Fabrication of biomimetic photocatalytic systems consisting of PI-COFs and molecular Ni complexes for selective reduction of CO2 is demonstrated. 2 into a given product is a great challenge but desirable. Inspired by natural photosynthesis occurring in hierarchical networks over non-precious molecular metal catalysts, we demonstrate an integration of single Ni sites into the hexagonal pores of polyimide covalent organic frameworks (PI-COFs) for selective photoreduction of CO2 to CO. The single Ni sites in the hexagonal pores of the COFs serve as active sites for CO2 activation and conversion, while the PI-COFs not only act as a photosensitizer to generate charge carriers but also exert a promoting effect on the selectivity. The optimized PI-COF with a triazine ring exhibits excellent activity and selectivity. A possible intra- and inter-molecular charge-transfer mechanism was proposed, in which the photogenerated electrons in PI-COFs are efficiently separated from the central ring to the diimide linkage, and then transferred to the single Ni active sites, as evidenced by theoretical calculations.Selective photoreduction of CO Molecular complexes as precursors of single active sites with tailorable and versatile coordination possess maximum efficiency of catalytic sites in the reaction.2.Selective photoconversion of CO2.The development of covalent organic frameworks (COFs)2 to CO  for selective photoreduction of CO2 to CO . The exc13C NMR spectroscopy and powder X-ray diffraction (PXRD). FT-IR spectra of all three COFs showed strong peaks at 1720–1725 cm–1 , which is in agreement with the pore sizes predicted from the theoretical crystal structures. The Brunauer–Emmett–Teller (BET) surface areas of PI-COF-1, PI-COF-2 and PI-COF-TT are calculated to be 475, 1175 and 825 m2 g–1, respectively  (29.76 kJ mol–1) for CO2 adsorption at low coverage  were synthesized by coupling pyromellitic dianhydride (PMDA) with tris(4-aminophenyl)amine (TAPA), 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 1,3,5-tris(4-aminophenyl)triazine (TAPT), respectively, and are denoted as PI-COF-1, PI-COF-2  and PI-Cm Fig. S4.43 The om Fig. S4.41 All P Fig. S11. This ma2 to various hydrocarbon fuels, making them suitable for reduction of CO2.UV-vis diffuse reflectance spectra (DRS) show that all three PI-COFs absorb light in the ultraviolet and parts of the visible region , suggest2,3]2+ complex has been shown to reduce CO2 actively in electrocatalysis3]2+ as photocatalytic systems may offer a viable approach for conversion of CO2. The photocatalytic activity of PI-COFs toward reduction of CO2 in aqueous solution was studied by using self-assembled [Ni(bpy)3]2+  as a precursor of active sites. The [Ni(bpy)3]2+ complex can be easily formed upon addition of a bpy ligand and Ni(ii) salt as evidenced by UV-vis absorption spectra. A remarkable red-shift of bpy in UV-vis absorption spectra indicates the coordination of bpy with Ni ions  facet weakened, due to the disorder induced by the Ni complexes inside the channels of Ni@PI-COF-TT 3]2+ removal 3]2+   of PI-COF-TT was estimated to be 0.55% at 380 nm. Acetonitrile was found to be a favourable reaction solvent for CO2 reduction with a relatively high activity and selectivity 3]2+ . Cyclingbpy)3]2+  and S26†n of CO2 . There w Fig. S27, suggestectivity , mainly ectivity . The resecreases , probablve sites .–2 at 0.1 V vs. RHE compared to PI-COF-1 and PI-COF-2  and transient photocurrent measurements. Nafion solution was used as an additive to PI-COF powder to form the active layer, Fig. S29. The cur Fig. S29, which p Fig. S29. All PI-2 over PI-COFs  emission spectra of PI-COFs show negligible peaks around 400–700 nm compared to the strong peak of PMDA at about 430 nm pyromellitimide, triphenylamine, triphenylbenzene and triphenyltriazine as model compounds of building blocks were performed to reveal their relative energy levels pyromellitimide are very close to those of triphenylamine, triphenylbenzene and triphenyltriazine, which means that the connection of pyromellitic diimide and triphenylamine, triphenylbenzene or triphenyltriazine affording an extended conjugation framework could presumably contribute to the electron transfer. When a physical mixture of pyromellitic diimide and triphenyltriazine was employed in the catalytic reaction, no reduction products such as CO, CH4 and H2 were observed 3]2+ 3]2+ case, the assembly of Ni ions and the bpy ligand occurred in the pores of PI-COFs, resulting in the uniform distribution of [Ni(bpy)3]2+ in PI-COFs. However, in the direct impregnation of [Ni(bpy)3]2+ case, the [Ni(bpy)3]2+ complexes with a relatively large molecular volume cannot diffuse effectively into the pores of PI-COFs, leading to the inefficient catalytic activity. The recovered PI-COF-TT does not generate the CO product in the additional reaction run in the absence of Ni complexes, further confirming that Ni active sites are free in the channel and only physically adsorbed on the pore surfaces.2 was not detected under similar reaction conditions (2]0 formed from the reduction of [Ni(bpy)3]2+ in the photocatalytic reaction serves as the active site for the activation and conversion of CO2.2]0 active sites are produced from the reduction of [Ni(bpy)3]2+ by photogenerated electrons of PI-COF-TT under UV-vis light irradiation 2]0via coordination of bpy to Ni.It is noteworthy that an obvious induction period accompanies the photochromic phenomenon. Thus, DFT calculations and control experiments were carried out to further explore the reaction mechanism. The ulations . Howeverulations  and EDX ulations  and S37†nditions . This co2 products, the adsorption energies of CO2 and H2O onto [Ni(bpy)2]0 with and without PI-COF-TT were calculated 2]0 in the presence of PI-COF-TT was significantly lower than that of H2O, implying the stronger affinity of molecular [Ni(bpy)2]0 toward CO2, which would facilitate the formation of the key intermediate Ni–CO2 adducts and afford selectivity for the CO2 reduction product rather than the H2 product. It is noteworthy that the adsorption energy of CO2 on Ni active sites was obviously reduced by PI-COF-TT through the hydrogen bonding interactions between the hydrogen atom in the PI unit and the activated CO2 molecule 2]0 active sites, which can couple with CO2 to produce CO. The open channel of PI-COF-TT facilitates the processes of photogeneration of molecular Ni active sites and the subsequent activation and conversion of CO2. Besides, the strong adsorption affinity of PI-COF-TT for CO2 may increase the local concentration of CO2 in the channels, thereby promoting the formation of Ni–CO2 adducts and facilitating the selective reduction of CO2.Based on the above results, a possible mechanism for the selective photoreduction of COproposed . Light i2. The electronic properties of PI-COFs can be facilely tuned for photocatalysis. The photogenerated electron–hole pairs in PI-COFs under irradiation can be efficiently separated through an intra- and inter-molecular charge-transfer mechanism to drive the reduction of CO2. The excellent catalytic performance of the COF-based catalytic system mainly arises from the synergistic effects of the photoactive PI-COF and single Ni sites, in which the PI-COF with a nature-mimicking architecture serves as the functionalized host for single Ni sites to promote the reaction. This work presents an integration of Ni single sites in PI-COFs for CO2 reduction and a deep understanding of the electron-transfer mechanism in COFs. Based on the diversity of COFs and molecular metal complexes, we believe, inspired by our work, there would be more functionalized COFs developed for robust and efficient catalytic systems for sustainable energy conversion.In summary, we demonstrated a design of an integration of PI-COFs with Ni single sites for selective photoreduction of CO2 bath) and degassed by pump–thaw three times. The tube was sealed and heated at 200 °C for 5 days, giving a yellow precipitate. The precipitate was purified by Soxhlet extraction using tetrahydrofuran overnight, and finally dried under vacuum at 80 °C to give PI-COF-TT (yield 66%).TAPT  and PMDA  were placed in a mixed solution of mesitylene/NMP/isoquinoline (0.5 mL/0.5 mL/0.05 mL). The tube was flash frozen at 77 K (liquid N2O and triethanolamine (TEOA)  containing PI-COFs (10 mg), Ni(ClO4)2·6H2O  and 2,2′-bipyridyl  was purged with CO2 for 15 min. The solution was then irradiated under UV-Vis light  at 313 K. After each reaction time, the generated gas in the headspace of the reaction vessel was sampled with a gas-tight syringe and determined by gas chromatography (Agilent 7890B) with both TCD and FID detectors. H2 was detected using a TCD detector. CO was converted to CH4 in a methanation reactor and then analyzed using an FID detector. The apparent quantum efficiency (AQE) at 380 nm was calculated by the following equation: AQE = (2 × amount of CO molecules evolved in 1 h/number of incident photons in 1 h) × 100%.A mixed solution of acetonitrile, HThere are no conflicts to declare.Supplementary informationClick here for additional data file.


