Bi2GaSbO7 and Bi2InSbO7 were crystallized with a pyrochlore-type structure and the lattice parameter of Bi2GaSbO7 or Bi2InSbO7 was 10.356497 Å or 10.666031 Å. The band gap of Bi2GaSbO7 or Bi2InSbO7 was estimated to be 2.59 eV or 2.54 eV. Compared with nitrogen doped TiO2, Bi2GaSbO7 and Bi2InSbO7, both showed excellent photocatalytic activities for degrading methylene blue during visible light irradiation due to their narrower band gaps and higher crystallization perfection. Bi2GaSbO7 showed higher catalytic activity compared with Bi2InSbO7. The photocatalytic degradation of methylene blue followed by the first-order reaction kinetics and the first-order rate constant was 0.01470 min−1, 0.00967 min−1 or 0.00259 min−1 with Bi2GaSbO7, Bi2InSbO7 or nitrogen doped TiO2 as a catalyst. The evolution of CO2 and the removal of total organic carbon were successfully measured and these results indicated continuous mineralization of methylene blue during the photocatalytic process. The possible degradation scheme and pathway of methylene blue was also analyzed. Bi2GaSbO7 and Bi2InSbO7 photocatalysts both had great potential to purify textile industry wastewater.In order to develop original and efficient visible light response photocatalysts for degrading organic pollutants in wastewater, new photocatalysts Bi Dye contaminants from textile wastewater were difficult to treat for their high chroma, high chemical oxygen demand content and complicated ingredients. Some conventional methods including biodegradation ,2,3, ele2 was investigated most repeatedly owing to its non-toxic property, excellent stability and low cost. However, with a wider band gap (3.2 eV), anatase TiO2 only efficiently absorbed ultraviolet light which occupied only 5% of the solar energy, and thus failed to make good use of optical energy. In order to make the best use of visible light which occupied 43% of sunlight, developing visible light responsive photocatalysts was an inevitable tendency in the field of photocatalysis research, which could be embodied from abundant endeavors of previous scholars in realizing the degradation of the pollutants during visible light irradiation by the method of iron doping [4 and A2B2O7, had great potential for photocatalytic H2 production during visible light irradiation. It was well known that minute changes in internal structure of the semiconductor photocatalysts would presumably promote the separation of photogenerated electrons and holes and thus improve photocatalytic activities. Zou et al. synthetized Bi2MNbO7  [2B2O7 compounds with the A3+2B4+2O7 pyrochlore structure by substituting B4+ sites in A3+2B4+2O7 for M3+  and Nb5+. Similarly, previous studies had reported Bi2GaVO7 [2SbVO7 [2 production. Previous works indicated that the Ga3+ and In3+ ions could influence the band gap and the electronic structure of the compound photocatalysts, which was expected to cause the different photocatalytic activity [Photocatalysis had gained great development since photocatalytic reaction was found in 1972 . Photocan doping ,35,36, fn doping ,40,41,42n doping ,46,47,48n doping ,50 foundY or Fe) ,52,53 whBi2GaVO7  and Bi2S [2SbVO7  by elemeactivity ,57.3+ ions ≤ 0.15) for the photodegradation of MB. In addition, the acquired results showed that doping the ZnO nanostructures with 0.03 mol fraction of Sb3+ ions increased the reaction rate by about three times, indicating that the decreasing recombination of charge carriers could enhance the photocatalytic activity. Al-Hamdi et al. [2) nanoparticles with different Sb concentrations , which was prepared by a sol–gel method, could degrade 12%, 45%, 71% and 97% of phenol in the mineralization process under UV irradiation for 120 min, which showed higher photocatalytic activity than the undoped SnO2 catalyst. These previous reports have shown that moderate Sb doped on the photocatalysts could greatly enhance the photocatalytic activity.As an important element with higher electron drift velocity and mobility, antimony (Sb) has been extensively studied as a good dopant candidate for enhancing the electron transfer rate of semiconductors . Omidi ei et al.  reported2GaSbO7 and Bi2InSbO7, were synthetized by doping element Ga or In with a solid-state reaction method. Meanwhile, the structural properties of Bi2GaSbO7 and Bi2InSbO7 were also characterized and their photocatalytic activities were also examined in degrading MB solution compared with N-doped TiO2, which had achieved the visible light response.In this paper, new photocatalysts, Bi2GaSbO7 and Bi2InSbO7 samples were firstly synthesized by a solid-state reaction method. Firstly, for the sake of the synthesis of Bi2GaSbO7, Bi2O3, Ga2O3 and Sb2O5 with a purity of 99.99%  were obtained by an atomic ratio of 2:1:1 to serve as raw materials. All powders were dried at 200 °C for 4 h before synthesis. In order to synthesize Bi2GaSbO7, the precursors were fully mingled with each other, then pressed into small columns and put into an alumina crucible . Eventually, calcination was performed at 1100 °C for 40 h in an electric furnace . Accordingly, Bi2O3, In2O3 and Sb2O5 with a purity of 99.99%  were obtained by an atomic ratio of 2:1:1 for the preparation of Bi2InSbO7. The synthesization procedure of Bi2InSbO7 was similar to that of Bi2GaSbO7, just the calcination was performed at 1070 °C for 30 h during mixed powder in the alumina crucible. The preparation of N-doped TiO2 was by the sol–gel method which was mentioned in our previous studies [New Bi studies .λ = 1.54056 angstrom) to confirm the crystal structures of Bi2GaSbO7 and Bi2InSbO7. The patterns of Bi2GaSbO7 and Bi2InSbO7 were recorded at 295 K with a step–scan procedure in the range of 2θ = 10°–100° (for Bi2GaSbO7) or 10°–95° (for Bi2InSbO7). The step interval was 0.02° and the time per step was 1 s. The transmission electron microscopy  was used to observe the surface state and structure of the photocatalysts. The Malvern’s mastersize-2000 particle size analyzer  was utilized to measure the particle size of the photocatalysts. We also utilized X-ray photoelectron spectroscopy  to determine the Bi3+ content, Ga3+ content, Sb5+ content, In3+ content and O2− content of Bi2GaSbO7 and Bi2InSbO7. The chemical composition of Bi2GaSbO7 and Bi2InSbO7 was determined by scanning the electron microscope-X-ray energy dispersion spectrum . The surface areas of Bi2GaSbO7 and Bi2InSbO7 were measured by the Brunauere–Emmette–Teller (BET) method  with N2 adsorption at liquid nitrogen temperature. Their diffuse reflectance spectrums were analyzed by a UV-visible spectrophotometer .In our paper, we adopted the X-ray diffraction method  with Cu Ka radiation (16H18ClN3S)  served as our objective pollutant. The whole photocatalytic activity process was as follows: firstly, we prepared 300 mL MB aqueous solution in quartz tubes whose initial concentration was 0.025 mmol·L−1 and initial PH value was 7.0. Then, 0.8 g photocatalyst powder of N-doped TiO2, Bi2GaSbO7 or Bi2InSbO7 was placed into every quartz tube, respectively. In order to ensure the establishment of an adsorption/desorption equilibrium among photocatalysts, the MB dye and atmospheric oxygen, above per solution was magnetically stirred in the dark for 45 min. In our paper, we employed a 500 W Xenon lamp (λ > 420 nm), which utilized a 420 nm cutoff filter as a visible-light source. The photoreaction was carried out in a photochemical reaction apparatus . During visible light illumination, the MB dye pollution was stirred by a magnetic stirrer and the photocatalyst powder was kept suspended in the solution. The filtrate was subsequently measured by a Shimadzu UV-2450 UV-visible spectrometer  with the detecting wavelength at 665 nm. The identification of MB and the degradation intermediate products of MB were measured by a liquid chromatograph-mass spectrometer . Here, post-photocatalysis solution (20 μL) was injected automatically into the LC–MS system. The eluent contained 60% methanol and 40% water, and the flow rate was 0.2 mL·min−1. MS conditions included an electrospray ionization interface, a capillary temperature of 27 °C with a voltage of 19.00 V, a spray voltage of 5000 V and a constant sheath gas flow rate. The spectrum was acquired in the negative ion scan mode, sweeping the m/z range from 50 to 600. Evolution of CO2 was analyzed with an intersmat™ IGC120-MB gas chromatograph  equipped with a porapack Q column (3 m in length and with an inner diameter of 0.25 in.), which was connected to a catharometer detector. The total organic carbon (TOC) concentration was determined with a TOC analyzer . The photonic efficiency was calculated according to the following equation [ξ was the photonic efficiency (%), and R was the rate of MB degradation (mol·L−1·s−1), and I0 was the incident photon flux (Einstein·L−1·s−1). The incident photon flux I0 which was measured by a radiometer  was determined to be 4.76 × 106 Einstein·L−1·s−1 under visible light irradiation (wavelength range of 400–700 nm).MB ξ2GaSbO7 and Bi2InSbO7 with high magnification. We could observe from the images of Bi2GaSbO7 and Bi2InSbO7 that their particles presented a similar oblate spheroid appearance and that their distribution was relatively uniform. The average particle size of Bi2GaSbO7 approached 190 nm, which was smaller than that of Bi2InSbO7, whose average particle size approached 390 nm. We could observe from the BET results that the specific surface area of Bi2GaSbO7 approached 2.36 m2/g, which was bigger than that of Bi2InSbO7, whose specific surface area approached 1.82 m2/g. It was clear that the BET results were consistent with the TEM results, indicating that the samples with small average particle size would have a higher specific surface area. 2GaSbO7 and Bi2InSbO7. It could be seen from 2GaSbO7 and Bi2InSbO7, meaning that Bi2GaSbO7 and Bi2InSbO7 crystals were both pure phase without impure elements.2GaSbO7 and Bi2InSbO7. The various elemental peaks which are corresponding to specific binding energies are given in 2GaSbO7 sample contained Bi, Ga, Sb and O elements. Similarly, the prepared Bi2InSbO7 sample contained Bi, In, Sb and O elements. These results also uncovered that Bi2GaSbO7 crystal or Bi2InSbO7 crystal were both at a high pure phase. Moreover, the analysis results of the XPS spectra also manifested that the valence of Bi, Ga, Sb, In or O from Bi2GaSbO7 and Bi2InSbO7 was +3, +3, +5, +3 or −2, respectively. Eventually, according to our comprehensive XPS and SEM–EDS analyses, as for Bi2GaSbO7, the mean atomic ratio of Bi, Ga, Sb and O was 2.00:0.98:1.02:6.98. As for Bi2InSbO7, the mean atomic ratio of Bi, In, Sb and O was 2.00:0.99:1.01:6.99.In this paper, X-ray photoelectron spectroscopy analysis techniques were utilized to reveal the surface chemical compositions and the valence states of various elements in Bi2GaSbO7 and Bi2InSbO7, respectively. We could judge from 2GaSbO7 crystal or Bi2InSbO7 crystal was single phase. 