pO2) and water vapor (pH2O), is not described. The purpose of this study is to examine conductivity, defects concentration and ionic transport numbers of La1−xCaxScO3−α .The transport properties of Lag0.1O3−α ,11; LaScxMgxO3−α , La1−xSrrxScO3−α ,14,15,16rxScO3−α  reported0.97Ca0.03ScO3−α (LCS3), La0.95Ca0.05ScO3−α (LCS5), La0.9Ca0.1ScO3−α (LCS10) and La0.9Sr0.1ScO3−α (LSS10) materials were prepared by the citric-nitrate combustion synthesis. The synthesis technique is given in detail in our previous work [2O3, Sc2O3  CaCO3 and SrCO3  with purity higher than 99.5% were mixed with consideration of the calcination coefficients and then dissolved in a solution containing the required amounts of nitric acid. After the dissolution of all precursors was completed, citric acid was added, and the mixture evaporated prior to the combustion reaction. The obtained powders were calcined at 1200 °C for 2 h. The calcined powders were reground and pressed to obtain samples in a form of pellets and bars. These samples were sintered at 1650 °C for 5 h in air. The estimated density of the as-sintered samples was determined by the Archimedes method in kerosene. The theoretical density was determined from the ratio of volumes and molar mass of unit cells of the materials under study.The Laous work . The stoThe phase composition and structure features of LCS powders were studied by the X-ray diffraction (XRD) analysis. XRD patterns were measured with a D/MAX-22 diffractometer  at room temperature in the 2θ range 20–70° with a step of 0.02°. The phase composition of the samples was identified with the use of the PDF-2ICDD database. Crystal structure calculations were carried out using software MDIJade 6.5  and Match-DEMO . −2 atmospheres), grinding and polishing using Allied MetPrep 4/PH-4  System grinding and polishing machine, as well as diamond suspensions.The surface and cross-section morphology of the ceramic samples was investigated by the scanning electron microscopy (SEM) on a TESCAN MIRA 3 LMU microscope . Maps of elements distribution were obtained for the cross-sections of the ceramic samples by Energy-Dispersive X-ray (EDX) spectroscopy using Oxford Instruments  INCA Energy 350 X-Max 80 equipment. The preparation procedure for the SEM characterization of the samples cross sections included an epoxy impregnation in rough vacuum  using an equipment based on vacuum chamber, heating device and radio frequency mass-spectrometer, which are described in detail in [−5 Pa and the heating rate was 42 °C min−1. The water saturation of the dense ceramic samples was carried out by annealing at 500 °C and pH2O = 4.5 kPa during 15 h.Studies of the release of Hetail in ,18. The −1 in dry (pH2O < 0.1 kPa) and wet (pH2O = 3.2 kPa) air atmospheres.The thermal expansion of the dense ceramic bars was studied using a quartz dilatometer and TESA Tesatronic TT-80  equipment from room temperature to 900 °C with a heating/cooling rate of 2 °C minsp) of 1.1 ± 0.2 m2/g using a Netzsch STA Jupiter 449 F3  analyzer with water vapor generator Adrop Asteam DV2MK. Powders specific surface area for TGA measurements was determined by Brunauer-Emmett-Teller method using Sorbi N.4.1 . The as prepared powders were heated up to 950 °C and held at this temperature for 8 h under the dry argon . Afterwards the carrier gas was saturated with water vapor (pH2O = 24.3 kPa) and then the increase in weight was recorded upon cooling from 950 °C to 300 °C with a cooling rate of 30 °C h−1 and exposure at every 100 °C during 2 h. The proton concentration was obtained from the weight change during TGA as follows:oxideM and 2OHM are molecular weights of the oxides and water, Δm and oxidem are weight change and weight of the specimen, respectively.The thermogravimetric analysis (TGA) was performed on the powder samples with specific surface area (SpO2 = 2.1 × 104–10−18 Pa) and water partial pressure (pH2O < 0.1 kPa and 4.5 kPa) by both two-probe impedance and four-probe DC methods. Porous platinum electrodes were fabricated by firing in air at 1000 °C for 1 h. Electrochemical impedance spectroscopy (EIS) was used to estimate the contributions of bulk and grain boundary resistances to the total resistance of ceramic the samples under study. The complex impedance was measured in the frequency range from 100 mHz to 8 MHz using electrochemical