Ds in the nanomolar range. The use of truncated C1q fragments and recombinant mutated C1q allowed to localize more precisely the binding site for LRP1 on the collagen-like regions of C1q (CLRs), nearby the site that is implicated in the interaction with the cognate protease tetramer C1r2s2. This site could be a common anchorage for other ligands of C1q CLRs such as sulfated proteoglycans and Complement receptor type 1. The use of a cellular model, consisting in CHO LRP1-null cells transfected with full-length LRP1 or a cluster IV minireceptor (mini IV) confirmed that mini IV interacts with C1q at the cell membrane as well as full-length LRP1. Further cellular interaction studies finally highlighted that mini IV can endorse the full-length LRP1 binding efficiency for apoptotic cells and that C1q has no impact on this interaction.LRP1 is a large endocytic modular receptor that plays a crucial role in the scavenging of apoptotic material through binding to pattern-recognition molecules. It is a membrane anchored receptor of the LDL receptor family with 4 extracellular clusters of ligand binding modules called cysteine rich complement-type repeats that are involved in the interaction of LRP1 with its numerous ligands. Complement C1q was shown to interact with LRP1 and to be implicated in the phagocytosis of apoptotic cells. The present work aimed at exploring how these two large molecules interact at the molecular level using a dissection strategy. For that purpose, recombinant LRP1 clusters II, III and IV were produced in mammalian HEK293F cells and their binding properties were investigated. Clusters II and IV were found to interact specifically and efficiently with C1q with K C1q is a defense collagen that is known for decades for its implication in the elimination of pathogens or altered-self bodies through the classical cascade of complement. In this context C1q recognizes targets and triggers the complement cascade through activation of an associated protease tetramer C1r2s2. Nowadays, a large body of research also highlights some widely diverse, non-complement related functions of C1q. As examples, C1q can act as an opsonin bridging targets and membrane receptors, C1q is implicated in the modulation of immune cells differentiation and it has an essential role in the enhancement of apoptotic cells phagocytosis . C1q is 1%, 1 cm were respectively for C1q   , CLR 1800; 6.8) , C1r2s2 00; 6.8 ,Full-length LRP1 Myc DDK clone (RC218369) was purchased from Origene. The pET22B-RAP plasmid was kindly provided by Søren Moestrup, Aahrus University, Denmark. pcDNA3.1 mini IV HA-tag was cloned as previously indicated .BamHI restriction site were introduced on the 3′-end of the sequence coding for the signal peptide in order to subclone soluble cluster sequences between BamHI and XhoI sites.The DNA sequence encoding the signal peptide for human LRP1 was inserted in pcDNA3.1/Neo by site directed mutagenesis using the QuickChange II XL kit (Agilent Technologies) according to an optimized procedure . A nucleThe cDNA for each cluster II, III and IV corresponding respectively to the mature LRP1 amino acid fragments 833-1163 (II), 2503-2921 (III), and 3313-3759 (IV) with a supplementary sequence coding for a 7 (cluster III) or 8 His-TAG (cluster II and IV) on the 5′ end, was generated by PCR amplification of full-length LRP1 Myc DDK and cloned in pcDNA3.1/LRP1 signal peptide vector as mentioned above.® 5/50 GL  and elution was achieved through a NaCl gradient (50 - 500 mM). Concentration was finally carried out to reach around 0.5 mg/ml and the buffer changed into 20 mM Tris, 150 mM NaCl, pH 7.5.pcDNA 3.1 plasmids coding for each cluster II, III and IV were transfected in Freestyle HEK293F (293-F) cells using 293fectin, according to the manufacturer’s protocol (Invitrogen) and stabilized by G418 selection (400 μg/ml). Around 500 ml of expression medium were harvested and submitted to two-step purification. First, the medium was dialyzed in 20 mM Tris, 150 mM NaCl, pH 7.5 and loaded on a HiTrap™ Chelating HP column . The fractions containing the clusters were then concentrated/diluted in 50 mM sodium acetate, 50 mM NaCl, 10 mM EDTA, pH 6 before loading on an anion exchange column Mono Q1%, 1 cm at 280 nm calculated using the PROTPARAM program on the Expasy server, and an experimental molecular weight determined by MALDI mass spectrometry. Their A1%, 1 cm at 280 nm and molecular weight were respectively 11.78 and 50,372 for cluster II, 9.96 and 63,734 for cluster III and 10.16 and 62,528 for cluster IV.The concentration of the purified soluble LRP1 clusters was estimated using the absorption coefficient AMass spectrometry analyses were performed on a Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometer , operated in linear positive mode. The proteins (1 mg/ml) were diluted 1:2 to 1:10 in SA matrix [sinapinic acid (Sigma-Aldrich) 10 mg/ml in acetonitrile/water/trifluoroacetic acid [50/50/0.1 (v/v/v)] and 2 μL were deposited directly on the target.Escherichia coli BL21(DE3) using conventional IPTG induction (1 mM) in LB medium for 3 h at 37°C. Bacteria were lysed by sonication in 100 mM NaCl, 20 mM Tris, 10 mM MgCl2, pH 8.5 supplemented with Complete® protease inhibitor cocktail (Roche Diagnostics). The lysate was then purified by nickel-affinity chromatography  followed by gel filtration on Superdex®S75 10/300 . Purification was performed in 100 mM NaCl, 20 mM Tris, pH 8.0. For concentration determination, a molecular mass of 36,440 Da corresponding to the RAP amino acid sequence preceded by a thrombin cleavage site and a 6His-Tag (MHHHHHHLVPRGS … Y) and A1%, 1 cm at 280 nm of 9.26 were calculated by the PROTPARAM program.RAP was overexpressed by pET22B-RAP transformed For all surface plasmon resonance experiments, protein ligands were immobilized on CM5 sensor chips using the amine coupling chemistry according to the manufacturer’s instructions . Immobilizations were performed at 10 µl/min in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005%, surfactant P20, pH 7.4  or the same buffer supplemented with 0.05% surfactant P20 . Regeneration of the surfaces was achieved by 15 μL injections of 1 M NaCl, 10 mM EDTA.RAP interaction with full-length LRP1 and clusters II, III, and IV was determined on a BIAcore 3000 instrument . Full-length LRP1 was diluted at 50 µg/ml in 10 mM sodium acetate pH 3.5 to get an immobilization level of 7,400 RU. Clusters were diluted at 10 µg/ml in 10 mM sodium acetate pH 4.0 (clusters II and III) and 4.5 (cluster IV). For interaction measurements, RAP (ranging from 0.25 to 16 nM) was injected over immobilized clusters II , III  and IV  in 50 mM triethanolamine-HCl (TEA), 150 mM NaCl, 1 mM CaCl2, 0.005% P20, pH 7.4 at 20 µl/min. Kinetic data were analyzed by global fitting to a 1:1 Langmuir binding model of both the association and dissociation phases using the BIAevaluation 3.2 software .Interaction of soluble clusters II, III, and IV with immobilized C1q was also performed on a BIAcore 3000. For that, serum C1q was diluted in 10 mM sodium acetate, pH 5.5 at 23 μg/ml and injected over the CM5 chip to get the immobilization level of 17,500 RU. Cluster II, III and IV (500 nM) interaction was done in 50 mM TEA, 150 mM NaCl, 1 mM CaCl2, 0.005% P20 pH 7.4 at 20 µl/min.Serum C1q, C1q GR, and CLR binding to immobilized clusters II and IV and C1r2s2 competition was performed on a T200 instrument. Both clusters were diluted at 50 µg/ml in 10 mM sodium acetate, pH 4 for immobilization. The interaction of C1q or CLR or GR on immobilized LRP1 cluster II  or cluster IV  was measured in 50 mM Tris, 150 mM NaCl, 2 mM CaCl2, 0.05% P20, pH 7.4 at 30 µl/min with association and dissociation of 180 s. For C1r2s2 competition, C1q was pre-incubated (15 min at 25°C) with C1r2s2 before injection. The equilibrium dissociation constants (KD) for serum C1q binding to clusters II and IV were determined by injection of concentrations ranging from 0.125 nM to 8 nM. The KDs were calculated from measured binding levels at equilibrium (Req) by fitting plots of Req versus concentration using steady state analysis .KDs for the interaction of clusters II and IV with immobilized serum C1q, rC1q WT, and rC1q ABCDetermination of the  was performed on a T200 instrument on immobilized serum C1q , recombinant C1q  and C1q mutant LysA59Ala/LysB61Ala/LysC58Ala . All C1q samples were diluted in 10 mM sodium acetate, pH 5. The interaction of clusters II and IV was measured by injection of indicated concentrations  were calculated as mentioned above for serum C1q interaction with immobilized clusters.®. CHO-null and Jurkat cells were respectively cultured in DMEM-F12 (Dulbecco’s modified Eagle’s medium), or in RPMI , supplemented with 10% (v/v) Fetal Bovine Serum (FBS) at 37°C with an humidified atmosphere and 5% CO2. For CHO clones expressing LRP1 receptors (full-length or mini IV), the media were supplemented with G418 (geneticin sulfate) at 400 µg/ml.LRP1-deficient CHO cells called in this study CHO-null cells  were obt6 cells per dish, in 1.5 ml of culture medium without G418, to reach 70% confluency at transfection. Four µg of plasmid DNA and 10 µl of lipofectamine 2000 were separately diluted in 250 µl of OptiMEM and mixed. After 15 min incubation, the mix was added to each well, and the cells were further incubated for 72 h at 37°C before adding 400 µg/ml of G418 for selection.Plasmid DNAs were transfected into CHO-null cells by lipofectamine 2000 following manufacturer’s instructions (Invitrogen™). Briefly, 24 h before transfection, cells were plated in 35 mm dishes (or 6-well plates) at 0.5 10Monoclonal cell populations were isolated and amplified after a series of 3 limit dilutions. Briefly, transfected cells were counted and diluted to inoculate unique cells into a 96-well plate. After 10 to 12 days in culture, full-length LRP1 or cluster IV mini-receptor expression and cell population homogeneity were tested by flow cytometry and immunofluorescence microscopy as detailed below.