2GaSbO7 and Bi2InSbO7. The refined outcomes from 2GaSbO7 or Bi2InSbO7 were both highly in accordance with the intensities of the pyrochlore-type structure with a cubic crystal system and a space group Fd3m (O atoms were included in the model), indicating that Bi2GaSbO7 and Bi2InSbO7 indeed formed the same crystal structure. The atomic coordinates and structural parameters of Bi2GaSbO7 and Bi2InSbO7 are listed in a of Bi2GaSbO7 was 10.356497 Å, which was slightly lower than that of Bi2InSbO7 whose lattice parameter a was 10.666031 Å. From the SEM–EDS spectra and XPS spectra which were taken from Bi2GaSbO7 and Bi2InSbO7, we had known that Bi2GaSbO7 crystal or Bi2InSbO7 crystal was both pure phase. Therefore, excluding the effects of impurities, we could deduce that the difference between the lattice parameter a for Bi2GaSbO7 and Bi2InSbO7 was perhaps concerned with M ionic radii which belonged to Bi2MSbO7. The reason was that the ionic radii of Ga3+ (0.62 Å) was minutely lower than that of In3+ (0.92 Å). Lastly, all the diffraction peaks (222), (400), (440), (622), (444), (800), (662), (840), (844) for Bi2GaSbO7 and Bi2InSbO7 were successfully indexed according to the lattice constant and above space group.2GaSbO7 and Bi2InSbO7, respectively. Compared with N-doped TiO2 whose absorption edge was about 445 nm, the absorption spectrum of newly prepared photocatalyst Bi2GaSbO7 or Bi2InSbO7 was estimated to be 480 nm or 490 nm, respectively, implicating that they had sizable potential to realize visible light response. The maximum absorption wavelength of MB was detected by an ultraviolet spectrophotometer, while the diffuse reflection spectra of Bi2GaSbO7, Bi2InSbO7 or N-doped TiO2 was detected by ultraviolet spectrophotometer with integrating sphere. In addition, the above two testing methods were totally different. Furthermore, the absorbance was obtained from the reflectance data and scattering should also be taken into consideration in data conversion from reflectance into absorbance, which was the reason why the ordinate of the diffuse reflection spectra in We realized that absorbance could not be proportional to 1-transmission, thus the absorbance was calculated using the Kubelka–Munk transformation method in our experiment. For a crystalline semiconductor compound, the optical absorption near the band edge followed the equation ,65: α, Eg and ν denoted proportional constant, absorption coefficient, band gap and light frequency, respectively. In this equation, n determined the character of the transition in a semiconductor compound. Eg and n could be calculated by the following steps: (i) plotting ln(αhν) versus ln(hν − Eg) assuming an approximate value of Eg; (ii) deducing the value of n according to the slope in this graph; (iii) refining the value of Eg by plotting (αhν)n1/ versus hν and extrapolating the plot to (αhν)n1/ = 0. According to this method, we first estimated that the value of n for Bi2GaSbO7 or Bi2InSbO7 was 2, indicating that the optical transition for Bi2GaSbO7 or Bi2InSbO7 is indirectly allowed. αhν)n1/ versus hν for Bi2GaSbO7 and Bi2InSbO7. It could be found that the value of Eg for Bi2GaSbO7, Bi2InSbO7 or N-doped TiO2 was calculated to be 2.59 eV, 2.54 eV or 2.78 eV.Here, A, 2GaSbO7 and Bi2InSbO7, we had analyzed that both of the novel photocatalysts sent a strong absorption signal in the visible light region. Therefore, we expected that they could have the potential to degrade organic pollutants under visible light irradiation. In order to evaluate their visible light photocatalytic degradation capabilities, we listed N-doped TiO2 as a referential photocatalyst. 2GaSbO7, Bi2InSbO7, N-doped TiO2 as well as in the absence of a photocatalyst under visible light irradiation (>420 nm). Consistent with our expectations, as time went by, the color of the MB solution gradually shallowed and the concentration of MB gradually declined in our measurements in the absence of a photocatalyst. After visible light irradiation for 400 min, the removal rate of MB was estimated to be 99.75%, 98.95%, 59.92% or 40.6% with Bi2GaSbO7, Bi2InSbO7, N-doped TiO2 as catalyst, as well as in the absence of a photocatalyst, respectively. The sharp decrease in the concentration of MB under visible light irradiation from 0 to 120 min was mainly due to the adsorption of MB on the surface of Bi2GaSbO7, Bi2InSbO7 or N-doped TiO2 as a photocatalyst [2GaSbO7, Bi2InSbO7 or N-doped TiO2 as a catalyst also played a significant role compared with the absence of a photocatalyst under visible light irradiation in this sharp decrease. In addition, the slower speed of MB degradation by using Bi2GaSbO7, Bi2InSbO7 or N-doped TiO2 as a photocatalyst during the later reaction process could be the result of as-prepared samples surface blocking by adsorbed MB degradation byproducts [−9 mol·L−1·s−1, 1.031 × 10−9 mol·L−1·s−1 or 0.624 × 10−9 mol·L−1·s−1 with Bi2GaSbO7, Bi2InSbO7 or N-doped TiO2 as a catalyst during 400 min of visible light irradiation, respectively. The self-degradation rate of MB was 0.422 × 10−9 mol·L−1·s−1 without a catalyst. Furthermore, the photonic efficiency was estimated to be 0.0218% (λ = 420 nm), 0.0217% (λ = 420 nm) or 0.0131% (λ = 420 nm) with Bi2GaSbO7, Bi2InSbO7 or N-doped TiO2 as a catalyst, indicating that the sufficient use of a large number of photons could lead to the production of a large number of electron/hole pairs which were responsible for the photocatalytic degradation reaction directly and/or indirectly [2GaSbO7 and Bi2InSbO7 harvested the highest photocatalytic degradation rate and photonic efficiency compared with N-doped TiO2 for degrading MB. The decolored MB solution and the decrease of MB concentration reflected from 2 during MB degradation. From the UV-vis spectra of Bicatalyst . In the products . Moreovedirectly . Accordi2GaSbO7 as a photocatalyst. Noticeably, we could observe a subtle blue shift in the maximum absorbance of MB in the spectral changes by using Bi2GaSbO7 as a photocatalyst under visible light irradiation, indicating the rather facile cleavage of the whole conjugated chromophore structure [2GaSbO7.tructure . This bl2GaSbO7, Bi2InSbO7 or N-doped TiO2 as a photocatalyst, which is consistent with the tendency shown in 2GaSbO7, Bi2InSbO7 or N-doped TiO2 was exposed under visible light irradiation and the removal rate of TOC was 98.23%, 96.42% or 58.08% with Bi2GaSbO7, Bi2InSbO7 or N-doped TiO2 as a catalyst after visible light irradiation for 400 min. In addition, the reactions stopped when the light was turned off in this experiment, which showed the obvious light response, suggesting that MB had been converted to other kinds of byproducts and the organic carbon in the MB had not been decomposed to CO2 [d to CO2 .2 produced during the photocatalytic degradation of MB by using Bi2GaSbO7, Bi2InSbO7 or N-doped TiO2 as a photocatalyst under visible light irradiation. It could be distinctly seen from 2 gradually augmented along the light irradiation time and increased less during the last 100 min when much TOC was eliminated according to the results of 2 production of 0.11711 mmol or 0.11512 mmol with Bi2GaSbO7 or Bi2InSbO7 as a catalyst was higher than that of 0.06875 mmol with N-doped TiO2 as a catalyst. In addition, the amount of CO2 production was nearly equivalent to that of the removed TOC; at the same time, the amount of CO2 production or the removed TOC was slightly lower than the amount of reduced MB by using different catalysts with respect to the C element equilibrium, which indicated that MB was mainly degraded into some inorganic products including CO2 and eventually H2O.2GaSbO7, Bi2InSbO7 or N-doped TiO2 as a catalyst, which exhibits a linear correlation between ln(C/C0) or ln(TOC/TOC0) and the irradiation time for the photocatalytic degradation of MB under visible light irradiation by using the aforementioned catalysts. The pseudo-first-order kinetic curves of MB photodegradation were plotted to quantitatively compare the degradation rate of MB [C and TOC represented the MB concentration and the total organic carbon concentration at time t, respectively. Likewise, C0 and TOC0 represented the initial concentration of MB and the initial total organic carbon concentration, respectively. By a linear fit for the relationship between ln(C/C0) and the irradiation time, the first-order rate constant kC was estimated to be 0.01470 min−1 with Bi2GaSbO7 as a catalyst, 0.00967 min−1 with Bi2InSbO7 as a catalyst or 0.00259 min−1 with N-doped TiO2 as a catalyst, which distinctly showed that Bi2GaSbO7 and Bi2InSbO7, with the highest and the second highest value of kC, respectively, exhibited more excellent visible light photocatalytic activities for degrading MB compared with N-doped TiO2. Similarly, by a linear fit for the relationship between ln(TOC/TOC0) and the irradiation time, the first-order rate constant kTOC was estimated to be 0.00881 min−1 with Bi2GaSbO7 as a catalyst, 0.00745 min−1 with Bi2InSbO7 as a catalyst or 0.00239 min−1 with N-doped TiO2 as a catalyst. The difference between kC and kTOC reflected that there might be some photodegradation intermediate products of MB which were produced during the photocatalytic degradation of MB under visible light irradiation.te of MB . In the 2GaSbO7, Bi2InSbO7 or N-doped TiO2 as a photocatalyst with respect to time. It could be seen from 2GaSbO7 or Bi2InSbO7 as a photocatalyst in comparison with the N-doped TiO2. The photocatalytic degradation efficiency of phenol by using Bi2GaSbO7, Bi2InSbO7 or N-doped TiO2 as a photocatalyst under visible light irradiation after 400 min was estimated to be 75.00%, 69.76% or 47.08%, respectively, indicating that Bi2GaSbO7 or Bi2InSbO7 itself had photocatalytic activity and that the photodegradation process of MB by using Bi2GaSbO7 or Bi2InSbO7 as a photocatalyst was not mainly due to the photosensitive effect [2GaSbO7 or Bi2InSbO7 was much higher than that in the presence of N-doped TiO2, meaning that the visible-light photocatalytic activity of Bi2GaSbO7 or Bi2InSbO7 was higher than that of N-doped TiO2.e effect . Moreove2GaSbO7 or Bi2InSbO7 was measured to be 2.36 m2·g−1 or 1.82 m2·g−1, which was much smaller than that of N-doped TiO2, whose specific surface area was 45.53 m2·g−1. Generally speaking, a larger specific surface area would facilitate higher photocatalytic activities at the same experimental condition [2GaSbO7 and Bi2InSbO7 showed higher activities than N-doped TiO2 for degrading MB during visible light irradiation, which sufficiently highlighted the excellent photocatalytic properties of Bi2GaSbO7 and Bi2InSbO7, and the above results might