analyzer IM6 . At temperatures above 600 °C, the impedance spectra are shifted towards higher frequencies, so the semi-circle corresponding to the bulk resistances become indistinguishable. The impedance spectra were analyzed using an equivalent circuit model (EC) , oxygen partial pressure (del (EC) .3-type orthorhombic distorted perovskite-type unit cell  than the volume of [Ca2O2 samples have uniform distribution of all elements, as well as La1−xSrxScO3−α (x = 0.05–0.1) [The relative density of LCS ceramic samples reaches 97%–98% . To asse.05–0.1) . Accordi2+ into La3+ is located within the range of x = 0.05–0.1. At Ca concentration of x = 0.1 second phase with higher Ca content could be observed. On the other hand, Fujji et al. [1−xCaxScO3−α (x = 0.05–0.2) as single-phase according only XRD data. Unfortunately, no further details were provided. Thus, according to our data the solubility limit Cai et al.  presenteIt is well-known that oxygen vacancies induce incorporation of water molecules and formation of OH-defects:tes OHO• ,23.1−xCaxScO3−α is determined by the concentration of the Ca acceptor dopant, which formed according to the following equation:The content of oxygen vacancies in La−1. Results of the TDS study of water release from hydrated LCS and LSS are presented in 2 release is observed only in LCS10  and wet (pH2O = 4.2 kPa) air is nearly linear and monotonic (−6 K−1 and (8.52 ± 0.03) × 10−6 K−1 for LCS5 and LCS10, respectively. In wet air conditions, TEC slightly decreases for LCS5 to (8.49 ± 0.03) × 10−6 K−1 and increases for LCS10 to (8.68 ± 0.03) × 10−6 K−1. Obtained values of TEC for LCS are close to those for LSS electrolytes [The temperature behavior of dilatometric curves for LCS ceramic samples in the heating regime in dry  × 10−6 K−1 at 500 °C [The thermal expansion of hydrated LCS5 and LCS10 samples in dry air a demonstthors of . Measuret 500 °C .pH2O = 24.3 kPa of the single-phase LCS5 powder (Ssp= 1.1 ± 0.2 m2/g), obtained by trituration of ceramic sample.KH of hydration reaction is expressed asKH, the following electroneutrality condition and site restriction for proton conducting perovskite oxide are employed by neglecting concentration of electronic holes:x in LCS5 oxide. Equilibrium constant KH (4) of hydration reaction (2) can be described in terms of enthalpy ΔhydrH and entropy ΔhydrS:R is the universal gas constant and T is the absolute temperature. The standard enthalpy ΔhydrH and entropy ΔhydrS were −132 ± 5 kJ mol–1 and −126 ± 5 J mol−1 K−1, respectively. The obtained values are comparable with the same values for the best-known perovskite-like oxides with orthorhombic type structure such us BaCex1−YxO3−α [x1−SrxYbO3−α [x1−SrxScO3−α [According to Equation (2), the equilibrium constant −xYxO3−α , La1−xSrrxYbO3−α  and La1−rxScO3−α ,26.x unlike Lax1−SrxScO3−α [3-based oxides, this phenomenon can be associated with not fully filled oxygen vacancies due to non-equality of O1 and O2 oxygen positions [According to Equations (2) and (3), the value of maximal water uptake (saturation level) has to be equal to the acceptor doping concentration: rxScO3−α , Figure rxScO3−α . In the ositions ,27.3 are mixed ion-hole conductors in oxidizing conditions [•) occurs at interaction atmospheric oxygen with oxygen vacancies:Acceptor-doped materials based on the LaScOnditions ,12,13,148 is the equilibrium constant of Equation (8).The concentration of electronic holes is equal tox1−CaxScO3−α in wet air isThus, the general condition of electroneutrality for LaIn reducing atmospheres, these materials are ionic conductors ,11,14; tpH2O = 4.5 kPa). The total conductivity of LCS5 sample even exceeds LCS10 conductivity . The total conductivity of LCS samples was measured depending on pO2 at 700 °C under wet (pH2O = 4.5 kPa) conditions, as shown in pO2 decreases, the total conductivity of all these samples decreases and reaches a constant value that corresponds to the ionic conductivity. The evaluated ion transfer numbers for LCS3, LCS5 and LCS10 are 0.45, 0.32 and 0.37, respectively. The value of hole conductivity was calculated as the difference between the total and ionic conductivities , the hole and proton concentration increases along with the concentration of oxygen vacancies and according to Equation (3), that is, in the order of LCS3–LCS5–LCS10. However, it was found that the hole and ionic conductivity in LCS5 is slightly higher than that of LCS10, despite the lower concentration of the Ca acceptor dopant. These experimental facts could be explained by reduced calcium content or by a decrease in the charge carriers mobility due to the presence of an impurity phase or the formation of associate defect complex (•-CaLa/) . 