Based on the framework of density functional theory and Boltzmann transport equation, we revealed that the monolayers possess high stability and narrow band gaps of 0.57~1.10 eV. Moreover, the high carrier mobilities (102~103 cm2·V−1·s−1) of these monolayers lead to high conductivities (106~107 Ω−1·m−1) and high-power factors (18.04~47.34 mW/mK2). Besides, as the strong phonon-phonon anharmonic scattering, the monolayers also show ultra-low lattice thermal conductivities of 0.23~3.30 W/mK at 300 K. As results show, all the monolayers for both p-type and n-type simultaneously show high thermoelectric figure of merit (ZT) of about 0.91~1.53 at room temperature.2D copper-based semiconductors generally possess low lattice thermal conductivity due to their strong anharmonic scattering and quantum confinement effect, making them promising candidate materials in the field of high-performance thermoelectric devices. In this work, we proposed four 2D copper-based materials, namely CuSbS S and T presents the temperature, n), L is Lorenz number.Thermoelectric generators can directly convert heat into electrical power, thus attracting wide research interest. Generally, the thermal-electric conversion capacity can be ruled by the dimensionless figure of merit, S2Tκe+κl , here S time τik ,4. Besidℏ2π3n2/3 . Additio, κe=LσT , where L2/Vs at 1.3 K, thus it was found to be a high-quality semiconductor ideal for thermoelectric applications [3Sb2 monolayer was proved to have a favorable ZT value of 2.5 at 900 K, which is higher than that of its bulk structure. These theoretical results also revealed that nano-engineering can effectively improve the thermoelectric conversion efficiency [ZT values of 0.46~0.60 at 750 K, indicating that 2D GeSbTe is a promising mid-temperature thermoelectric material.In fact, the thermoelectric performance of traditional thermoelectric materials has been effectively improved over the past few decades. Among them, two-dimensional layered (2D) materials, as unique mechanical, electronic, thermal, and optoelectronic properties, as well as quantum confinement effects, make them as promising thermoelectric materials in a variety of applications. For example, quasi-two-dimensional SnSe transistors were revealed to have high Seebeck coefficient, and a field effect mobility of about 250 cmications . The 2D ficiency . Additioσ-Cu2S, which has a low lattice thermal conductivity of 0.10 W/mK, and a high ZT value of 1.33 at 800 K. Cao et al. [β-Cu2Se under strain of −4~4%, they found that its ZT values can reach 1.65~1.71 at 800 K. Other materials, such as Fm-3m Cu2S [2Se [2S [ZT value. In fact, back in 2013 and 2016, Ma and Deng et al. [0.4Sb1.6Te3 alloy via an atomic-layer deposition (ALD) technique, and found that it possesses high ZT values of 1.50 at 329 K. Wu et al. [β-BiAs and β-BiSb monolayers by first-principles calculation and Boltzmann transport theory. They concluded that the monolayers simultaneously exhibit ultra-low lattice thermal conductivities (0.6~0.8 W/mK), and high ZT values (0.78~0.82) at 300 K.Recently, research on the thermoelectric properties of copper-based semiconductors has attracted much attention. For example, Yu et al.  proposedo et al.  investig-3m Cu2S , multi-su2S [2Se , and Cu-[2Se [2S , all exhg et al. ,16 invesg et al. ,18, whicg et al.  prepared2  are layered materials with narrow band gaps within ~1.38 eV. [2 exhibited the low lattice thermal conductivity of about 1.5~2 W/mK [2 and CuSbSe2 were revealed to have an excellent thermoelectric power factor at 300 K, reaching about 0.2~1.0 mW/mK2 at constant relaxation time approximation (CRTA) of 11 fs [2, CuSbSe2, CuBiS2, and CuBiSe2, can also deliver good thermoelectric performance. To this end, using first principles calculations, we investigated the electronic structures, mechanical, and transport properties, of these four 2D materials. The calculations revealed that all the monolayers possess narrow band-gaps (0.57~1.10 eV), high power factors (18.04~47.34 mW/mK2), and also low lattice thermal conductivities (0.23~3.30 W/mK). As a result, all the monolayers for both p-type and n-type simultaneously show high ZT values of about 0.91~1.53 at room temperature.CuMN1.38 eV.  