ascribe to two explanations. Firstly, as already mentioned, the calculated band gap for Bi2GaSbO7, Bi2InSbO7 or N-doped TiO2 was 2.59 eV, 2.54 eV or 2.78 eV. Apparently, Bi2GaSbO7 or Bi2InSbO7 possessed a narrower band gap than N-doped TiO2, meaning that Bi2GaSbO7 or Bi2InSbO7 could utilize more visible light energy than N-doped TiO2 [2GaSbO7 and Bi2InSbO7, we could find that Bi2GaSbO7 and Bi2InSbO7 were both obtained with high crystallization perfection, which might more efficiently inhibit the recombination of photoinduced electrons and holes than N-doped TiO2.The specific surface area of Biondition ,75. Howeped TiO2 ,77. Seco2GaSbO7 were slightly higher than that of Bi2InSbO7. There were perhaps two probable reasons to explain it. As we all know, the greater mobility of the photoinduced electrons and holes indicated the greater chance that the photoinduced electrons and holes would reach the reactive sites of the catalyst surface, which would bring higher photocatalytic activities. As we previously mentioned, the lattice parameter α = 10.356497 Å for Bi2GaSbO7 was lower than the lattice parameter α = 10.666031 Å for Bi2InSbO7. Generally speaking, the smaller the ionic radius was, the smaller the size of the particles could be; and the lower the lattice parameter was, the larger the specific surface area could be, which could increase more reactive sites on the photocatalyst surface and absorb more reactive species to improve the photocatalytic activities [2GaSbO7, the Ga–O–Ga bond angle was 131.302°; accordingly, for Bi2InSbO7, the In–O–In bond angle was 128.640°. Obviously, the bond angle of the Ga–O–Ga bond angle of Bi2GaSbO7 was larger than the bond angle of Bi2InSbO7, which induced that Bi2GaSbO7 exhibited higher photocatalytic activity than Bi2InSbO7.Meanwhile, the photocatalytic degradation rate and photonic efficiency of Bitivities . In additivities . As a reN,N-dimethyl-p-phenylenediamine, benzenesulfonic acid, phenol and aniline. There generated holes h+, ·O2− and ·OH radicals, as oxidative agents in the photocatalytic reactions. According to previous studies [+ = C functional group bonds to open the central aromatic ring which contained both heteroatoms S and N. Therefore, according to previous studies and our test results, a possible photocatalytic degradation pathway for MB was proposed. 2GaSbO7 or Bi2InSbO7 as a catalyst. The MB molecule was converted to small organic species, which were subsequently mineralized into inorganic products such as SO42− ions, NO3− ions, CO2 and ultimately water.The photodegradation intermediate products of MB in our experiment were identified as azure A, azure C, thionine, phenothiazine, leucomethylene blue,  studies ,81, the  studies  which po2GaSbO7 or Bi2InSbO7 as a catalyst under visible light irradiation. A clear photonic efficiency  at wavelengths which corresponded to sub-Eg energies of the photocatalysts (λ from 480 to 700 nm for Bi2GaSbO7 and λ from 490 to 700 nm for Bi2InSbO7) was observed. The existence of photonic efficiency at this region revealed that the photons were not absorbed by the photocatalysts. Enlightened by the correlation between the low-energy action spectrum and the absorption spectrum of MB, we speculated that any photodegradation which results at wavelengths above 480 nm, should be attributed to photosensitization effect by the dye MB itself  and the valence band (VB) were studied by calculating the electronic band structure of Bi2GaSbO7 or Bi2InSbO7 with the plane-wave-based density functional method. The band structure calculations of Bi2GaSbO7 and Bi2InSbO7 were carried out with the program of Cambridge serial total energy package (CASTEP) and first-principles simulation. It could be seen from 2GaSbO7 was composed of Ga 4p and Sb 5p orbital component, meanwhile, the valence band of Bi2GaSbO7 was composed of a small dominant O 2p and Bi 6s orbital component. Similarly, the conduction band of Bi2InSbO7 was composed of In 5p and Sb 5p orbital component. In addition, the valence band of Bi2InSbO7 was composed of a small dominant O 2p and Bi 6s orbital component. Direct absorption of photons by Bi2GaSbO7 or Bi2InSbO7 could produce electron–hole pairs within the catalyst, indicating that the larger energy than the band gap of Bi2GaSbO7 or Bi2InSbO7 was necessary for decomposing MB by the photocatalysis method. 2GaSbO7 and Bi2InSbO7 were firstly prepared by the solid-state reaction method. The structural properties and optical absorption properties of Bi2GaSbO7 and Bi2InSbO7 were characterized by some material characterization methods, the photocatalytic properties of Bi2GaSbO7 and Bi2InSbO7 were also verified in comparison with N-doped TiO2. XRD results indicated that Bi2GaSbO7 and Bi2InSbO7 crystallized with the pyrochlore-type structure, cubic crystal system and space group Fd3m. The lattice parameter a for Bi2GaSbO7 or Bi2InSbO7 was a = 10.356497 Å or a = 10.666031 Å. According to the results from the UV-vis absorption spectra of Bi2GaSbO7 and Bi2InSbO7, the band gap of Bi2GaSbO7 or Bi2InSbO7 was estimated to be about 2.59 eV or 2.54 eV, indicating that Bi2GaSbO7 and Bi2InSbO7 showed a strong optical absorption in the visible light region (λ > 420 nm). Photocatalytic degradation of aqueous MB was realized under visible light irradiation in the presence of Bi2GaSbO7 or Bi2InSbO7 accompanied with the formation of final products such as CO2 and water. The complete removal of organic carbon from MB was obtained as indicated from TOC and CO2 yield measurements with Bi2GaSbO7 or Bi2InSbO7 as a catalyst under visible light irradiation. Compared with N-doped TiO2, Bi2GaSbO7 and Bi2InSbO7 exhibited higher photocatalytic activities for MB degradation under visible light irradiation. Consequently, according to the above analyses, Bi2GaSbO7 and Bi2InSbO7 both had great potential to degrade MB in textile industry wastewater. In addition, Bi2GaSbO7 exhibited slightly higher photocatalytic activities for the degradation of MB than Bi2InSbO7.New photocatalysts Bi


To determine potential mitochondrial and oxidative alterations in colon biopsies from idiopathic REM sleep behavior disorder (iRBD) and Parkinson's disease (PD) subjects. Colonic biopsies from 7 iRBD subjects, 9 subjects with clinically diagnosed PD, and 9 healthy controls were homogenized in 5% w/v mannitol. Citrate synthase (CS) and complex I (CI) were analyzed spectrophotometrically. Oxidative damage was assessed either by lipid peroxidation, through malondialdehyde and hydroxyalkenal content by spectrophotometry, or through antioxidant enzyme levels of superoxide dismutase-2 (SOD2), glutathione peroxidase-1 (Gpx1), and catalase (CAT) by western blot. The presence of mitochondrial DNA (mtDNA) deletions was assessed by long PCR and electrophoresis.p: NS). Lipid peroxidation was maintained among groups . Antioxidant enzymes SOD2  and Gpx1  did not show significant differences between groups. CAT was only detected in 2 controls and 1 iRBD subject. One iRBD patient presented a single mtDNA deletion. Nonsignificant trends to CI decrease in both iRBD  and PD patients  were found compared to controls (59.51 ± 12.52,  Lewy type pathology also emerges in the peripheral autonomous nervous system (PANS)  an internal reference pork muscle homogenate at 2 mg/ml was used. This is the gold standard material to confirm the validation of such spectrophotometric measurements, to better characterize the optimal velocity while maintaining linear kinetics relative to time and enzyme concentration.Mitochondrial content was measured spectrophotometrically in cuvettes through CS enzymatic activity involved in the Krebs cycle, considered a reliable marker of the mitochondrial mass . This asThe CI enzymatic activity of MRC was measured spectrophotometrically in cuvettes following the quantification of NADH substrate oxidation and using the specific inhibitor rotenone at 340 nm and at 37°C, as reported elsewhere .Either protein content or CS or CI enzymatic assays were performed by standardized procedures following protocols from the national Spanish Network CIBERER (unpublished protocols).The oxidative damage was determined by the quantification of lipid peroxidation derived products: malondialdehyde and 4-hydroxyalkenals, using the assay kit OxisResearch LPO586 at 586 nm, following manufacturer instructions .µM: GE Healthcare). Membranes were blocked using 5% powdered skim milk-PBS solution for 1 hour (RT) and then incubated with primary antibody in 1 : 1 blocking buffer: PBS 0.4% Tween 20 solution. SOD2 (SIGMA), CAT (AbCam), and Gpx1  were used for immunostaining by 1-hour incubation (RT), followed by 1 : 2000 horseradish peroxidase- (HRP-) conjugated secondary antibodies (Biorad) for 1 hour in 1 : 1 blocking buffer (PBS 0.4% Tween 20 solution). Enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific) was used to detect immunoreactive proteins on X-ray film further scanned to quantify band intensity.SOD2, Gpx1, and CAT antioxidant enzyme protein levels were assessed by western blot as follows. Sample DTT-reduced homogenates were loaded equally using SDS-PAGE  and transferred by wet transfer onto PVDF membranes  whereas the difference was not significant when compared to control subjects . As expected, UPDRS showed a more severe involvement in PD compared to iRBD and control subjects. iRBD and control subjects with a score higher than 0 at motor evaluation (UPDRS III) were known to suffer from nonneurological movement impairment, mainly due to arthrosis. PD patients were more likely to suffer from gastrointestinal symptoms than iRBD .Demographic and clinical data of the participants are summarized in https://doi.org/10.1155/2017/9816095).Cryopreserved homogenates were used for experimental procedures. The presence of neuronal axons was proved in the tissue explants by fluorescence confocal microscopy .No significant differential trends were observed in some of the parameters among groups. Interestingly, mitochondrial CI of MRC was slightly decreased in iRBD and PD patients compared to controls. This trend to decrease was more evident in clinically diagnosed PD patients . OxidatiNo changes in antioxidant enzyme levels of SOD2 and Gpx1 were found between groups Figures , resp.. MtDNA deletions were absent, although the presence of a unique mtDNA deletion in a iRBD subject was remarkable, as shown in Studies on the identification of surrogate in vivo peripheral prognostic markers for PD so far are relevant as they may allow minimally invasive diagnostic procedures and provide a window to early preclinical diagnosis and putative neuroprotective therapies during the prodromal phase of PD . ConstipDespite the wide amount of data on mitochondrial dysfunction associated with PD, our study, reported here, in colonic biopsies of