® Versene buffer and further washed in PBS  supplemented with 1% BSA . For each sample, 1 × 106 cells were incubated on ice for 45 min in 100 µl of anti-CD91-PE  for full-length LRP1 receptor, or 100 µl of anti-HA-PE  for mini IV LRP1 truncated receptor. Cells were then washed twice in PBS 1% BSA before analysis  using the 561 nm excitation and 586(15) nm emission channel (Y1). PE positive populations were estimated after forward scatter (FSC) and side scatter (SSC) gating on the cells. For each condition, at least 20,000 events were analyzed.Adherent CHO-K1 transfected cells were recovered using GibcoNaïve or transfected CHO-null cells cultured on coverslip were fixed in 4% (w/v) paraformaldehyde (PFA) and processed for immunofluorescence using LRP1 full-length or mini IV receptors labeling respectively with anti-CD91-PE  or anti-HA-PE . Alternatively, secondary labeling with Cy3-conjugated anti-mouse antibody (dilution 1/250 in PBS 1% BSA) of anti-CD91  and of anti-HA (anti-HA.11 Biolegend 1/1000 in PBS 1% BSA) were used for LRP1 and mini IV receptors, respectively. Coverslips were mounted on slides using Vectashield with DAPI (Vector laboratory). Pictures were acquired with IQ software (Andor™), using a spinning disk confocal microscope (Yokogawa CSU-X1–IX81 Olympus) with an iXon EMCCD camera (Andor™), and the appropriate channels for PE and DAPI visualization.6 cells were incubated 30 min on ice with 8 µg of C1q in 100 µl PBS 1% BSA. After 2 washes with 1 ml of PBS 1% BSA, bound C1q was detected by immunostaining using a monoclonal anti C1q antibody  for 45 min on ice, followed by Cy3-conjugated goat anti-mouse antibody (dilution 1/250 in PBS 1% BSA for 30 min on ice). Flow cytometry analyses were performed on a MACSQuant VYB cytometer as already described, using the 561/586(15) nm Y1 channel for Cy3.CHO-null, or expressing either full-length LRP1 or mini IV LRP1 truncated receptors were detached using Versene, washed once in PBS 1% BSA and resuspended in the same buffer. For each condition, 1 × 105 cells per ml culture medium in 12-well plate. PKH67 labeled Jurkat cells were resuspended in complete RPMI medium at 1 × 106 cells per ml and when required, apoptosis was induced by UVB irradiation at 312 nm for 5 min (500 mJ/cm2) in 60 mm cell culture dish (5 ml/dish). 16 h after labeling healthy or late apoptotic Jurkat cells were centrifuged and counted. This treatment yields around 74% of apoptotic Jurkat cells. When required, Jurkat cells (2 × 106 cells) were incubated with 15 µg of C1q in 100 µl DPBS  for 1 h on ice. For interaction tests, 2 × 106 PKH67 labeled Jurkat cells were washed in 1 ml DPBS, resuspended in 1 ml of complete DMEM-F12 and added to the monolayer of CHO-K1 cells labeled with PKH26 in the 12-well plates. After incubation at 37°C , the CHO-K1 cells in a monolayer were washed with 1 ml DPBS to remove the non-attached Jurkat cells. PKH26-labeled CHO-K1 cells decorated with PKH67 labeled Jurkat cell were recovered by gentle scraping or using 100 ml of trypsin solution  before analysis by flow cytometry as described above, using the 561/586(15) nm Y1 channel for PKH26 and the 488/525(50) nm B1 channel for PKH67.The cell-cell interaction assay was performed using flow cytometry of differentially labeled Jurkat and CHO-null cells expressing either none, full-length LRP1, or mini IV receptors. In brief, 24 h before the assay, cells were harvested and labeled using PKH26 for CHO clones or PKH67 for Jurkat cells, according to manufacturer instructions (Sigma-Aldrich). The labeling reaction was stopped after 5 min incubation in the dark by adding pure FBS. After washing, CHO control and clones expressing LRP1 constructs were plated at 2 × 10Materials and Methods. These clusters were designed to get soluble fragments restricted to the CR modules described as functional interacting LRP1 regions. In our study, clusters II, III and IV are therefore respectively composed of 8, 10, and 11 CR modules  (data not shown). Nevertheless, the purification procedure leads to pure secreted fragments as observed by SDS-PAGE analysis . The apparent molecular mass of purified RAP observed by SDS-PAGE analysis , 1.06 ± 0.1 nM (III) and 1.28 ± 0.12 nM (IV)  –26 indicKDs in a sub-micromolar range  or collagen regions (CLR) obtained from purified serum C1q. The results of In order to confirm at the cell surface the results obtained with purified proteins, we expressed full-length LRP1 or the minireceptor IV (mini IV) at the surface of LRP1-null CHO cells and monitored their ability to bind C1q. CHO-null cells were chosen for their non-phagocytic properties in order to get a simple adsorption cellular model . LRP1 miin vitro . Since C1r2s2 binding to C1q involves ionic, calcium-dependent interactions with C1q lysine residues, one could easily assume these lysines to serve as ligands for LRP1 clusters. To our surprise, a recombinant mutant of C1q lacking these specific lysines still retained its full-binding abilities for both clusters with an affinity in the same nanomolar range as for wild type C1q. These findings suggest that other basic residues in the surrounding could be potential candidates for LRP1 interaction. Contrarily to C1q, in the case of the interaction of LRP1 with MBL, Duus and collaborators observed that the K55 lysine that is implicated in the interaction with the MBL associated proteases MASPs is also responsible for the interaction with LRP1 . Taken all together these data could indicate that the interaction mode of LRP1 could be enlarged to other members of the defense collagen family.The comparable affinity of LRP1 ligands for cluster II and IV binding is explained in some studies by a cluster replication that is specific to LRP1. LRP1 is the largest receptor of the LDL-receptor family with multiple biological functions. Since LRP1 such as C1q is a highly flexible protein that interacts with a wide variety of structurally unrelated ligands, in a multivalent manner, it is difficult to imagine a simple model for C1q interaction. Nevertheless, if C1q, like many other ligands, shares affinities for clusters II and IV, it is not evidenced in our study that they interact on both clusters synergically. This might be difficult if we consider that the potential residues for LRP1 interaction are oriented towards the center of the C1q cone , 44. Froin vitro experiments. Since C1q and LRP1 were reported to be important in efferocytosis (data not shown).We then aimed at confirming that C1q interaction was also happening at the cell surface using a simple non-phagocytic cellular model. For that purpose, LRP1-null cells were transfected with DNA coding for full-length LRP1 and a mini receptor IV that was chosen preferentially because of its natural location close to the membrane. Our results show that C1q interacts indeed with both LRP1 and minireceptor IV and that the interaction of mini IV receptor is nearly as important as that of full-length LRP1 (85% of the binding compared to LRP1). These results also suggest that other sites might contribute (but to a lesser extent) to LRP1 binding to C1q that could possibly be located on cluster II, the second site identified by our The implication of C1q in the removal of apoptotic cells through binding to LRP1 is controversial. While Ogden and Vanvidier et al. , 5 obserOverall our data indicate that similarly to many other LRP1 ligands, C1q binds to both clusters II and IV. The site responsible for LRP1 binding on C1q is located on its CLR, nearby the interaction site of the cognate protease tetramer C1r2s2. We propose a common canonical site for other C1q receptors and ligands, such as fucoidan and CR1. From a functional point of view, we show that C1q binds to both full-length and mini IV LRP1 receptors but that the first step of the uptake of late apoptotic cells by LRP1 is not influenced by C1q. Our results also highlight that cluster IV receptor can endorse most of LRP1 full-length binding capacities.The datasets generated for this study are available on request to the corresponding authors.JPK, VR, and NT designed the study. GF, EG, CW-P, IB, AC, JPK, and VR performed the research. GF, EG, CW-P, IB, JPK, VR, and NT analyzed the data. CDN and SD contributed key reagents. CG contributed to discussions and structural implements. VR, JPK, EG, and NT wrote the manuscript draft. All authors contributed to the article and approved the submitted version.This work has been supported by the French National Research Agency (grant ANR-16-CE11-0019) and by the University Grenoble Alpes (grant AGIR). This work used the platforms of the Grenoble Instruct-ERIC Center  with support from the French Infrastructure for Integrated Structural Biology  and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) within the Grenoble Partnership for Structural Biology.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.