Chromodomain-helicase-DNA-binding protein 9 (CHD9) has been implicated in the regulation of gene expression, yet its precise role in the maintenance of mammalian embryonic stem cell (ESC) remains unclear. In the present study, we demonstrated that mouse CHD9 controls the cell cycle of ESCs in a cell culture condition-dependent manner by modulating the accessibility of transcription factors to their target genomic elements. Our study, therefore, has not only established how CHD9 finetunes chromatin structure during animal development but provided a potential target for genetic screening of aberrant development in in vitro produced embryos.Chd9 knockdown (KD) in mouse embryonic stem cells (ESCs) cultured in normal serum media, not in 2i-leukemia inhibitory factor (LIF) media, causes rapid cell proliferation. This is caused by transcriptional regulation related to the cell cycle and the response to growth factors. Our analysis showed that, unlike the serum cultured-Chd9 KD ESCs, the 2i-LIF-cultured-Chd9 KO ESCs displayed elevated levels of critical G1 phase regulators such as p21 and p27. Consistently, the DNA binding sites of CHD9 overlap with some transcription factor DNA motifs that are associated with genes regulating the cell cycle and growth pathways. These transcription factors include the cycle gene homology region (CHR), Arid5a, and LIN54. Collectively, our results provide new insights into CHD9-mediated gene transcription for controlling the cell cycle of ESCs.Emerging evidence suggests that chromodomain-helicase-DNA-binding (CHD) proteins are involved in stem cell maintenance and differentiation via the coordination of chromatin structure and gene expression. However, the molecular function of some CHD proteins in stem cell regulation is still poorly understood. Herein, we show that  Chromodomain-helicase-DNA-binding (CHD) proteins, a subfamily member of the ATP-dependent chromatin-remodeling families, are largely evolutionary conserved. Eukaryotic CHDs have been implicated in diverse developmental processes including zygotic reprogramming, gastrulation and organogenesis ,2,3. CHDThe subfamily II proteins (CHD3-5) additionally contain a couple of PHD domains and form the nucleosome remodeling and deacetylase (NuRD) complex, a chromatin-remodeling complex that has histone deacetylase activity. CHD4 and 5 contribute to ESC differentiation into neural lineages through the transcriptional regulation of neuronal genes ,9,10,11.The subfamily III proteins (CHD6–9) are structurally similar to the subfamily II proteins, but they additionally contain SANT and BRK domains at the C-terminus . MutatioThe duration of the cell cycle and its turnover from one phase to the next in eukaryotes vary depending on the cell type. It has been shown that the cell cycle in mouse somatic cells takes approximately >16 h, whereas that in mouse ESCs takes ~8–10 h ,20. The Herein, we show that CHD9 silencing enhances ESC proliferation by changing the duration of the cell cycle phases. Our analysis also provides evidence that CHD9 modulates the status of chromatins in which transcription factors regulating ESC proliferation are accessible. Therefore, our study establishes new insights into CHD9-mediated gene transcription for controlling the cell cycle of ESCs.Chd genes during mouse ESC differentiation, a publicly available RNA-Seq dataset (GSE114219) was obtained and re-analyzed to compare the fragments per kilobase of transcripts per million mapped reads (FPKM) values of Chd1~9  RNA KD approach. To this end, two shChd9 were designed, tested for their KD efficiency, and found to repress ~80% of endogenous Chd9 expression  or 2i-LIF media (“2i-ESCs”). As shown in Chd9 KD serum-ESCs grew around two-times faster than the control serum-ESCs, whereas the Chd9 KD 2i-ESCs grew at a normal rate. To confirm these findings, the Chd9 KD ESCs were subjected to flow cytometric analysis. As shown in Intriguingly, we found that the Chd9 KD results in different patterns of cell proliferation depending on the culture conditions, publicly available data of a microarray (GSE64819) performed in Chd9 KD serum-ESCs and RNA-Seq (GSE148803) performed in Chd9 KO 2i-ESCs were re-analyzed. In the analysis of GSE64819, 569 downregulated DEGs and 527 upregulated DEGs were identified in the Chd9 KD serum-ESCs. In contrast, 488 downregulated DEGs and 150 upregulated DEGs were identified in the Chd9 KO 2i-ESCs (Cdkn1a) and p27 (encoded by Cdkn1b) is required for the extension of the G1 phase in the 2i-ESCs, we examined the expression of these genes. As shown in Chd9 KO 2i-ESCs, whereas both genes were not changed in the Chd9 KD serum-ESCs. A biological processes network analysis of upregulated DEGs showed that, among the various terms, genes related to “regulation of cell population proliferation” and “response to growth factor” were altered in the Chd9 KD serum-ESCs  carried out in mouse ESCs was re-analyzed. Our analysis identified 2614 genomic regions that Chd9 directly binds to. Further downstream analyses, including gene ontology biological process (GOBP) and KEGG pathway, were performed. As shown in cyclin B1 (Ccnb1) promoter. CCNB1 is essential for the regulation of the cell cycle; it interacts with other proteins such as the dimerization partner (DP), RB-like, E2F4, and MuvB (DREAM) complex [Chd9 silencing. Thirty-three CHD9 target genes repressed by Chd9 KD and 55 enhanced by Chd9 KD were identified in the comparison between the microarray and ChIP-Seq data. Our GOBP analysis showed that genes associated with chromatin modification such as “nucleosome assembly,” “chromatin silencing,” and “regulation of transcription” were significantly affected  regulates the cell cycle depending on the composition of the complex subunits [Ccnb1, Ccnb2, Cdk1, and Cdc20 [CHD9 expression by interfering with miR-197-5p in gastric cancer, and—interestingly—knocking down CHD9 in gastric cancer cells decreases cell growth [Chd7 KD causes the inhibition of cell proliferation and neurogenesis in vitro by repressing Hes5 and N-myc expression, whereas Chd7 overexpression leads to the acceleration of cell proliferation in human ESC-derived neural stem/progenitor cells [Chd8 mutation leads to induced apoptosis during early embryo development in mice by the activation of p53 [Besides the regulatory TFs, the epigenetic landscape in the 2i-ESCs differs from that in the serum-ESCs. For example, the DNA methylation level is globally reduced in the 2i-ESCs compared to that in the serum-ESCs. Furthermore, histone H3K9me3 is enriched at the hypermethylated genomic regions, including endogenous retroviruses (ERVs) and imprinting genes . Recentlsubunits . ARID1B subunits . The consubunits . Furthernd Cdc20 . Anotherl growth . Additioor cells . CHD7 isor cells ,39. Chd8n of p53 . CHD8 ren of p53 . It is gn of p53 . Liu et n of p53 .Ccnb1 promoter and that p53-p21-DREAM functions through CHR to regulate the expression of some critical cell cycle regulators, including CCNA2, CCNB1, and POLD1 [Our de novo motif analysis in CHD9 ChIP-Seq data showed that the CHD9 protein binds to genomic regions that overlap with cell cycle-related TFs binding DNA motifs, including CHR, LIN54, and Arid5a. It has been shown that CHR is present at the nd POLD1 ,45. Morend POLD1 ,47,48. Tnd POLD1 . Therefo® CRL-1821™, Manassas, VA, USA), and used for Chd9 knockdown (KD) studies. ESCs were cultured in DMEM  supplemented with 15% fetal bovine serum (FBS) , L-glutamine , MEM non-essential amino acids , penicillin/streptomycin , β-mercaptoethanol , and LIF  in a gelatin-coated dish. For the serum-free 2i culture conditions, the ESCs were maintained in DMEM/F12 : Neurobasal medium  (1:1) with N2 supplement , B27 supplement , penicillin/streptomycin , L-glutamine , 0.05% bovine serum albumin , 1 µm PD03259010 , 3 µm CHIR99021 , 150 µM monothioglycerol , and LIF  in a gelatin-coated dish.E14TG2a ESCs were obtained from ATCC  RNA of Chd9 were purchased . The shRNA sequences used in the study are as follows; KD1 (5′–3′) AAAGCCTGATTTGTATGACCG, and KD2 (5′–3′) TAATCTGTAGACTTTAACTGC. The synthesized oligos were duplexed, sequenced, and transfected into 293T cells to produce lenti-shChd9 viral particles using the SuperFect Transfection reagent . The supernatants harvested from the transfected 293T cells were pooled and concentrated using Amicon® Ultra-15 Centrifugal Filter Unit, 100K . Then, the ESCs were transduced using 10 μg/mL polybrene  