prodromal and clinically diagnosed PD patients did not detect conclusive mitochondrial alterations.In the colonic tissue of our study subjects mitochondrial mass, estimated by CS enzymatic activity, remained similar in all the groups, suggesting that no relevant changes in mitochondrial biogenesis of the samples occur.Although CI MRC did not present any significant changes among groups, a trend of decrease of this activity was observable in both prodromal and clinically diagnosed PD groups with respect to controls. These CI enzymatic activity levels were slightly lower in patients with manifest PD. On the other hand, oxidative damage measured by lipid peroxidation tended to not significantly increase in enteric tissue from patients but not in iRBD subjects compared to controls. These tendencies may suggest that decrease of CI enzymatic activity could occur prior to oxidative damage in PD. It is widely known that a MRC dysfunction, especially at CI level, may lead to oxidative damage due to electron leaks in the MRC and the consequent formation of ROS that promotes oxidative stress. In the present study the slight trends in CI enzymatic activity changes are observed in both iRBD and PD patients, suggesting that oxidative damage changes over time could be secondary to CI dysfunction. It is of note that previous studies in PD have pointed out to the presence of oxidative damage in the colonic submucosa either in physiological and pathological conditions .Antioxidant enzyme protein levels did not show significant changes between mitochondrial SOD2 and Gpx1 enzymatic levels between groups and CAT levels were not detectable in most cases, leaving an opened question about whether this was due to an art factual issue or due to a potential relevant molecular meaning relaying on such differential data. Although our findings point out to a lack of an association between endogenous antioxidant changes in PD peripheral tissue, a recent study has described specific plasmatic changes in antioxidant levels and lipid peroxidation as potential promising PD biomarkers . substantia nigra of PD patients or in in vitro models of PD, but they have not been detected in other areas of the brain and it is uncommon to find mtDNA deletions in peripheral tissues. Given the use of colonic biopsies in the present study, it is not surprising that most of the patients did not show any mtDNA deletion and, indeed, the presence of a unique mtDNA deletion of a iRBD subject is remarkable.MtDNA deletions have been found in theα-synuclein pathology , and the depth of the biopsy, which is critical for an appropriate assessment of submucosal autonomic plexus. Unfortunately, due to these possible limitations, any adequate correlation analysis between mitochondrial function and abnormal α-synuclein aggregation could be assessed.In the present study, which included part of the cohort of a previous work , only onIn summary, our findings in enteric samples do not demonstrate clear mitochondrial abnormalities in colon biopsies of iRBD or established PD patients. Such lack of mitochondrial dysfunction could be due to the type of tissue studied. Cryopreserved homogenates from colonic biopsies were used in the experimental procedures. In preliminary studies, only a few cells could be cultured and could have undergone cell death before reaching optimal conference. Other authors were successful in obtaining neuronal cultures from the enteric system by using the entire myenteric plexus, in human fetal tissue  or in moOur gastrointestinal biopsies contain epithelial cells, enteric glial cells, and enteric neurons. It is important to note that, aside from neurons, the enteric glial cells and epithelial cells, which are present in a gastrointestinal biopsy, are also dysregulated in PD . We haveDespite the negative results obtained, the trends observed towards suboptimal CI enzymatic activity in both groups of patients (iRBD and PD) point to the necessity of further research in mitochondrial studies in PANS. Such studies should address the role of mitochondrial disturbances as potential PD in vivo biomarkers in different sites and depths of enteric PD biopsies in larger cohorts of patients.The axonal presence in a colonic sample is shown in the image from confocal microscopy after immunohystochemistry (PGP.9.5 antibody).


Drosophila expressing the steroid-dependent GeneSwitch transcriptional activator under the control of the ubiquitous α-tubulin promoter was found to produce extensive pupal lethality, as well as a range of dysmorphic adult phenotypes, in the presence of high concentrations of the inducing drug RU486. Prominent among these was cleft thorax, seen previously in flies bearing mutant alleles of the nuclear receptor Ultraspiracle and many other mutants, as well as notched wings, leg malformations, and bristle abnormalities. Neither the α-tubulin-GeneSwitch driver nor the inducing drug on their own produced any of these effects. A second GeneSwitch driver, under the control of the daughterless promoter, which gave much lower and more tissue-restricted transgene expression, exhibited only mild bristle abnormalities in the presence of high levels of RU486. Coexpression of the alternative oxidase (AOX) from Ciona intestinalis produced a substantial shift in the developmental outcome toward a wild-type phenotype, which was dependent on the AOX expression level. Neither an enzymatically inactivated variant of AOX, nor GFP, or the alternative NADH dehydrogenase Ndi1 from yeast gave any such rescue. Users of the GeneSwitch system should be aware of the potential confounding effects of its application in developmental studies.Culture of  Drosophila in a graded fashion. GS comprises a modified form of the yeast transcriptional activator Gal4, which is covalently linked to the hormone-binding fragment of the progesterone receptor, rendering its transcriptional activity dependent on an exogenously supplied progesterone analog, RU486 or mifepristone (-tubulin-GS (tubGS) and actin5C-GS drivers confer ubiquitous, RU486-dependent transgene expression when crossed to lines bearing a UAS-governed transgene. Tissue-specific drivers such as the neuron-specific elav-GS enable transgene expression in just one tissue, but again at a level and timing that can be manipulated over a wide range. The use of this system is predicated on the assumption that the expression of GeneSwitch and exposure to RU486 do not themselves produce measurable effects on fly physiology and development, which is supported by controls in many studies.The GeneSwitch (GS) system is commonly used to activate transgenes in Drosophila, foreign transgenes coding for nonproton-motive alternative respiratory chain enzymes derived from simpler eukaryotes, such as the alternative oxidase (AOX) from Ciona intestinalis  of 100–200 μM  are exposed to RU486 concentrations in excess of those sufficient to produce maximal transgene expression. We detected a variety of developmental abnormalities dependent on driver expression and drug dose. Surprisingly, expression of AOX, but not other transgenes such as GFP or the yeast alternative NADH dehydrogenase Ndi1, mitigated these effects.1118w and DAHw (Dahomey) and the UAS-GFP (Stinger) line (insertion on chromosome 2) were obtained from stock centers. The tubGS driver line with insertion on chromosome 3 (daughterless-GS (daGS) line  mutUAS-AOX , and B20UAS-Ndi1] were as described previously (Wild-type (Oregon R), standard transgenic host strains mosome 3  was a kiCrosses were conducted in a minimum of three, usually four to five replicates, as described previously . Z projection images were generated using Carl Zeiss Zen 2012 software.Total protein was extracted from batches of 20 pupae crushed in homogenization buffer, and processed as described previously . Clefting also extended along the abdomen in some cases  and spatially using a UAS-GFP reporter . Expression of UAS-AOX driven by daGS was quantitatively much less than when driven by tubGS, even at high RU486 concentrations . Furthermore, unlike tubGS, which was able to drive expression ubiquitously in the developing larva, daGS produced transgene expression only in a minority of cells , including salivary glands, parts of the trachea, some epithelial cells, and segmentally reiterated cell clusters.In order to determine whether the induction of these developmental defects was a general property of GeneSwitch drivers, or a phenomenon specific to ncreased . HowevertubGS in the presence of RU486 was able to modify the developmental phenotypes resulting from the driver and drug alone . However, five copies of the tub-AOX transgene, when present simultaneously, did produce a rescue comparable with that of UAS-AOX. Coexpression of UAS-AOX with tubGS plus drug, in either of two backgrounds commonly used in transgenic studies (1118w and DAHw) also increased the proportion of pupae eclosing  expressing a catalytically inactive variant of AOX , produced only very subtle abnormalities in bristle organization.In this study, we identified a range of developmental abnormalities associated with the use of the tubGS or RU486 on their own, we can exclude the possibility that RU486 binds to or interferes with the activity of known nuclear receptors in Drosophila  and kayak , as well as in the JNK signaling pathway that links AP-1 activity to various upstream developmental signals, cause cleft thorax  results in pupal lethality and cleft thorax . Finally, wing disc-specific knockdown of Tap42, a key regulator of protein phosphatases, gives rise to cleft thorax and to wing abnormalities similar to some that we observed , primarily in segmentally reiterated clusters of cells that might represent larval sense organs  produced a less dramatic alleviation of the phenotypes studied than the corresponding high-expressors . Rescue was dependent on the enzymatic activity of AOX and was not seen with an inert reporter protein (GFP) or a different mitochondrially localized electron-transfer protein, yeast Ndi, which appeared to exacerbate some phenotypes. AOX maintains ATP production, redox homeostasis, and metabolic flux under physiological conditions where respiratory complexes III and IV are limiting due to overload, toxins, or genetic damage, and concomitantly limits mitochondrial ROS production consequent upon overreduction of the quinone pool  transcription factor, and were rescued by a gene from a distant phylum, the effects were systematic in both cases, indicating meaningful underlying biological processes. Thus, the extent of AOX rescue of pupal lethality, cleft thorax, and other dysmorphologies was dependent on the AOX expression level, since strains expressing only at a low level  have been brought into use for inducing broad expression, both in adults and larvae , the daGS driver is clearly not ubiquitous, despite the fact that the daughterless gene itself, as well as the “standard” daGAL4 drivers, do show widespread expression.We would recommend that future users of all GeneSwitch drivers should routinely include otherwise nontransgenic controls bearing the drivers, plus and minus drug, in all experiments. Based on our findings (