In a previous study, we reported that mitogen-activated protein kinases, especially MPK4, are important downstream components of H2S involved in alleviating cold stress; however the underlying mechanism is unclear. In this study, we determined that the ability of H2S to alleviate cold stress is impaired in mpk4 mutants, but not in the upstream mek2 and crlk1 mutants. MPK4 was basically persulfidated, and NaHS (H2S donor) further increased the persulfidation level of MPK4. MEK2 was not persulfidated by H2S. NaHS treatments increased the MPK4 activity level nearly tenfold. The persulfidation signal of MPK4 did not disappear after eight cystein residues in MPK4 were site-mutated, respectively. Above all, our results suggested that H2S alleviates cold stress directly by persulfidating MPK4 and increasing the MPK4 kinase activity.Hydrogen sulfide (H H2S is toxic at high concentrations; however, at low concentrations, H2S plays important roles in both animals and plants as a gasotransmitter, along with nitric oxide and carbon oxide  by persulfidation, which alters the -SH of Cys to -SSH, to regulate the activity or cell location of a protein. Persulfidation was first reported in mice, in which about 10%–25% of liver proteins, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin and tubulin, are persulfidated under physiological conditions. Persulfidation increases the activtiy of GAPDH and actin polymerization (Hydrogen sulfide (Hon oxide . H2S parrization . In planrization . Aroca erization .Mitogen-activated protein kinases (MAPKs) are crucial components of cell signaling transduction in eukaryotes. MAPKs consist of three cascades: MEKK, MEK and MPK which phosphorylate and thereby activated next cascade respectively. When the cell membrane received the signal from outside, the receptor on the membrane was activated firstly, then MEKK→MEK→MPK cascades was activated, MPK activated downstream target such as enzymes, transcription factor leading to cell response . MAPK is2S plays important role in plant response to cold stress. In a previous study we found that MAPKs especially MPK4 are important downstream components in alleviating cold stress. MPK4 is required for H2S to alleviate damage to roots caused by cold stresses and regulate stomatal movement. However, the mechanisms underlying these functions remain unknown  was kindly provided by John Mundy of Copenhagen University, in which Ds was integrated eight nucleotides upstream of the first intron of MPK4, and the expression of MPK4 was not detectable (mek2 (Garlic-511-H01.b.1a.Lb3Fa) of Columbia background (Col) (crlk1 (SALK-016240C) of Col (A Ds insertion mutant of tectable . A T-DNAnd (Col)  was prov) of Col  was boug−2s−1 light illumination.The growth conditions were described previously . Briefly2S donor), while the control group was not fumigated. Then, the seedlings were transferred to new 1/2 MS plates and put into cold (4°C) or normal (23°C) conditions vertically and inversely. All the growth condition parameters of growth except temperature were the same.The root tip-bending experiments were performed as previously described . BrieflyMEK2 was PCR amplified and cloned downstream of a GST tag using EcoRI and XhoI restriction site in pGEX4T1. The CDS of MPK4 was PCR amplified and cloned downstream of a His6 tag using NheI and BamHI restriction sites in pET28a. For site-mutated MPK4 and MEK2EE, primers with corresponding mutated sites (PM) were designed, PM/PF (forward primer of CDS) and PM/PR (reverse primer of CDS) were used to generate the mutant PCR fragment using the pET28a-MPK4 or pGEX4T1-MEK2 plasmid as the template. It was important to determine whether to use PM or its complement. Then, two PCR products were used as templates to generate the mutated MPK4 or MEK2EE using the PF/PR as primers. Cystein was substituted with alanine in MPK4, and MEK2EE were generated by changing both putative phosphorylation sites to glutamate residues (T220E and T226E), which resulted in a constitutively active kinase. The vectors were transformed into Escherichia coli DH5α competent cells, and the sequences were confirmed by sequencing. The primers used are listed in The CDS of E. coli strain JM109. pET28a-MPK4 and its Cys-mutant vector were transformed into E. coli strain BL21. A single clone was incubated in 5 ml LB liquid medium supplemented with the appropriate antibiotics overnight at 37°C. Then, 2 ml solution was transferred into 100 ml LB liquid medium supplemented with appropriate antibiotics and incubated approxiamately 2.5 h at 37°C until the OD600 was 0.6–0.8. Then, 0.1 mM isopropyl β-D-1-thiogalactropyranoside was added to induce protein expression, and the E. coli cultures harboring MEK2 and MPK4 were incubated approximately 20 h at 28°C and 16°C, respectively. MEK2 and MEK2EE were purified using GST Selfinose resin and the MPK4 and Cys-mutant MPK4 were purified using Ni-NTA Sefinose resin. The purified protein was mixed with loading buffer containing β-mercaptoethanol and boiled for 10 min. 10% SDS-PAGE gel electrophoresis was performed and stained the gel with Coomassie Brilliant Blue.pGEX4T1-MEK2 and pGEX4T1-MEK2EE were transformed into The methods were described previously . Briefly2, and 1 mM DTT) containing 50 μM ATP at room temperature for 0.5 h. Activated MPK4 was treated with NaHS at 4°C for 30 min and then incubated with MBP (1:10 enzyme substrate ration) in the reaction buffer containing 50 μM ATP at room temperature for 0.5 h. The control was not treated with NaHS. The MPK4 activity was calculated based on the conversion of ATP to ADP using an ADP-Glo Kinase Assay (Promega).His-MPK4 (0.2 μg) was activated by incubating with MEK2EE (0.05 μg) in a reaction buffer . The determination of significant differences was performed with SPSS version 17.0  using one way analysis of variance (ANOVA) followed by Duncan’s test or student’2S’s alleviation of cold stress requires MPK4. To determine whether H2S functions directly through MPK4 or MEK2, the cascade upstream of MPK4, we performed root tip-bending experiments using homozygous mek2 (Col) and mpk4 (Ler) seedlings  seeds  seedlings  seeds  and perfeedlings .2S modifies the proteins through persulfidation to regulate the proteins’ function. Consequently, we investigated the persulfidation of MPK4 and MEK2. Firstly, we cloned the CDS sequence of MPK4 into the pET28a vector, purified recombinant His-tagged MPK4 , and all of them were site-mutated to alanine, respectively. Then, the biotin-switch assay was performed. However, the persulfidation signal was detected in all eight Cys residues in MPK4, which had no significant difference with WT MPK4 , therefore we treated the purified-protein of MPK4 and MEK2 with NaHS at 0–100 μM and higher concentration to detect the persulfidation of H2S to the MPK4 and MEK2. The results showed that MPK4 was basically persulfidated, and the 100 μM NaHS treatment increased the MPK4 persulfidation level (2S, and MPK4 is the direct target of H2S-mediated persulfidation. Our results showed that MPK4, GST-MEK2 and GST were basically persulfidated. Endogenous persulfidation can be observed in many proteins without H2S treatment, and H2S treatment increase the persulfidation level, such as GADPH, Tublin in HEK293 cell (2S treatment. H2S treatment can persulfidate the Cys at a specific site therefore increase the persulfidation level of the protein.The physiological range of Hon level . MEK2 ison level . Thus, M293 cell , MEK1 in293 cell , and RBO293 cell . We infe2S persulfidated MEK1; therefore, it increased the acitvity of MEK1, leading to PARP-1 activation, which attenuates DNA damage (2S on the activities of other MAPK members have not been reported in plants. Here, our result showed that H2S increased the MPK4 kinase acitivity (Zhao et al. reported that HA damage . The effcitivity .2S persulfidated MEK1 at Cys residue 341 (2S persulfidated pyruvate carboxylase at Cys265 (We also investigated the Cys sites that were persulfidated in MPK4. Thus, we mutated the Cys residues in MPK4 and performed biotin switch assays using Cys-mutated MPK4. The persulfidation level of MPK4 did not disappeared after the Cys residues were mutated . In mostidue 341 , H2S pert Cys265 , and mutt Cys265 . Shen ett Cys265 . Thus, t2S in cold stress and explored the underlying mechanisms. We found that H2S alleviates cold stress through MPK4, not MEK2 or CRLK1, and that H2S increases the MPK4 persulfidation and improve the kinase activity level. However, we did not identify the Cys sites of persulfidation in MPK4, possibly because the persulfidation of MPK4 occurred on two or more Cys residues. In future studies, we will analyze the protein structure of MPK4 and use mass spectrometry to investigate this subject, which will be a very interesting study.In this study, we investigated the functions of H