and selected by puromycin (2 μg/mL) treatment at 48 h after transduction for more than 7 d.To silence For immunofluorescence staining, the cells were fixed with 4% paraformaldehyde, blocked using blocking solution (3% BSA and 2% serum of host secondary antibodies with 0.1% Tween-20 in PBS), and incubated with primary antibodies overnight. Then, the cells were washed with 0.1% Tween-20 in PBS and incubated with fluorescent-conjugated secondary antibodies. After washing, the cell nuclei were counterstained with DAPI. The antibodies used in the study were; SSEA-1 , SOX2 , Oct-3/4 , NANOG , donkey anti-rabbit or anti-goat IgG conjugated with Alexa Fluor 488, and anti-rabbit conjugated with Alexa Fluor 546 . For AP staining of the ESCs, the samples were fixed and stained using an Alkaline Phosphatase Staining Kit II  following the manufacturer’s instructions.Chd9 forward 5′-ACC CTT AAG GTT TCC ATC TG-3′, Chd9 reverse 5′-TGA AGT TTC TGT ACC TGT TCC-3′; Nanog forward 5′- AAG CAG AAG ATG CGG ACT GT-3′, Nanog reverse 5′- ATC TGC TGG AGG CTG AGG TA-3′; Oct3/4 forward 5′- CCA ATC AGC TTG GGC TAG AG-3′, Oct3/4 reverse 5′- CCT GGG AAA GGT GTC CTG TA-3′; and Sox2 forward 5′- GAA CGC CTT CAT GGT ATG GT-3′, Sox2 reverse 5′- TTG CTG ATC TCC GAG TTG TG-3′.For the cell proliferation assays, 1000 cells were seeded in a cell culture dish and counted after 3, 5, and 7 d. For cell cycle analysis, the cells were fixed with 80% ethanol at 4 °C for more than 16 h and treated with RNase A solution  for 2 h after washing with PBS. Then, the cells were stained with propidium iodide (PI) and analyzed flow cytometrically using CytoFLEX . For quantitative real-time PCR (qPCR) analysis, total RNA was extracted from the cells  and used for the synthesis of cDNA . qPCR was performed using SYBR green mix  on a StepOnePlus™ System . The primers used in this study were: Chd9 knockout ESCs (GSE148803) were obtained from Gene Expression Omnibus (GEO). The quality of the sequencing reads of the data was controlled using FastQC [RNA-Seq data performed in mouse embryoid bodies (GSE114219) and g FastQC , and theg FastQC  options:g FastQC  were useChd9 KD ESCs (GSE64819) were obtained from GEO. The microarray data were normalized and fitted into a linear model using the LIMMA  tool [Chd9 KD and control ESCs using LIMMA’s fold change and p-value data. With the list of DEGs, GO analysis was performed using the DAVID tool  [Microarray data performed in ia) tool  in R pacia) tool . Gene seia) tool  and the ia) tool , respectia) tool  package MD, USA) . ClueGo MD, USA)  plugged-MD, USA)  was usedA publicly available CHD9 ChIP-Seq dataset (GSE64825) was downloaded from GEO. The read quality of ChIP-Seq was assessed using FastQC. Bowtie2   was usedp-value lower than 0.05(*) or 0.001(***).All the bar graph data were generated using Prism 5.0 . Normality tests and equal variance tests were performed using SigmaPlot 12.0 . If the data failed the normality test, a rank-sum test was used and plotted in a scatter plot. One-way ANOVA and two-way ANOVA with a Holm–Sidak test as post hoc analysis were used for the qRT-PCR data of the ectoderm marker and proliferation data, respectively. Unpaired t-test analysis was performed on the rest of the data. The error bars in the data represent the standard error of the mean (SEM). Statistical significance was determined at a In summary, our study demonstrates that CHD9 regulates ESC proliferation by regulating the expression of cell cycle-related genes in a cell culture condition-dependent manner. In addition, our analysis suggests that CHD9 directly modulates the accessibility of cell cycle-related TFs to their target genes in the serum-ESCs. The analysis of the exact mechanism and partner proteins of the CHD9 machinery, which regulates cell cycle-related genes, is warranted in follow-up studies.


Lipid rafts serve as anchoring platforms for membrane proteins. Thus far they escaped direct observation by light microscopy due to their small size. Here we used differently colored dyes as reporters for the registration of both ordered and disordered lipids from the two leaves of a freestanding bilayer. Photoswitchable lipids dissolved or reformed the domains. Measurements of domain mobility indicated the presence of 120 nm wide ordered and 40 nm wide disordered domains. These sizes are in line with the predicted roles of line tension and membrane undulation as driving forces for alignment. Cell plasma membranes often display lateral inhomogeneities . Such coBecause of their small size, rafts are below the diffraction limit of light microscopy. Consequently, optical observations were thus far limited to model membranes, where domains reach larger sizes . Two majα) nanometer sized LODs or (β) rafts in a plasma membrane. However, proof for their registration is extremely scarce. There are four lines of support for the notion: (i) AFM experiments, (ii) theoretical considerations, (iii) molecular dynamics simulations, and (iv) simulation aided time resolved fluorescence resonance energy transfer experiments: (i)The AFM experiments were carried out on supported lipid bilayers . Yet the(ii)γ along the LOD’s rim, thereby minimizing the total energy stored in the system [Domain registration reduces e system . The gaie system . Yet, co(iii)Cholesterol’s preference for saturated tails drives phase separation in ternary lipid mixtures . Coarse-(iv)Exploiting fluorescence lifetime imaging of Förster resonance energy transfer in combination with Monte Carlo simulations suggested nanodomain registration in giant unilamellar vesicles . These dMicrometer sized LODs from the two monolayers of an unsupported lipid bilayer appear to be always in register . Conceivμm in diameter) in a Teflon diaphragm was lowered beneath lipid monolayers on top of the adjacent preheated aqueous solutions. The diaphragm was pretreated with 0.5% hexadecane in hexane. The monolayers differed in the lipid anchored dyes that they harbored: Atto565–DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, or Atto633–PPE, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine . Since both dyes preferentially partition into LDDs, the colocalizations of (i) dark membrane areas (LODs) from both leaflets with each other and (ii) bright membrane patches (LDDs) from the two leaflets with each other indicate domain registration  to confirm the assembly of membrane spanning nanometer-sized LODs in a minimal (protein-free) system. Therefore, we formed solvent-depleted asymmetric planar membranes as previously described . In briestration .cis or trans conformations  domain movement during image acquisition (ad for a <d 1.7 μm.D of one kind of domain (either LOD or LDD) in the other phase appears feasible. For the analysis of domain diffusion we used the Mosaic/Particle Tracking 2D/3D plugin [D depended on ad (D on ad (3D/hη) <ε = , water viscosity η3D = 10−3 Pa s, and domain diameter d = 1 μm, we find β = 3Duη/(hη) and u = 1 μm. In consequence we used the so-called generalized Saffman–Delbrück equation [−3 < ε < 103, i.e., for the diffusion of micrometer-sized domains [BT/(4πhη), γe =A = k 0.5772, p = 2.74819, q = 0.61465, v = 0.73761, and w = 0.52119.Inferring d from the diffusion coefficient D plugin ,31 of ImD plugin ,32  < 0.1 . Consideequation  that has domains : (2)D=Alβ, and δ by fitting Eq.  = η3D. The accordingly calculated η3D values did not significantly differ for LDDs and LODs. Yet the error was comparatively large. This prompted us to validate the parameters A and β by (i) predicting single lipid diffusion from the fit and (ii) measuring D of labeled lipids in LDDs and LODs by fluorescence correlation spectroscopy. The respective experimental values of 7.8 and 0.9 μm2/s (d = 0.9 nm): 6.2 and 1.6 μm2/s, respectively. This calculation neglects the height differences between a lipid and a domain, because for lipids that span one or two leaflets differs D by only about 30% [We treated .9 μm2/s  agree rebout 30% .Even the smallest LODs and LDDs span the whole bilayer as indicated by the fluorescence intensity of the dyes in the two monolayers , i.e., dd ~ 0.9 nm to d ~ 20 μm was the only major supposition made to introduce δ The assumed invariance of δ on domain size was experimentally confirmed by extrapolating from domain diffusion to the mobility of single lipids  × 10−9 and dhη = (0.43 ± 0.14) × 10−9 Ns/m, respectively. They agree well with published data (3.3 ± 1.1) × 10−9 [−9 Ns/m [Additional support for the approach comes from a rough assessment of membrane viscosity data. Using Eq.  and the ) × 10−9  and ≈0.5[−9 Ns/m .