2pO on total conductivity is considered in the example of the LCS5 sample (pH2O < 0.1 kPa) and wet (pH2O = 4.5 kPa) air, and even exceeds the latter. This behavior is explained by the domination of conductivity of electronic holes, which decreases with increasing humidity and decreasing temperature (The influence of humidity and 5 sample a. At temperature b. In drypH2O = 4.5 kPa) reducing atmosphere does not contain the contribution of hole conductivity   . Therefo1−xCaxScO3−α  materials were investigated.In the present work, phase composition, microstructure features, thermal expansion behavior, processes of water uptake and release and electrical conductivity of La1−xCaxScO3−α  dense ceramic materials were synthesized by the citrate-nitrate combustion technique. An X-ray diffraction analysis demonstrated that samples under study are single-phase, whereas according to the SEM study LCS10 sample contains a Ca-enriched impurity together with the main perovskite phase. The presence of the impurity leads to lower calcium content in the main phase and a decrease in water release on TD-spectrum, compared with La0.9Sr0.1ScO3−α. This also results in an interaction with CO2. No evidence of CO2 release was detected in LCS5 and LSS10, indicating good chemical stability of these materials in CO2-containing atmospheres. The LaThe concentration of the dopant and the composition of the gas phase have a slight effect on the thermal linear expansion coefficient of the LCS5 and LCS10 samples. For the hydrated samples, observed inflections on the linear expansion temperature dependencies are in good agreement with those obtained by TDS temperature at which desorption begins. Proton concentrations, obtained by TGA, for LCS5 does not reach dopant level. The LCS materials under study have mixed ion-hole conductivity in wet air. The higher total and bulk conductivities are obtained for LCS5 samples. LCS3 grain boundary conductivity is up to two orders of magnitude lower compared with other samples. The hole conductivity of LCS5 is slightly higher than that of LCS10, despite the lower concentration of the Ca acceptor dopant. The decrease in the electrical conductivity of LCS10 is possibly due to presence of Ca-enriched impurity phases or formation of associated defects.The contribution of ionic conductivity of LCS rises with decreasing temperature and increasing humidity of the atmosphere. Under reducing atmospheres, LCS materials are pure ionic conductors. At temperatures below 500 °C, the protonic conductivity is dominant in all atmospheres. The results obtained here allow the LCS5 to be considered as the promising proton-conducting electrolyte for electrochemical devices.


Mean DIN reduction rates with and without microbial activity were 96.0 ± 46.4 mg·(m3·d)−1 and 288.1 ± 67.8 mg·(m3·d)−1, respectively, with microbial activity rates accounting for 29.7% of the DIN load and 2.2% of the N load. Results of correlation and principal component analysis showed that the main factors influencing N processing were the concentrations of different N forms and multiple environmental factors in spring, N concentrations, DO and pH in summer, N concentrations and water velocity in autumn and N concentrations in winter.Understanding spatio-temporal differences in nitrogen (N) transformation, transport and reduction rates in water bodies is critical to achieve effective mitigation of river eutrophication. We performed culture experiments in six rivers in the Taihu Basin using a custom made in-situ experimental apparatus. We investigated spatio-temporal differences in reduce processes and rates of different N forms and assessed the contribution of biological processes to dissolved inorganic N (DIN) reduce. Results showed that biological processes played a major role in N reduction in summer, while non-microbial processes were dominant in winter. We observed significant spatial and temporal differences in the studied mechanisms, with