Both cytocompatibility and antibacterial activity have been evaluated. Data show that both nano-functionalized scaffolds exhibit a good antibacterial activity towards Staphylococcus aureus, reducing colony number to 99.4% (Ti_TiO2) and 99.9% (Ti_γFe2O3), respectively. In addition, an increase of both human adipose-derived mesenchymal stem cells (hADSCs) cell proliferation (up to 4.3-fold for Ti_TiO2 and 3.7-fold for Ti_γFe2O3) and differentiation has been observed. These data suggest that these nano-functionalized titanium substrates represent promising prototypes for new antimicrobial and osteoconductive biomaterials to be used in the orthopedic field to reconstruct significant bone defect.Implantable biomaterials play a key role for the success of orthopedic surgery procedures. However, infections remain one of the most damaging post-operative complications that lead to the implant failure. Recently, several approaches have been proposed to avoid or manage implant-associated infections. Among these, an appropriate surface functionalization to confer intrinsic antibacterial properties preserving the osteo-integration ability represents an appealing strategy for the development of innovative implant materials. Titanium and its alloys are the most used materials for manufacturing of both articular and bone skull prostheses as well as dental implants. However, to date there is still a significant clinical need to improve their bioactivity, osseointegration and antibacterial activity. In this study, titanium biomimetic scaffolds are prepared by nano-functionalization with TiO Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis, and Gram-negative bacilli including Pseudomonas aeruginosa and Escherichia coli [In recent years, the number of people undergoing surgery for osteoarticular problems, such as osteoarthritis and osteoporotic fractures, is greatly grown due to an overall increase in life expectancy. Biomaterials play a key role for the success of orthopedic surgery procedures. However, despite significant advances in the patient’s quality of life, implant-associated infections remain one of the most damaging post-operative complications . Actuallhia coli . The infhia coli . This lehia coli ,8. Therehia coli .Titanium (Ti) and its alloys are the most used materials for orthopedic implants due to their exceptional mechanical and chemical properties, good biocompatibility, corrosion resistance, good bone affinity and osteoconductivity ,11. SeveHowever, although Ti shows these excellent properties, to date there is still a significant clinical need to improve its antibacterial properties preserving osteo-integration ability. Therefore, the development of new biomaterials with bactericidal/osteoinductive function is still a challenge. Several approaches have been proposed in the literature for Ti substrates mainly involving: (1) the use of materials with intrinsic antibacterial properties  ,18,19 an2 NPs are among the most studied due to their efficiency and versatility based on photo-catalytic mechanism upon light irradiation depending on crystal structure, doping metals and irradiation wavelength [2 is mainly due to Reactive Oxygen Species (ROS) generation and lipid oxidation of bacterial cell wall. The ROS generation has been also found in the absence of light in presence of species undergoing catalytic decomposition (like H2O2) [In particular, metal or metal oxide nanoparticles (NPs) are increasing their use as alternative to antibiotics due to their broad spectrum of action against both gram +(ve) and gram −(ve) bacteria . Among tvelength ,30. The ke H2O2) ,32. Amonke H2O2) .2O3 and as Fe3O4 due to multiple mechanisms including ROS species, superoxide radical’s generation and singlet oxygen formation [Iron (III) oxide NPs are also appealing to nano-materials for tissue regeneration, since iron is an essential micro nutrient for cell growth and can also exhibit magnetic properties promoting cell proliferation ,35. Addiormation ,37. Howeormation , requiriormation . Therefo2 and γFe2O3 NPs (Ti_TiO2 and Ti_γFe2O3) by wet chemistry. Both the antibacterial properties towards S. aureus and the cell growth and differentiation using hADSCs have been assessed by in vitro studies and discussed.In this paper we proposed the preparation of new dual functional material with antibacterial and osteo-integration ability based on surface nano-functionalization of Ti alloy with TiO6Al4V alloy and circular shaped with diameter of 8 mm and height of 4 mm.All chemicals were obtained from commercial sources at the highest possible purity and were used as received. All solvents used were spectrophotometric grade. Milli-Q-grade water was used in all preparations. Ti supports were provided by Istituto Ortopedico Rizzoli . The supports are made of trabecular Ti(a)2 (Ti_TiO2). Ti supports were dipped in a titanium isopropoxide (0.1 M) isopropyl alcohol solution for 5 min and heated in air at 600 °C for three times. After that, the Ti_TiO2 scaffolds were rinsed with isopropyl alcohol and dried under nitrogen.Nano-functionalization with TiO(b)2O3 NPs (Ti_γFe2O3). Ti supports were cleaned in ethanolic ultrasound bath for 10 min and after, they were placed in a quartz tube containing ethyl alcohol, iron (III) acetylacetonate and acetone as photosensitizers. After 15 min of degassing with N2, the solution was irradiated with four lamps (254 nm 16 W) for 90 min. NaOH 1 M was added to the resulting yellow solution and stirred in air for 60 min. Finally, the red-brown Ti_γFe2O3 substrates were rinsed three times with deionized water and dried with nitrogen flow.Nano-functionalization with γFe2 window energy-dispersive X-rays spectrometer was used. Atomic force measurements (AFM) were performed using a Solver P47  in contact mode. We used standard silicon AFM probes  having cantilever force constant in the 0.01–0.5 Nm range. The zeta potential was evaluated using a ZetaSizer NanoZS90 , equipped with a 633 nm laser, at the scattering angle of 90 °C and 25 °C temperature. Z-potential measurements were performed in aqueous dispersion at pH 7.2. Each measurement was performed three times.The morphological and microstructural properties of nano-derivatised Ti scaffold surfaces were evaluated by Scanning Electron Microscopy (SEM-EDX) performed on a SEM-LEO 438 VP  and a Zeiss EVO MA10  equipped with a LaB6 electron gun. Transmission electron microscopy (TEM) analysis was performed using the bright field in conventional parallel beam (CTEM) mode (BF). An ATEMJEOL JEM-2010  equipped with a 30-mm(a)Cell culture and proliferation analysis.hADSCs were acquired from Lonza  and cultured as previously reported  until 806 cell were plated on Ti scaffolds (Ti_CTRL (not-treated Titanium substrate), Ti_TiO2, Ti_γFe2O3) in culture medium and incubated in a humidified atmosphere containing 5% CO2 at 37 °C for 1, 3 and 7 days. After, hADSCs were detached from Ti-derivatized supports with trypsin , stained with trypan blue  and counted by using a Leica DMI 4000B fluorescence microscope .Trypan blue test. 1 × 106 hADSCs were cultured on each scaffold for 1, 3 and 7 days, fixed in 4% PFA  and washed three times in PBS . After, each scaffold was permeabilized in 0.3% Triton X-100 for 10 min, washed in PBS and the nuclei stained with DAPI (1:5000) in PBS for 5 min. Finally, the images (20 for each scaffold) were acquired using a Leica DMI 4000B fluorescence microscope and the nuclei counted by Fiji Image J recognition software. The proliferation rates differences were assessed using One-way ANOVA test with Holm test as post-hoc for multiple comparisons. Each biological test was conducted in triplicate for each time points.4′,6-Diamidino-2-Phenylindole  staining. 1 × 10(b)Osteogenic Differentiation.6 hADSCs were cultured on each scaffold for 1, 3 and 7 days in 6-well plates. After that, the cells on the scaffolds were washed with PBS and fixed in 4% PFA at room temperature for 15 min and then washed twice with excess dH2O prior to addition of 1 mL of 40 mM ARS (pH 4.1) per well. The scaffolds were incubated at room temperature for 20 min under shaking. After aspiration of the unincorporated dye, they were washed four times with 4 mL dH2O while shaking for 5 min, re-aspirated and stored at −20 °C prior to dye extraction. For the quantification of ARS, 800 µL 10% (v/v) acetic acid was added to each well, and the plate was incubated at room temperature for 30 min with shaking. The cells were then detached from the scaffolds, transferred with 10% (v/v) acetic acid to a 1.5-mL microcentrifuge tube and vortexed for 30 s. After, the samples were sealed with parafilm, heated to 85 °C for 10 min and transferred to ice for 5 min. They were then centrifuged at 20,000× g for 15 min and 500 µL of the supernatant was removed to a new 1.5-mL microcentrifuge tube. Then 200 µL of 10% (v/v) ammonium hydroxide was added to neutralize the acid. In total, 150 µL of the supernatant for each sample were read in triplicate at 405 nm by a Microplate Reader . Alizarin Red S (ARS) staining quantification assay. ARS staining  was performed to evaluate calcium deposits in cells culture according to the manufacturer’s instructions. Specifically, 1 × 106 hADSCs were cultured on each scaffold for 1, 3 and 7 days and the culture media collected and used to measure ALP activity. This was enzymatically determined using an alkaline phosphatase assay kit  according to the manufacturer’s instructions. The ALP level was measured by reading the absorbance at 405 nm by a Microplate Reader . All experiments were performed in triplicate.Alkaline phosphatase (ALP) activity evaluation. ALP activity was evaluated by colorimetric assay performed on cell culture supernatants. Specifically, 1 × 10(c)Bacteria strain and viability assay.S. aureus (ATCC 29213) was purchased from American Type Culture Collection  and cultured in Tryptone Soya Broth . Ti scaffolds were sterilized in autoclave at 121 ± 1 °C for 20 min and placed in 24-well plates (one scaffold per well). Bactericidal activity was evaluated according to Hu et al. [S. aureus cultured overnight in Tryptone Soya Agar , was inoculated in Mueller–Hinton Broth  and incubated at 150 rpm on a rotary shaker for 6–8 h at 37 °C. After incubation period, the bacteria suspension (107 cells/mL) was placed onto the titanium scaffold surfaces with a 60 µL/cm2 density, and incubated in a humidified atmosphere at 37 °C for 24 h. Then, the scaffolds were placed in tubes containing phosphate-buffered saline (PBS) solution and, vigorously stirred for 60 s to detach the bacteria from the coating surface. Finally, a Colony Forming Units (CFU) assay was performed by using an untreated titanium scaffold as control, as following. Specifically, 100 µL of bacterial suspension were serially diluted in 900 µL of PBS, 100 µL of each dilution spread on solid medium and incubated overnight at 37 °C. After incubation, colonies in the range of 30–300 were counted to determine the number of CFU:u et al.  with som2 e γFe2O3 nano-functionalized scaffolds were evaluated by live/dead cell staining kit . Briefly, after bacteria incubation, the scaffolds were stained with a mixture