γ at the domain border in a way that stabilized domain size over an extended observation period. This is important because γ acts as the major driving force for registration of nanometer sized domains [w stored in the rim of every LOD (LDD) [w: LOD’s hydrophobic thickness per monolayer R =h 1.8 nm, LDD’s hydrophobic thickness per monolayer s =h 1.3 nm, splay modulus of the LOD monolayer R =B 20 BTk, splay modulus of LDD monolayer S =B 10 BTk, and lateral compression-stretching modulus A =K 120 mN/m (per monolayer). The spontaneous curvatures RJ and SJ of coexisting phases were taken as indicated in cis-trans photoswitching, the PhoDAG–1 molecular geometry  is expected to change substantially from slightly conical in the trans state to strongly conical in the cis state. Since γ strongly depends on curvatures RJ and SJ [w per unit length of the boundary as Δcis=γ 0.2 BT/nmk (for cis-PhoDAG–1) and Δγtrans = 0.07 BTk/nm (for trans–PhoDAG–1) [warea upon registration of ordered domains driven by membrane shape undulations [a is the ultraviolet cutoff parameter of the undulations, which is of the order of 1 nm. We find warea = 0.013 2kBT/nm for splay moduli R = 2BSB.An important technical advancement that allowed us to optically observe nanometer-sized domains is the use of photoswitchable lipids. They (i) triggered both domain dissolution and domain induction, and (ii) altered  domains ,43. The OD (LDD) ,43. We uR and JS , the phohoDAG–1) ,43. The ulations  is givenulations : warea=kd = 40 nm, cisw = 24.33 BTk, wtrans = 8.33 BTk , and Warea = 15.56 BTk. Thus, registration of these small domains is mainly driven by a term proportional to d. Considering solely the undulation related energy gain, Warea results in underestimated probabilities of domain registration.For domains of w led to theoretical predictions of antiregistration [Neglecting stration ,47. Neglstration .d = 120 nm): cisw = 73.2 BTk, wtrans = 25.2 BTk, and Warea = 144 BTk. That is, for trans–PhoDAG–1 containing domains we see a transition in the driving force: now undulations make the major contribution to coupling. The criterion for the transition can be calculated by requiring that γ-and undulation-driven energies be equal to each other. This is the case for the critical diameter d* = 4Δγ/warea. For cis-PhoDAG-1 and trans-PhoDAG-1 we find cisd* = 60 nm and d*trans = 20 nm, respectively.The situation is different for larger domains  in cell membranes may arise: By observing domains that are too small to be in register according to midplane coupling ,22, i.e.Supplementary material


The Cu7S4@SiO2/DOX was constructed by firstly synthesizing Cu7S4 nanocrystals, then in situ growing SiO2 shell on the surface of Cu7S4 nanocrystals, and finally loading DOX within SiO2 shell. The Cu7S4@SiO2/DOX was composed of Cu7S4 core as the photothermal transducer, SiO2 shell as DOX carrier and DOX as the model of anticancer drug. Once exposed to a 1064 nm laser, the Cu7S4@SiO2/DOX could simultaneous generate heat for photothermal therapy and accelerate the DOX release. When the Cu7S4@SiO2/DOX was injected into the center of tumor, the tumor exhibit rapid temperature elevation once exposed to the NIR laser and the tumor growth is significantly inhibited through the synergistic photothermal-chemo therapy, in comparison to the limited therapeutical effect of photothermal therapy or chemotherapy alone. Therefore, the Cu7S4@SiO2/DOX with photothermal-chemo function can be used as excellent nanoplatforms for treating solid tumor with high theoretical effect.The integration of photothermal therapy and chemotherapy has been recognized to be an efficient strategy through the instant thermally ablation and long-term chemical inhibition, thus achieving high therapeutical effect. In the present work, we designed and prepared Cu Among tcrystals  and oxygcrystals , and theructures  and Bi2Snanodots . For exaer cells . However2 nanosheets , oleic acid (AR), oleylamine (80–90%), cetyltrimethylammonium bromide (CTAB), sodium hydroxide , doxorubicin hydrochloride (DOX) and tetraethylorthosilicate  were brought from Sigma Aldrich.Sodium diethyldithiocarbamate (SDEDTC), CuCl7S4 nanocrystals were prepared by a typical thermolysis method (2⋅2H2O (20 mmol) was dissolved into 10 mL deionized water, which was then dropwise added into aqueous solution of SDEDTC  under magnetically stirring. After stirring for 2 h, the above solution was centrifuged  and washed with deionized water. The precipitate [Cu(DEDTC)2 precursor] was dried in vacuum at 50°C for further use. Secondly, oleic acid (15 mL) and oleylamine (10 mL) were added into three-neck bottle and heated to 280°C within 30 min under the continuous nitrogen flow to remove any moisture and oxygen. The Cu(DEDTC)2 precursor (1 mmol) dissolved in 2 mL oleic acid was injected to the three-neck bottle and heated at 280°C for 10 min. The dark green mixture was quickly cooled to 60°C by air flow and then 20 mL ethanol was introduced to precipitate Cu7S4 nanocrystals. Finally, the Cu7S4 nanocrystals were dispersed in ethanol followed by centrifuging and washed with ethanol for three times.The Cus method . Firstly7S4@SiO2 nanoplatforms were prepared by in situ growing SiO2 shell on the surface of Cu7S4 nanocrystals. Firstly, the hydrophobic Cu7S4 nanocrystals were converted into hydrophilic Cu7S4 through the surface-modification with CTAB. The Cu7S4 nanocrystals in chloroform solution  were added into aqueous solution of CTAB , which was stirred vigorously at 40°C for 24 h and then centrifuged to collect hydrophilic Cu7S4 nanocrystals. Secondly, the hydrophilic Cu7S4 nanocrystals were dispersed into 50 mL deionized water, followed by the introduction of NaOH solution  and TEOS (0.1 mL) under sonication. After 1 h of sonication, then 100 μL of PEG-silane was added and the mixture was maintained at 40°C for another 8 h. The products were centrifuged and washed with deionized water for three times for collecting Cu7S4@SiO2 nanoplatforms.The Cu7S4@SiO2 nanoplatforms were characterized by using JEOL 2100F transmission electron microscopy (TEM). The photoabsorption of Cu7S4@SiO2 nanoplatforms was studied on a Shimadzu UV-1900 spectrophotometer. The concentration of copper irons released from the Cu7S4@SiO2 nanoplatforms was determined by an inductively coupled plasma atomic emission spectroscopy (ICP-AES). The specific surface area and pore diameter of Cu7S4@SiO2 powder were investigated on the Autosorb-iQ/Autosorb-iQ Brunauer-Emmett-Teller (BET).The size, morphology, phase of Cu7S4@SiO2 nanoplatforms was investigated by illuminating their aqueous dispersion at a series of concentrations under a 1064 nm laser with the output power density of 0.6 Wcm–2. The temperature change was recorded by using a thermal imaging camera. The photothermal conversion efficiency of Cu7S4@SiO2 was calculated according to the previous report  and the heat capacity (CD) of deionized water.Where 7S4@SiO2  and DOX (10 mg) were dispersed into PBS solution, which was magnetically stirred in the dark. After 24 h of stir, the mixture was centrifuged  and the supernatant was collected. The DOX loading content was calculated based on (load weight of DOX/Cu7S4@SiO2/DOX) × 100%, in which the load weight of DOX was determined by (10 mg – DOX in the supernatant). For DOX releasing, Cu7S4@SiO2/DOX was dispersed into PBS at pH 7.4 or pH 5.4 and divided into two groups. One group was used as control and 0.5 mL solution were taken out which was centrifuged  at each time point. Another group was irradiated by a 1064 nm NIR laser (0.6 Wcm–2) at a specific time. The DOX releasing rate was calculated by using (the release weight of DOX/the load weight of DOX) × 100%.For loading DOX, Cu4 cells per well) at 37°C in the presence of 5% CO2 for 24 h. After incubation, the cell medium was removed, the cells were divided into four groups: (1) control, (2) DOX, (3) Cu7S4@SiO2/DOX+NIR, (4) Cu7S4@SiO2+NIR. Hundred microliter of the dispersion at varied concentrations was then added into the wells. After incubation for another 24 h, the cells were washed with PBS buffer solution for three times. Then the cells were irradiated with/without a 1064 nm laser. Cell viability was measured using the CCK-8 assay.Melanoma cells were seeded into a 96-well plate  and propidium iodide (dead cells) to distinguish live cells with green fluorescence and dead cells with red fluorescence.To visually compare the viability difference in cellular level among four groups, cells were seeded into a 24-well plate at a density of 1.2 × 10n = 3): (1) The control group; (2) DOX group; (3) Cu7S4@SiO2+NIR group; (4) Cu7S4@SiO2/DOX+NIR group. The mice in (2) group were intratumorally injected with DOX PBS solution , and mice in (3 and 4) group were, respectively, injected with Cu7S4@SiO2  or Cu7S4@SiO2/DOX PBS solution . The tumors on mice in  group were exposed to a 1064 nm NIR laser (0.6 Wcm–2) at the 0.5 h post-injection and mice body. After treatments, mice in all groups were observed regarding their body weight and tumor sizes. When a tumor size was beyond 1.0 cm, mice in all groups were sacrificed and tumors were extracted for histological examination.The BALB/c mice  with 4T1 tumors (the surface diameter of 0.3∼0.5 cm) on the back were divided into four groups 2 precursor with Cu source and S source was heated to 280°C for 10 min to produce uniform Cu7S4 nanocrystals. Secondly, the Cu7S4 nanocrystals were served as the core to allow the coating of SiO2 shell through the hydrolysis of TEOS. During the SiO2 coating, CTAB were used as a soft template. SiO2 grew around the template due to electrostatic interactions. 2 nanoplatforms, thus the nanoplatforms could be realized the bioapplication directly. The as-obtained Cu7S4@SiO2 nanoplatforms have a uniform morphology and the average size of 100 nm, as shown in the TEM image  plane of the orthorhombic Cu7S4 (JCPDS card no. 22-0250), which verifies the core is Cu7S4 nanocrystals. Subsequently, the phase of Cu7S4@SiO2 powders was characterized by using XRD. The XRD pattern (7S4 (JCPDS card no. 22-0250). It should be noted that there is a broad peak between 10° and 40°, which should be attributed to the characteristic peak originating from the amorphous SiO2 shell. 7S4@SiO2 nanoparticles. The binding energy peaks at 932.8 and 954.6 eV can be assigned to Cu+ coordinated to Cu in Cu7S4@SiO2 nanoparticles, whereas the binding energy peak at 943.2 eV is formally described as Cu2+. The coexistence of Cu+ and Cu2+ indicated the Cu vacancies on the surface of Cu7S4 nanocrystals  and the absorption intensity goes up with the increase of wavelength up to 1100 nm. Compared to the bandgap-induced UV-vis absorption, this kind of NIR absorption should be attributed to the localized surface plasmon resonances (LSPR) effect because of Cu vacancies on the surface of Cu7S4 nanocrystals, which has been reported for other Cu2–xS nanocrystals  nanocrystals  and NIR-II (1000–1350 nm). Compared to the commonly used 808 nm, 915 m and 980 nm NIR laser, 1064 nm NIR laser can offer more efficient tissue penetration depth by considering absorption and scattering effects  within 5 min of irradiation, which confirmed the negligible photothermal effect from deionized water , as shown in 7S4@SiO2 nanoparticles is 48.2% which is comparable to the reported CuS nanomaterials  surface area and pore size were investigated by using nitrogen adsorption-desorption curves. The nitrogen adsorption/desorption isotherms  was studied by dispersing Cu7S4@SiO2/DOX into PBS solution. At the time point of 1, 2, 3, 4, 6, 8, and 10 h, part of solution was taken out from the original 10 mL dispersion and centrifuged for collecting the released DOX in the supernatant, and the amount of DOX in the supernatant was calculated by applying absorption-concentration curve. 7S4@SiO2/DOX dispersion at the time point of 1, 2, 3, 4, 6, 8, and 10 h by using a 1064 nm NIR laser . For instance, after the firstly irradiation, the DOX releasing rate is calculated to be 45.8% which is much higher than that (28.4%) at the 2 h, indicating NIR laser-induced photothermal effect can significantly enhance the DOX releasing rate. After the irradiation cycles, 75.6% of DOX is released from Cu7S4@SiO2/DOX with the help of 1064 nm NIR laser in comparison to 47.4% of the released DOX without irradiation, within 10 h. Thereby, it is concluded that the Cu7S4 nanocrystals within Cu7S4@SiO2/DOX can produce heat to accelerate the DOX release. To study the DOX release behavior from Cu7S4@SiO2/DOX at the simulating tumor microenvironment, the release of the DOX against buffer solution at pH 5.4 was measured. It was found that the drug release rate became much faster at pH 5.4  Cu7S4@SiO2/DOX+NIR group. The mice in (2) group were intratumorally injected with DOX PBS solution , and mice in (3 and 4) group were intratumorally injected with Cu7S4@SiO2  and Cu7S4@SiO2/DOX PBS solution . –2), in which the tumor treated with Cu7S4@SiO2/DOX shows the bright red while the tumor received PBS exhibits the normal color. The surface temperature of tumor treated with Cu7S4@SiO2/DOX increases from ∼34.2°C to the balanced ∼57.9 °C at 300 s, resulting in temperature elevation of 23.7°C which was much higher than that (3.5°C) for the tumor received PBS (7S4@SiO2/DOX within tumor remain high photothermal conversion effect, which can convert 1064 nm NIR laser energy into enough heat to thermally ablate cancer cells.Before realizing bioapplication, the Cuvery low , which cfference . To perfived PBS . Thus, t7S4@SiO2+NIR group, their growth has been inhibited, due to the cytotoxicity of DOX for DOX group and the photothermal therapy for Cu7S4@SiO2+NIR group. Interestingly, in the case of the tumors in the Cu7S4@SiO2/DOX+NIR group, their volume goes down continuously with the significant inhibition efficiency compared to the other three groups. The high inhibition efficiency should be due to the synergistic photothermal-chemo effect from the combination of Cu7S4@SiO2/DOX and NIR laser irradiation. On the one side, the photothermal therapy can instantly kill part of cancer cells through high temperature, and on the other side, the load DOX within Cu7S4@SiO2/DOX can continuous release to kill the remaining cancer cells, thus achieving the synergistic effect on melanoma tumors. In addition, there was no any sign of loss in body weight for all groups (7S4@SiO2/DOX and 1064 nm NIR laser, while no obvious side effect for mice.After different treatments, mice in all groups were raised under the standard condition for the long-term observation of cancer treatment efficacy. The tumor sizes and body weights were recorded. The change of relative tumor volumes is demonstrated in l groups . Therefo7S4@SiO2/DOX+NIR group (2+NIR group (Furthermore, mice in all groups were sacrificed when a tumor size was beyond 1.0 cm . The tumIR group , includiIR group  and DOX IR group , there aIR group . The abo7S4@SiO2/DOX and used them as efficient nanoplatforms for synergistic photothermal-chemo therapy on melanoma tumors. The Cu7S4@SiO2/DOX was prepared by firstly synthesizing Cu7S4 nanocrystals, then in situ growing SiO2 shell on the surface of Cu7S4 nanocrystals, and finally loading DOX. The Cu7S4@SiO2 consisted of Cu7S4 core with the average diameter of 50 nm and SiO2 shell with the average thickness of 25 nm. The Cu7S4@SiO2 nanoplatforms exhibited the strong and broad NIR absorption and rapidly converted 1064 nm laser energy into heat, and they also demonstrated high specific surface area and a large amount of pores with high DOX-loading content of 59.8%. Importantly, under the irradiation of 1064 nm laser, Cu7S4@SiO2/DOX simultaneous generated heat and accelerated the DOX release. Give these advantages, mice were intratumorally injected with Cu7S4@SiO2/DOX and irradiated with1064 nm laser, which achieved the highest therapeutical effect through synergistic photothermal-chemo therapy compared to photothermal therapy or chemotherapy alone. Therefore, the Cu7S4@SiO2/DOX can be served as novel and efficient photothermal-chemo nanoagents for efficient tumor therapy.In summary, we prepared the CuAll datasets presented in this study are included in the article/Supplementary Material.The animal study was reviewed and approved by the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine.LZ and HP contributed equally to this work. LZ, HP, and XH designed the project and wrote the manuscript. LZ, HP, and YL carried out the experiment. LZ and FL performed the experimental data analysis. All the authors contributed to discussion of the results.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. The reviewer XH declared a shared affiliation, with no collaboration, with the authors to the handling editor.