HTLV-1 appears transcriptionally latent in freshly isolated cells; however, the chronically active anti-HTLV-1 cytotoxic T cell response observed in infected individuals indicates frequent proviral expression in vivo. The kinetics and regulation of HTLV-1 proviral expression in vivo are poorly understood. By using hypoxia, small-molecule hypoxia mimics, and inhibitors of specific metabolic pathways, we show that physiologically relevant levels of hypoxia, as routinely encountered by circulating T cells in the lymphoid organs and bone marrow, significantly enhance HTLV-1 reactivation from latency. Furthermore, culturing naturally infected CD4+ T cells in glucose-free medium or chemical inhibition of glycolysis or the mitochondrial electron transport chain strongly suppresses HTLV-1 plus-strand transcription. We conclude that glucose metabolism and oxygen tension regulate HTLV-1 proviral latency and reactivation in vivo.The human retrovirus HTLV-1 causes a hematological malignancy or neuroinflammatory disease in ∼10% of infected individuals. HTLV-1 primarily infects CD4  •Physiological (1%) hypoxia enhances HTLV-1 plus-strand transcription•HTLV-1 transcription is hypoxia regulated but HIF independent•Inhibition of glycolysis or the mitochondrial ETC suppresses HTLV-1 transcription•Extracellular glucose concentration regulates HTLV-1 reactivation from latency The human leukemia virus HTLV-1 remains dormant most of the time in the infected person, but is intermittently reactivated by unknown mechanisms. Kulkarni et al. show that fluctuations in glucose metabolism and oxygen availability are two major factors that govern the reactivation of HTLV-1 from dormancy. It is a single-stranded positive-sense RNA virus that reverse transcribes its RNA genome and integrates the resulting double-stranded DNA copy of its genome into the host cellular chromatin (as a provirus), thereby establishing a persistent infection in the cells. It is estimated that 5–10 million people worldwide are HTLV-1 carriers. HTLV-1 infection is asymptomatic in most cases. In a subset of infected individuals (∼5%–10%), however, HTLV-1 infection progresses to either a CD4+ T cell malignancy known as adult T cell leukemia/lymphoma or to HTLV-1-associated myelopathy, a progressive inflammatory disease of the spinal cord.HTLV-1 is a human retrovirus that primarily infects CD4in vivo. This understanding might suggest strategies to reactivate the dormant virus and make it accessible to attack by the immune system and antiretroviral drugs.HTLV-1 infection is considered to be largely latent in infected individuals, because the viral structural RNA and protein products are usually undetectable in freshly isolated infected peripheral blood mononuclear cells (PBMCs) . HoweverPrevious work on HTLV-1 proviral latency and reactivation has largely focused on identifying the viral factors involved, such as the HTLV-1 integration site . Spontanin vivo. While pulmonary alveolar cells experience oxygen concentrations of ∼15%, embryonic, neural, mesenchymal, and hematopoietic stem cells are exposed to profoundly hypoxic environments (∼1% O2)  . Viruses(∼1% O2) . Circulain vitro selection and adaptation, we employed primary PBMCs isolated from HTLV-1-infected individuals to study the effects of various stresses and inhibitors on HTLV-1 reactivation from latency. Plus-strand HTLV-1 gene expression is typically undetectable in fresh PBMCs obtained from HTLV-1-infected individuals, but there is a strong spontaneous increase in the expression of Tax, the viral transactivator of plus-strand gene transcription, within a few hours of isolation (To minimize artifacts due to solation . An incrsolation . These otax (surrogate for HTLV-1 plus-strand transcription), HTLV-1 sHBZ (surrogate for minus-strand HTLV-1 transcription), and host cellular VEGF (hypoxia-inducible positive control). There was a significant increase in plus-strand transcription in PBMCs cultured under physiological hypoxia when compared with PBMCs cultured under ambient oxygen conditions  . However, lymphocytes spend most of their time in the highly hypoxic environment of the lymph circulation or solid lymphoid organs (1%–2% oxygen) . To studnditions A. No sucor (HIF) A.Figure Next, we tested whether the hypoxia-mediated induction of HTLV-1 plus-strand transcription is associated with a specific epigenetic signature in the provirus, by analyzing changes in histone methylation status. HTLV-1-infected frozen PBMCs were thawed and either fixed immediately or cultured overnight under either hypoxia (1% oxygen) or normoxia (20% oxygen), and fixed subsequently at 17 hr. Chromatin from the respective samples was fragmented by sonication and subjected to chromatin immunoprecipitation (ChIP), using antibodies directed against H3K4me3, which is associated with promoters and enhancers; H3K36me3, which is associated with actively transcribed gene bodies; and H3K27me3, a repressive epigenetic mark associated with heterochromatin.gag, pol, env, and vCTCF regions of the HTLV-1 provirus in cells cultured in 1% as well as 20% oxygen. By contrast, there was no significant change (within the limits of detection) in H3K4me3 at the 3′-LTR junction  junction, junction B. Similajunction C. Howeveed PBMCs C. Althoued PBMCs . Howevered PBMCs A, so was2). Under hypoxic conditions, PHD activities are inhibited and α,β-HIF is stabilized, thus enabling it to orchestrate the cellular response to hypoxia through its role as a transcriptional activator  in the 5′-LTR and the gag region of the provirus , in addition to being a co-substrate for 2-OG oxygenases, is also an important intermediate in metabolism, specifically the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and amino acid metabolism. To test the hypothesis that DMOG influences HTLV-1 transactivation through perturbation of these cellular metabolic pathways (in an HIF-independent manner), we treated HTLV-1-infected patients' PBMCs overnight with either 0.5 mM DMOG in DMSO, or DMSO alone (control). The cells were lysed and the extracts subjected to ion-exchange liquid chromatography coupled directly to tandem mass spectrometry (LC-MS/MS), to identify the metabolic pathways modulated by DMOG. Untargeted metabolite profiling measured 4,261 molecular species with a unique mass-to-charge ratio. The identified metabolites (n = 137) were sorted according to the maximum fold change in the normalized abundance between DMSO-treated and DMOG-treated samples, respectively. Any change that was statistically significant (p < 0.05) and exceeded 1.3-fold was analyzed further (raw data with identified metabolites are provided in (1)Glycolysis. A significant reduction was observed in the levels of most of the measured glycolytic intermediates in response to DMOG treatment. In particular, intermediates toward the end of glycolysis: 2,3-diphosphoglycerate and 3-phosphoglycerate were highly depleted in DMOG-treated cells . Glycera(2)cis-aconitate, isocitrate, 2-OG, and malate were significantly lower in DMOG-treated HTLV-1-infected PBMCs than in control (DMSO-treated) cells. Only succinate levels remained unchanged among the measured TCA-cycle metabolites in response to DMOG treatment. DMOG inhibits mitochondrial respiration independently of its hypoxia mimic effect Pentose phosphate pathway. Higher levels of glyceraldehyde-3-phosphate, α-D-glucose 6-phosphate, 6-phosphogluconate, ribulose 5-phosphate, and D-ribose in combination with low levels of 1-deoxy-D-xylulose 5-phosphate, deoxyribose 5-phosphate, and sedoheptulose 1,7-bisphosphate in DMOG-treated samples when compared with control pointed toward a downstream inhibition in the pentose phosphate pathway in response to DMOG treatment.(4)Redox metabolism. The pentose phosphate pathway is responsible for synthesis of NADPH, a reducing equivalent in cell metabolism , e.g., i(5)Purine and pyrimidine metabolism. While levels of purine metabolites adenosine and guanosine monophosphate (AMP and GMP) were significantly higher in DMOG-treated cells when compared with controls, the corresponding nucleoside triphosphates (ATP and GTP) were significantly depleted. All pyrimidine metabolites measured  were depleted in DMOG-treated cells when compared with control. dCTP, a precursor for DNA synthesis, was strongly inhibited by DMOG treatment.(6)Amino acid, carbohydrate, and lipid metabolism. DMOG treatment resulted in a significant increase in L-tryptophan levels. This might be expected given the strong concomitant reduction in tryptophan degradation products, quinolinic acid and 2-aminomuconic acid semialdehyde, observed in the DMOG-treated samples. Inhibition of glucose metabolism by DMOG might in turn inhibit many anabolic processes, because of the reduction in ATP and NADH. The observed significant decrease in the levels of N-acetylated metabolites could be due to the limited availability of acetyl-coenzyme A resulting from an inhibition of glycolysis and the TCA cycle.The results showed significant changes in six metabolic pathways:tax transcription in HTLV-1-infected primary PBMCs.Thus most of the observed metabolic effects of DMOG, discussed above, are secondary to its perturbation of glycolysis, the TCA cycle, and oxidative phosphorylation . Hence wTo investigate the influence of glucose metabolism on HTLV-1 transcription, we tested the effects of the TCA-cycle inhibitor sodium arsenite  and the tax mRNA transcription, whereas arsenite caused no change in tax mRNA levels at the concentration tested   or 0 mM glucose with 10% FBS in the presence or absence of the glycolysis inhibitor 2-deoxy-D-glucose (2-DG)  and the tax mRNA than did PBMCs cultured in RPMI with physiological (5.5 mM) glucose +10% FBS. Addition of the glycolysis inhibitor 2-DG to the 0-mM glucose medium led to a further significant suppression of HTLV-1 plus-strand transcription. Further addition of the TCA-cycle inducer sodium pyruvate (0 mM glucose + 2-DG + pyruvate) did not rescue the inhibitory effect of 2-DG on HTLV-1 tax transcription. These observations are consistent with the data shown in VEGF mRNA levels. Inhibition of glycolysis by 2-DG also resulted in a significant decrease in the mRNA of lactate dehydrogenase A, a surrogate marker of the rate of glycolysis (sHBZ transcription upon either inhibition of glycolysis (by incubation in 0 mM glucose or 2-DG) or TCA-cycle induction  when compared with control  