of SYTO 9 (green-fluorescent staining viable cells) and propidium iodide (red-fluorescent staining dead cells) for 15 min. After, they were visualized under fluorescence microscopy by using Leica DMRE epifluorescence microscope  with Leica C Plan 63xobjective and BP 515–560 nm excitation filter in combination with a LP 590 nm suppression filter.The bacterial adhesion on the surface TiO(d)ROS evaluation.2DCFDA, Sigma-Aldrich, Milan, Italy) at 10 µM final concentration and incubated at 37 °C for 20 min. After the incubation time, bacteria were washed twice with PBS, to remove the excess of dye, and finally images were acquired by using a Leica DMRE epifluorescence microscope with Leica C Plan 100x objective and BP 515–560 nm excitation filter in combination with a LP 590 nm suppression filter.For ROS evaluation, bacteria were detached, washed twice with PBS, stained with 2′, 7’-dichlorodihydrofluorescein diacetate (H(e)Statistical analysis.p < 0.05 were considered to be significant.Data were analyzed either as raw data or as mean ± standard error (SE), as appropriate. Differences between the two time points and the different types of scaffolds were evaluated by using one- and two-way ANOVA with post-hoc Holm test, where appropriate. The values 2 and Ti_γFe2O3 scaffolds were prepared by surface in-situ growth with wet chemistry. Ti-isopropoxide precursor were converted in nanosized TiO2 on scaffold surface trough thermal growth 3 precursor in the presence of photosensitizers, followed by pH-basic oxidation to obtain nanosized γFe2O3 on the scaffold surface (Both Ti_TiOl growth a. Simila surface b.4 and FeIII(acac)3) into TiO2 and γFe2O3 NPs at the scaffold surface. The nano-derivatized biomimetic scaffolds were morphologically and chemically characterized trough AFM, SEM, EDX and TEM inspections. 2 nano-functionalization lization c,d. The diameter c,d. 2 nanostructures. In the case of Ti_γFe2O3 it was not possible to detect the presence of γFe2O3 NPs in the SEM images because of their nanosize : 2.95 Å (28%), 2.54 Å (100%), 2.09 Å (20%), 1.73 Å (10%), 1.64 Å (25%) and 1.47 Å (42%)  compared to the untreated Ti_CTRL titanium scaffold see , confirmnanosize a. The prnanosize b. The si42%) see . AFM (Fi42%) see c, perfor4 ± 3 nm d. The av2 and γFe2O3 NPs in solution. Our results indicate the presence of negative charged nanoparticles with Z-potential values of −11.8 ± 1.4 mV for TiO2 and −39.2 ± 0.9 mV for γFe2O3. Surface negative charges that can promote the adsorption of hydrophilic proteins  leading to increased bone cell adhesion [In addition, we performed Z-potential investigation on TiOadhesion .2 and Ti_γFe2O3, cell proliferation was investigated by observing the viability of hADSCs for 1, 3 and 7 days on nano-functionalized scaffolds  compared to not treated Ti_CTRL scaffold (2 and Ti_γFe2O3 scaffolds was considerably higher than of Ti_CTRL (2 and Ti_γFe2O3 scaffolds was comparable with that of the Ti_CTRL (control) , after 3 days (D3) the cell numbers on Ti_TiO2 and Ti_γFe2O3 scaffolds were 1,4-fold and 1,3-fold higher than Ti_CTRL control and at D7 these values increased to 2.3-fold and 2.6-fold, respectively, .To gain insight on the osteo-integration ability of both Ti_TiOscaffold A. The ce Ti_CTRL A and tha2 and Ti_γFe2O3 scaffolds a considerable cell number increasing of for 3.7-fold Ti_TiO2 and 4.3-fold for Ti_γFe2O3, compared to the Ti_CTRL control was found , showed no significant difference in the cell number in the nano-functionalized scaffolds compared to Ti_CTRL. After 3 and 7 days of culture in both Ti_TiOound see . Contrar2 and Ti_γFe2O3 were measured. Data obtained from ARS staining quantification show a higher calcium deposits content in both Ti_TiO2 and Ti_γFe2O3 scaffolds than Ti_CTRL control at the same incubation times .In addition to the osteo-integration, we also evaluated another important aspect for implantable-devices, the osteogenic differentiation. To this aim, ARS staining quantification and ALP activities after 1, 3, and 7 days of culture on both Ti_TiOon times B. In det2 and Ti_γFe2O3 scaffolds than Ti_CTRL control at the same incubation times . These data together indicate that both nano-functionalizations are able to induce osteogenic differentiation.In accordance with ARS staining quantification data, ALP results show values significantly higher in both Ti_TiOon times C. Specif2  spread onto the micrometric trabecular microstructure of the pristine Ti substrate. These results, therefore, evidence that a combined micro-nanotopography can promote and induce the cellular integration.The above results highlight that both Ti_TiOegration ,43. In olication ,34. Thenlication . Our fin2 and γFe2O3 nano-systems exhibit negative charges at the surface  indicating the possibility to improve the adsorption of hydrophilic proteins  leading to increased bone cell adhesion [Another important factor for cell–biomaterial interaction is the surface hydrophilicity, that improve adhesion and spread of cells, osteoblastic differentiation and maturation . Both Tiadhesion .2 and Ti_γFe2O3 scaffolds, S. aureus bacteria were incubated on them for 24 h, and after CFU assay and live/dead staining were performed. Results are reported in 2 and Ti_γFe2O3 scaffolds compared to the Ti_CTRL scaffold. Specifically, the CFU assay reveals that TiO2 and γFe2O3 NPs incorporated on the titanium scaffold surfaces are able to reduce the colony number of about 99.4% for Ti_TiO2 (3.2 ± 0.7 × 105 CFU/mL) and 99.9% for Ti_γFe2O3 (4.3 ± 0.9 × 102 CFU/mL) compared to the Ti_CTRL (3.7 ± 1.4 × 107 CFU/mL)  and many dead (red fluorescent) cells for Ti_TiO2, while almost all of them were red for Ti_γFe2O3 compared to the Ti_CTRL, demonstrating their strong biocidal activity . In fact, bactericidal effect is increased in nanoparticles with smaller size, due to cell uptake, intracellular distribution and interaction with biological macromolecules  [In order to assess the antibacterial properties of both Ti_TiO CFU/mL) A. In addactivity B. Conseqmbranes) .2 and γFe2O3 is reported to mainly due to generation of free radicals and liberation of metal ion [2O3, it has been found that ROS generation induces membrane depolarization, lipid peroxidation and DNA damage, while ion release negatively affects metabolic activities, cell homeostasis and protein functions [2, its antibacterial effect is closely related to generation of ROS under UV-radiation or in dark condition in presence of oxidizing species [The antibacterial activity of both TiOetal ion ,47. Abouunctions ,49. Furtunctions . In term species ,30,31. I species ,52.2DCFDA) staining was performed after 1, 2 and 4 h post bacteria incubation and fluorescence images detected. Specifically, ROS signal was evaluated in the early hours , because ROS-positive cells decrease due to the reduction of viable cells [2, and Ti_γFe2O3 scaffolds after 2 h  and to gain more insight on the regeneration and bactericidal mechanisms with in vitro studies to evaluate the specific genes and cellular pathways to be finally validated by in vivo study.The above results highlight that both Ti_TiO2 and γFe2O3 NPs by surface in situ growing method and their osteo-integrative/inductive capability and antimicrobial activity were assessed. Morphological characterization of both Ti_TiO2 and Ti_γFe2O3 scaffolds showed the presence of TiO2 NPs with dimensions between 300 nm and 800 nm in diameter spread onto Ti surface, while in the case of γFe2O3 both TEM and AFM measurements highlight the presence of NPs with a diameter in the range of 10–20 nm. Both Ti_TiO2 and Ti_γFe2O3 scaffolds exhibited good osteo-integration ability. In particular, a considerable increase of hADSCs cell growth after 7 days of culture equal to 2.3-fold for Ti_TiO2 and 2.6-fold for Ti_γFe2O3, compared to the control, was found. Further, an improved osteogenic differentiation was observed with an ALP activity increasing up to 17%. SEM analyses highlighted good cell adhesion and colonization over the scaffold porosities.In this study, we prepared Ti scaffold nano-functionalized with TiO2 and Ti_γFe2O3 scaffolds with a reduction of 99.4% and 99.9% of S. aureus colony number, respectively, compared to the not-treated sample. H2DCFDA staining investigation reveals the presence of ROS occurring in presence of both TiO2 and γFe2O3 nano-functionalization. These data prove that both Ti_TiO2 and Ti_γFe2O3 scaffolds are able to match the double function to promote osteo-integration and deplete bacterial invasion. Further in vitro investigations are planned to optimize the nano-functionalized coating  and gain more insight on mechanisms inducing bone regeneration and antibacterial activity, before final validation by in vivo studies. Results here presented pave the way for future use of Ti_TiO2 and Ti_γFe2O3 biomimetic scaffolds to reconstruct large bone defect in orthopedic fields.Good antibacterial activities were exhibited from both Ti_TiO


In combination of molecule dynamics (MD) simulation, docking and site-directed mutagenesis, our structure reveals that a hydrogen-bond network of water molecules and key polar residues is the key molecular determinant for LTB4 binding. We also find that the displacement of residues M1013.36 and I2717.39 to the center of receptor, which unlock the ion lock of the lower part of pocket, is the key mechanism of receptor activation. In addition, we reveal a binding site of phosphatidylinositol (PI) and discover that the widely open ligand binding pocket may contribute the lack of specificity and efficacy for current BLT1-targeting drug design. Taken together, our structural analysis provides a scaffold for understanding BLT1 activation and a rational basis for designing anti-leukotriene drugs.Leukotriene B4 receptor 1 (BLT1) plays crucial roles in the acute inflammatory responses and is a valuable target for anti-inflammation treatment, however, the mechanism by which leukotriene B4 (LTB4) activates receptor remains unclear. Here, we report the cryo-electron microscopy (cryo-EM) structure of the LTB4 -bound human BLT1 in complex with a G In the paper, Dr. Wang et al reported a cryo-EM structure of the human leukotriene B4 receptor 1 (BLT1) in complex with its native ligand leukotriene B4 (LTB4) in an active conformation complexed with Gi protein. The structure reveals the molecule determinant of LTB4 binding and the mechanism of receptor activation. These structural information will boost the understanding of LTB4-BLT1 signaling and provide a rational basis for designing novel anti-leukotriene drugs. LTB4 acts as a chemoattractant for neutrophils and macrophages to vascular endothelium2. The binding of LTB4 to its receptors (BLT1 and BLT2) activates leukocytes and prolongs their survival3. BLT1 expresses exclusively in the immune system while BLT2 has a broad spectrum of expression profile. In general, BLT1 has a higher affinity for LTB4 than BLT24. On the other side the heptadecanoid 12(S)-hydroxyheptade catrienoic acid (12-HHT) has been discovered as the endogenous ligand for BLT2, which has similar affinity to BLT2 as LTB4 to BLT15. 