Among th5~2 W/mK . Additioof 11 fs . TherefoWe carried out the calculations in the Vienna Ab initio Simulation Package (VASP) , in whicZT value, are directly related to carrier relaxation time, therefore, we used the deformation potential theory (DPT) to calculate carrier mobility, and further corrected it by the acoustic phonon-limited method (APM), which is more suitable for anisotropic materials. See The transport properties, such as electrical conductivity, electron thermal conductivity and 2  for bulk and single-layer are shown in 2 are layered structures, similar to graphite and MoS2. Therefore, we first investigated the cleavage energy of their single-layers, and its calculation method was based on the latest Rigorous Method proposed by Jung et al. [2, which are higher than those of graphene (0.33 J/m2), black phosphorus (BP) (0.36 J/m2), and MoS2 (0.27 J/m2) [2N (1.09 J/m2) [3 (1.14 J/m2), and InP3 (1.32 J/m2) [2 by mechanical exfoliation in experiments. The lattice constants and thicknesses of the monolayers after structural relaxation are listed in a/b) and thickness (h) follow the order of CuSbS2 < CuBiS2 < CuSbSe2 < CuBiSe2.The crystal structures of CuMNg et al. . Ef=Eiso27 J/m2) , but stiaphene 0. J/m2, bl32 J/m2) . All of a-axis. As can be seen, their Young’s moduli are relatively close, showing the maximum values of 58.20~66.52 N/m in the direction of 0° (180°) and the minimum values of 28.34~33.04 N/m in the direction of 90° (270°), which are obviously lower than those of graphene (350 ± 3.15 N/m) [2 (200 N/m) [2 and CuBiS2 are rare auxetic materials with negative Poisson’s ratio (NPR) of −0.02 and −0.04 at 0° (180°). Such interesting NPR phenomenon is also observed in PN (−0.08) [In general, for a new 2D material, we can identify its mechanical stability by its elastic constants nd C66>0 , indicataterials , as show.15 N/m) , h-BN ((.15 N/m)  and MoS2200 N/m) . Such lo200 N/m) . On the  (−0.08) , and tet (−0.08) , which h (−0.08) .e, indicating that they have relatively weak bond strength and thus exhibit low Young’s moduli. Besides, we analyzed the thermal stabilities of these four materials at different temperatures by using ab initio molecular dynamics (AIMD) simulations [We also investigated the bonding strength of materials by calculating their electron localization function (ELF) and Bader charge analysis  on their electronic band structures. As shown in a- and b-axis of the monolayers  of electrons are higher than those of holes (mh) along a-axis, corresponding to their flatter band near CBMs than VBMs along the Γ–X direction. However, the opposite is true for CuBiSe2, the me (0.93 m0) is indeed lower than mh (2.83 m0), which can be interpreted as the steeper dispersion curve corresponding to its VBM between Γ and X. Besides, for all monolayers, their elastic modulus (C2D) along the a-axis is larger than that along b-axis, which is consistent with their elastic constants. Further, the deformation potential constant (lE) fluctuates widely from 0.75 eV to 4.48 eV, and the lower lE occur simultaneously for the hole along the a-axis. As a result, the hole mobilities in the a-axis are higher than those in other cases, and the highest is even up to 4562.47 cm2·V−1·s−1 for CuSbS2. Since DPT method tends to overestimate the mobility of semiconductor, especially when the lE is relatively small [lE is relatively large, and decreases otherwise. As can be seen, the mobilities of both electrons (eμ) and holes (hμ) are basically in the range of 102~103 cm2·V−1·s−1, in which CuSbS2 and CuSbSe2 exhibit the highest hμ and eμ of 1661.49 and 937.12 cm2·V−1·s−1, which are far higher than that of MoS2 (μh ~200 cm2·V−1·s−1) [μe ~105 cm2·V−1·s−1) [μh ~104 cm2·V−1·s−1) [Next, we