reduction rates of different N compounds being significantly higher in summer and autumn than spring and winter. Reduction rates ranged from 105.4 ± 25.3 to 1458.8 ± 98.4 mg·(m Nitrogen (N) is an essential nutrient element for all organisms, but excessive inputs of bioavailable N from anthropogenic activities have severely impacted aquatic ecosystems ,2,3. Exc2 production, with other processes acting as only temporary N storage mechanisms [−1·d−1 in Poyang Lake’s suspended sediments, 0–56.4 mg N·m−2·d−1 in sediments in the Taihu Basin and 274–2111 mg N·m−2·d−1 in the water of a Lake Superior estuary; N2O emission rates were 0.04–56.47 mg N·m−2·d−1 in urban rivers and 0.01–80.9 mg N·m−2·d−1 in runoff rivers; N2 production rates ranged between 19.08 and 375.44 mg N2-N·m−2·d−1 in western Lake Taihu [3−-N, dissolved oxygen (DO) and suspended solids [When point source pollutants discharged directly into water bodies are controlled effectively, rivers are the main sources of pollutants to lakes and oceans. Aquatic ecosystems naturally possess self-purification capacities. After N enters aquatic ecosystems, part of it is removed through biological processes  and abiotic processes  the biochemical activity can significantly reduce N in natural water within one day, and (2) the device can attain the same environmental conditions found in each river.The Lake Taihu Basin is located in the center of the Yangtze River Delta in southeastern China, one of China’s most economically developed regions. The region is characterized by a dense river network and is heavily populated, with a large portion of the population residing along rivers and leading to developed industry and intensive agriculture. The region is dominated by a subtropical monsoon climate, with a rainy season  occurring from May to September and alternating with a dry season . Annual mean air temperature varies between 14.9 °C and 16.2 °C.−1 (56.68% as NH4+-N and 41.07% as NO3−-N). Similarly, JX is also mainly used for shipping and N pollution to this river derives from sewage and industrial wastewater, resulting in average DIN concentrations of 3.48 mg·L−1 (18.84% as NH4+-N and 77.73% as NO3−-N). NX is located in the city of Yixing, where it supports agricultural activities and shipping, and it exhibits average DIN concentrations of 3.01 mg·L−1 (20.08% as NH4+-N and 76.32% as NO3−-N). TG is located in the town of Fenshui, the main N sources to this river are domestic sewage and industrial wastewater and the average DIN concentrations are 4.01 mg·L−1 (35.20% as NH4+-N and 60.98% as NO3−-N). PW and TP are located in the Wujiang District, Suzhou, and their average DIN concentrations amount to 3.44 mg·L−1 (38.73% as NH4+-N and 58.54% as NO3−-N) and 1.93 mg·L−1 (21.02% as NH4+-N and 76.59% as NO3−-N), respectively. Both sampling sites are located in the main channel, but PW is surrounded by an urban area, while TP is located in a predominantly rural area.We sampled six rivers in the Lake Taihu region  in July We developed an experimental apparatus (Patent No. ZL201520816420.7) to measure in-situ N reduction rates . The dev2. For the controls, which reflected N reduce under typical riverine conditions, three culture bottles were filled with river water collected near both river shores and from the open channel at a depth of 50 cm. For the inhibition treatments, three culture bottles were filled with water and added with 2 mL 0.5% HgCl2, thereby reflecting N reduce in river water without microbial activity. The water bottles were sealed with a rubber stopper to prevent air exchanges through the bottle top and hanged in the apparatus  on N reduction rates under in-situ riverine conditions. The experiment consisted of two treatments, a control and a microbial inhibition treatment, the latter consisting in the addition of 2 mL 0.5% HgClpparatus , and theTo avoid disturbing suspended solids settled at the bottom of the bottles, the experimental apparatus was taken out of the water slowly after reaching the preset incubation time (1 day) and then water samples were immediately collected from the culture bottles in the field. Samples of culture water from the upper layer of each bottle were collected from a side sampling port, while samples of water and suspended solids from the bottom of each bottle were collected from a bottom sampling