Here, by employing Micrococcal nuclease (MNase) digestion of crosslinked chromatin followed by chromatin immunoprecipitation (ChIP) and paired-end sequencing (MNase-X-ChIP-seq), we mapped unwrapping states of nucleosomes containing histone variant H2A.Z in mouse embryonic stem (ES) cells. We found that H2A.Z nucleosomes are more enriched with unwrapping states compared with canonical nucleosomes. Interestingly, +1 H2A.Z nucleosomes with 30–80 bp DNA is correlated with less active genes compared with +1 H2A.Z nucleosomes with 120–140 bp DNA. We confirmed the unwrapping of H2A.Z nucleosomes under native condition by re-ChIP of H2A.Z and H2A after CTCF CUT&RUN in mouse ES cells. Importantly, we found that depletion of H2A.Z results in decreased unwrapping of H3.3 nucleosomes and increased CTCF binding. Taken together, through MNase-X-ChIP-seq, we showed that histone variant H2A.Z regulates nucleosome unwrapping in vivo and that its function in regulating transcription or CTCF binding is correlated with unwrapping states of H2A.Z nucleosomes.Nucleosome is the basic structural unit of chromatin, and its dynamics plays critical roles in the regulation of genome functions. However, how the nucleosome structure is regulated by histone variants  The crystal structure of the nucleosome core particle showed that the DNA was wrapped on the octamer by about 1.65 superhelix turn in a left-hand manner with periodic interaction with histones  has been used to analyze the organization of individual histones within a nucleosome at genome-wide in yeast. Subnucleosome structures and asymmetries of histone compositions were found to be widespread across the yeast genome  to analyze the genome-wide unwrapping states of H2A.Z nucleosomes in mouse ES cells. Our results showed that H2A.Z is enriched with nucleosome unwrapping compared with canonical nucleosomes, and H2A.Z could function in gene regulation and CTCF binding regulation through modulating the unwrapping states of nucleosomes.The unwrapping states of nucleosomes may exit due to nucleosome dynamics and maturation during transcription and replication leosomes . During leosomes ,12. Apart genome . Microcot genome . Recentlt genome ,16, moust genome , and Drola cells . Howeverla cells ,16, therl-glutamine , Nucleosides , Pen/Strep  and 1000 U/ml leukemia inhibitory factor (LIF)  in standard incubator with 5% CO2 at 37°C. Plasmids or siRNA oligos were transfected into mouse ES cells by Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.Mouse ES cells were cultured in the medium with 80% DMEM , 15% FBS , Nonessential amino acids , 2-Mercaptoethanol , To generate H2A.Z depletion cells, H2A.Z was knocked down by the siH2A.Z oligo: 5′-GGTAAGGCTGGAAAGGACT-3′. Knock down efficiency was confirmed by western blot.2, 0.5% NP-40, 1 mM CaCl2) , and the chromatin particles before crosslink reversion are ∼1000–2000 bp after resolved on 1% agarose, which is the typical chromatin particle size for ChIP-seq.For MNase X-ChIP, mouse ES cells were crosslinked with 1% formaldehyde in DMEM for 10 min at room temperature, then quenched by 125 mM glycine. Cells were washed with cold DPBS for twice, and then resuspended in lysis buffer   with proFor H2A.Z, H2A or H3 ChIP, chromatin was first incubated with H2A.Z , H2A  or H3  antibody in RIPE-150  at 4°C, then the BSA blocked protein A/G Dynabeads were added and incubated overnight at 4°C. The Dynabeads were washed by RIPE-150 with 0.1% SDS for 5 times, and eluted with Direct Elution Buffer .The ChIPed DNAs were extracted using a standard phenol-chloroform extraction procedure. For paired-end sequencing, libraries without size selection were prepared as described in  using NEP-40, 0.7% sodium deoxycholate, 0.5M LiCl2) for once and 1× TE Buffer, pH 8.0 for twice. Libraries were prepared and sequenced using Illumina HiSeq2000 platform, and sequencing data were processed as previously described  on ice for 10 min. The nuclei were spin down at 600 × g for 3 min at 4°C, and re-suspended in Buffer I  on ice for 5 min. The nuclei were spin down at 600 × g for 3 min at 4°C, and washed once by Buffer II  and re-suspended in Buffer II. 15 ul CTCF antibody  was used for each 2 × 107 cells and incubated at 4°C for 2 h on a mixing platform. The nuclei were washed with Buffer II for three times and re-suspended at 1 × 107 cells/600 ul Buffer II /6 ug pA-MNase , and incubated at 4°C for 1 h on a mixing platform. The nuclei were washed with Buffer II for three times and re-suspended at 1 × 107 cells/1 ml Buffer II with 2 mM CaCl2. The digestion was performed at 25°C for 20 min without mixing. The digestion was stopped with 10 mM EGTA. The nuclei were incubated on a thermal incubator at 37°C for 30 min with 900 rmp angitation. The nuclei were centrifuged at 15 000 rpm for 10 min at 4°C. The supernatant were taken out without disturbing the pellet. 10% percentage of the supernatant was kept for input of CTCF CUT&RUN. Protein A and Protein G Dynabeads were blocked with 0.1% BSA and then incubated with H2A.Z antibody  or H2A antibody . The resulting Dynabeads/antibody complexes were incubated with the remaining supernatant at 4°C for 6 h on a mixing platform. The beads were washed for three times by Buffer II with 0.1% Triton X-100. The chromatin was eluted with Direct Elution Buffer . The ChIPed DNAs were extracted using a standard phenol-chloroform extraction procedure. For paired-end sequencing, libraries without size selection were prepared as described in . Motif analysis and gene ontology analysis were performed with HOMER (http://www.r-project.org/) or Microsoft Excel.Paired-end reads were trimmed for adaptor sequence using cutadapt  with parsoftware . The reath HOMER . Other pDrosophila cells  and ‘longMN’ (32 min digestion) conditions . We founN’ input . PreviouN’ input . TogetheN’ input ,16. Few N’ input  (data noN’ input . SurprisN’ input . This ren = 20 935), overlap peaks (n = 11 908) and 80–168 bp specific peaks (n = 21 445). We found that the overlap peaks showed higher level of H2A.Z    .1, where bp DNA) .4, S2E.5 bp DNA) . Cluster bp DNA) . However bp DNA) . Thus, wSnx24 and Nt5c3); and the absence of unwrapped H2A.Z nucleosomes with 30–80 bp DNA is associated with high gene expression (Snx2 and Fkbp9). These results confirm that the regulatory function of the +1 H2A.Z nucleosomes in transcription is correlated the unwrapping states. To test whether the Cluster-A and the Cluster-D unwrapped H2A.Z nucleosomes are involved in the regulation of genes with distinct biological functions, we performed Gene Ontology analysis with the genes associated with Cluster-A and Cluster-D H2A.Z nucleosomes by HOMER  , and areSs , and s Figure .We also observed moderate signals of 30–80 bp DNA fragments of both H2A.Z nucleosomes Figure . Thus, oin vivo  with higher signal to noise ratio compared with ChIP-seq . We re-aTo explore whether H2A.Z regulates the CTCF binding, we performed crosslink ChIP-seq for CTCF after depletion of H2A.Z. Interestingly, we found that the CTCF binding is globally increased after H2A.Z knockdown Figure , G, suggin vivo when controlled properly. Meanwhile, we are aware that the FLPs derived by X-MNase-ChIP-seq are dependent on the digestion condition. Thus, fragment length observed under a certain digestion condition cannot be taken as direct evidence for specific unwrapping state of nucleosome. In addition, although the positioning and pattern of small DNA fragments of H2A.Z nucleosomes at +1 nucleosomes  re-ChIPed by H2A.Z after CTCF CUT&RUN under native condition  associated with H3–H4 tetramer than H2A-H2B dimers. However, we observed that histones H2A and H3 have similar FLPs, indicating that H2A–H2B dimers may be tethered to the tetrasome intermediate during the nucleosome remodeling in vivo. Consistent with this hypothesis, we and others found that histone chaperone FACT can facilitate the tethering of H2A-H2B dimers onto the tetrasome immediately and prevent the loss of H2A–H2B dimers from the nucleosome  response genes and set a chromatin structure to potentiate these genes for activation . CTCF and H2A CUT&RUN data were downloaded from GSE84474. Coordinates for transcription state sites (TSSs) were retrieved from RefSeq Genes (mm9) downloaded from UCSC genome browser (http://genome.ucsc.edu/cgi-bin/hgTables). The code used for V-plot analyses is available at https://github.com/WenZengqi/nucleosome_unwrapping. The materials used during the current study are available from the corresponding author on reasonable request.The datasets generated and analyzed during the current study are submitted to the Gene Expression Omnibus (GEO) repository under the GEO accession GSE146082. Mouse ES cell gene expression data and total Pol II ChIP-seq data were downloaded from GSE117035 . H3K4me3gkaa360_Supplemental_FilesClick here for additional data file.