explored the carrier mobility of electrons and holes along the ly small , we adopS) satisfy CuSbS2 > CuBiS2 > CuSbSe2 > CuBiSe2, which consist with their band-gaps ordering, as small gap implies high carrier concentration. Coincidentally, the p-type Seebeck coefficients are higher than those of n-type in a-axis, but opposite in the b-axis, which may be caused by the anisotropy of the density of states effective mass. Besides, constrained by the Wiedemann-Franz-Lorenz’s law [e are carrier effective mass, mobility, and electron charge, as listed in 6~107 Ω−1·m−1, also high PF of 18.04~47.34 mW·K−2·m−1 at 300 K (see 2) [2Se3 (1.21~1.61 mW·K−2·m−1) [2 (5~25 mW·K−2·m−1) [2O (10~33 mW·K−2·m−1) [Furthermore, we explored the electron transport properties of the monolayers by solving Boltzmann transport equation, as show in nz’s law , the elea τ=m*μe , here m*00 K see , which a (see 2) , Pd2Se3 K−2·m−1) , and Tl2K−2·m−1) . 2 and CuBiSe2, their phonon frequencies are relatively lower, and more coupling occurs in the low frequency range, resulting in their scattering free path is shorter, and thus have lower phonon lifetimes.The phonon dispersions are shown in q [2 (~6.5 km/s) [2 and CuBiSe2, their phonon lifetimes are significantly shorter than those of the others, which is mainly due to their strong coupling at low frequency phonons, as analyzed above.The lattice thermal conductivity, vector q . As seenvector q , BP  . Althoug2 and CuBiSe2 exhibited larger values than those of CuSbS2 and CuBiS2, and thus have inherently stronger anharmonic interactions, as well as lower Generally, we can use the Grüneisen parameters γ to describe the anharmonic interactions of a material, which is effective mean to analyze the physical nature of lattice thermal conductivity. It can be obtained from the relationship of phonon frequency Vωq∂ωq∂V . For a l= S, Se) . Obviousoperties . −1·K−1, with CuSbSe2 and CuBiSe2 having lower values due to stronger phonon anharmonic interactions and low phonon lifetimes. Additionally, we can notice that the a-axis, which can be attributed to the stronger bonding, as well as higher Young’s moduli in this direction, and thus better heat transport. As shown in −1·K−1 at 300 K, which are comparable to or lower than those of bilayer SnSe (0.9 W·m−1·K−1) [2O (0.9~1.2 W·m−1·K−1) [−1·K−1) [−1·K−1) [Although the volumetric specific heat m−1·K−1) , Tl2O (0m−1·K−1) , Tetrady[−1·K−1) , and Ant[−1·K−1) . ZT) of these four monolayers at 300 K, as shown in ZT values in a-axis, while higher n-type ZT in b-axis, which is consistent with the results of higher hole mobility in the a-axis, while higher electron mobility in b-axis. As listed in ZT values for p-type of 0.91~1.17, and n-type of 0.74~1.53 at 300 K, which are higher than or comparable to those of many 2D thermoelectric materials, such as Pd2Se3 (0.9) [Finally, we fitted the thermoelectric figure of merit (e3 (0.9) , Telluree3 (0.9) , InSe (0e3 (0.9) , and SnSe3 (0.9) .2 . We found that monolayers possess the acceptable cleavage energies of 0.68~0.93 J/m2, and narrow band-gaps of 0.57~1.10 eV, respectively. Based on the acoustic phonon-limited method, we revealed that the electron and hole mobility are basically in the range of 102~103 cm2·V−1·s−1. Besides, they also have high electrical conductivity of 106~107 Ω−1·m−1, high PF of 18.04~47.34 mW·K−2·m−1 at 300 K. Furthermore, due to the stronger phonon anharmonic interactions and low phonon lifetimes, their lattice thermal conductivities are as low as 0.23~3.30 Wm−1 K−1. As a result, all the monolayers simultaneously exhibit high ZT values for p-type of 0.91~1.17, and n-type of 0.74~1.53 at 300 K, indicating that they have potential applications in nano-electronic and thermoelectric devices.In this work, we investigated the stability, mechanical, electrical, and phonon transport properties of 2D CuMN