port. The volume of the upper and lower parts of water was measured with a measuring cylinder after being transported back to the laboratory. Water samples were placed in ice for transport to the laboratory, filtered  and frozen until analysis.4+-N, NO3−-N, NO2−-N and a set of physico-chemical parameters were measured in each river in spring, summer, autumn and winter. The original water was analyzed for TN using potassium persulfate oxidation with detection limits of 0.001 mg·L−1, and suspended solids were measured by weight method with detection limits of 0.0001 mg·L−1. Filtered water was analyzed for NH4+-N, NO3−-N and NO2−-N using a flow injection analyzer with detection limits of 0.1, 0.01 and 0.01 mg·L−1, respectively . The DIN was calculated by summing the NH4+-N, NO3−-N and NO2−-N. At each sampling site, a multi-parameter water quality probe  was used to measure water temperature, DO and pH, and the River Surveyor M9  was used to measure water velocity and flow across each river’s cross-section. The water temperature, DO and pH were measured only once at the beginning of the experiment, and the water velocity and flow were measured at the beginning and the end of the experiment.Total N (TN), NHcR) in the upper and bottom layer of each bottle was calculated using Equation (1). In the absence of microbial activity, a theoretical rate of change can be calculated for the bottom layer (R2) was calculated using Equation (3). The absolute N reductions rates (aR) was calculated using Equation (4), which quantifies the total reduction rates of different N forms.The rate of change in N concentration  and bottom  layer of the culture bottle and V1 and V2 are the upper and bottom sampled water volume.In the above equations, Data were checked for normality and homogeneity of variance prior to statistical analyses. Spatial and temporal differences in mean reduction rates among sites and seasons were tested with one-way analysis of variance (ANOVA) followed by Fisher’s Least Significant Difference (LSD) test. Relationships between reduction rates and measured environmental variables were examined with correlation and principal component analysis (PCA). All statistical analyses were performed in SPSS version 19.0 .Concentrations of TN in the upper and bottom layers changed significantly after incubation . CompareA scatterplot of the theoretical vs. measured rate of change for the bottom layer shows that most spring, autumn and winter data points are distributed in quadrant II, close to the y = −x line, while summer data points are located in quadrant I . These r4+-N concentrations showed significant differences among seasons and sampling sites, although no clear seasonal and spatial pattern emerged  and II (19 points), with only two points appearing in each of quadrant III and IV, respectively , autumn and winter   .Most data points illustrating the relationship between theoretical and observed bottom rates of change lie in quadrant I (19 points) and quadrant II (27 points), with only two points in quadrants III and IV . Points 4+-N, NO3−-N and NO2−-N reduction rates ranged from 105.4 ± 25.3 to 1458.8 ± 98.4 mg·(m3·d)−1, 33.1 ± 12.3 to 440.9 ± 33.1 mg·(m3·d)−1, 56.3 ± 22.7 to 332.1 ± 61.9 mg·(m3·d)−1 and 0.4 ± 0.3 to 31.8 ± 9.0 mg·(m3·d)−1, respectively (p < 0.05) at all sites but TP site. No significant differences of seasonal average TN reduction rates were observed across sampling sites except TP site (p > 0.05). Reduction rates of NH4+-N, NO3−-N and NO2−-N were higher in summer and autumn than in the remaining seasons at most sites, while in DC high spring NH4+-N and NO3−-N concentrations were observed. All six sites had significantly higher average NO3−-N reduction rates in autumn compared to spring, while NO2−-N reduction rates were highest in summer (p < 0.05).Across the six rivers and four sampled seasons, TN, NHectively . TN redu4+-N, NO3−-N and NO2−-N reduction rates within the same season across rivers or within the same river across seasons. In spring, the reduction rates of NH4+-N in DC and TG and NO3−-N in DC were significantly higher than in other rivers (p < 0.05), while no significant difference was observed in NO2−-N reduction rates among rivers (p > 0.05). In summer, reduction rates of NH4+-N in TG, NO3−-N in NX and TG were significantly higher than in other