12-HHT is an enzymatic product of thromboxane A2 (TXA2) synthase in the conversion of prostaglandin H 2 (PGH2) to TXA2, 12-HHT and malondialdehyde. A major difference between 12-HHT and LTB4 chemical structure is that the 12-HHT only has a hydroxyl group at the C12 position, while LTB4 has two hydroxyl groups, one at the C12 position and one at the C5 position. Whether this difference determines the specificity of LTB1 and LTB2 is an interesting question to ask. The LTB4-BLT1 signaling pathway is crucial for many inflammatory diseases, such as infectious diseases, allergy, autoimmune diseases, and metabolic disease6. Emerging evidence also suggested a complex role of the LTB4-BLT1 signaling in cancers. Comparing to cysteinyl leukotriene (CysLTs) which mainly act in the airway, LTB4 is regarding as one of the most potent neutrophil chemoattractants in the host defense system against infection and invasion from foreign bodies6.LTB4 is an inflammatory lipid mediator produced from arachidonic acid, and is one of the most important leukotrienes in the onset of acute inflammatory responsesi and Gq pathways. BLT1 is an attractive drug target for allergic airway inflammation, inflammatory arthritis, atherosclerosis, and psoriasis3. Despite extensive efforts in developing BLT-targeting compounds, none of them have yet made to the market due to the high incidence of side effects, low specificity, and poor efficacy. For instance, etalocib (LY293111), has been demonstrated to activate peroxisome proliferator activator receptor gamma (PPARγ)7. Part of the reason for the unsuccessful drug design may be due to the limited structural information of BLT available, particularly, the mechanistic insights into receptor activation. Currently, there are only two antagonist-bound BLT1 receptor structures, one is BIIL260-bound guinea pig BLT18, and the other is MK-D-046-bound human BLT19. The absence of agonist-bound BLT1 structures has limited mechanistic understanding of the ligand recognition and receptor activation. Here, we report the cryo-EM structure of the endogenous ligand LTB4-bound human BLT1 in complex with Gi protein in an active conformation. Together with MD simulation, docking, and mutational studies, the structural information of our study provides a framework for understanding LTB4 signaling and a rational basis for designing novel anti-leukotriene drugs.BLT1 and BLT2 belong to the class A G-protein coupled receptors (GPCRs), and can be further classified as members of lipid receptor subfamily which includes prostanoid receptors, sphingosine receptors, lysophospholipid receptors, cannabinoid receptors etc. BLT1 and BLT2 are mainly coupled with G3.41W, A1965.53I, C2877.55F, and S310A, superscripts refer to the Ballesteros-Weinstein numbering) adopted from the MK-D-046-bound human BLT1 crystal structure  reporter assay10, an established reporter assay for Gi signaling , and the C-terminus of Gβ was fused to the renovated 13-amino acid peptide of NanoBiT (HiBiT). The introduction of the LgBiT to receptor do not affect the overall receptor activation is evidenced by the similar transactivation activity compared with the wild-type receptor  of the Gα subunit as a main interface to interact with the open intracellular core of the receptor  forms a hydrogen bond network that connect R1564.64 and R2677.35 to the C5 hydroxyl group of LTB4 . In addition, water molecule 6 interacts with N2687.36 and is connected to water molecule 7. We also measure the distance between those water molecules and the connected LTB4 and the key residues of the receptor. The distances vary from 3.0 Å to 3.3 Å, in a good range to form ideal hydrogen bond, therefore these densities are highly likely to be water molecules that connect LTB4 to the receptor. Together, these water molecules form a hydrogen bond network that connects polar residue N2687.36, R1564.64, R2677.35, and H943.29 to LTB4 to lock the ligand in the pocket. We use MD simulations to examine the hypothesis. If the ligand-binding pocket is energy unfavorable, the ligand LTB4 will quickly jump out of the pocket. The 200 ns triplicated runs all show that the LTB4 molecule sticks within the pocket, in poses very similar to the original LTB4 position we observed in our cryo-EM structure, throughout whole simulations , is consistent with their roles in forming the skeleton of the hydrophobic tunnel in the pocket. Conversely, the great decrease of receptor activity by the “small to large” mutants (L782.64F and G1895.46F) is consistent with their roles in forming the beginning and the ending of the pocket, as the bulky side chain of phenylalanine may block the entry or disturb the docking pose of LTB4 in the pocket. Interestingly, the dramatic decrease of activity is seen on the polar mutant R1564.64A, R2677.35A, and H943.29A, which almost completely abrogate the receptor activity. These data are consistent with our structural observation and the MD simulation outcome. The mutation data of R1564.64A and H943.29A is also consistent with a previous study by Basu et al.16.We use an SRE-reporter assay to examine the contributions of those residues in the pocket. The data shows that F74TB4 Fig. . Our MD TB4 Fig. . More im18. Interestingly, we identified a cluster of density that resembles a PI molecule in the middle of TM3, TM4, and intracellular loop 2 (ICL2), facing the membrane side  molecule, a component of the membrane, into the pocket. Interestingly, we found that the amine head group of the PE molecule inserted into the same tunnel previously discovered by the MK-D-046-bound BLT1 crystal structure9, where a nonaethylene glycol (P2E) molecule snuggled in  toward the center of the receptor core  pack against the center of the ligand-binding pocket  for comparison. The Alpha Fold BLT2 aligns well with BLT1  cutting site and 2 x maltose-binding protein (MBP) was cloned in pFastBac1 baculovirus expression vector. Four thermostabilizing mutations L1063.41W, A1965.53I, C2877.55F, and S310A in BLT1 adopted from the MK-D-046-bound BLT1 crystal paper9 was introduced the receptor. The C-terminus HiBiT fusion of human Gβ1 was cloned into pFastBac plasmid as the VIP1R paper11. The human dominant-negative Gαi1 34, wild-type human Gβ1, wild-type human Gγ2 were cloned into pFastBac plasmid. The scFv16 encoding the single-chain variable fragment of mAb1613 as described before35.The codon-optimized human BLT1R gene with a HA-signal peptide sequence at its N-terminus and a LgBiTi1, Gβ1, Gγ2, and scFv16 protein were co-infected into the Spodoptera frugiperda (Sf9) cells (2 × 106 cells per ml) at a ratio of 1:100 (virus volume vs cells volume) and cells were harvested 48 h postinfection. Cell pellets were resuspended in 20 mM Hepes buffer, 150 mM NaCl, 10 mM MgCl2, 20 mM KCl, 5 mM CaCl2, pH 7.5, with 0.5 mU/ml apyrase and homogenized by douncing ~30 times. To keep the complex stable, ligands was added at final concentration of 300 nM all through the purification. After 1 h incubation of the lysis at room temperature, 0.5% (w/v) lauryl maltose neopentylglycol , 0.1% (w/v) cholesteryl hemisuccinate TRIS salt (CHS) were added to solubilize the membrane at 4 °C for 2 h. Then the lysis was ultracentrifuged at 65,000 g at 4 °C for 40 min. The supernatant was incubated with amylose column for 2 h then washed with a buffer of 25 mM Hepes, pH 7.5, 150 mM NaCl and 0.01% LMNG/0.002% CHS, and eluted with the same buffer plus 10 mM maltose. The elute was concentrated and cut with home-made TEV overnight at 4 °C, then separated on a Superdex 200 Increase 10/300 GL  gel infiltration column with a buffer of 25 mM Hepes, pH 7.5, 150 mM NaCl and 0.00075% (w/v) LMNG, 0.00025% glyco-diosgenin (GDN), 0.0002% (w/v) CHS (Anatrace). The BLT1/Gi complex corresponding peak was concentrated at about 10 mg/ml and snap frozen for later cryo-EM grid preparation.For expression, baculovirus encoding the BLT1, Gαi complex sample (~10 mg/ml) was applied to a glow-charged quantifoil R1.2/1.3 Cu holey carbon grids (Quantifoil GmbH). The grids were vitrified in liquid ethane on a Vitrobot Mark IV (Thermo Fisher Scientific) instrument at the setting of blot force of 10, blot time of 5 sec, the humidity of 100%, the temperature of 4 °C. Grids were first screened on a FEI 200 kV Arctica transmission electron microscope (TEM) and then grids with evenly distributed particles in thin ice were transferred to a FEI 300 kV Titan Krios TEM equipped with a Gatan Quantum energy filter. Images were taken by a Gatan K2 direct electron detector at the magnitude of 64,000, super-resolution counting model at pixel size of 0.55 Å, the energy filter slit was set to 20 eV. Each image was dose-fractionated in 40 frames using a total exposure time of 7.3 sec at a dose rate of 1.5 e/Å2/s . All image stacks were collected by the FEI EPU program, nominal defocus value varied from −1.2 to −2.2 µm.A 3 µl BLT1/G35. A total of 3158 raw movies (0.55 Å) were binned (1.1 Å) and motion-corrected using MotionCor236, followed by CTF estimation by CTFFIND 4.137. Particles (1.5 million) were picked by crYOLO38 and extracted by RELION (version 3.1)40 and subjected to reference-free 2D classification in RELION. 12 classes , which of well-defined features were passed to the next round for initial model generation and 3D classification. The initial model was generated by cryoSPARC41 ab initio. The model was used as reference in RELION 3D classification (5 classes). One class  showed clear secondary structure features was selected for a 3D refinement in RELION, followed by a Baysian polishing42, then a 3D refinement and a CTF refinement in RELION. The refined particles were subjected to a second-round 3D classification (4 classed) with fine angular sampling to yield a class of about 410,000 particles for final refinement by the cryoSPARC Nonuniform Refinement, which generated a map of 2.91 Å, based on the gold standard Fourier Shell Correlation (FSC) = 0.143 criterion. Local resolution estimations were performed using an implemented program in cryoSPARC.We use a similar pipeline to process data as descripted before9 and the Gi protein complex from the DRD2 (PDB 6vms)23 were used as initial models for model rebuilding and refinement against the electron microscopy map. All models were docked into the electron microscopy density map using UCSF Chimera43 then subjected to iterative manual adjustment in Coot44, followed by a rosetta cryoEM refinement45 at relax model and Phenix real space refinement46. The model statistics were validated using MolProbity47. Structural Figures were prepared in UCSF Chimera, ChimeraX48, and PyMOL (https://pymol.org/2/).The crystal structures of MK-D-046-bound human BLT1 (PDB 7k15)49. Sequence alignment by the Clustal Omega sever50 and the representation of sequence alignment was generated using the ESPript website51 (http://espript.ibcp.fr). The generic residue numbering of GPCR is based on the GPCRdb4 (https://gpcrdb.org/)52.The calculation of the pocket volume was done by the CASTp 3.0 sever2. 