Solid tumors subjected to intermittent hypoxia are characterized by resistance to chemotherapy and immune-killing by effector T-lymphocytes, particularly tumor-infiltrating Vγ9Vδ2 T-lymphocytes. The molecular circuitries determining this double resistance are not known.2 consumption were monitored by spectrophotometer and electronic sensors. The role of ROS/HIF-1α/LAP axis was validated in knocked-out or overexpressing cells, and in humanized (Hu-CD34+NSG) mice bearing LAP-overexpressing tumors. The clinical meaning of LAP was assessed in 60 NSCLC patients prospectively enrolled, treated with chemotherapy.We analyzed a panel of 28 human non-small cell lung cancer (NSCLC) lines, using an in vitro system simulating continuous and intermittent hypoxia. Chemosensitivity to cisplatin and docetaxel was evaluated by chemiluminescence, ex vivo Vγ9Vδ2 T-lymphocyte expansion and immune-killing by flow cytometry. Targeted transcriptomics identified efflux transporters and nuclear factors involved in this chemo-immuno-resistance. The molecular mechanism linking Hypoxia-inducible factor-1α (HIF-1α), CCAAT/Enhancer Binding Protein-β (C/EBP-β) isoforms LAP and LIP, ABCB1, ABCC1 and ABCA1 transporters were evaluated by immunoblotting, RT-PCR, RNA-IP, ChIP. Oxidative phosphorylation, mitochondrial ATP, ROS, depolarization, O2 consumption, increasing mitochondrial ROS that favor the stabilization of C/EBP-β mRNA mediated by HIF-1α. HIF-1α/C/EBP-β mRNA binding increases the splicing of C/EBP-β toward the production of LAP isoform that transcriptionally induces ABCB1 and ABCC1, promoting the efflux of cisplatin and docetaxel. LAP also decreases ABCA1, limiting the efflux of isopentenyl pyrophosphate, i.e. the endogenous activator of Vγ9Vδ2 T-cells, and reducing the immune-killing. In NSCLC patients subjected to cisplatin-based chemotherapy, C/EBP-β LAP was abundant in hypoxic tumors and was associated with lower response to treatment and survival. LAP-overexpressing tumors in Hu-CD34+NSG mice recapitulated the patients’ chemo-immuno-resistant phenotype. Interestingly, the ROS scavenger mitoquinol chemo-immuno-sensitized immuno-xenografts, by disrupting the ROS/HIF-1α/LAP cascade.By up-regulating ABCB1 and ABCC1, and down-regulating ABCA1, intermittent hypoxia induced a stronger chemo-immuno-resistance than continuous hypoxia in NSCLC cells. Intermittent hypoxia impaired the electron transport chain and reduced OThe impairment of mitochondrial metabolism induced by intermittent hypoxia increases the ROS-dependent stabilization of HIF-1α/LAP complex in NSCLC, producing chemo-immuno-resistance. Clinically used mitochondrial ROS scavengers may counteract such double resistance. Moreover, we suggest C/EBP-β LAP as a new predictive and prognostic factor in NSCLC patients.The online version contains supplementary material available at 10.1186/s13046-022-02447-6. Hypoxic conditions subtract the hypoxia induced factor α subunit (HIF-α) from the O2-dependent hydroxylation by prolyl hydroxylase domain (PHD) enzymes and subsequent degradation triphenyl-phosphonium, monomethanesulfonate (mitoquinol or mitoQ) was from Cayman Chemical .2. The experimental conditions were set as it followed: 24 h at 20% O2 (normoxia), 24 h at 1% O2 (hypoxia), 12 h at 1% O2 followed by 12 h at 20% O2 (hypoxia/normoxia), 12 h at 1% O2 followed by 12 h at 20% O2 and 12 h at 1% O2 (hypoxia/normoxia/hypoxia or intermittent hypoxia).Human NSCLC cells  were purchased from ATCC . Cells were maintained in the respective medium supplemented with 10% v/v FBS, 1% v/v penicillin-streptomycin, 1% v/v L-glutamine, in a Heracell incubator  with different pO2), with increasing concentrations (from 1 × 10− 9 to 1 × 10− 5 M) of cisplatin or docetaxel. Cell viability was assessed by the ATPlite Luminescence Assay System , as per manufacturer’s instructions, using a Synergy HT Multi-Detection Microplate Reader  to measure the relative luminescence units (RLUs). The RLUs of untreated cells were considered as 100% viability; the results were expressed as a percentage of viable cells versus