rivers (p < 0.05), while NO2−-N reduction rates were significantly lower in JX and TP compared to others (p < 0.05). In autumn, DC had a significantly higher NH4+-N reduction rate than other rivers, while reduction rates of NO3−-N in JX, TG and NO2−-N in JX were significantly higher compared to other rivers (p < 0.05). In winter, the highest NH4+N, NO3−-N and NO2−-N reduction rates were observed in NX, PW and NX, respectively, while TN reduction rates were higher in DC than in other sites across the four seasons.We observed remarkable differences in NH3·d)−1, with an average of 96.0 ± 46.4 mg·(m3·d)−1, while non-microbial DIN (nMDIN) reduction rates ranged from 75.6 to 582.3 mg·(m3·d)−1, with an average of 288.1 ± 67.8 mg·(m3·d)−1. These results suggest that non-microbial processes were generally the primary DIN reduction mechanism.Different N reduce processes occurring in natural freshwater ecosystems, including microbial and non-microbial mechanisms, result in spatial and seasonal differences in DIN reduction rates. The relative contributions of microbial and non-microbial processes to DIN reduce differed among rivers and seasons . Across In spring, MDIN and nMDIN reduction rates were higher in DC than in other sites. In summer, nMDIN reduction rates were generally higher than MDIN rates at all sites except TP, where nMDIN and MDIN rates were similar. In autumn and winter, nMDIN reduction rates were higher than MDIN rates at all sites. Negative MDIN reduction rates observed in PW, DC and NX might be due to sediment resuspension offsetting the effect of microbial reduce. The proportions of DIN reduction rates and N load attributable to microbial and non-microbial processes are shown in We investigated environmental factors that may have affected N reduction rates observed in our in-situ incubation experiments. We found significant correlations between N reduction rates and initial N concentrations, DO, pH, suspended solids, water velocity and flow rate . SpecifiWe performed PCA analysis to explore correlations among variables and assess the relative importance of different factors. PCA results showed three significant components illustrating the effects of different environmental variables on N reduce in each season . In spri4+-N, NO3--N and NO2−-N migration and transformation processes across different seasons , which can promote nitrification in rivers [4+-N  are complex and include microbial and non-microbial mechanisms. Nitrification, the production of NO3− from NH4+, and denitrification of NO3− to gaseous N are the main microbial reduce processes. DIN can also be adsorbed by suspended solids and leave the water through deposition. These processes are affected by different river conditions.Processes contributing to observed decreases in DIN −1, 56.3 ± 22.7 to 332.1 ± 61.9 mg·(m3·d)−1 and 0.4 ± 0.3 to 31.8 ± 9.0 mg·(m3·d)−1, respectively, across four seasons. Reduction rates of NH4+-N and NO2−-N were positively correlated with NH4+-N and NO2−-N initial concentrations, while a similar correlation was not observed between NO3−-N reduction rates and initial NO3− concentrations (3 oxidation rate of 224 mg (m3·d)−1 was measured in relation to an NH3 concentrations of 0.85 mg/L [4+-N reduction rates measured in this study including microbial (nitrification and anaerobic NH3 oxidation) and non-microbial processes showed that 39.62% of the reduce was attributable to microbial activity, indicating that our results are plausible. Several studies have reported nitrification rates in different aquatic systems. For example, nitrification rates of 249 mg N (m2·d)−1 and 370 mg N (m2·d)−1 were measured in sediments of a Lake Superior estuary and Onondaga Lake, respectively [3-N, with NH3 oxidizing bacteria increasing significantly at high NH3-N levels, thereby promoting NH3 oxidation [4+ from the water column through bottom deposition. NH4+-N reduction rates were negatively correlated with water velocity and flow rate. Previous studies have shown that heavy navigation traffic and wider river can accelerate pollutant degradation and dilution, thereby decreasing pollutant concentrations [3−-N reduction rates were not correlated with NO3−-N concentrations, but a positive correlation was observed between microbial NO3−-N reduction rates and suspended solid content. In Poyang