Background: Variations in the viral receptor human angiotensin-converting enzyme 2 (ACE2) may specify the susceptibility of a certain population to severe acute respiratory syndrome coronavirus 2. Objective: Evaluation of the affinity of severe acute respiratory syndrome coronavirus 2 spike glycoprotein to the Iranian genetic variants of ACE2. Materials & methods: Single nucleotide polymorphisms of ACE2 among the Iranian population were collected from the Iranome database. Missense mutations in the N-terminal peptidase domain were selected for in silico analysis. Results: 17 missense single nucleotide polymorphisms were found at ACE2. Viral glycoprotein had the lowest affinity to ACE2 mutant V485L. Discussion: The V485L variant of ACE2 could be a natural resistance mutation among the Iranian population. In addition, variant S331F can increase slightly the susceptibility to infection with the virus. Accordiavirus/) . S proteavirus/) ,6.The ACE2 gene is located on chromosome Xp22 and its product is expressed in large amounts in human lung and small intestine epithelia ,8. ACE2 ACE2 contains a structural transmembrane domain that connects the protein to the plasma membrane. In addition, SARS-CoV-2 S protein interacts with the extracellular domain of ACE2 as a receptor . Interachttp://zhang.bioinformatics.ku.edu/I-TASSER) [Prediction of 3D protein structures from amino acid sequence SARS-CoV-2 glycoprotein (accession numbers: YP_009724390.1) was performed by using I-TASSER server (-TASSER) . In compwww.uniprot.org) and protein databank  with ID:1r42, respectively [The sequences and validated crystallographic structure of ACE2 was obtained from UniProt  [Iranome (ome.com) , a databDunbrak rotamer library was used to substitute targeted amino acids residues with those that are prevalent in Iranian population ,24. The https://cluspro.org/login.php) [The ClusPro web server (gin.php)  was usedThe results of docking SARS-CoV-2 glycoprotein with the ACE2 variants were analyzed by MGLTools 1.5.6 software  for determining ACE2 amino acids residues involved in interaction with the viral glycoprotein. The interaction analysis were done according to visual molecular dynamics (VDW) scaling factor 1 Å and the amino acid position(s) of all binding sites were investigated and validated in UniProt database regarding the molecular domains .polymorphism, Population, ACE2 or ‘angiotensin I converting enzyme 2’, SNO or ‘Single nucleotide polymorphism’ (SNP). Furthermore, ClinVar [The scholar databases, including Google Scholar, PubMed and Scopus. The search terms were  ClinVar  and sing ClinVar  databaseNative human ACE2 crystallographic structure with 615 amino acid length containing SARS binding domains were retrieved from PDB database. The protein comprised three regions responsible for interaction with SARS-CoV spike glycoprotein. These were amino acids 30–41, 82–84 and 353–357 . Therefo-1). It was also found that fewer close contacts were established between SARS-CoV-2 spike glycoprotein and ACE2 V485L mutant. Supplementary Tables 1 & 2.The interaction energy and affinity of SARS-CoV-2 to mentioned ACE2 variants are demonstrated in The known SNPs of ACE2 among some populations is given in The SARS-CoV-2 is a novel virus that can cause severe respiratory disease in humans. The virus uses its glycoprotein to bind to the cell surface receptor, ACE2 . ACE2 iset al. found no associations between ACE2 polymorphism and the disease outcome [The receptor binding domain of SARS-CoV-2 spike glycoprotein, S1 (residues 318–510) has a higher ACE2 affinity than the complete S1 domain (residues 12–672) . Nonethe outcome . Neverthet al. investigated variants, including S19P, I21V, E23 K, K26R, T27A, N64 K, T92I, Q102P and H378R, which increase susceptibility to virus infection. On the other hand, other ACE2 variants such as K31R, N33I, H34R, E35 K, E37 K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L and D509Y reduced the binding affinity of SARS-CoV-2 spike glycoprotein to ACE2. It should be noted that these variants are rare in populations [In a systematic study of 1700 variants of ACE2 from the China Metabolic Analytics Project under review and 1KGP (1000 Genome Project) databases, the authors suggested that there was no proof of a natural resistant ACE2 mutant in different populations . In anotulations ,41. On tulations ,43. It iulations .-1). S331F may therefore increase the susceptibility of a population to SARS-CoV-2 infection. In addition, it was observed that viral glycoprotein had a far lower affinity to human ACE2 polymorphism V485L . There was a less close interaction between SARS-CoV-2 spike glycoprotein and ACE2 V485L mutant in this regard. We propose that substitution of V485L for ACE2 may be a natural resistance mutation among the Iranian population. This may be due to conformational changes in the binding site within peptidase domain. All the studied variants of ACE2 were located at the N-terminal peptidase domain of ACE2. Our findings are presented based on other studies, which have been conducted by cryo–electron microscopy of the full-length human ACE2.We examined the affinity and interaction site of SARS-CoV-2 spike glycoprotein with human wild-type and ACE2 mutants. According to our results, most ACE2 variants have the same interaction energy with SARS-CoV-2 spike glycoprotein. Nonetheless, the virus glycoprotein has a slightly higher affinity to ACE2 variant S331F .The V485L variant of ACE2 could be a natural resistance mutation among the Iranian population.


Meyerozyma guilliermondii strain SMB. This yeast-like fungus strain SMB was characterized to assess its suitability as an expression host. Lipase activity was absent in this host (when assayed at 30 °C and 70 °C) and Hygromycin B (50 μg/mL) was found to be its best selection marker. Then, the hyg gene (Hygromycin B) was used to replace the sh ble gene (Zeocin) expression cassette in a Komagataella phaffii expression vector (designated as pFLDhα). A gene encoding the mature thermostable lipase from Bacillus sp. L2 was cloned into pFLDhα, followed by transformation into strain SMB. The optimal expression of L2 lipase was achieved using YPTM (Yeast Extract-Peptone-Tryptic-Methanol) medium after 48 h with 0.5% (v/v) methanol induction, which was 3 times faster than another K. phaffii expression system. In conclusion, a new host-vector system was established as a platform to express L2 lipase under the regulation of PFLD1. It could also be promising to express other recombinant proteins without inducers.Screening for a new yeast as an alternative host is expected to solve the limitations in the present yeast expression system. A yeast sample which was isolated from the traditional food starter ‘ragi’ from Malaysia was identified to contain  Komagataella phaffii, which was previously known as Pichia pastoris, is the most common host for recombinant protein expression in industries. Most foreign genes are expressed under the control of the K. phaffii alcohol oxidase 1 promoter (PAOX1), where methanol is a vital inducer. Some recombinant proteins require a longer time for optimal production and high methanol induction when expressed under the regulation of PAOX1  every 24 h.Lipases are remarkable biocatalysts for high-value application in various industries . Productf et al. . From rer, Sabri  showed tMeyerozyma guilliermondii . The recombinant strain SMB and Komagataella phaffii strain GS115 (pPαG2) containing recombinant L2 lipase were grown in YPD supplemented with Hygromycin B  (50 μg/mL) and Zeocin  (100 μg/mL), respectively. Escherichia coli JM109 harbouring pFLDα and E. coli containing DH5α pSELECT-hygro-mcs plasmids were grown in Luria-Bertani medium supplemented with 50 μg/mL ampicillin . The vectors were purchased from Invitrogen, USA and InvivoGen, USA, respectively.Local yeast isolate (strain SMB) collected from a traditional fermented food starter (ragi) in Malaysia was cultured in YPD medium  of olive oil  and 50 mM phosphate buffer (pH 7.0) with a homogenizer until it was emulsified. The reaction mixture  was shaken for 30 min at 30 °C and 70 °C, separately. Then, the reaction was stopped by the addition of 1.0 mL of 6 M HCL and 5.0 mL isooctane  and vigorously vortexed for 1 min. The upper layer (3.0 mL) containing the fatty acids was transferred to another test tube consisting 1.0 mL of 5% (w/v) copper pyridine for free fatty acids colour detection. Then, the mixture was vortexed and left to settle for 30 min. Finally, 1.0 mL of the upper layer isooctane was taken for OD715nm reading using a UV-visible spectrophotometer . Unit (U) definition for lipase activity was defined as the rate of fatty acid formation (μmole) per min (standard assay condition).Strain