untreated cells. The IC50 was calculated applying the [inhibitor] vs. normalized dose-response equation .After the growth in normoxic or hypoxic conditions indicated, cells were incubated 48 h in normoxia , pH 7.2, 0.5% bovine serum albumin (BSA) and 2 mM EDTA, centrifuged at 300×g for 10 min, incubated 20 min at room temperature in the dark with 250 μl of Inside Fix reagent , centrifuged at 300×g for 5 minutes, washed with 1 ml of Inside Perm (Inside Stain Kit), centrifuged at 300×g for 5 minutes, and incubated 30 minutes at room temperature with the following antibodies : anti-CD243/ABCB1 antibody (PE-Vio® 770-conjugated); anti-MRP1/ABCC1 antibody (PE-conjugated); anti-ABCA1(DyLight 488-conjugated). Cells were washed with 1 ml of Inside Perm reagent, centrifuged at 300×g for 5 minutes and read using a Guava® easyCyte flow cytometer , equipped with the InCyte software (Millipore).1 × 10http://primerdepot.nci.nih.gov/), were reported in the Additional File S14, using the Bio-Rad Software Gene Expression Quantitation (Bio-Rad Laboratories).Total RNA was extracted and reverse-transcribed using the iScript™ cDNA Synthesis Kit . The PCR arrays were performed on 1 μg cDNA, using customized Plus PCR Arrays (Bio-Rad Laboratories) pre-coated with primers for the ABC transporters or the transcription factors indicated in the Results section, as per manufacturer’s instructions. Data analysis was performed with the PrimePCR™ Analysis Software (Bio-Rad Laboratories). The qRT-PCR was performed with the IQ SYBR Green Supermix (Bio-Rad Laboratories). The primer sequences, designed using the qPrimerDepot database (https://epd.epfl.ch//index.php), using as inputs: https://epd.epfl.ch/cgi-bin/get_doc?db=hgEpdNew&format=genome&entry=ABCB1_1 (RefSeq NM_001348945) for ABCB1, https://epd.epfl.ch/search_EPDnew.php?query=ABCC1&db=human (RefSeq NM_004996) for ABCC1,https://epd.epfl.ch/cgi-bin/get_doc?db=hgEpdNew&format=genome&entry=ABCA1_1 (RefSeq NM_080282) for ABCA1. The transcription factors predicted to bind ABCB1, ABCC1 and ABCA1 promoters  -IPP , extracting lipids, isolating the IPP by thin layer chromatography and counting radiolabelled IPP by liquid scintillation  vs. normalized dose-response curves and relative IC50, obtained with the GraphPad Prism 9 software. Fig. S2. Effects of intermittent hypoxia on ABC transporters expression. NCI-H2228 cells were cultured in the following conditions: normoxia , hypoxia , hypoxia/normoxia , hypoxia/normoxia/hypoxia or intermittent hypoxia . The expression level of the indicated ABC transporters was measured by a RT-PCR array and is represented in a colorimetric scale. The expression of each ABC transporter in normoxic cells was considered 1; the relative expression in the other experimental conditions is indicated in the heatmap. Supplemental Fig. S3. Overexpression of C/EBP-β LAP and LIP. Normoxic NCI-H2228 cells, wild-type (wt), overexpressing C/EBP-β LAP (LAP+) or overexpressing C/EBP-β LIP (LIP+) were lysed and immunoblotted with an anti-C/EBP-β antibody, recognizing both LAP and LIP isoforms. Actin is included as control of equal protein loading. The image is representative of 1 out of 3 experiments. Supplemental Fig. S4. C/EBP-β mRNA sequence. Sequence of C/EBP-β LAP (a) and LIP (b) mRNA. The hypoxia-response element (HRE) is highlighted in cyano, the ATG codon of LIP mRNA in green. Supplemental Fig. S5. ABCB1, ABCC1 and ABCA1 promoter sequences. Sequences of ABCB1 (a), ABCC1 (b) and ABCA1 (a) promoters. The putative binding sites for C/EBP-β (CAAT boxes) are highlighted in yellow. Supplemental Fig. S6. Effects of C/EBP-β silencing on sensitivity to cisplatin and docetaxel. NCI-H2228 cells, stably silenced with the C/EBP-β-targeting sequence #2 (sh2), were cultured in the following conditions: normoxia , hypoxia , hypoxia/normoxia , hypoxia/normoxia/hypoxia or intermittent hypoxia . After normoxic and hypoxic cultures, cells were treated for 48 h in normoxia (20% O2) with increasing