Lake, denitrification rates ranging from 0.25 ± 0.18 to 0.63 ± 0.24 μmol N·L−1, corresponding to ca. 3.5 to 8.8 mg N (m3·d)−1, suggested the occurrence of denitrifying processes on the surface of suspended solids [3− content. In this study, NO3−-N reduction rates were negatively correlated with DO concentrations, similar to the findings of Wang (2015), who reported a significant correlation between N2O production rates and DO. Denitrification can be affected by suspended solids, whose surface can provide microscopic interstices characterized by anaerobic conditions for denitrification to occur. In addition, NO3−-N adsorbed on the surface of suspended solids can be removed from the water column through deposition. NO2−-N reduction rates showed a significant positive correlation with initial NO2− concentrations and a negative correlation with DO. As the substrate for the second part of the nitrification process, NO2− concentrations can directly affect nitrification rates. NO2− concentration is generally the primary factor affecting nitrification rates under limiting NO2− levels, with other environmental factors becoming significant when NO2− is not limiting.According to the results of our in-situ experiment, reduction rates of NHtrations . In Lake.85 mg/L , which iectively . Both stxidation . At the xidation  and can trations . Water td solids . PreviouIn this study, the fraction of DIN reduction rates associated with non-microbial processes was higher than that accounted for by microbial activities, suggesting that adsorption and sedimentation had a stronger effect than biotransformation on DIN reduction rates. However, sedimentation of suspended matter is expected to be less prominent in a natural river setting than what we observed in our study. Because our experiment was conducted in a glass culture flask and no external water was supplied continuously, we may have overestimated the relative contribution of non-microbial processes on DIN reduce. Nevertheless, our results on the main DIN transformation processes and the overall effects of non-biological and microbial processes on spatio-temporal differences are realistic, because we ensured identical in-situ experimental conditions among seasons and sampling sites. Spring and summer DIN reduction rates in the absence of microbial activity varied greatly because of strong differences in flow velocity among sampling sites , as indiWe estimated that N reduce by microbial activities accounted for 7.9–61.2% of the DIN load (average: 29.7%) and 0–6.4% of the N load (average: 2.2%). Our results are consistent with previous studies, which estimated that the amount of N removed by denitrification accounted for 4.6% and 6.3% of the N load in the Mara River Basin and in Poyang Lake, respectively ,34.In natural conditions, N transformation and transport mechanisms mediated by biological and non-biological processes can reduce the N content in river water. In order to determine the differences in reduce processes and rate of different N forms and assessed the contribution of biological processes to dissolved inorganic N (DIN) reduce and the effects of different environmental factors on N reduce processes, we designed an experimental apparatus to conduct in-situ culture experiments in six rivers in the Taihu Lake Basin. We found that reduction rates of different N forms were higher in summer and autumn than spring and winter, with biological and non-biological processes playing a stronger role in summer and winter, respectively. Biological and non-biological DIN reduction rates differed spatially and temporally. Although non-biological processes played a major role in N reduce, biological processes are the main mechanism that can permanently remove N from the water column. Various factors affected the observed spatio-temporal differences in N reduction rates. The main influencing factors were the concentrations of different N forms and multiple environmental factors in spring, N concentrations, DO and pH in summer, N concentrations and water velocity in autumn and N concentrations in winter. Although our results may overestimate the effects of non-microbial processes in removing N forms due to the design characteristics of our experimental apparatus, the observed temporal and spatial patterns were not affected and can be considered realistic.