HER2, a member of the Erythroblastosis Protein B/Human Epidermal Growth Factor Receptor (ErbB/HER) family of receptor tyrosine kinase, is overexpressed in 20~30% of human breast cancers. Trastuzumab, a HER2-targeted therapeutic monoclonal antibody, was developed to interfere with the homodimerization of HER2 in HER2-overexpressing breast cancer cells, which attenuates HER2-mediated signaling. Trastuzumab binds to the domain IV of the HER2 extracellular domain and does not directly block the dimerization interface of HER2-HER2 molecules. The three-dimensional structures of the tyrosine kinase domains of ErbB/HER family receptors show asymmetrical packing of the two monomers with distinct conformations. One monomer functions as an activator, whereas the other acts as a receiver. Once activated, the receiver monomer phosphorylates the activator or other proteins. Interestingly, in our previous work, we found that the binding of trastuzumab induced phosphorylation of HER2 with the phosphorylation pattern of HER2 that is different from that mediated by epidermal growth factor (EGF) in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Binding of trastuzumab to HER2 promoted an allosteric effect of HER2, in both tyrosine kinase domain and ectodomain of HER2 although details of allosteric regulation were missing. In this study, we utilized molecular dynamics (MD) simulations to model the allosteric consequences of trastuzumab binding to HER2 homodimers and heterodimers, along with the apo forms as controls. We focused on the conformational changes of HER2 in its monomeric and dimeric forms. The data indicated the apparent dual role of trastuzumab as an antagonist and an agonist. The molecular details of the simulation provide an atomic level description and molecular insight into the action of HER2-targeted antibody therapeutics. The ErbB family of receptor tyrosine kinases, including ErbB1/EGFR/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4, share common structural features. They consist of a ligand-binding extracellular domain and an intracellular domain connected by a single transmembrane helix. The extracellular domain consists of four domains, I-IV, and the intracellular domain consists of a short juxtamembrane segment, a tyrosine kinase domain, and a long unstructured C-terminal tail. The activation of EGFR depends on EGF-induced receptor dimerization and the structural arrangement of the ligand-induced dimers ,6 promotTrastuzumab is a monoclonal antibody specifically designed to target the extracellular domain IV of HER2. The mechanisms of actions of trastuzumab include antibody dependent cellular cytotoxicity (ADCC), antibody-mediated HER2 internalization followed by receptor degradation, inhibition of HER2 dimerization, and protection of the HER2 extracellular domain from proteolysis . TherapeThe sequence of HER2 was obtained from UniProt.org . First, homology modeling was used to obtain the different domains of HER2 separately. Domains I, II, and III of the ectodomain dimer were built based on the structure of the HER2 dimer  obtained3+ and COO− groups, respectively. HER2 Tyr877 was phosphorylated. The above HER2 related complexes were inserted into a POPC/POPS bilayer using the CHARMM-GUI membrane builder generator [The conserved disulfide bonds of HER2 and the antibody Fabs were constructed according to UniProt and the PDB files of trastuzumab and pertuzumab. As the non-sequential Kabat numbering scheme is used in the crystal structures of Fabs, the residues were renumbered for convenience in the simulation. The N- and C-termini were charged as NHenerator . The lipxy plane (membrane plane) remained constant with a volume change in the z direction. A switch function with a twin-range cutoff at 12 and 14 Å was used to evaluate the van der Waals (vdW) interactions. The Particle Mesh Ewald (PME) method was used for long-range electrostatic interactions evaluation. Each simulation system was run twice using the same strand configuration, with different lipid conformations randomly selected from the lipid library, and different initial velocities for all atoms. All MD simulations were performed using NAMD software [The systems were then solvated by TIP3P water molecules, and sodium and chlorides were added to neutralize the system and to achieve a total concentration of ~150 mM. The systems were minimized for 5000 conjugate gradient steps, with the protein backbone atoms and lipid head group atoms fixed followed by an additional 5000 conjugate gradient minimization steps that were applied to the systems. In the equilibration run, each system was gradually relaxed by a series of dynamic cycles, in which the harmonic restraints on the proteins and lipid head groups were gradually released to optimize the protein-water, protein-lipid, and water-lipid interactions. In the production stage, NPAT  ensemble at 310 K was used. The surface area in the software  with a CTo evaluate the total potential energy of the system, the trajectory for each system was extracted from the last 20 ns of explicit solvent MD without water molecules and ions. The solvation energies of all systems were calculated using the generalized Born method with molecular volume (GBMV)  after 50http://www.ks.uiuc.edu/Research/vmd/ (accessed on 26 October 2020)).Correlations between all the residues in the different clusters from four systems were analyzed using the normalized covariance to characterize the correlation in motion of protein residues ,21,22,23To evaluate the TM distance, the residues on the N- and C-termini were selected: N-terminal Thr652 and C-terminal Ile 673 were selected for the HER2 homodimer; N-terminal Thr652 and C-terminal Ile 673 of HER2 and N-terminal Ile622 and C-terminal Phe643 of EGFR were selected for the EGFR-HER2 heterodimer; N-terminal Thr652 and C-terminal Ile 673 of HER2 and N-terminal Leu634 and C-terminal Val654 of HER3 were selected for the HER3-HER2 heterodimer. The α carbons of above the residues were selected to measure the distance. The distance during the simulation was recorded and averaged, listed in 5 in 6-well plates and treated with trastuzumab, heregulin, trastuzumab + heregulin or left untreated. After treatments, whole cell lysate was collected and subjected to Western blotting to detect the phosphorylated and total levels of HER3.SKBR3 cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM media (Lonza) containing 10% fetal bovine serum and 1% antibiotics/antimycotics (Invitrogen). Antibodies directed against total HER3 and phosphorylated HER3 were purchased from Cell Signaling Technologies. Trastuzumab was obtained from FDA-Designated pharmacy and heregulin was purchased from Sigma Aldrich. For Western blot analysis, cells were seeded at 5 × 10The overall structure of EGFR showed symmetry in the extracellular domain and transmembrane helix and asymmetry in tyrosine kinase domain . The asyThe stability of the HER2-containing homodimers and heterodimers was evaluated after trastuzumab bound to HER2. After a 500 ns simulation, two transmembrane helices detached from each other when trastuzumab bound HER2 with a receiver conformation in both HER2-HER2 and EGFR-HER2 dimers  HER2 (activator) heterodimer . MoreoveIn this work, the unstructured long C-terminal tails were not included as these structures have not been determined. Although some structural models have been proposed for the C-terminal tails , the lon


Saccharomyces cerevisiae-based DFM and fermentation products on the plasma concentrations of carbonyl-containing metabolites via a metabolomics approach, and fecal bacterial community, via 16S rRNA gene sequencing, of beef steers during a 42-day receiving period. Forty newly weaned steers were randomly assigned to receive a basal diet with no additive  or a basal diet supplemented with 19 g of Commence™  for a 42-day period. Commence™  is a blend of 6.2 × 1011 cfu/g of S. cerevisiae, 3.5 × 1010 cfu/g of a mixture of Enterococcus lactis, Bacillus subtilis, Enterococcus faecium, and Lactobacillus casei, and the fermentation products of these aforementioned microorganisms and those of Aspergillus oryzae and Aspergillus niger. On d 0 and 40, rectal fecal samples were collected randomly from 10 steers from each treatment group. On d 42, blood was collected for plasma preparation.Previous studies have evaluated the metabolic status of animals fed direct-fed microbial (DFM) using enzyme-based assays which are time-consuming and limited to a few metabolites. In addition, little emphasis has been placed on investigating the effects of DFM on hindgut microbiota. We examined the effects of dietary supplementation of a blend of P ≤ 0.05) the relative abundance of Prevotellaceae UCG-003, Megasphaera, Dorea, Acetitomaculum, and Blautia. In contrast, the relative abundance of Elusimicrobium, Moheibacter, Stenotrophomonas, Comamonas, and uncultured bacterium