concentrations (from 1 × 10− 9 to 1 × 10− 5 M) of cisplatin  and docetaxel . Cell viability was measured by a chemiluminescence-based assay, in technical quadruplicates . Representative [inhibitor] vs. normalized dose-response curves and relative IC50, obtained with the GraphPad Prism 9 software. The dose-response curves and relative IC50 in NCI-H2228 cells treated with a non-targeting sequence (scr) shRNA sequence are reported in Fig. Supplemental Fig. S7. Effects of intermittent hypoxia on HIF-1α/C-EBP-β/ABC transporter axis and chemo-immuno-resistance in NCI-H1563 cells. NCI-H1563 cells were cultured in the following conditions: normoxia , hypoxia , hypoxia/normoxia , hypoxia/normoxia/hypoxia or intermittent hypoxia . a. Immunoblot of HIF-1α in whole cell extracts. Actin is included as control of equal protein loading. The image is representative of 1 out of 3 experiments. b. EPO1, VEGFA, GLUT1, ABCB1, ABCC1, ABCA1 mRNAs, measured by RT-PCR, in technical triplicates. Data are means±SD . *p < 0.05,**p < 0.01,***p < 0.001: H, H/N, H/N/H versus N cells; °°°p < 0.001: H/N cells versus H cells. c. RNA-IP with an anti-HIF-1α antibody, followed by RT-PCR amplification  with primers for C/EBP-β LAP (upper panel) or LIP (lower panel) isoforms. Data are means±SD . *p < 0.05,**p < 0.01,***p < 0.001: H, H/N, H/N/H versus N cells. d. Immunoblot of C/EBP-β LAP in whole cell extracts. Actin is included as control of equal protein loading. The image is representative of 1 out of 3 experiments. e. ChIP of C/EBP-β on ABCB1, ABCC1 and ABCA1 promoters, in technical triplicates. Data are means±SD . *p < 0.05,**p < 0.01,***p < 0.001: H, H/N, H/N/H versus N cells. f. After normoxic and hypoxic cultures, cells were treated for 48 h in normoxia (20% O2) with increasing concentrations (from 1 × 10− 9 to 1 × 10− 5 M) of cisplatin (Pt) and docetaxel (Dx). Cell viability was measured by a chemiluminescence-based assay, in technical quadruplicates . Representative [inhibitor] vs. normalized dose-response curves and relative IC50, obtained with the GraphPad Prism 9 software. g. Amount of released [14C]-IPP, considered an index of efflux, measured by liquid scintillation, in technical triplicates. Data are means±SD . ***p < 0.001: H, H/N, H/N/H versus N cells. h. Percentage of Ki67+IFN-γ+ Vγ9Vδ2 T cells collected after the co-cultures with the NCI-H1563 cells, evaluated by flow cytometry, in technical duplicates. Data are means±SD . *p < 0.05,***p < 0.001: H, H/N, H/N/H versus N cells. i. Percentage of annexin V+PI+ NCI-H1563 cells, as index of tumor cells killed by Vγ9Vδ2 T-cells, evaluated by flow cytometry, in technical duplicates. Data are means±SD . ***p < 0.001: H, H/N, H/N/H versus N cells. Supplemental Fig. S8. Antioxidants defenses in NCI-H2228 cells subjected to hypoxia. NCI-H2228 cells were cultured in the following conditions: normoxia , hypoxia , hypoxia/normoxia , hypoxia/normoxia/hypoxia or intermittent hypoxia . a-e. Superoxide dismutase 2 (SOD2) and superoxide dismutase 1 (SOD1) activity (a-b), catalase (c) activity, GSH/GSSG ratio (d) and glutathione peroxidase (GPX) activity (e) were measured spectrophotometrically, in technical triplicates. Data are means±SD . *p < 0.05,**p < 0.01,***p < 0.001: H, H/N, H/N/H cells versus N cells. f. Cell viability was measured after the different culture conditions indicated, by a chemiluminescence-based assay, in technical quadruplicates . Supplemental Fig. S9. Effects of elesclomol and mitoQ on mitochondrial ROS. NCI-H2228 cells were cultured in the following conditions: normoxia , hypoxia , hypoxia/normoxia , hypoxia/rnormoxia/hypoxia or intermittent hypoxia . Normoxic cells were treated with or without (−) 5, 10, 50 μM elesclomol , hypoxic cells were treated with or without (−) 0.1, 0.2, 0.4 μM mitoquinol . Mitochondrial ROS, measured spectrofluorimetrically, in technical triplicates. Data are means±SD . *p < 0.05,**p < 0.01,***p < 0.001: H, H/N, H/N/H cells or Es/−mQ-treated cells versus