SMB underwent lipase screening procedures prior to utilizing it as a host for bacterial L2 lipase expression. For the quantitative assay, lipase activity was measured colourimetrically by the method described by Kwon and Rhee . The subA variety of antibiotics was added to the YPD agar to assess the antibiotic resistance for strain SMB. A single colony was inoculated into 10 mL YPD broth (incubated at 30 °C with 250 rpm) and 100 µL of the overnight yeast culture was spread on several YPD plates supplemented with different types of antibiotics at respective concentrations for 3 days. The antibiotics and the recommended concentrations used were as follows: Blasticidine-S  (50 μg/mL), Zeocin (500 μg/mL), Hygromycin B (50 μg/mL), Puromycin  (25 μg/mL), Geneticin  (400 μg/mL), and Phleomycin  (25 μg/mL).AOX1), and formaldehyde dehydrogenase 1 (PFLD1). Amplification of the genes was performed using the specific promoter primers, as listed in AOX1 (51 °C) and PFLD1 (50 °C).The presence of methanol-inducible promoters in the local isolate was screened using PCR. Two methanol-inducible promoters tested were alcohol oxidase 1 (Psh ble gene deletion was amplified using the PCR. The primers used were forward (CYC1 HindIII F) and reverse (pTEF1 PstI R). The amplified pFLDα without the sh ble gene was designated as pFLDα-Z. The pFLDα-Z was amplified using PCR in a 50 μL reaction mixture containing 10 μL of 5× Hi-Fi buffer, 0.5 μL of 100 mM dNTP, 1 μL (20 pmol/μL) of each forward and reverse primers, 3 U of VELOCITY DNA polymerase, 2 μL of plasmid DNA (100 ng/μL), 2 μL of DMSO, and topped up with distilled water. The gene was amplified in 30 PCR cycles with the following parameters: pre-denaturation at 96 °C for 2 min, 30 repeated cycles for denaturation at 96 °C (30 s), annealing at 64 °C (30 s), and extension at 72 °C (1 min). The final extension was run at 72 °C for 7 min. Then, the Hygromycin B (hyg) gene (CMV-em7-hyg) was amplified from pSELECT-hygro-mcs vector in 50 μL reaction mixture using forward (hyg-cmvF) and reverse (hyg-cmvR) primers, respectively. The same PCR mixture and condition were used to amplify the hyg gene, except that the annealing temperature was set at 66 °C and the final extension time was 24.5 min.pFLDα vector with hyg were digested with HindIII and PstI followed by overnight ligation using DNA T4 ligase at 16 °C. Then, the ligation mixture was transformed into E. coli JM109 according to the method suggested by Sambrook et al. , and cultivated under the same condition until OD600nm = 4. Then, the cells were harvested at 3000× g and room temperature (RT) for 5 min. The cell pellet was resuspended in 20 mL MMAs [same composition as MGAs with the exception that glycerol was replaced with 0.5% (v/v) methanol ] and cultivation was continued for 5 days. Methanol [0.5% (v/v)] was added every 24 h for induction. The strain SMB containing the empty vector (pFLDhα) was used as the control. Aliquots (15 mL) of the cultures were harvested at 8000× g and RT for 3 min every 24 h of post-induction. Finally, the supernatant was assayed colourimetrically using the previously described method at 70 °C. Recombinant strain SMB/pFLDhα/L2lipase (pFαS3) with the highest lipase expression was selected for further optimization.A single colony of the recombinant strain SMB carrying the pFLDhα/L2 was inoculated in 10 mL YPD broth at 30 °C and 250 rpm overnight. Then, 100 μL of the culture were inoculated into 100 mL of MGAs , and YPTG  media. Then, the cells were cultivated until the culture reached an OD600nm = 4. Next, the cells were harvested at 3000× g and RT for 5 min followed by resuspension to a final volume of 100 mL in induction media MMAs, BMMY [same composition as BMGY with the exception the glycerol was replaced with 0.5% (v/v) methanol], and YPTM [same composition as YPTG with the exception that glycerol was replaced with 0.5% (v/v) methanol], respectively. Methanol [0.5% (v/v)] was added every 24 h and cultivation was stopped after 48 h. Cell growth was monitored and the supernatant from each flask was assayed for lipase activity for the optimization of each factors.The effect of different medium formulations was studied using three different media. Recombinant strain (pFαS3) culture from the YPD medium was inoculated into 100 mL MGAs, BMGY  (for induction with methylamine) and MMMa medium [same composition as MGMa with the exception that glycerol was replaced by 0.5% (v/v) methanol] (for induction with methanol plus methylamine), respectively. The respective inducers were added every 24 h for 48 h cultivation.For the effect of different types of inducers, recombinant pFαS3 culture was grown in 100 mL MGAs. Then, the cells were harvested and resuspended in 20 mL of MMAs medium (for induction with methanol), MGMa medium [0.17% (600nm). The effect of different growth temperatures on L2 lipase production was studied at 20 °C, 25 °C, 30 °C, and 35 °C. Recombinant pFαS3 culture was grown in 100 mL of YPTG, followed by YPTM supplemented with 0.5% (v/v) methanol every 24 h for 48 h in respective temperatures. Finally, for time-course study, the recombinant pFαS3 culture was grown in YPTM medium supplemented with 0.5% (v/v) methanol at 25 °C for 120 h. A 5 mL culture was harvested every 24 h for lipase assay and cell growth was monitored. Then, 12% SDS-PAGE [Next, the effect of different inducer concentrations was investigated. Recombinant pFαS3 culture was grown in YPTG, followed by YPTM media. During the induction phase, different methanol concentrations  were used to induce the cultures every 24 h. At 48 h, the cultivation was stopped, followed by lipase assay and growth measurement  could be used to inhibit its growth [K. phaffii.In this study, ibiotics . The ress growth . HygromyPichia genera for foreign protein expression are linked to the use of strong methanol-inducible promoters derived from genes of the methanol utilization pathway, such as alcohol oxidase 1 (AOX1), and formaldehyde dehydrogenase 1 (FLD1). The availability of the promoters for these genes were investigated in strain SMB. The amplified PFLD1 and PAOX1 using the designed primers is shown in AOX1 (550 bp) over its full sequence (941 bp). In this study, PFLD1 was chosen to be used as a promoter to regulate the recombinant L2 lipase expression in strain SMB. FLD1 gene is the key enzyme required for the catabolism of methanol as a carbon source and methylamine as nitrogen sources in methylotrophic yeasts [FLD1 gene is useful for regulating the expression of foreign genes under conditions where methanol is inappropriate. A sequence of PFLD1 from strain SMB showed 99% similarity to PFLD1 from K. phaffii.The applications of methylotrophic hosts such as c yeasts . In addiL2 lipase gene was cloned into pFLDhα  exhibited the highest activity when the o pFLDhα . Thus, iPichia transformants. From v/v) methanol gave the highest expression (0.64 U/mL) with the optimum cell biomass. Increasing the methanol concentration resulted in increased expression level, up to the optimum methanol concentration; whereby too much supply showed an inhibition effect with a decrease in expression level. Significant lipase activity was also detected in the absence of methanol. A similar trend was reported by Abu et al. [M. guilliermondii strain SO could also express the recombinant lipase without methanol.All three types of inductions were compared to assess the best inducer for u et al. , where M600nm = ~37).K. phaffii (144 h) [M. guilliermondii strain SO, where the T1 lipase activity was optimally produced after 30 h of cultivation in YPTM with 1.5% (v/v) methanol [FLD1 in M. guilliermondii strain SO to express the recombinant protein without the presence of an inducer, where its production was subsequently optimized. Similar optimization strategies could also be performed for strain SMB in expressing L2 lipase.Referring to  (144 h) . Furthermethanol . Optimizmethanol  also repM. guilliermondii strain SMB was identified as a new host isolated from a traditional food starter; it was developed as a new host-vector system for bacterial enzyme expression. The newly developed host-vector system demonstrated the ability to express bacterial L2 lipase extracellularly, three times faster than a commercial yeast system. It was also suggested that this system could be used as an alternative workhorse for other recombinant protein expression with and without methanol induction.In this study,