16 h after transfection, cells were induced by LTB4 (300 nM) or vehicle. Cells were harvested and lysed by addition of 1× Passive Lysis Buffer (Promega) 6 h after induction. The luciferase activity was assessed by the Dual-Glo Luciferase system (Promega). Data were plotted as firefly luciferase activity normalized to Renilla luciferase activity in Relative Luciferase Units (RLU).The SRE reporter assays (Promega) were performed by the Promega instruction as described before. Briefly, AD293 cells were split into 24 well plate at a density of 40,000 per well then transfected with 100 ng of SRE-Luc, 10 ng of pcDNA3-BLT1 wild-type or mutations, 10 ng of phRGtkRenilla plasmids (Promega) by X-tremeGENE HP (Roche) at a ratio 3:1 to DNA amount after one day of growth on 37 °C at 5% COThe purified BLT1 sample (78 µg) was mixed with 1365 µL ice-cold solution containing methyl tert-butyl ether/methanol/2 M hydrochloric acid solution  and vortexed for 1 min. Then, the solution was added with 250 µL hydrochloric acid (0.1 M) and vortexed for 5 min. After 5 min centrifugation (6000 g), the upper layer of the solution, which contained the extracted lipids was pipetted out and dried under nitrogen flow. The lipid extracts were dissolved in 100 µL solution containing isopropanol/methanol/13 M ammonia solution/chloroform  for subsequent LC-MS analysis. LC-MS was conducted on a Shimadzu LC-20AD system  hyphenated with a QTRAP 4500 mass spectrometer . The injection volume was 2 µL. A hydrophilic interaction chromatography column  was used for the separation of glycerophospholipids by class. A binary mobile phase system was used with A being 10 mM ammonium acetate aqueous solution and B being acetonitrile. The flow rate was set at 0.2 mL/min and the oven temperature was kept at 32 °C. The mobile phase gradient was as follows: 90% B for 0–2 min, 90–85 % B for 2–5 min, 85–80% B for 5–8 min, 80% for 8–15 min, 80–70% for 15–16 min, 70% for 16–20 min, 70–90% for 20–21 min, and 90% for 23–25 min. The MS parameters were as follows: voltage for electrospray ionization, −4400 V; curtain gas, 30 psi; interface heater temperature, 450 °C; nebulizing gas 1 and gas 2, 30 psi; declustering potential, −100 V; CID energy at −40 eV.54, the complex was inserted into a bilayer lipid contain POPC  and cholesterol at ratio of 4:1, the membrane size is 65 × 65 Å with 22.5 Å water and ion 0.15 M KCl in the top and bottom, temperature 303.15 K. The Amber force fields were set to protein FF19SB, lipid LIPID17, water TIP3P and ligand GAFF2. The simulations were performed by Amber20 package55. The system was first energy minimized for solvent and all atoms, heat to 300 K in 300 ps and then equilibrated for 700 ps, followed by three independent production runs of 200 ns with a timestep of 2 fs. During simulations, Particle mesh Ewald algorithm were applied for the calculation of long-range electrostatic interaction and a cutoff of 10 Å were applied for short-range electrostatic interaction and van der Waals interactions. All bonds with hydrogens are constrained by SHAKE algorithm. The system temperature (300 K) and pressure (1 atm) were controlled by Langevin thermostat and Berendsen barostat, respectively. The trajectories were analyzed by CPPTRAJ56 and visualized in VMD57. The video was recorded by VMD.The cryo-EM structure of BLT1 (receptor only) was used to initial model in the MD simulation. The five water molecules and LTB4 were kept in the model while CHS and PI were deleted. Using CHARMM-GUIBased on the cryo-EM structure, LTB4 were re-docked to its pocket. After the preparation and minimization of BLT1 with waters, LTB4 were firstly placed in the pocket using the triangle matcher with London docking scoring. Then, refinement was employed based on a rigid receptor and GBVI/WSA docking scoring. The top-scored pose was shown in Supplementary Fig. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of additional Supplementary Filesupplementary movie 1Reporting Summary


Background: 11β-Hydroxysteroid dehydrogenase one is responsible for activating inert glucocorticoid cortisone into biologically active cortisol in humans and may be a novel target for the treatment of nonalcoholic fatty liver disease.Methods: A series of benzylidene cyclopentanone derivatives were synthesized, and the selective inhibitory effects on rat, mouse and human 11β-hydroxysteroid dehydrogenase one and two were screened. The most potent compound [5-bis--cyclopentanone] (WZS08), was used to treat nonalcoholic fatty liver disease in mice fed a high-fat-diet for 100 days.Results: WZS08 was the most potent inhibitor of rat, mouse, and human 11β-hydroxysteroid dehydrogenase 1, with half maximum inhibitory concentrations of 378.0, 244.1, and 621.1 nM, respectively, and it did not affect 11β-hydroxysteroid dehydrogenase two at 100 μM. When mice were fed WZS08  for 100 days, WZS08 significantly lowered the serum insulin levels and insulin index at 4 mg/kg. WZS08 significantly reduced the levels of serum triglycerides, cholesterol, low-density lipoprotein, and hepatic fat ratio at low concentration of 1 mg/kg. It down-regulated Plin2 expression and up-regulated Fabp4 expression at low concentration of 1 mg/kg. It significantly improved the morphology of the non-alcoholic fatty liver.Conclusion: WZS08 selectively inhibits rat, mouse, and human 11β-hydroxysteroid dehydrogenase 1, and can treat non-alcoholic fatty liver disease in a mouse model. Non-alcoholic fatty liver disease (NAFLD) is a common disease in humans and a liver manifestation of metabolic syndrome . It is cPlin2) and PLIN3 (encoded by Plin3) are involved in the formation of lipid droplets and in the pathophysiological process of NAFLD, which is characterized by excessive accumulation of lipids in hepatocytes (Some lipid-binding proteins may be associated with the progression of NAFLD. One family of these proteins is perilipins (PLINs). PLINs are associated with the surface of lipid droplets . Recent atocytes .Hsd11b1) is considered to be related to NAFLD. 11β-HSD1 in fat and liver tissue converts inactive 11-ketoglucocorticoids [cortisone in humans or 11-dehydrocorticosterone (11DHC) in rodents] into biologically active 11β-hydroxy glucocorticoid cortisol or corticosterone (CORT)  . It has e (CORT) . It was e (CORT) . Indeed,e (CORT) .In addition to 11β-HSD1, there is another isoform, 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), which is mainly present in the kidney to regulate the role of mineralocorticoid receptors at pre-receptor level . 11β-HSD3H]-Cortisone, [3H]-CORT, and [3H]-cortisol were purchased from DuPont-New England Nuclear  [3H]-11DHC was prepared from [3H]-CORT according to a previous method (High-fat diet (HFD) containing 10% fat, 20% sucrose, and 2.5% cholesterol  was purchased from Beijing HFK Bioscience Co. . Regular chow containing 3% fat, 10% sucrose, and 1% cholesterol  was purchased from Zhejiang Experimental Animal Center . Ultra-sensitive mouse insulin ELISA kit was obtained from CrystalChem . Oil Red O solution was purchased from Sigma-Aldrich . Corticosterone ELISA kit was purchased from Abnova (Taiwan). . ESI-M1H-NMR (CDCl3) δ: 3.05 , 7.06–7.10 , 7.73 . ESI-MS m/z: 687.3 (2M + Na)+, calculated for C25H28O7: 332.29. Purity > 95%.Yellow powder, 29.4% yield, mp 208.1–209.8°C. 1H-NMR (CDCl3) δ: 2.73 , 6.92–6.95 , 7.29–7.34 , 7.50 . ESI-MS m/z: 334.4 (M + 1)+, 687.4 (M + Na), calculated for C25H28O7: 332.29. Purity > 95%.Yellow powder, 79.2% yield, mp 146.8149.6°C. 1H-NMR (CDCl3) δ: 7.74 , 7.19 , 7.13 , 6.98 , 5.35 , 3.96 × , 2.78 . ESI-MS m/z: 351.2 (M − H)−, calculated for C22H14F8O: 352.38. Purity > 95%.Green powder, 61.2% yield, mp 120.1e122.7C. +, calculated for C21H12F8O: 432.31. Purity > 95%.Yellow powder, 58.1% yield, mp 221.8e222.4°C . ESI-MSThe liver and kidney microsomes were prepared as previously described . Briefly2). The testis was removed. The testis was perfused with collagenase solution through the testicular artery. The testis was digested with collagenase and DNase for 15 min, and the digested cells were filtered with nylon mesh, and the cells were separated under a Percoll gradient. The cells with density of 1.070–1.088 g/ml were collected and washed. The purity of the Leydig cell fraction was evaluated by histochemical staining of 3β-hydroxysteroid dehydrogenase using 0.4 mM etiocholanolone as a steroid substrate and 2 mM NAD + as a cofactor and tetranitroblue tetrazolium as the H+ acceptor as previously described  and viable (not stained) cells were counted. Viability was calculated as the percentage of viable cells divided by the total number of cells, and the viability of Leydig cells (intact Leydig cells) was over 99%. After incubation with chemicals, intact Leydig cells were used for the measurement of 11β-HSD1 activity.Leydig cells were isolated as previously described . Brieflyescribed . More thescribed . In brie−10–10−5 M) of drug candidates at 37 °C for 60–90 min. The inhibitory ability of the chemical relative to the control (DMSO solvent) was measured. The chemical was dissolved in DMSO to a final concentration of 0.4%, at which DMSO did not inhibit enzyme activity. The reaction was stopped by adding 10 µL of 1 mM glycyrrhetinic acid and 1 ml of ice-cold ether. The steroid was extracted with ether, and the organic layer was dried under nitrogen. The steroids in chloroform and methanol  were chromatographically separated on a thin layer plate, and radioactivity was measured using a scanning radiometer . The percentage of 11DHC being converted to CORT or cortisone being converted to cortisol can be calculated by dividing the radioactivity count of 11-OH-steroids by the total count. 11β-HSD1 is abundantly expressed in the livers of all species.The reductive activity of 11β-HSD1 in rat, mouse, and human liver microsomes was measured as previously described . In shor6 intact Leydig cells. Other procedures were described above.To test whether chemicals can penetrate cell membranes to inhibit 11β-HSD1 activity, intact rat Leydig cells were used because this cell type contains the highest level of 11β-HSD1 of all cell types . The red+ for 30 min. Other procedures were described above. The percentage conversion of cortisol to cortisone (humans) or CORT to 11DHC (rats and mice) was calculated by dividing the radioactive count of an 11keto-steroid product by the total count associated with both substrate and product.The 11β-HSD2 assay was based on rat, mouse, and human kidney microsomes, as previously described . In brie50 was determined by adding 25 nM steroid substrate with 0.2 mM cofactor and various concentrations (10 nM–100 μM) of WZS08 to 250 μL PBS  containing 11β-HSD1 or 11β-HSD2 microsomal protein and each reaction mixture was incubated for 60 min. In addition, when Leydig cells were involved, 2000 nM of steroid substrate with 0.4 mM cofactor and various concentrations (10 nM–100 μM) of WZS08 was added to 500 μL of reaction buffer (0.1 mM phosphate buffer) to determine the IC50 value.The ICThe KM mice were randomly divided into six groups, including a regular chow (Group C), HFD (Group M) as a model control group, HFD supplemented with various doses of WZS08, 1 (Group M + W1), 2 (Group M + W2), and 4 (Group + W4) mg/kg, 20 mice per group. Mice were housed five animals per cage. Mice were maintained at a constant temperature (25 ± 3°C) for a 12-h light-dark