on HTLV-1 plus-strand transcription. PBMCs from HTLV-1-infected individuals were incubated overnight in the presence or absence of inhibitors of mitochondrial ETC complex II  showed a significant enhancement in HTLV-1 plus-strand transcription when compared with those cultured under ambient oxygen conditions (∼20% oxygen) . HypoxiaIn contrast to the effect of hypoxia, the hypoxia mimic DMOG potently inhibited proviral plus-strand transcription in HTLV-1-infected primary PBMCs C. We theChemical inhibition of glycolysis by either iodoacetic acid or 2-DG significantly reduced plus-strand HTLV-1 transcription . HoweverVEGF and the glycolytic enzyme LDHA, suggesting that their effect on HTLV-1 transcription is independent of hypoxia and glycolysis. Ciminale and colleagues have shown that the p13 accessory protein of HTLV-1 modulates mitochondrial membrane potential by facilitating an inward K+ current, thereby increasing ETC activity and reactive oxygen species production, consistent with a link between mitochondrial function and the HTLV-1 life cycle , as present in the lymphoid organs and bone marrow, enhances HTLV-1 transcription. Second, inhibition of glycolysis or the mitochondrial electron transport chain suppresses plus-strand HTLV-1 transcription. We conclude that both glucose metabolism and oxygen availability regulate HTLV-1 transcription. The significance of these results is twofold. First, the identification of two microenvironmental factors that regulate HTLV-1 expression constitutes a basic advance in the understanding of HTLV-1 persistence and pathogenesis. Second, targeting these pathways with currently available as well as novel therapies could complement existing antiretrovirals and improve treatment efficiency.Retroviruses such as HTLV-1 persist life-long in their host by integrating a copy of their genetic material into the host cellular genomic DNA. Although HTLV-1 infection is asymptomatic in most cases, in a subset of infected individuals (∼10%) it causes an aggressive hematological malignancy or a debilitating neuroinflammatory condition. Infection is considered largely latent due to the absence of viral RNA and proteins in fresh blood samples. However, when blood obtained from HTLV-1-infected individuals is cultured c.bangham@imperial.ac.uk )Further information and requests for reagents may be directed to, and will be fulfilled by, the corresponding author Charles R.M. Bangham , using the manufacturer’s instructions. Equal volumes of RPMI with L-Glutamine without Glucose  and RPMI with L-Glutamine and 11mM Glucose (BE12-702F) were mixed to make RPMI with L-Glutamine and 5.5mM Glucose. This medium was used for cell culture with 10% FBS (Gibco) in all experiments, unless otherwise stated.In situ dissolved oxygen in the culture media was measured using the integrated OxyLite™ (Oxford Optronix) oxygen sensor to accurately monitor oxygen availability in the cellular microenvironment.A ‘Hypoxylab’  - hypoxia workstation and incubator was employed for all hypoxia-related experiments. All media and consumables used in hypoxic culture were conditioned to the target oxygen concentration (1% or 2%) before use. Primary PBMCs were manipulated and cultured in the HypoxyLab at the desired oxygen concentration for the indicated time. Multiple chemical inhibitors of different cellular pathways were employed in this study. A comprehensive list of all the compounds used along with the corresponding references is provided in RNA was extracted from cultured PBMCs using the RNeasy Plus Mini kit (Qiagen). cDNA was synthesised from the extracted RNA using the Transcriptor First Strand cDNA Synthesis kit (Roche) by following the manufacturer instructions. An additional no-RT control was included for each cDNA sample synthesised. The kinetic PCR amplification was carried out using the Viia7 Real-time PCR system (Applied Biosystems) with gene-specific primers and Fast SYBR Green Master Mix. The list of primers used is given in Gag, Pol, Env, vCTCF, Tax and 3’-LTR junction of the HTLV-1 provirus and corresponding qPCR TaqMan probes . Subsequently nuclei were pelleted by centrifugation at 2000rpm for 5 minutes and subjected to lysis with 130 μl Nuclear lysis buffer . Nuclear lysates were sonicated using the Covaris S220 sonicator in a microTUBE (Covaris), with the following Sonolab7.2 program parameters: Peak Incident Power: 105 Watts; Duty Factor: 10%; Cycles per burst: 200; Treatment time: 480 sec; Distilled Water Temperature: 4-6°C. Sonicated lysates were subjected to immunoprecipitation (IP) using the following antibodies: ChIPAb+Trimethyl-Histone H3 Lys 4  and ChIPAb+Trimethyl-Histone H3 Lys 36 , ChIPAb+ Trimethyl-Histone H3 Lys 27  and corresponding control IgG overnight at 4°C in the presence of MagnaChIP A+G magnetic beads (Millipore). A 10% IP input sample was collected separately as a reference for relative quantification. The bead-bound immunoprecipitated DNA was washed sequentially for 10 minutes each at 4°C with the following wash buffers- Low salt wash buffer , High salt wash buffer , LiCl wash buffer ; 1mM EDTA; 10mM Tris-HCl pH8.0) and 2 washes with TE buffer . The DNA was eluted from the beads by IP elution buffer . The eluted DNA was reverse cross-linked at 65°C overnight in the presence of 300mM NaCl and thereafter subjected to proteinase K digestion at 45°C for 2 hours. The immunoprecipitated and input DNAs were purified by using the QIAquick PCR Purification Kit (Qiagen). The DNA enrichment in ChIP samples was quantified using region-specific primers for 5’-LTR junction, Genomic DNA from PBMCs was extracted using a QIAamp DNA Mini kit (Qiagen), following the manufacturer’s instructions. The extracted DNA was sonicated using a Covaris S220 machine . MeDIP assays were carried out using the MethylCollector Ultra kit (Active Motif) according to the manufacturer’s protocol. Immunoprecipitated DNA was quantified by qPCR as described above for ChIP, using the same primers and probes.PBMCs isolated from HTLV-1 infected individuals were cultured overnight in the presence of 0.5mM DMOG or DMSO (control). Briefly, pelleted cells were lysed with ice-cold 80% methanol (MS grade). 0.2ml of each lysate was then filtered using a 10kd molecular weight cut-off filter . The liquid which had passed though the filter was then placed in an autosampler vial (Waters) and stored at -80°C. On the day of analysis the extract was allowed to warm to 4°C in the chilled autosampler and then analysed directly by LC/MS/MS.Metabolite analyses were performed using a Thermo Scientific ICS-5000+ ion chromatography system coupled directly to a Q-Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer with a HESI II electrospray ionisation source . The ICS-5000+ HPLC system incorporated an electrolytic anion generator (KOH) which was programmed to produce an OH- gradient from 5-100mM over 37 minutes. An inline electrolytic suppressor removed the OH- ions and cations from the post-column eluent prior to eluent delivery to the electrospray ion source of the MS system (Thermo Scientific Dionex AERS 500). A 10 μL partial loop injection was used for all analyses and the chromatographic separation was performed using a Thermo Scientific Dionex IonPac AS11-HC 2 × 250 mm, 4 μm particle size column with a Dionex Ionpac AG11-HC 4 μm 2x50 guard column inline. The IC flow rate was 0.250 mL/min. The total run time was 37 minutes and the hydroxide ion gradient comprised as follows: 0mins, 0mM; 1min, 0mM; 15mins, 60mM; 25mins, 100mM; 30mins, 100mM; 30.1mins, 0mM; 37mins, 0mM. Analysis was performed in negative ion mode using ascan range from 80-900 and resolution set to 70,000. The tune file source parameters were set as follows: Sheath gas flow 60; Aux gas flow 20; Spray voltage 3.6; Capillary temperature 320; S-lens RF value 70; Heater temperature 450. AGC target was set to 1e6 and the Max IT value was 250ms. The column temperature was kept at 30°C throughout the experiment. Full scan data were acquired in, continuum mode across the mass range m/z 60-900.Raw data was processed using Progenesis QI for small molecules . Briefly this encompassed chromatographic peak alignment, isotope cluster recognition (peak picking) and compound identification. Identification of compounds in experimental samples was based on matching to an in-house library of authentic standards, using four measured parameters for each compound from the database. These were: accurate mass measurement (<5ppm) based on theoretical mass derived from the chemical formula, experimental retention time window of 0.5mins, isotope pattern recognition  and matching with fragmentation patterns from an authentic standard where these were available from survey scans. All values in the database were obtained from the analysis of authentic standard compounds.Statistical analysis was performed using Progenesis QI and the EZ info plugin for Progenesis QI developed by Umertrics. Supervised and unsupervised modelling of the data was performed (PCA and OPLS-DA). Volcano plots, S-plots and VIP values were extracted from OPLS-DA models to help identify highly significant compounds and potential biomarkers. p-values, %CV and fold-changes associated with the statistical comparison of experimental groups were calculated for each metabolite using progenesis QI. These data were used to identify and evaluate potential metabolic biomarkers. The identified metabolites were sorted according to the maximum fold difference in normalized abundance between DMSO-treated and DMOG-treated samples respectively. Any difference in metabolite abundance, which was statistically significant (p<0.05), and fold change >1.3-fold, was analysed further. The resultant metabolite list was mapped according to the metabolic pathways they were associated with.Ct. All values were normalized to their respective 18S rRNA levels, which was the internal PCR control.LinRegPCR-Ct method  was usedΔCt)*10, where ΔCt= Ctinput- Ctsample. LinRegPCR software was used to determine the mean efficiency of the reaction for each primer pair.DNA enrichment was calculated as % Input = (ETwo-tailed Student’s T test, Wilcoxon matched pairs signed rank test and 1-way ANOVA with post-test for linear trend were employed for statistical analysis of data as described in the corresponding figure legends.The raw metabolomics data with the identified metabolites has been provided in Conceived and designed the experiments: A.K., C.C.T., C.J.S., and C.R.M.B.; Performed the experiments: A.K., M.M., and J.S.M.; Analyzed the data: A.K., J.S.M., C.J.S., and C.R.M.B.; Contributed reagents/materials/analysis tools: C.C.T., C.J.S., and J.S.M.; Writing – original draft: A.K. and C.R.M.B.; Writing – review and editing: A.K., C.R.M.B., C.J.S., J.S.M., and G.P.T.; Recruited patients: G.P.T.


Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a critical mediator of tumor necrosis factor-α (TNF-α) signaling. However, the regulatory mechanisms of TRAF2 are not fully understood. Here we show evidence that TRAF2 requires brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) to be recruited into TNF receptor 1 (TNFR1) signaling complexes. In BIG1 knockdown cells, TNF-α-induced c-Jun N-terminal kinase (JNK) activation was attenuated and the sensitivity to TNF-α-induced apoptosis was increased. Since these trends correlated well with those of TRAF2 deficient cells as previously demonstrated, we tested whether BIG1 functions as an upstream regulator of TRAF2 in TNFR1 signaling. As expected, we found that knockdown of BIG1 suppressed TNF-α-dependent ubiquitination of TRAF2 that is required for JNK activation, and impaired the recruitment of TRAF2 to the TNFR1 signaling complex (complex I). Moreover, we found that the recruitment of TRAF2 to the death-inducing signaling complex termed complex II was also impaired in BIG1 knockdown cells. These results suggest that BIG1 is a key component of the machinery that drives TRAF2 to the signaling complexes formed after TNFR1 activation. Thus, our data demonstrate a novel and unexpected function of BIG1 that regulates TNFR1 signaling by targeting TRAF2. Adenosine diphosphate (ADP)-ribosylation factor (ARF) is a small guanosine triphosphate (GTP)-binding protein that plays a key role in the regulation of membrane trafficking and the organization of the cytoskeleton ,2. The aTumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that induces a wide variety of cellular responses including cell growth, differentiation, immune responses, and cell death . Two difTumor necrosis factor receptor-associated factor 2 (TRAF2), belonging to the TRAF family of proteins, is a really interesting new gene (RING) finger protein that participates in TNF-α-induced cellular responses as a signaling component of both complex I and complex II ,21. UponCompelling evidence indicates that cellular responses mediated by TNFR1 are highly associated with the membrane trafficking of TNFR1 ,28,29. TIn order to examine whether BIG1 is involved in TNFR1 signaling, we performed BIG1 knockdown experiments using HT1080 fibrosarcoma or HeLa cells that predominantly express TNFR1 rather than TNFR2 . Both inp = 0.16 vs. control) in the cells knocked down by BIG1 #2 siRNA , cIAP1/2 and TRAF2/5, the activation of cell death pathways is strongly suppressed ,34. HoweWe next examined whether knockdown of BIG1 affects the sensitivity to TNF-α-induced apoptosis. When siRNA-transfected cells were treated with TNF-α, we found that knockdown of BIG1 significantly reduced cell viability to a similar extent as TRAF2 knockdown cells  were co-immunoprecipitated with TRAF2 peaked at 15 min after the treatment with TNF-α, whereas BIG1 was not evident in the co-immunoprecipitates of TRAF2 A. MoreovGiven that BIG1 regulates the TRAF2 recruitment to the complex I, TRAF2 may fail to assemble into the death-inducing complex (complex II) in BIG1 knockdown cells. To explore this possibility, we analyzed the complex II formation using caspase-8 antibody. To promote formation of the complex II, HT1080 cells were treated with TNF-α in combination with the protein-synthesis inhibitor cycloheximide (CHX) which blocks NF-κB-dependent induction of anti-apoptotic proteins that interfere with complex II formation. As shown in In the present study, we provide evidence that BIG1, one of the high-molecular-weight ARF-GEFs, participates in TNFR1 signaling through the mechanism governing TRAF2 recruitment to the TNFR1 signaling complexes. In BIG1 knockdown cells, TNF-α-induced JNK activation was attenuated whereas the degradation of IκBα was unaltered. Moreover, knockdown of BIG1 promoted TNF-α-induced caspase-8 and subsequent caspase-3 activation, leading to cell apoptosis. These signaling properties observed in BIG1 knockdown cells were similar to those in TRAF2 knockdown cells, which alluded to the possibility that BIG1 is an upstream regulator of TRAF2. Indeed, we found that BIG1 knockdown abrogated TNF-α-induced TRAF2 ubiquitination. Moreover, we also found that BIG1 knockdown impaired the recruitment of TRAF2 to both complex I and complex II. Collectively, these findings suggest that BIG1 upregulates the activities of TRAF2 by governing the TRAF2 recruitment to TNFR1 signaling complexes. On the other hand, BIG2 knockdown failed to promote TNF-α-induced apoptosis as shown in TNFR1 favors both survival and death signaling pathways. Its signaling diversity seems to be exquisitely determined by intracellular trafficking that controls the correct intracellular distribution of TNFR1. At the plasma membrane, TNFR1 forms complex I, which principally activates the survival signaling pathways. In turn, the complex I changes from a plasma membrane complex to the receptosome as a signaling vesicle after its internalization, which is essential for the late-phase process including apoptosis and necroptosis ,29,38. T2 atmosphere. FLAG-TNF-α was produced and purified as described [HT1080 and HeLa cells were grown in Dulbecco’s Modified Eagle Medium (DMEM), 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin-streptomycin solution, at 37 °C under a 5% COescribed ,40. TNF-Cells were lysed in lysis buffer ), containing one cOmplete Protease Inhibitor Cocktail tablet  per 50 mL. Cell extracts were resolved by SDS-PAGE, transferred to nitrocellulose or polyvinylidene difluoride (PVDF) membranes, blocked with phosphate buffered saline, 0.5% Tween 20, 5% powdered skim milk, probed with the indicated antibodies and revealed using the ECL reagent .NF-κB reporter assays were performed essentially as described . Cells wv/v) mixture of 0.9 mg/mL PMS (phenazine methosulfate) and 2 mg/mL MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium),  solution was added into each well. Following color development for 2–4 h, absorbance was measured at 492 nm.At 60 h post-transfection, siRNA-transfected cells were plated in 96-well plates at 20–30,000 cells per well in 100 μL of medium. Then, 12 h later, cells were treated with TNF-α in 100 μL of medium for about 16 h. Following this, 20 μL of a 1:20  for 15 min on ice. Lysates were immunoprecipitated with 20 μL anti-FLAG M2 magnetic beads for 4 h at 4 °C. Beads were recovered by the magnet and washed five times with 1 mL of lysis buffer before analysis by SDS-PAGE and immunoblotting. To analyse complex II, HT1080 cells were lysed in lysis buffer. After centrifugation, supernatants were precleared with 20 µL of Sepharose 6B  for 1 h at 4 °C, and then immunoprecipitated with caspase-8 antibody using protein G-Sepharose . Beads were extensively washed with lysis buffer before immunoblot analysis.In this study, we found that BIG1 knockdown cells exhibited similar properties to those of TRAF2 in TNFR1-mediated cellular responses. These data thus prompted us to examine whether BIG1 functions an upstream regulator of TRAF2 in TNFR1 signaling. Indeed, knockdown of BIG1 suppressed TNF-α-dependent ubiquitination of TRAF2, and impaired the recruitment of TRAF2 to the TNFR1 signaling complexes. These results suggest that BIG1 is required for the recruitment of TRAF2 to the signaling complexes formed after TNFR1 activation. Altogether, our data demonstrate a novel function of BIG1 that regulates TNFR1 signaling by targeting TRAF2.


Plants confront multifarious environmental stresses widely divided into abiotic and biotic stresses, of which heavy metal stress represents one of the most damaging abiotic stresses. Heavy metals cause toxicity by targeting crucial molecules and vital processes in the plant cell. One of the approaches by which heavy metals act in plants is by over production of reactive oxygen species (ROS) either directly or indirectly. Plants act against such overdose of metal in the environment by boosting the defense responses like metal chelation, sequestration into vacuole, regulation of metal intake by transporters, and intensification of antioxidative mechanisms. This response shown by plants is the result of intricate signaling networks functioning in the cell in order to transmit the extracellular stimuli into an intracellular response. The crucial signaling components involved are calcium signaling, hormone signaling, and mitogen activated protein kinase (MAPK) signaling that are discussed in this review. Apart from signaling components other regulators like microRNAs and transcription factors also have a major contribution in regulating heavy metal stress. This review demonstrates the key role of MAPKs in synchronously controlling the other signaling components and regulators in metal stress. Further, attempts have been made to focus on metal transporters and chelators that are regulated by MAPK signaling. III), arsenate (AsV), cadmium (Cd), chromium (Cr), lead (Pb), copper (Cu), mercury (Hg), aluminum (Al), etc. These metals have not only known to perturb animal kingdom but also plant kingdom. Their damaging impact on our agriculture has also been very well-documented  that bind to Ca2+ and trigger different downstream signaling pathways , arsenite , heavy metal transport ATPase (CPx- and P1B-ATPase), natural resistant associated macrophage protein (NRAMP), cation diffusion facilitator (CDF), and ATP-binding cassette (ABC) transporters, present at plasma membrane and on tonoplast membrane of cell  are the metal binding SOD and require Fe3+ metal ion as cofactor  as corroborated by different studies acts as a universal secondary messenger in the normal functioning of plants as well as in response to various environmental stresses  that bind to Ca2+ and trigger different downstream signaling pathways  by blocking the Ca2+ channels  of vacuolar and Golgi membrane in coping with Cd toxicity. This is achieved through cooperation with a Glutathione-conjugated transporter Ycf1p whose activity is controlled by phosphorylation once again insinuating an interface between different signaling pathways in response to environmental stresses   exhibit higher levels of tolerance to Ni toxicity. Contrastingly, the same plants were found to be hypersensitive to Pb2+, depicting an exclusion of Ni2+ and augmented accumulation of Pb2+ as compared to wild type plants , hydrogen sulfide was found to interact with Ca2+ signaling in imparting improved tolerance to Chromium (Cr VIIV)-mediated heavy metal stress. It has also been discerned that Ca2+ provides tolerance against Cr stress by enhancing the activity of antioxidant enzymes  dose and acid rain strength. The adverse effects on the roots caused by acid rain could be alleviated by low concentrations of LaIII and synergistic effects on the roots were observed upon combined exposures at higher concentrations of La(III) and acid rain  resulted in increased IAA concentration in Arabidopsis roots promoting lateral root growth, thus protecting roots from Cd  are N6-prenylated adenine derivatives involved in the regulation of plant growth and development and in biotic and abiotic stresses  is a gaseous plant hormone regulating various important growth aspects. It is biosynthesized by ACC synthase (ACCS) that convert AdoMet to ACC, while ACC oxidase (ACCO) catalyzes the conversion of ACC to ethylene. ACCS and ACCO are encoded by multigene families and regulated by many biotic and abiotic factors Kende, . SeveralERF1, ERF2, and ERF5  is being considered as a major limiting factor for plant development interfering with cellular redox equilibrium. Similar to other metals, Al stress also downregulates most of the miRNAs in rice such as miR156, miR395, miR398, miR159 except miR399, miR166, miR168 which showed upregulation , bZIP (basic region leucine ZIPper), AP2 (Activator Protein 2), ERF (ethylene-responsive factor), and DREB (dehydration responsive element-binding protein), have been identified as potential downstream targets of MAPKs  that regulates As-associated transporters genes. He found that OsARM1 binds to the conserved MYB binding sites in the promoters of OsLsi1, OsLsi2, and OsLsi6, which encode key As transporters and affects their expression. Several studies suggest MYB TFs to be the downstream targets of MAPKs. Most recently, Li et al.  plant has showed that it positively regulates heavy metal stress tolerance by improving root growth upon overexpression , ZAT12 expression modulated upon short-term exposure to Cu while no such influence was observed upon long-term Cd exposure , NRAMP, CDF, and ABC transporters  by MEK1 (MAPKK) Table . This isApart from the transporters activating MAPKs or vice versa, there are also studies suggesting the activation of MAPKs by metal chelators. The important metal chelators are PCs and metallothioneins and these are small cystein rich peptides with metal binding capacity (Singh S. et al., From this review it is implied that metal exerts tremendous effect on plant by modulating its functioning at various levels. Metal stress activates several signaling pathways, known to have important role in imparting resistance against environmental stresses. Of these, an important signaling pathway contributing majorly in managing stress response is MAPK signaling pathway. Activation of signaling pathways magnifies the activation and functioning of various downstream components like transcription factors and other cytosolic protein thereby altering the expression of genes. In this review, the impact of heavy metals on activation of MAPK, calcium and hormone signaling along with other regulators like transcription factors and miRNAs is certainly