Aims: The analysis of the online databases revealed that CCND1 expression is correlated with poor prognosis in LSCC. We aimed to explore the function of CCND1 in tumor progression in LSCC.Main methods: The expression of mRNA was measured using qRT-PCR. Protein expression was assessed by Western blot. Cell migration and invasion were assessed by transwell assay.Key findings: CCND1 was co-overexpressed with FGFR1 in lung cancer patients. Overexpression of CCND1 promoted LSCC cell proliferation and metastasis. FGFR1 promoted the processes of EMT through AKT/MAPK signaling by targeting CCND1 in FGFR1-amplification cell lines.Significance: IIn conclusion, our study demonstrated the regulatory mechanism between CCND1 and FGFR1 in FGFR1 amplified LSCC. Co-targeting CCND1 and FGFR1 could provide greater clinical benefits. Lung cancer is the leading cause of cancer related death worldwide with only 16.8% 5-year survival rate after diagnosis . TargeteFibroblast Growth Factor Receptor 1 (FGFR1) is a transmembrane protein and member of the fibroblast growth factor receptor family . It is kThe D-type cyclins family members  along with its binding partners CDK 4/6 partially regulate G1 to S-phase transition of the cell cycle ,21. MoreWe hypothesis for the first time that CCND1 interacts with FGFR1 to promote EMT in LSCC in in vitro and in vivo. In this study, we observed that CCND1 and FGFR1 were co-overexpressed in LSCC.The human lung cancer cell lines (H1581 and HCC95) were obtained from the American Type Culture Collection. Both cell lines were identified by short tandem repeat (STR) profiling (Table.S1) ,32. The The CCND1 plasmid, siRNA and FGFR1 plasmid, siRNA were obtained from biolink . Plasmid and siRNA (50 nM) were transfected by Lipofectamine3000 kit . The sequences of siRNA was described by us before .2. After incubation of 2–3 weeks, cells were fixed with methanol and stained with 0.1% crystal violet. Colonies with the diameter larger than 100 µm were counted, and each Colony formation assay was repeated in triple.Cells were transfected into plasmid or siRNA. After 24 h, cells were digested by trypsin and then replanted at a density of 100 cells/1cmLung cancer tissues and non-tumor adjacent tissues (NATS) were obtained by Shanghai Chest Hospital, Jiao Tong University . Written consents approving the use of tissue samples for research purposes were provided from lung cancer patients. The study gained approval by the Institute Research Ethics Committee of Shanghai Jiao Tong University . Total RZO-1 (forward 5´-AGCGAAGCCACCTGAAGATA-3´and reverse 5´-GATGGCCAGCAGGAATATGT-3´)Snail (forward 5´- CGCGCTCTTTCCTCGTCAG −3´ and reverse 5´- TCCCAGATGAGCATTGGCAG −3´),CCND1(forward 5ʹ-GGAGCCTATTCTGCCCATTT-3′)(5ʹ-CGAGGTCATAGTTCCTGTTGGTG-3′reverse);GAPDH (forward 5ʹ- AGAAGGCTGGGGCTCATTTG −3′)(5ʹ- AGGGGCCATCCACAGTCTTC −3′reverse);Data were calculated by using the comparative threshold cycle (Ct) method, and the results were expressed as fold difference normalized to GAPDH. The experiment was repeated three times. The procedure other primer sequences were described by us before ,10.Cells (3 × 104) with 1% FBS medium were seeded into an 8-μm pore membrane with or without Matrigel , and medium with 10% FBS was set in the lower chamber. After 24 hours incubation, cells on the upper layer were erased with a cotton swab. Cells on the lower layer were fixed in Methanol for 20 min and stained with crystal violet dye (0.1%) for 20 min. Cells were then counted at three randomly selected views for the subsequent calculations.3 cells were seeded into a 96-cell plate. After transfected with plasmid or siRNA r for 24 h, 10 μl CCK8 reagent was put into the medium per well. The absorbance (450 nm) was measured by the microplate reader  after 1 to 3 hours co-incubation.CCK- 8 assay kit  was used to quantify the rate of proliferation under treatment. Briefly, 1–2*10Treated cells or tissues were lysed by RIPA with phosphatase inhibitor, protease inhibitor, and phenylmethylsulfonyl fluoride (PMSF).The procedure was described by us before . All theAfter being rehydrated, the tissues were incubated with primary antibody overnight. The secondary-antibody was donkey anti-rabbit IgG that conjugated with Alexa Fluor 594. Cell nuclei was stained with DAPI (Sigma). Cells were then photographed and then quantified by the immunofluorescence microscope (Leica DFC420C) ,32.After rehydrating, tissue specimens were incubated with primary antibodies for 1 hour, following by incubation with secondary antibodies. Diaminobenzidine-hydrogen peroxide (Sigma) was used as the chromogen. The counterstaining was conducted with 0.5% hematoxylin. The immune-histochemical grades are as follows: -, no staining; +, 10%, 10%, 50%, 50%, 50%, 50%, 50% -, no staining; +, 10%; ++, 10%–50%; and +++, >50%.6 cells) in a volume of 50 µL was injected into the right side of the nude mouse. AZD4547 (12.5 mg/kg/d) or siRNA were given to nude mice by gavage or Intravenous injection for 3 weeks. In the orthotropic lung tumor model, cell suspension of H1581(2 × 106 cells) in a total volume of 50 μL (Matrigel: PBS = 1:4) were injected directly into the left lung with insulin injection syringes . AZD4547 (12.5 mg/kg/d) was given by gavage for 2 weeks. All the nude mice were housed in the SPF (Specific Pathogen Free) animal room of Shanghai Jiao Tong University. All animal experiments were carried out in accordance with the approved scheme of the Shanghai Jiao Tong University Institutional Ethics Committee.In the subcutaneous xenograft model, H1581  with the Complete Protease Inhibitor Cocktail, and then centrifuged for 20 min . Soluble lysates were incubated with the antibodies at 4°C overnight, followed by incubation of Protein A/G Plus-Agarose for 2 hr. Complexes were isolated from the beads and boiled for 10 minutes. Thermo Scientific Pierce Co-IP kit (Thermo Fisher Scientific) was used for the Co-IP assay. These obtained proteins were tested by Western blotting analysis.www.kmplot.com/analysis) [Prognostic values of CCND1 level were downloaded from TCGA database and analyzed by Kaplan-Meier survival curves of NSCLC patients, using Kaplan-Meier Plotter  ,34.All the data were calculated as mean ± SEM or SD and analyzed using GraphPad Prism software  . All expWe analyzed the relationship between CCND1 and clinical features. CCND1 was not associated with shorter OS in LADC ), but reAltogether, the above results suggested that both CCND1 and FGFR1 might involve in lung cancer progression.To explore the role of CCND1 in FGFR1-amplified lung cancer cells, H1581, and HCC95 cells as lung squamous cell carcinoma cell lines, with high expression of FGFR1, were used and authenticated by STR profiling (Table.S1). Both cells were transfected with CCND1 overexpression plasmid or si-CCND1. First, CCK8 assay and colony formation assay showed that CCND1 promoted the proliferation of H1581 and HCC95 cells  and (b))Conversely, CCND1 knockdown inhibited the proliferation, as determined by CCK8 assay and colony formation assay  and (b))These results implied that CCND1 promoted the EMT process in FGFR1-amplified lung cancer as an oncogene.CCND1 had a sharp downregulation after AZD4547 treatment(1 μM) and a significant upregulation after bFGF treatment(20 ng/mL) for 24 hours, which was detected by western blot ) and PCRTo determine whether CCND1 acts a pivotal role in FGFR1-induced alterations in cell proliferation and metastasis, H1581 and HCC95 cells transfected with CCND1 overexpression plasmid and si-FGFR1 were used. The inhibitory effects of si-FGFR1 on proliferation  and (b))/ICx,A + CB,x/ICx,B). Combination index (CI) is widely used to evaluate the efficiency of combination between drugs [As the co-overexpression of CCND1 and FGFR1 in LSCC, we hypothesized that the co-target CCND1 and FGFR1 may provide higher efficiency. To verify this hypothesis, H1581 cells were treated with increasing concentration of AZD4547, ranging from 0.01–10 μM, in combination with si-CCND1 or siRNA-NC using at a weak, fixed concentration (0.3 nM). The IC50 for was 0.2433 μM and 11.81 μM for AZD4547 and si-CCND1, respectively. However, the IC50 for AZD4547 plus si-CCND1 was reduced to 0.0668 μM. To further explore the interaction of CCND1 and AZD4547, we calculated the CI index between them ). AfThe orthotopic lung cancer mouse models were established. The volume of primary lung cancer and the number of metastatic nodules both decreased in an orderly fashion for the following four groups: the NC group, the AZD4547 group, the sh-CCND1 group and the sh-CCND1 plus AZD4547 group  of lung cancer, and separately ,47–49. OCollectively, we discovered a regulatory axis between CCND1 and FGFR1. Si-CCND1 proved significant antitumor effect in the genetic background of FGFR1 amplified lung cancer. In addition, we found a synergistic antitumor effect between si-CCND1 and FGFR1 inhibitor AZD4547 in vitro and in vivo. These findings may provide new insights for the prognosis and treatment of patients with FGFR1-amplified lung cancer.