cycle with free access to food and water. After a week of diet adaptation, the mice were fed with WZS08 dissolved in 1% sodium carboxymethyl cellulose (CMC-Na) or 1% CMC-Na for 100 days alone. Each mouse was gavaged daily with 0.5 ml vehicle (1% CMC-Na) or drug in 1%CMC-Na. By the end of the treatment, the mice were killed by CO2, and the weights of body, liver, epididymal fat, mesenteric fat, and dorsal fat were measured.Oral glucose tolerance test was performed according to a previously described method with some modifications . The oraThe intraperitoneal insulin sensitivity test was performed at the last week, mice were fasted 4 h and then administered insulin intraperitoneally  as previously described . At 0, 1The energy intake of the mice in each experimental group is different. The weights of the HFD and regular chow consumed by mice in the experimental group were calculated within the next 45 days after drug treatment. HFD contained an energy of 329 KJ/100 g and regular chow contained an energy of 167 KJ/100 g. The calories consumed by mice in each experimental group during the 45 days was calculated.g for 5 min, and the supernatant was collected. An aliquot of water (1 ml) was added to the supernatant, and the mixture was vortexed and centrifuged at 500 ×g for 10 min. The lower chloroform layer was transferred to a pre-weighed tube, and the sample was dried by a nitrogen evaporator and weighed.The extraction of fecal and liver lipids was performed according to the previously published method with only minor modification . BrieflyOil red-O staining was performed as previously described . BrieflyGapdh (encoding GAPDH) and Rps16 as the internal controls. The mRNA levels of Acs2 (encoding acetyl-CoA synthetase2), Hsd11b1 (encoding 11β–HSD1), Ldlr (encoding LDLR), Plin2 (encoding PLIN2), and Plin3 (encoding PLIN3) were evaluated. There were no changes in Gapdh and Rps16 mRNA levels between groups and the test mRNA levels were adjusted to Gapdh. Primer information is listed in Total RNA was isolated from mouse liver using Trizol kit according to the manufacturer’s instruction. To reduce genomic DNA contamination, total RNAs were treated with ribonuclease-free deoxyribonuclease I  for 1 h at 37°C. 1 μg RNA was used to synthesize the first strand cDNA by reverse transcription kit. qPCR was carried out using SYBR GREEN as an indicator using Bio-Rad real time PCR system. The standard curve method was adopted as previously described . The Ct On the last day of the experiment, the mice were fasted for 6 h (with free access to drinking water), and then blood and tissues were sampled. Blood samples were collected. The blood sample was centrifuged at 5000 rpm for 10 min to collect the serum, and the serum was frozen at -40°C until use. According to standard clinical protocols, the levels of serum glucose, low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol, and triglyceride (TG) were measured using Hitachi 7600 biochemical analyzer . In addition, serum insulin and corticosterone levels were evaluated using the ultrasensitive mouse insulin ELISA kit  and the commercially available corticosterone ELISA kit  according to the manufacturers’ instructions.Total hepatic protein extract was obtained as previously described . The proBefore hematoxylin and eosin (HE) staining, the livers of KM mice were fixed in 10% formalin, dehydrated, and embedded in paraffin, as previously described . In briepost hoc Turkey’s test for diet and drug using GraphPad . Data in untreated HFD mice was compared to the results in STD control mice; data in treated HFD mice were compared to the results in untreated HFD mice. Statistical significance was set at p < 0.05.The data are expressed as mean ± SEM (standard errors). A mixed multiple regression model for repeated measurement was used to compare body weight, plasma insulin, and glucose resistance by SPSS . In additional to data that was mentioned above, the other results were subjected to two–way ANOVA and then According to our previous report  Liang e, a serie50 value of WZS08 by inhibiting 11β-HSD1 activity in all three species, and we demonstrated that WZS08 is a potent inhibitor of 11β-HSD1 with IC50 in the nanomolar range  and human (IC50 of 621 nM) 11β-HSD1.Our previous study showed that 5-bis-(2-bromo-benzylidene) cyclopentanone, an analog of WZS08, had ICectively . In the 11β-HSD1 converts cortisone or 11DHC (inactive) to cortisol or CORT (active) in the liver and adipose tissue in humans or rodents (rats and mice), respectively . Our datWZS08 treatment for three months resulted in a significant decrease in the mean liver fat content of HFD-induced NAFLD mice, reaching the level similar to normal chow treatment (C group) by the highest dose (4 mg/kg). We found that at doses of 2 and 4 mg/kg, WZS08 significantly decreased liver fat content after inhibiting 11β-HSD1 . We examAlthough blood glucose in HFD-treated mice did not show significant changes by oral administration of WZS08, the result indicates that WZS08 improved glucose tolerance and insulin sensitivity as judged by serum insulin levels  and HOMAThe immune response has been shown to be involved in the pathogenesis of NAFLD. Local signals related to microbial balance, bacterial translocation or signals derived from adipose tissue or the intestinal tract activate immune cells and recruit more immune cells into the liver. These effects result in inflammatory responses, which may lead to cell damage and death, thereby promoting NAFLD disease progression . It is win vitro NAFLD models indicate that PLIN2 is associated with NAFLD (Plin2 expression to prevent the formation of NAFLD. In fact, WZS08 reduced liver fat content (Acs2 was significantly down-regulated (Acs2 expression and its protein level were significantly reduced in the HFD-induced NAFLD mouse model (Recent studies have shown that PLIN2 and PLIN3 are involved in the formation of lipid droplets and are involved in the pathophysiology of NAFLD . In partth NAFLD . Whole-bth NAFLD  or liverth NAFLD  of PLIN2 content  and lipi content . This ef content  and feca content  did not egulated . Indeed,se model , while Wse model .In conclusion, we synthesized ten benzylidene cyclopentanone derivatives and found that WZS08 was the most potent selective inhibitor of 11β-HSD1 in rats, mice and humans. WZS08 can effectively prevent NAFLD in HFD-induced mouse models.


Saccharomyces cerevisiae, MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant met17 yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH. Exposure to dimethyldiselenide (DMDSe), a direct precursor of MeSeH, caused an endoplasmic reticulum (ER) stress, as evidenced by increased expression of the ER chaperone Kar2p. Mutant strains (∆ire1 and ∆hac1) unable to activate the unfolded protein response were hypersensitive to MeSeH precursors but not to selenomethionine. In contrast, deletion of YAP1 or SKN7, required to activate the oxidative stress response, did not affect cell growth in the presence of DMDSe. ER maturation of newly synthesized carboxypeptidase Y was impaired, indicating that MeSeH/DMDSe caused protein misfolding in the ER. Exposure to DMDSe resulted in induction of the expression of the ER oxidoreductase Ero1p with concomitant reduction of its regulatory disulfide bonds. These results suggest that MeSeH disturbs protein folding in the ER by generating a reductive stress in this compartment.Methylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in  During the last decades, extensive research has been focused on the identification of selenium (Se) compounds exhibiting cancer chemo-preventive properties . Apart f2Se), derived mainly from inorganic sources, and methylselenol (CH3SeH and MeSeH), derived from organic compounds, such as methylselenic acid (MSeA) and dimethylselenide (DMDSe), have been shown to be crucial for the anticancer properties of Se [Two key metabolites of Se, hydrogen selenide  and DMDSe in revisiae . We showmet17 S. cerevisiae strain. Inside cells, both Se compounds can be reduced to MeSeH through nonenzymatic and enzymatic reactions [We, first, determined the concentration of MeSeSG or DMDSe necessary to inhibit the growth of BY4741, a eactions . Exposurmet17 cells, we used chromosomally integrated GFP-tagged constructs to study the expression of specific targets of transcription factors controlling stress responses, in cells exposed to DMDSe. We used SSA4, TSA1 and SOD1 and KAR2 as fluorescent reporters for heat-shock, oxidative stress or ER stress, respectively. SSA4 encodes a chaperone belonging to the HSP70 family of heat-shock proteins. Ssa4p expression is not detectable in normal growth conditions, but is upregulated by the transcriptional activator Hsf1p upon heat shock [To gain insights into the mechanisms of MeSeH toxicity in at shock . The thiat shock , requireat shock . To moniat shock .2O2 or 5 mM DTT were used as positive controls for heat-shock, oxidative stress or ER stress, respectively. DMDSe failed to induce a heat-shock response in contrast to a temperature shift to 42 °C, indicating that protein homeostasis in the cytosol was not significantly disturbed by DMDSe. The expression of Tsa1p and Sod1p were slightly induced by exposure to DMDSe (1.3–1.5-fold) but less efficiently than by exposure to 4 mM H2O2 (2.1–2.4-fold). The Kar2p-sfGFP signal increased 2-fold after a 2 h treatment with DMDSe or nearly 3-fold with 5 mM DTT, suggesting that DMDSe caused ER stress. Since triggering UPR signaling in yeast strictly requires Ire1p, the UPR cannot be induced in ∆ire1 cells. Our results show that the induction of Kar2p by DMDSe or DTT was dependent on the presence of Ire1p, since no change in Kar2p-sfGFP fluorescence intensity was observed in cell extracts of a ∆ire1 strain , a protein requiring formation of five disulfide bonds for proper folding and ER to vacuole transport. Cpy1p is produced as a 67 kDa precursor in the ER (p1 form). After folding and formation of disulfide bonds, it is glycosylated and processed to the Golgi  followed by transport to the vacuole where cleavage of the propeptide results in the mature 63 kDa form of the protein (m form). Cpy1p was shown to be retained in the ER of DTT-treated cells due to a defect in the formation of disulfide bonds . We follDisulfide bonds are formed in the ER by dithiol/disulfide transfer reactions with the oxidized form of protein disulfide isomerase Pdi1p, which in turn is oxidized by the flavoprotein ER oxidase Ero1p . Ero1p cERO1 by the UPR. When DMDSe was removed, Ero1p regulatory bonds reformed within 20–40 min ) was prepared as a 20 mM stock solution in 100% ethanol and stored at −20 °C. MeSeSG was prepared according to . L-SeMetS. cerevisiae strains used in this study are derived from BY4741 (MATa his3∆1 leu2∆0 met15∆0 ura3∆0). The parental strain and all the single mutants were obtained from Euroscarf . The BY4741 strains containing GFP-tagged constructs of SSA4, TSA1 and SOD1 were purchased from ThermoFisher Scientific . Strains BY4741 Kar2p-sfGFP::HIS, BY4741 ire1Δ::KanMX4 Kar2p-sfGFP::HIS and BY4741 UPR-mCherry::URA were provided by P. Lajoie . The plasmid pAF84 (2 µ URA3-ERO1-myc) was obtained from C. Kaiser  and is described in [w/v) yeast nitrogen base , 2% (w/v) glucose and 80 mg/L of adenine, uracil and all amino acids (160 mg/mL leucine) except methionine and cysteine and was buffered at pH 6.0 by the addition of 50 mM MES-NaOH. Standard synthetic defined (SD) medium contained 0.67% (w/v) yeast nitrogen base, 2% (w/v) glucose and 50 mg/L of histidine, leucine and lysine and was buffered at pH 6.0 by the addition of 50 mM MES-NaOH.The ribed in . Standar600 in the same medium and left to grow for 2 h, after which DMDSe or MeSeSG were added. Cell growth was monitored by measuring the OD600 at various times. For stress tolerance analysis of deletion mutants, cells grown overnight at 30 °C in SC medium supplemented with 100 µM methionine were diluted in water to obtain samples with optical densities of 0.5, 0.05, 0.005 or 0.0005 at 600 nm. Five microliters of each sample were spotted onto SC + 100 µM methionine agar plates containing 25 µM SeMet, 10 mM DTT or 100 µM MeSeSG. Plates were analyzed after incubation for 2 days at 30 °C.For growth inhibition assays, cells were grown overnight at 30 °C in SC medium supplemented with 100 µM methionine. Exponentially growing cells were diluted to 0.1 OD600 of 0.5 in SC medium supplemented with 100 µM methionine followed by exposure to 5 mM DTT or to 50 µM DMDSe for 75 min. Cells were mounted on glass slides covered with a thin layer of 1% (w/v) agarose. Confocal images were acquired at room temperature using a commercial confocal laser scanning microscope  equipped with an inverted frame (Leica), a high NA oil immersion objective , and a white light laser . For the acquisitions we used the following settings: the excitation wavelength was set to 488 nm, the fluorescence was collected in the 500–550 nm band, the lateral pixel size was set to 92.35 nm, the line frequency to 400 Hz and the laser power to 16%.BY4741 cells expressing Kar2-sfGFP were grown at 30 °C to an OD600 of 0.5 in SC medium + 100 µM methionine, followed by 2 h exposure to 100 µM DMDSe, 4 mM H2O2, 5 mM DTT, 25 or 40 µM SeMet or transfer to 42 °C. Whole cell extracts from 1 to 3 OD600 units were prepared in 100 mM potassium phosphate, pH 7.4, 10 mM MgCl2, 100 mM KCl, 10 mM 2-mercaptoethanol, by vortexing cells at 4 °C for 10 × 30 s in the presence of an equal volume of glass beads (500–750 µm). After centrifugation at 10,000× g for 10 min, the supernatant was recovered and the optical density at 280 nm was determined. GFP fluorescence was recorded at 508 nm in a Jasco FP-8300 spectrofluorometer using an excitation wavelength of 487 nm (bandwidth 2.5 nm). Cells expressing UPR-mCherry were grown in SC medium without uracil supplemented with 100 µM methionine and exposed to 0, 2.5, 10, 25, 50 or 100 µM DMDSe for 2 h. GFP fluorescence in whole cell extracts was recorded at 610 nm using an excitation wavelength of 580 nm (bandwidth 2.5 nm).Cells expressing GFP constructs were grown at 30 °C to an OD600 of 0.5 in SC medium + 100 µM methionine. The culture was split in three and incubated with nothing, 6 mM DTT or 100 µM DMDSe at 30 °C for 20 min, harvested by centrifugation and resuspended at 5 OD600 units/mL in 0.8 mL SC medium lacking methionine. After a further incubation of 15 min, cells were pulse-labeled with 100 µCi (25 µCi/OD600) Express35S Protein Labelling Mix  for 10 min at 30 °C. Chase was performed by addition of 1 mM methionine and 1 mM cysteine. Cells (2 OD600 units) were collected in 20 mM NaN3, incubated at 4 °C for 10 min, resuspended in 200 µL of lysis buffer  and protease inhibitors ) and lyzed by vortexing at 4 °C for 10 × 30 s in the presence of an equal volume of glass beads. After 3 min incubation at 92 °C, extracts were diluted to 1 mL in IP buffer . Proteins were immunoprecipitated with polyclonal anti-CPY antibodies in the presence of protein A sepharose magnetic beads  for 3 h at 4 °C on a rotative wheel. The beads were washed 4 times with 500 μL of IP buffer without BSA, resuspended in 15 μL of Laemmli buffer and incubated at 94 °C for 5 min. Samples were resolved by SDS-PAGE (8%), and analyzed with a Typhoon imager .For carboxypeptidase Y maturation assays, BY4741 cells were grown at 30 °C to an OD600 of 1 in SD medium + 100 µM methionine. The culture was split in three and incubated with nothing, 2 mM DTT or 100 µM DMDSe at 30 °C for 30 min. Cells (10 OD600 units) were lyzed by vortexing at 4 °C for 10 × 30 s in the presence of an equal volume of glass beads in 10% (w/v) TCA at 4 °C. Proteins were collected by centrifugation, washed once with chilled acetone and resuspended in 50 μL of non-reducing Laemmli buffer containing 25 mM IAA. Samples were incubated 30 min at 37 °C, followed by 2 min at 90 °C. For reduced control samples, 20 mM DTT was added to an aliquot of the sample before heating at 90 °C. Samples were resolved by nonreducing SDS-PAGE (7.5%) and Ero1p was detected by Western blotting with monoclonal anti-myc antibodies.To analyze the redox state of Ero1p, BY4741 cells containing the plasmid pAF84 were grown at 30 °C to an OD


CMTM7 is a tumor suppressor that positively regulates EGFR degradation by promoting Rab5 activation, and plays a vital role in tumor progression. Rab5 forms complexes with Beclin1 and VPS34, and acts in the early stage of autophagy. However, the affects of CMTM7 on autophagy and its mechanism are still unclear.The effect of CMTM7 on autophagy induction was confirmed by western blotting, confocal microscopy and transmission electron microscopy. Co-immunoprecipitation was used to analyse the interaction of CMTM7 with autophagy initiation complex and Rab5. The xenograft model in nude mice was used to elucidate the function of CMTM7 in tumorigenicity and autophagy in vivo.In this study, we first demonstrated that CMTM7 facilitated the initiation of autophagosome formation, which consequently promoted the subsequent multistage process of autophagic flux, i.e. from autophagosome assembly till autolysosome formation and degradation. Confocal and co-immunoprecipitation showed that CMTM7 interacted with Rab5, VPS34, Beclin1, and ATG14L, but not with ULK1, UVRAG and LC3B. CMTM7 also increased the activity of ATG14L-linked VPS34 complex and its association with Rab5. Both in vitro and in vivo experiments demonstrated that knockdown of CMTM7 enhanced tumor growth by impairing autophagy.These findings highlighted the role of CMTM7 in the regulation of autophagy and tumorigenicity, revealing it as a novel molecule that is associated with the interaction of Rab5 and ATG14L-Beclin1-VPS34 complex.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-021-00720-3. CKLF and CMTM1-8. CMTM7, located on chromosome 3p22.3, is a tumor suppressor gene that is downregulated or absent in many cancer types [such as esophageal squamous cell carcinoma (ESCC) and non-small cell lung cancer (NSCLC)] due to promoter hypermethylation and loss of heterozygosity. CMTM7 inhibits the growth of ESCC and NSCLC cells by suppressing epidermal growth factor receptor-protein kinase B (EGFR-AKT) signaling [Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing proteins (CMTMs) is a family of proteins that structurally and functionally intermediate between classical chemokines and the transmembrane-4 superfamily –3. In huignaling . Moreoveignaling . These dRab5 is a small GTPase that belongs to the Ras superfamily. It acts as a marker of early endosome and is necessary for the biogenesis of endolysosomal system . SimilarAutophagy is like a double-edged sword that can either promote cancer cell survival or cell death, and could be determined by the level of autophagic flux . Basal aInitiation of autophagosome biogenesis is done by UNC-51-like kinase (ULK) complex, and is negatively regulated by mammalian target of rapamycin complex 1 (mTORC1) . InactivRab5 plays an important role in autophagy by inhibiting mTORC1 activation  or formiD60K and GFP-CMTM7 plasmids were also constructed in our laboratory. All plasmids were confirmed by DNA sequencing. siRNAs targeting Rab5 and VPS34 were designed and synthesized by GenePharma Co., Ltd. . The siRNA sequences were as follows: si-VPS34: 5′-GUGUGAUGAUAAGGAAUAUTT-3′, and negative control (si-NC): 5′-UUCUCCGAACGUGUCACGUTT-3′. Inhibition of autophagy was achieved by treating cells with 10 mM of 3-methyladenine  or 25 mM of chloroquine . Other reagents used in this study were as follows: DAPI , Rapamycin (Selleckchem), and DMSO .Human HeLa and A549 cells were purchased from the American Type Culture Collection (ATCC) and grown in RPMI-1640 medium supplemented with 10% fetal bovine serum . The stably transfected GFP-LC3B HeLa cell line was obtained as a gift from Yingyu Chen . Lentivirus-mediated shRNA knockdown of CMTM7 in A549 cells was done as described previously [Thr389, anti-ATG12, anti-4E-BP1, anti-phospho-4E-BP1Thr37/46, anti-AMPKα, anti-phospho-AMPKαThr172, anti-mTOR, anti-phospho-mTORSer2448, anti-ERK, anti-phospho-ERKThr202/Tyr204 , anti-LC3B, anti-β-actin , and anti-CMTM7 rabbit polyclonal antibody . The protein bands were visualized by using a DyLight800-conjugated secondary antibody, and the signals were detected by an Odyssey Infrared Imager .Co-immunoprecipitation assays were performed with control and CMTM7-myc-overexpressing HeLa cells. Cells were harvested after transfection for 36 h in a buffer containing 20 mM Tris–HCl, at pH 8.0, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 5 μg/ml pepstatin, and 1% protease inhibitor cocktail . Protein concentrations were determined by using BCA protein assays . Whole-cell lysates (1000 μg) were incubated with the indicated antibodies for 12 h at 4 °C and further incubated for another 2 h after adding pre-equilibrated protein-G Sepharose beads . The sepharose beads were then washed thrice with the same buffer at 4 °C. The beads were collected by centrifugation, and then resuspended in 2 × SDS loading buffer and heated to 100 °C for 5 min. The whole-cell lysates were then fractionated using 10% or 15% SDS–PAGE gels and then electrotransferred onto polyvinylidene difluoride membranes . Western blotting was performed according to the standard protocol as previously described [Cells were incubated with 50 nM LysoTracker Red  for 20 min at 37 °C, washed with PBS, fixed and permeabilized with 3% paraformaldehyde containing 0.1% Triton X-100 for 30 min at 4 °C. They were then washed with PBS and stained with 4′,6-diamidino-2-phenylindole   for 10 min before being imaged with a TCS-SP laser-scanning confocal microscope . The number of GFP-LC3B puncta per 20 cells was assessed, and statistical data were obtained from 3 independent experiments.6 cells in 100 μL PBS were injected subcutaneously into the right axilla of nude mice (four mice per group). Tumor diameters were measured with a caliper every 3 days, and tumor volumes were calculated according to length × width2 × 0.5. The mice were sacrificed after 4 weeks, when their tumors were dissected, weighed and fixed for immunohistochemistry and transmission electron microscopy analysis.All protocols regarding animals were reviewed and approved by the institutional Animal Research Ethics Board of Jilin University. Female BALB/c nude mice  were maintained in a germ-free environment in the animal facility. The tumorigenesis assay was performed as previously described . Briefly2O2 for 10 min, washed with PBS, blocked with 10% normal goat serum for 30 min, and then incubated with anti-Ki67 (1:800)  at 4 °C overnight. After being washed, the sections were stained with reagents of the Catalyzed Signal Amplification System Kit . Photos were acquired with a Nikon E800 camera.Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded tumor tissues as previously described . BrieflyA549 cells or tumor tissues were initially fixed in 0.1 M sodium phosphate buffer containing 3% glutaraldehyde (pH 7.4) at 4 °C and then fixed in 0.1 M sodium phosphate buffer containing 1% OsO4 (pH 7.2) for 2 h. The cells and tissues were dehydrated in an ascending series of acetone, embedded using an Ultracut microtome  and sliced into 60 nm sections. Ultrathin sections were stained with uranyl acetate and lead citrate before being observed under a transmission electron microscope  at an accelerated voltage of 75 kV.Total amount of PI(3)P was examined in a quantitative and competitive ELISA format assay according to the manufacture (Echelon Biosciences K-3000). Briefly, VPS34 containing complex was enriched by anti-ATG14L antibody and then mixed with phosphatidyl inositol (PI) substrates in an appropriate reaction buffer. After the PI3K reactions were complete and quenched, reaction products were diluted and added to the PI(3)P-coated microplate for competitive binding to a PI(3)P detector protein. The amount of PI(3)P detector protein bound to the plate was determined through colorimetric detection.t-tests in Prism 5.0 . Differences were considered significant if P < 0.05.The data are expressed as the mean ± s.d., and statistical analyses were performed using two-tailed Student's Two ubiquitin-like conjugation systems are mostly involved in elongation and closure of phagophore. The first involves covalent conjugation of ubiquitin-like ATG12 to ATG5. The ATG12-ATG5 conjugates are localized onto the phagophore assembly site (PAS) and dissociate after autophagosome formation. The second involves conjugation of phosphatidylethanolamine (PE) to microtubule-associated protein 1 light chain 3-I  to generate lipidated form of LC3, LC3-II, which in turn specifically targets ATG12-ATG5-associated phagophores by association with autophagosomes even after fusion with lysosomes , 26. We To determine the physiological effects of CMTM7 on autophagy regulation, experiments in stable CMTM7 knockdown A549 cells were conducted by using shRNA as identified in our previous study [According to the above data, CMTM7 promoted autophagic flux from autophagosome biogenesis to autolysosome formation and degradation, and immunofluorescence analyses were performed further to determine the intracellular distribution of GFP-CMTM7 in the regulation of autophagy. The results revealed that GFP-CMTM7 was co-localized with phagophore marker WIPI-1, but not with elongation marker ATG5, maturation marker LC3B, and lysosomal marker LysoTracker Red P), promoting nucleation of phagophore and recruitment of autophagy downstream effectors, such as DFCP1 and WIPI-1 . To inveThe above results suggested that CMTM7 showed association with VPS34-Beclin1-ATG14L complex and enhanced VPS34 kinase activity. As reported previously that CMTM7 can increase the activity of Rab5, which interacts with VPS34 to regulate VPS34 activity and autophagy, we hypothesized that the promoting effects of CMTM7 on VPS34 activity might involve Rab5. Initially, the interaction between CMTM7 and Rab5 was assessed by coIP assays. CMTM7-myc plasmid was transfected into HeLa cells, which were lysed after transfection for 48 h and immunoprecipitated by anti-myc and anti-Rab5 antibodies, respectively. Western blotting showed that CMTM7-myc showed co-precipitation with Rab5 and vice versa (Fig. D60K. This is used to inhibit the phosphorylation of RPS6KB1 and 4E-BP1 through inhibition of MTOR signaling [D60K overexpressing cells. Simultaneously, overexpression of RHEBD60K recovered LC3B lipidation inhibited by CMTM7 knockdown (Fig. CMTM7 has been identified as an activator of Rab5 . We hypoignaling . As showCMTM7 as a tumor suppressor demonstrated suppression of tumorigenicity  and promTo confirm the promoting role of CMTM7 in autophagy and its tumor-suppressor function in vivo, CMTM7 knockdown and control A549 cells were subcutaneously injected into the right axilla of BALB/c nude mice. On day 28 after inoculation, the mice were euthanized and their tumors were removed and then were photographed (Fig. CMTM7 belongs to CMTM family of proteins, and comprises of a MARVEL domain. The MARVEL domain-containing proteins participate in a variety of cellular processes, such as tumorigenesis and transport vesicle biogenesis , 31. OurIt has been demonstrated that several tumor suppressor proteins, such as BH3-only proteins, DAPK1, PTEN, TSC1 and TSC2, can induce autophagy , 33. OurIt has been reported that by interacting with and activating PI3-kinase VPS34, Rab5 becomes a part of Beclin1-VPS34 complex, regulating ATG12-ATG5 conjugation in the early stages of autophagosome formation. Rab5 inhibition and loss of activity of VPS34 decreases ATG12-ATG5 conjugation and suppresses autophagy . Our datOn the other hand, overexpression of Rab5 at the initial stage by inhibiting mTOR activation induces autophagy. Besides, hyperactivation of endogenous Rab5 by overexpressing GAPex, which is a guanine nucleotide exchange factor for Rab5, inhibits mTORC1 as well . The reaAccumulating evidence indicate that CMTM7 acts a dual modulator for both class I PI3K and class III PI3K by completely different regulatory patterns. As reported previously, CMTM7 decreases class I PI3K activity by repressing EGFR internalization and trafficking. But CMTM7 increased the activity of ATG14L-linked class III PI3K in this study. Thus, CMTM7 might be considered to play a crucial role as a tumor suppressor by inversely regulating the two types of PI3K. Namely, on one hand, CMTM7 inhibits over-activation of class I PI3K signaling induced by growth factors, such as EGF, and on the other hand, CMTM7 promotes autophagic flux to suppress cell growth. Therefore, through different regulator mechanisms on two classes of PI3K, CMTM7 more effectively exerts tumor-suppressive function.In summary, our results suggested that CMTM7 is mainly involved in the initiation of autophagosome formation through Rab5-mediated functional groups of ATG14L-Beclin1-VPS34 complex and mTOR signaling pathway Fig. . Both inAdditional file 1: Fig. S1. Effect of CMTM7 knockdown on AMPKα phosphorylation. Western blot analysis of total and phosphorylation levels of AMPKα in control and CMTM7 knockdown A549 cells. β-actin was used as an internal standard.


Pleurotus) are widespread and comprise the most commonly cultivated edible mushrooms in the world. Species identification of oyster mushroom spawn based on cultural, morphological, and cultivated characteristics is time consuming and can be extraordinarily difficult, which has impeded mushroom breeding and caused economic loss for mushroom growers. To explore a precise and concise approach for species identification, the nuclear ribosomal internal transcribed spacer (ITS), 28S rDNA, and the widely used protein-coding marker translation elongation factor 1α (EF-1α) gene were evaluated as candidate DNA barcode markers to investigate their feasibility in identifying 13 oyster mushroom species. A total of 160 sequences of the candidate loci were analyzed. Intra- and interspecific divergences and the ease of nucleotide sequence acquisition were the criteria used to evaluate the candidate genes. EF-1α showed the best intra- and interspecific variation among the candidate markers and discriminated 84.6% of the species tested, only being unable to distinguish two closely related species Pleurotus citrinopileatus and Pleurotus cornucopiae. Furthermore, EF-1α was more likely to be acquired than ITS or 28S rDNA, with an 84% success rate of PCR amplification and sequencing. For ITS and 28S rDNA, the intraspecific differences of several species were distinctly larger than the interspecific differences, and the species identification efficiency of the two candidate markers was worse . In addition, these markers had some sequencing problems, with 55 and 76% success rates of sequencing, respectively. Hence, we propose EF-1α as a possible DNA barcode marker for oyster mushroom spawn.Oyster mushrooms (genus  Pleurotaceae, Agaricales, Basidiomycota), commonly known as oyster mushrooms, are widespread and comprise the most commonly cultivated edible mushrooms in the world. These fungi can also be used for various medicinal, environmental, and biotechnological purposes , 1.25 μl of each primer (10 μmol/L), 0.5 μl of dNTPs (10 mmol/L each), 1.25 μl of DNA template, and 0.25 μl of Taq DNA polymerase (5 U/μl). For ITS, the PCR conditions were an initial step of 5 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at 53°C, and 30 s at 72°C, followed by 10 min at 72°C. For 28S rDNA, the PCR conditions were an initial step of 5 min at 94°C, 10 cycles of 30 s at 94°C, 30 s at 62°C (decreasing 1°C per cycle), 55 s at 72°C, plus 25 cycles of 30 s at 94°C, 30 s at 52°C, 55 s at 72°C, followed by 10 min at 72°C. For EF-1α, the PCR conditions were an initial step of 5 min at 94°C, 10 cycles of 30 s at 94°C, 55 s at 63 or 66°C (decreasing 1°C per cycle), 90 s at 72°C, plus 36 cycles of 30 s at 94°C, 55 s at 53, or 56°C, 90 s at 72°C, followed by 7 min at 72°C. The obtained amplicons were sequenced in both directions using an ABI 3730 XL DNA Sequencer .The genomic DNA of each strain was extracted from mycelium grown on potato dextrose agar (PDA) . The nucThe sequences were aligned using ClustalX 1.81  and manuThe success rates were assessed for the PCR amplification and sequencing of the DNA barcode loci considered for oyster mushroom spawn. The criterion for successful amplification was when a single PCR band was obtained. The criterion for successful sequencing was a high-quality chromatogram. The success rate of PCR amplification multiplied by the success rate of sequencing to determine the overall success rate of PCR amplification and sequencing .Neighbor-joining (NJ) trees inferred from ITS, 28S rDNA, and EF-1α gene sequences were reconstructed using MEGA 5.2  with thePleurotus species , Pleurotus nebrodensis and P. eryngii var. ferulae (0.9%), and Pleurotus fossulatus and P. eryngii var. ferulae and P. eryngii (1.2%), were all extraordinarily small  of the species  according to morphological similarities and mating test  approach combining molecular and phenotype data to continuously and critically revise existing knowledge is necessary to achieve high-quality taxa.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/PZ and TB conceived the project. PZ and S-PJ performed the experiments. PZ, X-HC, H-XD, and X-XG provided the resources. PZ analyzed the data and wrote the manuscript. All authors read 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.