reported. Several studies performed throughout different scientific groups suggest important role of MAPK signaling in heavy metal stress. However, a detailed evaluation of complete MAPK cascade working in combating heavy metal stress is required. Further, this review compiles the results revealing interplay of MAPK signaling with calcium, auxin, and ethylene signaling in response to heavy metal stress. All these findings outline the regulatory function of MAPKs acting either upstream or downstream to other signaling molecules. In animals this regulatory network has been elaborately studied in metal stress than in plants. Hence, it will be highly advantageous to study the importance of this signaling crosstalk in heavy metal stress in plants. Additionally, this review also summarizes interplay between MAPK signaling and other regulators like miRNAs and transcription factors in conveying a response against metal stress. However, scarce reports on regulatory network of MAPKs with transcription factors suggest a need for more in depth experiments in response to heavy metal stress in plants. Though, there are ample numbers of reports on activation of different signaling components, studies on deciphering of a complete regulatory network in heavy metal stress in plants are still lacking. Furthermore, the studies on activation of MAPKs by metals and metal transporters and in turn their regulation by MAPKs in animals and yeast, suggests occurrence of this phenomenon even in plants. However, the fragmentary work performed keeps this area mysterious in plants. Exploring the regulators of these metal transporters will contribute significantly in unraveling the mechanisms of metal stress tolerance in plants.SJ, PB, DV, SN, ST, and DS: wrote the manuscript; SJ: conceptualize the overall structure, prepared the illustrations, and edited the manuscript; KS and PB: prepared figures; AS: conceptualized, edited and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila and male mammals. Furthermore, the discovery of mammalian OGSCs challenged the traditional opinion that the number of primary follicles is fixed in postnatal mammals, which is of significance for the reproductive ability of female mammals and the treatment of diseases related to germ cells. Meanwhile, it still remains to be determined whether the Hh signaling pathway participates in the regulation of the behavior of OGSCs. Herein, we review the current research on the role of the Hh signaling pathway in mediating the behavior of GSCs. In addition, some suggestions for future research are proposed.Germline stem cells (GSCs) are adult stem cells that are responsible for the production of gametes and include spermatogonial stem cells (SSCs) and ovarian germline stem cells (OGSCs). GSCs are located in a specialized microenvironment in the gonads called the niche. Many recent studies have demonstrated that multiple signals in the niche jointly regulate the proliferation and differentiation of GSCs, which is of significance for reproductive function. Previous studies have demonstrated that the hedgehog (Hh) signaling pathway participates in the proliferation and differentiation of various stem cells, including GSCs in  Drosophila and male mammals, the migration of OGSCs from the ovarian surface epithelium (OSE) to the ovary cortex, and the development of OGSCs in the ovary cortex of mammals.Stem cells have many possible applications based on their ability for differentiation. At present, advances in regenerative treatments with stem cells are progressing dramatically, bringing hope for individuals with certain complicated and refractory diseases, such as maculopathy and nerve injury , 2. GSCsDrosophila embryos; thus, the gene was named Hh [Shh, Dhh, and Ihh, homologous to Hh, were found in vertebrates. The proteins encoded by all of the above genes can activate the Hh signaling pathway, which can regulate the function of stem cells, repair damaged cellular tissue, and maintain cellular structure [In 1980, when Nusslein-Volhard and Wieschaus were screening genes that affected the growth of fly larvae, they found that a certain mutated gene caused hedgehog-like spiked protrusions on the ventral side of named Hh . Afterwatructure , 5.Hh encodes the Hh precursor protein, which turns into the Hh ligand protein after self-cleavage and palmitoylation. The Hh ligand protein acts on target cells through paracrine or autocrine modes of action with the help of the dispatched (Disp) protein [The classical Hh signaling pathway is composed of the Hh ligand, a membrane protein receptor complex of patched (Ptc) and smoothened (Smo) proteins, nuclear transcription factors, and target genes.  protein , 7. Inte protein , 9. In tβ (glycogen synthase kinase 3 beta) and is phosphorylated by PKA, GSK3β, and CK1. Then, Gli3 is recognized by the E3 ubiquitin ligase βTrCP (beta-transducin repeat-containing proteins). Meanwhile, the transcriptional activation region at the C-terminal end is removed, and Gli3 becomes a transcription repressor (GliR), which enters the cell nucleus and inhibits the transcription of target genes. When the Hh ligand is present, activated Smo mediates the phosphorylation of SuFu/Gli/Kif7 complexes that release Gli (mainly Gli1), which blocks the hydrolysis process of the transcriptional activation region at the C-terminal end. Then, the full-length Gli acts like GliA to promote the transcription of target genes. It has been proven that Hh, Gli1, Ptc, and Bmp are target genes for the classical Hh signaling pathway.Generally, nuclear transcription factors are divided into transcription activators (CiA/GliA) and transcription repressors (CiR/GliR) . In mammGSCs are restoring cells in the gonads that have the ability to differentiate into germ cells. They exist in a microenvironment (niche) in the gonads that contain somatic cells. These somatic cells cooperatively regulate the proliferation and differentiation of GSCs. GSCs are usually called spermatogonial stem cells (SSCs) in males  and ovarDrosophila gonads are located in a niche composed of SSCs, hub cells (HCs), and cyst stem cells (CySCs). In Drosophila, at the apex of the bilateral testicle, there is a cluster of HCs. One side of the cluster connects with the basement membrane of the testicle; the other side is the binding site for SSCs and CySCs. Usually, there are six to nine SSCs on one side of the testicle, and each SSC is isolated by two irregular-shaped CySCs. During spermatogenesis, SSCs divide asymmetrically into two daughter cells. One cell adheres to HCs to maintain self-renewal, while the other separates from the HCs and forms a gonialblast (GB), which enters the differentiation stage. CySCs divide into two cyst cells, which surround the GB and regulate its proliferation and differentiation [SSCs in ntiation –15.In 1994, SSCs were first discovered in mammals in the basement membrane of the mouse testis seminiferous tubule, and they account for 0.02%–0.03% of the total cells in the testis . They arDrosophila are located in a niche composed of cap cells (CpCs), terminal filament cells (TFCs), and escort cells (ECs). The niche is located in the apex of the oviduct in the bilateral germarium. In general, there are two or three OGSCs in each germarium. They adhere to CpCs through the E-calcium adhesion protein. OGSCs can asymmetrically divide into two daughter cells: one cell turns into a new OGSC and continues to adhere to CpCs while the other one separates itself from CpCs and proceeds to becoming an oocyte latterly [OGSCs in latterly , 19. Furlatterly  first selatterly , 22, andlatterly  further latterly  purifiedDrosophila, the hedgehog signaling pathway regulates the proliferation and differentiation of SSCs via an indirect mechanism. Although SSCs are closely conglutinated with HCs, SSCs cannot express Ptc and Smo and fail to receive Hh signaling from HCs. Furthermore, the Hh ligand originating from HCs directly binds with Ptc in CySCs, which results in the upregulation of the transcription of target genes, including Hh and Bmp (bone morphogenetic protein) [bag-of-marbles (Bam), which encodes Bam (differentiation-associated factor), to sustain the undifferentiated status of SSCs [Drosophila spermatogenic cycle.In protein) . The inc of SSCs . Hence,  of SSCs . Hence, In mammals, the Hh signaling pathway regulates the proliferation and differentiation of SSCs in direct and indirect ways. Previous studies mainly considered that Hh signaling could indirectly affect the cellular behavior of SSCs through somatic cells in the niche. However, recent research revealed that SSCs could secrete Hh ligand and directly activate Hh signaling pathway in SSCs.In mice, SSCs directly adhere to adult SCs in the niche. During the adult SC period, the transcription and sustained expression of Dhh can be detected in SCs. After secreting Dhh, adult SCs accept Hh signaling and promote the expression of glial cell-derived neurotrophic factor (GDNF), bone morphogenetic protein (BMP), and stem cell factor (SCF). These proteins can further activate other signaling pathways in SSCs, such as BMP, PI3K-Akt, Src, and RAS/ERK1/2, which inhibit the differentiation of SSCs and sustain a certain amount of SSCs –29  and glass bottom boat (Gbb) (both belong to the Bmp family), in AECs [Dpp in CpCs. Eventually, BMP (Dpp) signaling in the OGSCs niche is intensified. As a result, Dpp suppresses the expression of Bam and inhibits the differentiation of OGSCs. As OGSCs differentiate, the expression of Fused (Fu), a positive regulator in the Hh pathway, is constantly increased. Moreover, Fu mediates the ubiquitination and proteolysis of thickveins  in OGSCs, which is beneficial for the differentiation of OGSCs [Hh signaling in the niche of OGSCs can suppress the differentiation of OGSCs in direct and indirect ways. Firstly, Hh signaling has been proposed to directly signal OGSCs and control the maintenance of OGSCs , A. Seco in AECs , 43. Hh of OGSCs , 44–47  derived but not CpC-derived Hh starts to participate in the differentiation process. It was discovered that maintaining the activation of the Hh signaling pathway and secretion of Hh ligand in PECs requires the presence of COP9  in ECs . Moreoveechanism  (Figure OGSCs in mammals are located in a single layer of epithelial cells with tight junctions in the OSE. In almost all species, it is not possible to observe obvious OGSC division in the normal OSE, and most research concerning OGSC function involves OGSC transplantation experiments. Thus, it is more reasonable to discuss the effect of Hh signaling pathway on OGSCs in normal OSE or in the ovary cortex after transplantation –52. MoreWe proposed a hypothesis that the special structure of the OSE restricts the activity of OGSCs, but Hh signaling could help OGSCs migrate into the ovary cortex. Under natural conditions, the outside of the OSE is covered by the peritoneum, and the underside of the OSE adjoins the ovary cortex –55. HoweHh, Ptc, and Gli are very low [Drosophila when Hh signaling stimulates other cells to secrete BMP protein.It is not clear whether somatic cells in the ovary cortex regulate the function of OGSCs through the Hh signaling pathway. During the initial forming period of the mammalian fetal ovary, the transcription levels of very low . After bvery low , inducinvery low –64. In tvery low , 65. Henvery low . Furthervery low . Then, tvery low  found thDrosophila? Grieve et al. [Can somatic cells in the ovary regulate OGSC development as in e et al.  found thAccording to the current research progress in this area, the following questions need to be thoroughly addressed in the near future.Drosophila, the JAK-STAT signaling pathway, whose functions overlap with those of the Hh signaling pathway, enhances the proliferation of SSCs and the overexpression of the antiapoptosis protein DIAPI in cells, which helps sustain the activity of SSCs [Currently, many studies have indicated that the Hh signaling pathway has a strong effect on the proliferation and differentiation of SSCs. What we are mostly interested is whether we can artificially alter the self-renewal or differentiation process via regulating Hh signaling in the SSC niche, thereby improving the reproductive ability of animals. For example, some animals with azoospermia cannot produce a sufficient number of sperm and lose their reproductive ability because of the dysfunction of SSCs or the low number of SSCs. A sufficient number of SSCs is a prerequisite for a normal spermatogenic ability in males. In 2014, it was determined in testicular biopsies that the ectopic expression of Shh results in the absence of spermatocytes and increased numbers of LCs in the testes . We disc of SSCs . Thus, tDrosophila ovary somatic cells, which eventually promotes the differentiation of OGSCs. Meanwhile, the Hippo-YAP signaling pathway has a negative regulatory effect on the generation and development of mammalian follicles [OGSCs are considered one of the possible cells in animals that can generate oocytes. Regulating OGSC function can be used to help those who wish to lengthen their reproductive life span or to treat animals or humans with germ cell dysfunction to restore their reproductive ability. Whether changing the Hh signaling pathway in OGSCs can affect the formation of primordial follicles in mammals and whether any effects on the proliferation and differentiation of OGSCs are caused by Hh signaling regulation still require further investigation. Previous studies have shown that Hh signaling can influence the development of TCs and hinder ovulation in mammals , but theollicles , which p