The electrical characteristics were investigated when the temperature changes from 298 K to 423 K. The barrier height ϕb increases, and the ideality factor n decreases as the temperature increases, indicating the presence of barrier height inhomogeneity between the polymer and β-Ga2O3 interface. The mean barrier height and the standard deviation are 1.57 eV and 0.212 eV, respectively, after taking the Gaussian barrier height distribution model into account. Moreover, a relatively fast response speed of less than 320 ms, high reponsivity of 0.6 A/W, and rejection ratio of R254 nm/R400 nm up to 1.26 × 103 are obtained, suggesting that the hybrid PEDOT:PSS/β-Ga2O3 Schottky barrier diodes can be used as deep ultraviolet (DUV) optical switches or photodetectors.In this paper, the hybrid β-Ga Furthermore, the overshooting feature can be observed from the shapes of photoresponse curves with a wedgy head at the lower Plight of 150 μW/cm2 than that occurred at the Plight of 600 μW/cm2 in [30] for the effective collection of photogenerated carriers under the reverse bias of − 1.2 V rather than 0 V.The time-dependent photoresponse characteristics of hybrid photodetector are studied by using square wave light with a period of 10 s under the 3 flakes , 42, 43 λ under the Vbias of − 1.2 V. The maximum responsivity Rmax of 0.62 A/W is achieved at a λ of 244 nm and the corresponding external quantum efficiency(EQE) of 3.16 × 102% calculated by the expression EQE = hcRmax/(eλ), much higher than that obtained in [Rmax is the peak responsivity, and h is the Plank constant. e and λ are the electronic charge and the illumination wavelength, respectively. As the wavelength is longer than 290 nm, the photoresponsivity is lower than 1 × 10−3, illustrating a much better spectral selectivity in the hybrid β-Ga2O3 devices. At the same time, the rejection ratio of R254 nm/R400 nm is determined to be 1.26 × 103. Compared with the reported inorganic Ga2O3 photodetector [Figure ained in , 38 for detector –49, the 2O3 hybrid Schottky barrier diode. The Schottky barrier height ϕb and ideality factor n are dependent on temperature, indicating that the Schottky barrier height was inhomogeneous at PEDOT:PSS/β-Ga2O3 interface. The mean barrier height and standard deviation can be evaluated to be 1.57 eV and 0.212 eV, respectively, based on the Gaussian barrier height distribution model. Furthermore, the characteristics of PEDOT:PSS/β-Ga2O3 DUV Schottky barrier photodetectors were also investigated. A higher responsivity of 0.6 A/W, rejection ratio of R254 nm/R400 nm = 1.26 × 103, EQE of 3.16 × 104% and a faster response speed of less than 320 ms are achieved, suggesting that the hybrid Schottky barrier diodes can be used as DUV optical switches or photodetectors.We have fabricated PEDOT:PSS/β-Ga


In Arabidopsis thaliana, the typical leaf chlorosis phenotype in the knockout mutant of calcineurin B-like interacting protein kinase 23 (CIPK23) is high-NH4+-dependent under low-K+ condition. However, the correlation of K+ and NH4+ in the occurrence of leaf chlorosis in the cipk23 mutant has not been deeply elucidated. Here, a modified hydroponic experimental system with different gradients of NH4+ and K+ was applied. Comparative treatments showed that NH4+ toxicity, which is triggered mainly by the high ratio of NH4+ to K+ (NH4+/K+ ≥ 10:1 for cipk23) but not by the absolute concentrations of the ions, results in leaf chlorosis. Under high NH4+/K+ ratios, CIPK23 is upregulated abundantly in leaves and roots, which efficiently reduces the leaf chlorosis by regulating the contents of NH4+ and K+ in plant shoots, while promoting the elongation of primary and lateral roots. Physiological data were obtained to further confirm the role CIPK23 in alleviating NH4+ toxicity. Taken all together, CIPK23 might function in different tissues to reduce stress-induced NH4+ toxicity associated with high NH4+/K+ ratios by regulating the NH4+–K+ balance in Arabidopsis.Ammonium (NH Pl. Pl3−) a4 for 5 min and twice with double-distilled water [The plant samples were washed thoroughly once with 10 mM CaSO+ contents, dry samples were digested in 6 mL (approximately 0.2 g DW) of 0.5 M HCl, shaken for 1 h at room temperature, filtered by filter paper into a new tube and measured by a flame photometer (6400A). The readings were used to calculate the K+ concentrations in the solution.−1); M is the relative molecular mass of K+; V is the reading volume (mL); m is the dry weight (g).To determine the K4+ content, the shoots and roots of each sample were washed with 10 mM CaSO4 and double-distilled water; the fresh weight was then obtained, and the samples were frozen in liquid nitrogen. To ensure that the samples were well mixed, the shoots and roots of every six plants were mixed with liquid nitrogen, and then extractions were conducted with 6 mL of 10 mM formic acid for the NH4+ content assay using the o-phthalaldehyde (OPA) method, which has been described in a previous report [4+.−1); M is the relative molecular mass of NH4+; V is the reading volume; m is fresh weight (g).To measure the NHs report . The sam2O2 was performed as previously described, with minor modifications [−1 3,3′-diaminobenzidine (DAB) solution was prepared, and the other steps remained unchanged. A ProCIPK23::GUS analysis of the plants was performed under multiple growth conditions for 7 days. Shoots and roots of promoter plants were cut off and transferred to tubes  containing a GUS staining solution (LEAGENE Lot.1127A19) and incubated at 37 °C for 24 and 2 h, respectively. The staining solution was then removed and the tissues were stored in 75% ethanol. GUS and H2O2 staining pictures of shoots were taken using Shineso 2.0. GUS-stained roots were photographed with a LEICA S8APO. Three replicates were completed for all experiments.Histochemical staining of Hications . The pH 2 and then ground using mortar and pestle. Homogenized sample was transferred into a 2 mL microcentrifuge tube and mixed with preheated (80 °C) 350 µL phenol water (Solarbio) and 350 µL RNA extraction buffer (100 mM Tris-HCl (pH 8.0), 0.1 M LiCl, 10 mM EDTA (pH 8.0)). The sample was vortexed for 20 s to be sure that the aqueous and organic phases were properly mixed and then placed for 5 min at room temperature. Next, 350 µL chloroform was added, and the sample was vortexed for 20 s before being placed for 5 min at room temperature again. The sample was then centrifuged for 15 min at 12,000 rpm, yielding three main phases. The aqueous phase was transferred to a new RNase-free microcentrifuge tube to which was added 1 volume of 4 M LiCl. This was then vortexed for 20 s and incubated for over 4 h at −20 °C. After incubation, the sample was centrifuged for 15 min at 12,000 rpm at 4 °C. The supernatant was discarded by decanting, and the pellet was washed twice with 70% ethanol. The ethanol was discarded by decanting, traces of ethanol by were eliminated by pipetting and the pellet was air-dried in a hood. The pellet was resuspended in 80 µL RNase-free H2O.Total RNA of seedlings was extracted with a phenol-based method. Samples were frozen in liquid NCIPK23 and some marker genes, the seedlings were treated for 7 days before being used to detect gene expression. cDNA was synthesized from 1 μg total RNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme) for RT-qPCR. qPCRs were performed using ChamQ SYBR qPCR Master Mix (Vazyme) on the LightCycler 96 (Roche). The amplification reactions were performed in a total volume of 20 µL, which contained 10 µL 2× ChamQ SYBR qPCR Master Mix, 7.2 µL ddH2O, 0.8 µL forward and reverse primers (10 µM), and 2 µL cDNA (which had been diluted 10 times after synthesis). PCR was conducted as follows: 95 °C for 1 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 min. Primers of qPCRs are listed in Actin2 was used as an internal control [To test the expression level of  control ,37. ThreTo measure the level of etiolation, the chlorophyll contents in the leaves were examined. The chlorophyll contents were measured according to the method described in a previous study . Seedlincipk23 mutant, the chlorophyll fluorescence under low NH4+/K+ (0.5 mM NH4+ and 0.5 mM K+) and high NH4+/K+ (10 mM NH4+ and 0.5 mM K+) ratios for 7 days was tested by analyzing the major fluorescence parameters , which revealed the PSII activity. The protocol selected was Quenching Act1. The parameters of the software were as follows: UV = 100, EI. Shutter = 1 and Sensitivity = 2.9%.To determine the photosynthetic performance of the wild type (Col-0) and the cipk23 under all conditions for 7 days were taken with a Shineso 2.0. Photos of the first and secondary lateral roots under low NH4+/K+ (0.5 mM NH4+ and 0.5 mM K+) and high NH4+/K+ (10 mM NH4+ and 0.5 mM K+) ratios were taken with a root scanner . Subsequently, the lengths of roots  were measured using the ImageJ software (https://imagej.en.softonic.com/).Photos of the primary roots of Col-0 and p < 0.05. The figures were drawn by GraphPad Prism 6.0. All graphs and images were arranged using Adobe Acrobat XI Pro.Statistical analysis of the data was developed by using the IBM SPSS Statistics 23 software. Significant differences among treatments were examined by one-way ANOVA using the LSD test at