Objective: Our previous study showed that glioma stem-like cells could be induced to undergo dedifferentiation under hypoxic conditions, but the mechanism requires further study. HIF1α and HIF2α are the main molecules involved in the response to hypoxia, and Sox2, as a retroelement, plays an important role in the formation of induced pluripotent stem cells, especially in hypoxic microenvironments. Therefore, we performed a series of experiments to verify whether HIF1α, HIF2α and Sox2 regulated glioma cell dedifferentiation under hypoxic conditions.Materials and methods: Sphere formation by single glioma cells was observed, and CD133 and CD15 expression was compared between the normoxic and hypoxic groups. HIF1α, HIF2α, and Sox2 expression was detected using the CGGA database, and the correlation among HIF1α, HIF2α and Sox2 levels was analyzed. We knocked out HIF1α, HIF2α and Sox2 in glioma cells and cultured them under hypoxic conditions to detect CD133 and CD15 expression. The above cells were implanted into mouse brains to analyze tumor volume and survival time.Results: New spheres were formed from single glioma cells in 1% O2, but no spheres were formed in 21% O2. The cells cultured in 1% O2 highly expressed CD133 and CD15 and had a lower apoptosis rate. The CGGA database showed HIF1α and HIF2α expression in glioma. Knocking out HIF1α or HIF2α led to a decrease in CD133 and CD15 expression and inhibited sphere formation under hypoxic conditions. Moreover, tumor volume and weight decreased after HIF1α or HIF2α knockout with the same temozolomide treatment. Sox2 was also highly expressed in glioma, and there was a positive correlation between the HIF1α/HIF2α and Sox2 expression levels. Sox2 was expressed at lower levels after HIF1α or HIF2α was knocked out. Then, Sox2 was knocked out, and we found that CD133 and CD15 expression was decreased. Moreover, a lower sphere formation rate, higher apoptosis rate, lower tumor formation rate and longer survival time after temozolomide treatment were detected in the Sox2 knockout cells.Conclusion: In a hypoxic microenvironment, the HIF1α/HIF2α-Sox2 network induced the formation of glioma stem cells through the dedifferentiation of differentiated glioma cells, thus promoting glioma cell chemoresistance. This study demonstrates that both HIF1α and HIF2α, as genes upstream of Sox2, regulate the malignant progression of glioma through dedifferentiation. In their opinion, two reasons led to this: one was that GSCs proliferated faster under hypoxic conditions than differentiated cancer cells, and the other was the dedifferentiation process that differentiated glioma cells undergo in a hypoxic environment. We agree with the opinion of BarGlioma stem cells (GSCs) were identified many years agoInduced pluripotent stem cells (iPSCs) have been successfully generated through reprogramming Sox-2, Oct-4, Klf-4, Nanog, lin28A and lin28B in cells, such as human dermal fibroblastshttp://cancergenome.nih.gov/, https://www.gtexportal.org/, https://portals.broadinstitute.org/ccle, and http://www.cgga.org.cn, respectively. The TCGA and CGGA databases were used to analyze the expression of HIF1α, HIF2α, Sox2, Oct4, KLF4, Nanog, Lin28A and Lin28B through Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/detail.php) and CGGA . The TCGA and GTEx databases were used to analyze the correlations among HIF1α, HIF2α and Sox2 through the dplyr, tibble and ggpolt2 packages in R.The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), Cancer Cell Line Encyclopedia (CCLE) and Chinese Glioma Genome Atlas (CGGA) databases can be found at 2. The surgical waste obtained from patients was anonymized. After culturing these cells for approximately 3~5 days, we sorted CD133-CD15- cells by magnetic activated cell sorting (MACS). In brief, cell suspensions were prepared after 0.25% trypsin digestion by resuspension in PBS containing 0.08% EDTA and 0.5% BSA  and then incubated at 4°C for 15 min with polyclonal rabbit anti-human/mouse CD133+ IgGs . The suspension was washed with PBS containing 1% BSA, resuspended in PBSE (108 cells/300 μl), labeled with goat anti-rabbit IgG microbeads , cultured at 10°C for another 15 min, washed and resuspended in 500 μl of PBSE. A cell separation column with a flow resistor was placed in a miniMACS magnet that was fixed on a MACS multistand and flushed with 500 μl of PBSE. The cell suspension was poured into the column reservoir, and unlabeled, nonmagnetic CD133- cells that passed through the column were collected. The above steps were repeated three times to increase the purity of the collected CD133- cells. Identical steps were used to sort CD15- cells from CD133- cells. Sorted CD133-CD15- cells were cultured in DMEM/F12 supplemented with 10% FBS (DMEM/F12+10% FBS) to maintain their differentiation status.GL261 cells were obtained from ATCC, and primary glioma cells were isolated from surgical waste. The primary glioma tissue samples were initially minced and digested with 0.25% trypsin  and 10 U/ml DNase I  at 37°C for 45~60 min. ACK lysis buffer  was used to lyse red blood cells. The suspension was washed with PBS two times and filtered through a 100-μm cell strainer. The obtained cells were cultivated in DMEM/F12  supplemented with 10% fetal bovine serum  to maintain growth in 21% O2, and the other was incubated in 21% O2. Sphere formation was observed, imaged and recorded on days 0, 3, 7, 14 and 21.Suspensions were made from glioblastoma multiforme (GBM) and GL261 cells in DMEM/F12+10% FBS with a density of 1,500 cells/1 ml medium. One microliter of cell suspension was placed into each well of 96-well plates that contained 170 μl of serum-free DMEM/F12. Six 96-well plates were utilized for each cell line, and two groups for each cell line were randomly grouped with three 96-well plates. One group was incubated in 1% O2 for 72 h were evaluated for HIF1α, HIF2α and Sox2 expression using immunofluorescence. Cells were fixed with 4% paraformaldehyde at 4°C for 30 min, washed with PBS containing 0.5% Triton X-100 , and blocked with 10% serum. The cells were incubated at 4°C for 24 h with primary antibodies against HIF1α , HIF2α , SOX-2 , CD133  and CD15 . The cells were then washed with PBS three times and incubated for 2 h with fluorophore-labeled secondary antibodies , and images were acquired using a laser scanning confocal microscope .New spheres formed by single glioma cells, GBM cells or GL261 cells cultured in 1% O-CD15- cells cultured in 21% O2 (control group) or 1% O2 for 72 h were evaluated for HIF1α, HIF2α, SOX-2, CD133 and CD15 expression by western blotting. Total protein was prepared using prechilled RIPA buffer , and the isolated proteins were subjected to SDS-PAGE, followed by transfer to nitrocellulose membranes. Then, the membranes were blocked with 5% nonfat milk and incubated with primary antibodies against HIF1α , HIF2α , SOX-2 , CD133  and CD15  at 4°C overnight. HRP-labeled secondary antibodies  were added to the membranes and incubated at 37°C for 1 h. Enhanced chemiluminescence was utilized for visualization.New spheres formed by single glioma cells and CD1332 for 12 h were used to detect RNA expression by RT-qPCR. Total RNA was prepared using TRIzol  according to the manufacturer's instructions. The melting conditions were 94°C for 5 min, the denaturing conditions were 94°C for 30 s, the annealing conditions were 57°C for 30 s, and the extension conditions were 72°C for 30 s. These conditions were used for a total of 40 cycles. The primer sequences are presented in Supplementary New spheres formed by single glioma cells and control cells in 21% O2 or 1% O2 for 48 h, and temozolomide (TMZ) (400 μM) was added to the culture medium for an additional 72 h before cell apoptosis was detected using FCM. Cells were digested with 0.25% trypsin and prepared as a single-cell suspension in PBS at a density of 1×106 cells/ml, and 100 μl of suspension was added to an Eppendorf tube. The cells were centrifuged, and 195 μl of 0.05% trisodium citrate-dihydrate and 5 μl of Annexin V-FITC were added to the cell suspension and incubated in the dark for 15 min. Then, 190 μl of 0.05% trisodium citrate-dihydrate and 10 μl of propidium iodide (PI) were added to the centrifuged cell suspension and incubated for 10 min in the dark. Samples were analyzed using a flow cytometer .Cells were exposed to 21% O4) were seeded into mouse brains, and the mice were housed in 10% O2 for 14 days. Tumor and normal tissues were collected to analyze the expression of HIF1α, HIF2α, Sox2, CD133 and CD15 using immunohistochemistry (IHC). The detailed steps for IHC are described below.Primary glioma cells , HIF2α , SOX-2 , CD133  and CD15 , and incubated at 4°C overnight. The slides were washed with PBS buffer again and covered with an HRP-labeled polymer for 2 h. Then, the tissue samples were covered with a DAB chromogen solution and incubated for ~1 min to allow staining reactions to occur, and images were acquired.http://crispr.mit.edu), annealed and cloned into the lentiCRISPRv2 vector ; the sgRNA oligonucleotide sequences are listed in Supplementary 2 before apoptosis was detected. The detailed steps were described earlier in the manuscript.HIF1α, HIF2α and Sox2 sgRNAs for plasmid constructs were designed based on the CRISPR design program (4) were injected into the brains of mice, and the mice were housed in 10% O2. Next, these mice were randomly grouped . TMZ (2 mg/kg) was injected into the enterocoelia each day from day 3 to day 17. Magnetic resonance imaging (MRI) was used to measure tumor volume on day 21 for five mice, and the other mice were used to record survival time.Next, the cells described above , and we used SPSS 19.0 for statistical analysis. The significance of differences between two groups was determined using Student's t test, and one-way analysis of variance (one-way ANOVA) was utilized for comparing at least three groups. The log-rank test was performed for survival analysis. Pearson's correlation was used to analyze the correlations among HIF1α, HIF2α and Sox2. -CD15- GL261 and primary glioma cells were sorted by MACS, and just one cell was plated in each well of 96-well plates and incubated in 21% O2 or 1% O2 for 21 days. We found that most of the cells died in 21% O2; however, the cells in 1% O2 appeared to be in a state of suspended spheres, and the sphere formation rate was over 95% after hypoxic exposure for 21 days according to the statistical analysis  was added to the culture medium for an additional 72 h, and cell apoptosis was detected using FCM. The results showed that there were higher early, late and total apoptosis rates for the cells cultured in 21% O2 than for those cultured in 1% O2 . Vector, HIF1α-knockout (ko), HIF2α-ko and HIF1α/HIF2α-ko cells were cultured in 1% O2 for 72 h, and the results showed that CD133 and CD15 expression decreased significantly after HIF1α or HIF2α knockout and that both HIF1α and HIF2α knockout cells presented the lowest expression of CD133 and CD15  to the culture medium for an additional 72 h before detecting apoptosis, and the results showed that there was more late and total apoptosis after HIF1α or HIF2α knockout alone. However, after HIF1α and HIF2α were knocked out simultaneously, the apoptosis rates for early, late and total apoptosis were significantly higher than those of the other groups  into mouse brains and treated them with TMZ (2 mg/kg). The results showed that tumor volume and weight were decreased with HIF1α or HIF2α knockout, and the group with simultaneous HIF1α and HIF2α knockout presented the smallest tumor volume and weight (Fig HIF1α and HIF2α were successfully knocked out in CD1332 for 72 h, and the spheres formed in 1% O2 by single glioma cells also highly expressed Sox2 (Fig Previous studies have revealed the important roles of Sox2, Oct4, KLF4, Nanog, Lin28A and Lin28B in cell pluripotencyIn a hypoxic environment, Sox2 was highly expressed. After HIF1α or HIF2α knockout, cells showed reduced expression of Sox2, and after knocking out both HIF1α and HIF2α simultaneously, cells presented the lowest expression of Sox2 Fig A. Then, et alet al found that hypoxia caused increased expression of stem cell markers and promoted tumor formation both in vitro and in vivoet al in vitro studies that showed hypoxia-induced "dedifferentiation" of differentiated glioma cells, which then acquired stemness features- differentiated glioma cells derived from GBM-derived neurospheres can be induced to become CD133+ GSCs in a hypoxic environment-CD15- glioma cells three times and cultured cells under hypoxic conditions without EGF and FGF2. According to our results, GSCs with high expression of CD133 and CD15 could be induced by dedifferentiation of differentiated CD133-CD15- cells in the hypoxic environment, and a relatively low apoptosis rate was observed after hypoxia exposure. In addition, the most significant result was found for sphere formation by single glioma cells in 96-well plates. Each well contained only one cell, and almost every survived cell in the hypoxic environment formed spheres with high expression of CD133 and CD15. Undoubtedly, some cells in the wells were GSCs before hypoxia exposure, but the GSC rate was extremely low, and we sorted CD133-CD15- cells three times through MACS to minimize this rate. Therefore, dedifferentiation indeed occurs in glioma in hypoxic environments. Previous studies have suggested that the response to hypoxia is mainly mediated by HIF1α and HIF2α, which have been found to have great effects on stemnessThere are two kinds of cells in glioma, namely, GSCs and differentiated tumor cells et alNext, how HIF1α and HIF2α induce stemness under hypoxic conditions was analyzed. Sox2, Oct4, KLF4, Nanog, Lin28A and Lin28B promote the formation of iPSCs from human dermal fibroblastsFinally, we have to consider new glioma treatment strategies Fig . PreviouBriefly, this study demonstrates that both HIF1α and HIF2α, as genes upstream of Sox2, regulate the malignant progression of glioma through dedifferentiation. Therefore, we unexpectedly identified both HIF1α and HIF2α as critical targets in glioma. In addition, Sox2, another factor, was studied in this article and found to be an ideal target for glioma treatment because it was highly expressed in glioma but expressed at low levels in normal tissues. Based on our results, we conclude that the dedifferentiation process is induced under hypoxic conditions via regulation by the HIF1α/HIF2α-Sox2 pathway, which provides new ideal targets for glioma treatment.Supplementary figure and tables.Click here for additional data file.