untreated N cells; °°p < 0.01,°°°p < 0.001: mQ-treated cells versus respective H, H/N, H/N/H untreated cells. Supplemental Fig. S10. Scavenging mitochondrial ROS sensitizes non-small cell lung cancer cells to docetaxel. NCI-H2228 cells were cultured in the following conditions: normoxia , hypoxia , hypoxia/normoxia , hypoxia/rnormoxia/hypoxia or intermittent hypoxia . After these incubation times, cells were treated for 48 h in normoxia (20% O2) with increasing concentrations (from 1 10− 9 to 1 × 10− 5 M) of docetaxel (Dx). When indicated, 0.4 μM mQ was added. Cell viability was measured by a chemiluminescence-based assay, in technical quadruplicates . Representative (inhibitor) vs. normalized dose-response curves and relative IC50, obtained with the GraphPad Prism 9 software. Supplemental Fig. S11. Chemo-immuno-sensitizing effects of mitoquinol in NCI-H1563 cells grown in intermittent hypoxia. NCI-H1563 cells were cultured in the following conditions: normoxia , hypoxia , hypoxia/normoxia , hypoxia/normoxia/hypoxia or intermittent hypoxia . a. Mitochondrial ROS, measured spectrofluorimetrically, in technical triplicates. Data are means±SD . ***p < 0.001: H, H/N/H versus N cells; °°°p < 0.001: H/N cells versus H cells. b. Hypoxic cells were treated with or without (−) 0.1, 0.2, 0.4 μM mitoquinol (mQ). Normoxic cells were included as internal control. Mitochondrial ROS were measured spectrofluorimetrically, in technical quadruplicates. Data are means±SD . **p < 0.01,***p < 0.001:H, H/N, H/N/H cells or mQ-treated cells versus untreated N cells; °p < 0.05,°°°p < 0.001 mQ-treated cells versus respective H, H/N, H/N/H untreated cells. c. After normoxic and hypoxic cultures, cells were treated for 48 h in normoxia (20% O2) with increasing concentrations (from 1 × 10− 9 to 1 × 10− 5 M) of cisplatin  and docetaxel . When indicated, 0.4 μM mQ was added. Cell viability was measured by a chemiluminescence-based assay, in technical quadruplicates . Representative [inhibitor] vs. normalized dose-response curves and relative IC50, obtained with the GraphPad Prism 9 software. d. Percentage of Ki67+IFN-γ+ Vγ9Vδ2 T cells collected after the co-cultures with the NCI-H1563 cells, evaluated by flow cytometry, in technical duplicates. Data are means±SD . *p < 0.05,***p < 0.001: H, H/N, H/N/H versus N cells; °p < 0.05,°°°p < 0.001 mQ-treated cells versus respective H, H/N, H/N/H untreated cells. e. Percentage of annexin V+PI+ NCI-H1563 cells, as index of tumor cells killed by Vγ9Vδ2 T-cells, evaluated by flow cytometry, in technical duplicates. Data are means±SD . ***p < 0.001: H, H/N, H/N/H versus N cells; °°°p < 0.001 mQ-treated cells versus respective H, H/N, H/N/H untreated cells. Supplemental Fig. S12. Dose-response effects of cisplatin and mitoquinol combination in LAP-overexpression NCI-H2228 tumors. 1 × 106 C/EBP-β LAP-overexpressing cells were injected subcutaneously in Hu-CD34+NSG mice. When tumor reached the volume of 50 mm3, animals (n = 4/group) were randomized and treated for 6 weeks as it follows: vehicle (0) group, treated with 0.1 ml saline solution intravenously (i.v.), once a week; mitoquinol (mQ) groups, treated with 10, 25, 50, 100 or 200 mg/kg daily via oral gavage, in the absence (−) or in the presence (+) of 2 mg/kg cisplatin (PT) i.v., once a week. To induce LAP intratumourally, 1 mg/ml doxycycline was added daily to the drinking water. Tumor growth was monitored by caliper. ***p < 0.001: PT + mQ-groups vs. PT-group (weeks 2–7). Supplemental Fig. S13. Tumors and post-mortem tissues characterization. 