belonging to family p-2534-18B5 gut group (phylum Bacteroidetes) were reduced (P ≤ 0.05).A total number of 812 plasma metabolites were detected. Up to 305 metabolites  including glucose, hippuric acid, and 5-hydroxykynurenamine were increased by PROB supplementation, whereas 199 metabolites  including acetoacetate were reduced. Supplementation of PROB increased (The results of this study demonstrated that supplementation of PROB altered both the plasma carbonyl metabolome towards increased glucose concentration suggesting an improved energy status, and fecal bacterial community, suggesting an increased hindgut fermentation of the beef steers. Saccharomyces cerevisiae and lactate-utilizing bacteria  were stratified by BW into 4 weight blocks. The steers were randomly assigned (within each weight block) to 1 of 2 treatments and housed in individual slatted floor pens (1 steer per pen). These calves were from a single source and were not transported. The treatments were a basal diet with no additive  approximately 4 h after feeding and immediately stored at − 80 °C until further analysis. On d 42, 15 mL of blood was taken before the morning feeding. The blood was collected from the coccygeal vessels into tubes containing sodium heparin  for preparation of plasma and immediately stored at − 80 °C until further analysis.12C- and 13C- dansylhydrazine (DnsHz) labeling to change the chemical and physical properties of carbonyl metabolites to enable them to be efficiently separated by LC and ionized by electrospray ionization MS [12C2-labeling was mixed with 20 μL of the individual plasma samples and the pooled sample (prepared by mixing 70 μL of each plasma sample). In addition, 30 μL of pH adjusting reagent and 30 μL of 13C2-labeling was mixed with another 20 μL of pooled sample. After vortexing and spinning down , the mixture was incubated at 40 °C for 60 min. Then the mixture was cooled down in − 80 °C freezer for 10 min to stop the labeling reaction. The mixture was then dried using a centrifugal vacuum concentrator. Finally, the dried mixture containing the labelled metabolites was re-dissolved in 100 μL of acetonitrile/Water . The 12C2-labeled individual sample was mixed with 13C2-labeled reference sample in equal amount according to carbonyl-labeling LC-UV normalization results. The solution was diluted and centrifuged at 15294×g for 10 min before injecting into Bruker Elute LC linked to Bruker Impact II QTOF MS for analysis. Quality control (QC) sample  was injected every 10 sample runs to monitor instrument performance.Relative quantification of metabolites containing a carbonyl group (carbonyl-metabolome) in plasma samples collected on d 42 was done using a CIL/LC-MS method. This method uses a differential ation MS . The woration MS . BrieflyTotal DNA was extracted from the feces with a PowerSoil DNA Isolation Kit . The integrity of the DNA was verified using 0.8% agarose gel electrophoresis. DNA concentration was determined using a NanoDrop 3300 . The DNA samples were prepared for sequencing according to the Illumina 16S Metagenomic Sequencing Library protocols to amplify the V3–V4 region. Amplification of the genomic DNA (10 ng) was performed using polymerase chain reaction (PCR) conditions: 94 °C for 3 min followed by 35 cycles of 94 °C for 15 s, 55 °C for 45 s, and 72 °C for 1 min, followed by a final elongation step of 8 min at 72 °C. 519F:5´-CCTACGGGNGGCWGCAG-3´ and 806R: 5´-GACTACHVGGGTATCTAATCC-3´ were used as the primer sequences. Quantification of the final purified product was done according to qPCR Quantification Protocol Guide , and the quality was checked using the LabChip GX HT DNA High Sensitivity Kit . Sequencing was performed on a paired-end Illumina MiSeq platform to generate 300-bp paired-end raw reads. Raw paired end reads were joined using FLASH2 v.c41a82e . The merVariables such as relative abundance of fecal bacteria and diversity indices were analyzed as a randomized complete block design using the GLIMMIX procedure of SAS 9.4 . The model included the fixed effects of treatment, random effects of block (BW as the block). Day 0 data were used as covariates. Each steer housed in an individual pen served as the experimental unit. Linear discriminant analysis effect size (LEfSe), which uses a Kruskal-Wallis (KW) test and the logarithmic linear discriminant analysis (LDA) score, was used to identify the most differentially abundant taxa . The sigFor the metabolomics data, 48 LC-MS data  were first exported to .csv file with Bruker DataAnalysis 4.4. The exported data were uploaded to IsoMS Pro 1.0.10. After format conversion, data quality check and data processing were performed according to procedures described by Mung and Li . MetabolP-value. Relative concentrations of metabolites with FC ≥ 1.5 or ≤ 0.67 having FDR ≤ 0.01 were differentially increased or decreased relative to CON, respectively. Pearson correlation analysis was used to examine the association between the relative concentrations of the identified plasma metabolites and growth performance indices (feed efficiency (FE) and average daily gain (ADG). The Pearson correlation coefficients were generated using R software (http://www.r-project.org). Pearson correlations were declared significant at P ≤ 0.10.Partial least squares discriminant analysis (PLS-DA) scores plot was used to visually show the differences between plasma metabolome of CON and PROB. Univariate (volcano plot) was generated using IsoMS Pro 1.0  by plottP = 0.01; SE = 2.67) and ADG . There was a tendency for improved FE with PROB supplementation . There was a tendency for lower fecal pH  in steers fed PROB diet.The effects of PROB supplementation on performance and health of the beef steers were reported in our previous study . BrieflyP = 0.01) and PLS-DA cross validation results  confirm the validity of the PLS-DA model  of 251.0849 was used as a background peak to check the mass accuracy of all the samples as well as 4 QC and 4 blank group samples. The appearance and mass of the peak was consistent for all runs and all scanned m/z were within the expected range, showing good stability and mass accuracy for the data acquisition  were increased by PROB supplementation, whereas 199 metabolites  were reduced Fig. . Among tPlasma concentrations of 5-oxopentanoate, isomer of (S)-2-aceto-2-hydroxybutanoate, and isomer of (S)-3-methyl-2-oxopentanoic acid were positively correlated with both FE and ADG while relative concentrations of 3-(4-hydroxyphenyl)pyruvate, (S)-2-aceto-2-hydroxybutanoate, (R)-3-hydroxy-3-methyl-2-oxopentanoate, and 2-dehydropantoate were positively correlated with ADG . The mean read length of quality checked and merged reads was 451 bp.P > 0.10) on alpha (Shannon index) and beta diversity (unweighted unifrac distance)  the relative abundance of Prevotellaceae UCG-003, Megasphaera, Dorea, Acetitomaculum, and Blautia. In contrast, the relative abundance of Elusimicrobium, Moheibacter, Stenotrophomonas, Comamonas, and uncultured bacterium belonging to family p-2534-18B5 gut group were reduced (P ≤ 0.05)  such as Enterococcus lactis, Enterococcus faecium, and Lactobacillus casei. Saccharomyces cerevisiae favors the activities of lactic acid utilizing bacteria (such as Selenomonas ruminantium and Megasphaera elsdenii) in the rumen [Enterococcus strains (5 × 109 cfu/g) and S. cerevisiae (2 × 109 cfu/g) to lactating Holstein cows during the transition period and reported improved performance associated with higher blood levels of glucose and lower serum levels of beta-hydroxybutyric acid. Similar results were reported by Nocek and Kautz [The increased relative concentrations of plasma monosaccharides such as glucose, galactose, fructose, and glyceraldehyde in steers fed PROB diet is evidence of an improved energy status of the animals. Glucose is the primary energy source and must be sufficient in the blood stream for optimum performance of animals , 22. In he rumen , 25. Prohe rumen , 26. Thehe rumen . Nocek ehe rumen  investignd Kautz .Compared to CON, greater plasma concentration of hippuric acid was observed in steers fed PROB diet. Hippuric acid is a gut-derived phenolic metabolite formed by the conjugation of benzoic acid  with glycine in the liver or kidney . StudiesRecent studies have identified tryptophan catabolism, through the kynurenine metabolic pathway, as one of the mechanisms by which the immune system modulates the balance between response to pathogens and tolerance to harmless antigens . The enzS. cerevisiae-based additive [Phenylacetylglycine levels in the blood is primarily driven by gut microbial metabolism . Formatiadditive –41, the Limited or no information is available in the literature on the significance of the other metabolites affected by PROB supplementation in this study. Some of these metabolites such as 5-oxopentanoate, isomer of (S)-2-aceto-2-hydroxybutanoate, isomer of (S)-3-methyl-2-oxopentanoic acid, 3-(4-hydroxyphenyl)pyruvate, (S)-2-aceto-2-hydroxybutanoate, (R)-3-hydroxy-3-methyl-2-oxopentanoate, and 2-dehydropantoate were positively correlated with performance parameters such as ADG and FE. More in-depth studies are needed to understand better how these metabolites are associated with improved animal performance.The primary limitation of this study was the use of an untargeted metabolomics approach which provides relative quantification of the metabolites based on intensity values. Nonetheless, this study confirms the benefits of PROB supplementation at improving the metabolic status of beef steers. Further studies that utilize a targeted metabolomics are needed to provide absolute quantification of some of the metabolites whose functions are currently unknown. Future advances in metabolomics technology should also focus on identifying those metabolites that were putatively matched in MCID library in this study.Prevotellaceae UCG-003 was increased in steers fed PROB diet and was the most altered taxa as revealed by LEfSe analysis. Prevotellaceae UCG-003 belongs to a group of bacteria, Prevotella. Prevotella plays a key role in the metabolisms of carbohydrate  and protein [Prevotella can grow effectively at low pH (< 6.0), especially in the rumen [Prevotellaceae UCG-003 in the feces of beef steers fed PROB diet is probably an increased production of organic acids in the hindgut due to increased fermentation of carbohydrates, which is associated with reduced fecal pH [S. cerevisiae and lactobacilli, both of which are constituents of PROB, throughout the gastrointestinal tract of beef cattle [Prevotellaceae UCG-003 in the feces is that PROB supplementation reduced ruminal fermentation and caused a shift in the site of fermentation to the large intestine, thereby increasing substrate availability for increased growth of Prevotellaceae UCG-003. This scenario would be expected to reduce the performance of the animals, which is contradictory to the growth performance results reported in our companion paper [In the present study, the relative abundance of  protein –44. Prevhe rumen , 46. Thufecal pH . This prfecal pH . These rf cattle , 49. An on paper , becauseDorea and Megasphera. This suggests the activities of the lactate-producing bacterial content of PROB. Increased colonization of Lactobacillus in the hindgut could result in increased production of lactic acid, which can favor the growth of lactate-utilizing bacteria. Blautia, a bacterial genus that can degrade complex carbohydrates, and Acetitomaculum, an acetogenic bacterial genus were also increased in feces of steers fed PROB. Conversely, the relative abundance of Elusimicrobium, Moheibacter, Stenotrophomonas, Comamonas, and uncultured bacterium belonging to family p-2534-18B5 gut group were reduced. The relative abundance of these aforementioned bacterial genera, including Dorea and Megasphera are low (≤ 0.1%), therefore, changes in their relative abundance may not be of biological relevance. Taken together, these results indicate that PROB supplementation altered the fecal bacterial community toward increased relative abundance of Prevotellaceae UCG-003 and lactate-utilizing bacteria.Supplementation of PROB increased the relative abundance of lactate-utilizing bacteria, such as Prevotellaceae UCG-003 and some.Supplementation of PROB improved the energy status of the beef steers by increasing the relative concentrations of plasma monosaccharides such as glucose, galactose, fructose, and glyceraldehyde, as well as others  with possible health benefits. In contrast, the relative concentration of acetoacetate was reduced. The significance of some metabolites, notably 5-oxopentanoate, isomer of (S)-2-aceto-2-hydroxybutanoate, isomer of (S)-3-methyl-2-oxopentanoic acid, 3-(4-hydroxyphenyl)pyruvate, (S)-2-aceto-2-hydroxybutanoate, (R)-3-hydroxy-3-methyl-2-oxopentanoate, and 2-dehydropantoate that showed positive correlations with performance measures merits further evaluation in future studies. Supplementation of PROB alters the fecal bacterial population towards increased relative abundance of Additional file 1:Table S1. Ingredient and chemical composition of the basal diet.Additional file 2: Figure S1. Mass accuracy checks of all the samples, including quality control and blank samples.Additional file 3:Table S2. Number of peak pairs for each sample.Additional file 4: Table S3. List of peak pairs detected from CIL LC-MS measurement of the samples.Additional file 5: Figure S2. A. PLS-DA cross validation results  and B. PLS-DA permutation test results .Additional file 6: Table S4. List of altered peak pairs.Additional file 7: Figure S3. A. Alpha  and B. Beta (unweighted unifrac distance) fecal samples from beef steers fed no (CON) or 19 g/d of a blend of S. cerevisiae-based direct-fed microbials and fermentation products .


Therefore, emerging attention has been applied to enzymatic production of d-mannose due to its advantage over chemical synthesis. The gene age of N-acetyl-d-glucosamine 2-epimerase family epimerase/isomerase (AGEase) derived from Pseudomonas geniculata was amplified, and the recombinant P. geniculata AGEase was characterized. The optimal temperature and pH of P. geniculata AGEase were 60 °C and 7.5, respectively. The Km, kcat, and kcat/Km of P. geniculata AGEase for d-mannose were 49.2 ± 8.5 mM, 476.3 ± 4.0 s−1, and 9.7 ± 0.5 s−1·mM−1, respectively. The recombinant P. geniculata AGEase was classified into the YihS enzyme subfamily in the AGE enzyme family by analyzing its substrate specificity and active center of the three-dimensional (3D) structure. Further studies on the kinetics of different substrates showed that the P. geniculata AGEase belongs to the d-mannose isomerase of the YihS enzyme. The P. geniculata AGEase catalyzed the synthesis of d-mannose with d-fructose as a substrate, and the conversion rate was as high as 39.3% with the d-mannose yield of 78.6 g·L−1 under optimal reaction conditions of 200 g·L−1d-fructose and 2.5 U·mL−1P. geniculata AGEase. This novel P. geniculata AGEase has potential applications in the industrial production of d-mannose. Th. Th6-barS enzyme  13]. Th. Th6-bard-glucose and d-fructose; d-fructose and d-mannose; and d-lyxose and d-xylulose. To further determine the categorization of P. geniculata AGEase, the kinetics for different substrates were investigated  with 100–300 g·L−1d-fructose . P. geniculata AGEase was confirmed to be the d-MIase belonging to the YihS enzyme of the AGE enzyme family. P. geniculata AGEase exhibited a high conversion rate of 39.3% for d-mannose production from d-fructose. This identified P. geniculata AGEase has great value for d-mannose production.The


Presence of both EF-hand motifs in TPK2 resulted in Ca2+ binding, but EF1 or EF2 alone failed to interact with Ca2+. The EF hands were not required for K+ transport activity. EF1 contains two cysteines separated by two amino acids. Replacement of both cysteines with serines in TPK2 increased Ca2+ binding. We generated a two-pore domain chimeric K+ channel by replacing the missing pore region in KCO3 with a pore domain of TPK2. Alternatively, we generated two versions of simple one-pore domain K+ channels by removal of an extra region from KCO3. The chimera and one of the simple one-pore variants were functional channels. This strongly suggests that KCO3 is not a pseudogene and KCO3 retains components required for the formation of a functional K+ channel and oligomerization. Our results contribute to our understanding of the structural properties required for K+ channel activity.Arabidopsis thaliana contains five tandem-pore domain potassium channels, TPK1-TPK5 and the related one-pore domain potassium channel, KCO3. Although KCO3 is unlikely to be an active channel, it still has a physiological role in plant cells. TPK2 is most similar to KCO3 and both are localized to the tonoplast. However, their function remains poorly understood. Here, taking advantage of the similarities between TPK2 and KCO3, we evaluated Ca The alignments were then combined and manually modified based on structural inspection taking helix continuity into consideration. When selecting the templates described above, no appropriate template sequence for the entire two-pore transmembrane domain was found by homology search (Blast); therefore, two subunits of a typical one-pore domain K+ channel were used. For modeling the EF-hand domain, only a few templates with weak similarity  were found. One template (GCAP1) containing a single Cys corresponding to C384 in TPK2 was selected among those. Model structures of the chimera dimer, the KCO3 dimer, and the KCO3ΔM’ tetramer were manually created based on structural insights gained from the topology of a TPK2 dimer.A model structure of TPK2 was created by homology modeling (Discovery Studio 2017 R2) using KClick here for additional data file.