1 × 106 C/EBP-β LAP-overexpressing cells were injected subcutaneously in Hu-CD34+NSG mice. When tumor reached the volume of 50 mm3, animals (n = 6/group) were randomized and treated for 5 weeks as it follows: 1) vehicle (ctrl) group, treated with 0.1 ml saline solution intravenously (i.v.), once a week; 2) cisplatin (PT) group, treated with 2 mg/kg cisplatin i.v., once a week; 3) mitoquinol (mQ) group, treated with 100 μg/kg daily via oral gavage; 4) cisplatin + mitoquinol (PT + mQ) group, treated with 2 mg/kg cisplatin i.v., once a week and 100 μg/kg mitoquinol daily via oral gavage. To induce LAP intratumourally, 1 mg/ml doxycycline was added daily to the drinking water (LAP+ mice). a. Weight of the excised tumors. Data are means±SD (n = 6 tumors). ***p < 0.001:LAP+PT + mQ-group vs. LAP−ctrl-group; °°°p < 0.001:LAP+PT + mQ-group vs LAP+ctrl-group; ###p < 0.001:LAP+PT + mQ-group vs LAP+PT-group. b. Immunohistochemical analysis quantification. The amount of cells positive for C/EBP-β LAP, ABCB1, ABCB1, ABCA1 and cleaved caspase 3 was calculated counting 200 ± 25 cells/field, analyzing 5 fields for each treatment group, derived from each tumor and using Photoshop program. ***p < 0.001:LAP−PT-group vs LAP−ctrl-group, LAP+ctrl/PT-group vs. LAP−ctrl-group; °°°p < 0.001: LAP+mQ/PT + mQ-group vs LAP+ctrl-group; ###p < 0.001: LAP+mQ/PT + mQ-group vs LAP+PT-group. c. Animals weight were monitored weekly. d. Representative hematoxylin-eosin staining of liver, kidneys and spleen examined post-mortem. For each experimental group a minimum of 5 field were examined. Liver, kidneys: 63× objective, 20× ocular, bar: 50 μm; spleen: 63× objective, 10× ocular, bar: 100 μm. Table S4. Hematochemical parameters of treated animals.


GT863 inhibited the Aβo-induced increase in plasma-membrane phospholipid peroxidation, decreased membrane fluidity and resistance, and decreased excessive [Ca2+]i influx, showing cytoprotective effects. The effects of GT863 on cell membranes may contribute in part to its neuroprotective effects against Aβo-induced toxicity. GT863 may be developed as a prophylactic agent for AD by targeting inhibition of membrane disruption caused by Aβo exposure.In Alzheimer’s disease (AD), accumulation of amyloid β-protein (Aβ) is one of the major mechanisms causing neuronal cell damage. Disruption of cell membranes by Aβ has been hypothesized to be the important event associated with neurotoxicity in AD. Curcumin has been shown to reduce Aβ-induced toxicity; however, due to its low bioavailability, clinical trials showed no remarkable effect on cognitive function. As a result, GT863, a derivative of curcumin with higher bioavailability, was synthesized. The purpose of this study is to clarify the mechanism of the protective action of GT863 against the neurotoxicity of highly toxic Aβ oligomers (Aβo), which include high-molecular-weight (HMW) Aβo, mainly composed of protofibrils in human neuroblastoma SH-SY5Y cells, focusing on the cell membrane. The effect of GT863 (1 μM) on Aβo-induced membrane damage was assessed by phospholipid peroxidation of the membrane, membrane fluidity, membrane phase state, membrane potential, membrane resistance, and changes in intracellular Ca There is a rapidly aging population worldwide, and with aging, the prevalence of dementia worldwide continues to increase. Recent estimates predict that 50.4 to 65.1 million people worldwide were affected by dementia in 2019, with the prevalence increasing to 130.8 to 175.6 million by 2050 i in SH-SY5Y cells immediately after exposure. Aβo exposure caused [Ca2+]i to peak at 60–70 s, and sustained high [Ca2+]i values were maintained until 300 s.Aβo exposure significantly increased i in the presence of L-type/T-type voltage-gated calcium-channel (VGCC) blockers (nicardipine), N-type VGCC blockers (PD173212), and T-type highly selective VGCC blockers (NNC55-0396). Pretreatment with nicardipine (10 µM) significantly suppressed the sustained elevation of [Ca2+]i from 60 to 300 s of Aβo exposure    A. Howeve 0.5271) A. Under 2+]i in response to Aβo stimulation was inhibited by 1 µM curcumin and 1 µM GT863 pretreatment .The increase in i was measured using the FLIPR Calcium 5 Assay Kit (Molecular Devices). SH-SY5Y cells were loaded with FLIPR reagent diluted in buffer  for 60 min at 37 °C. Aβo, Aβo + curcumin or Aβo + GT863 was then added to the loaded cells and the changes in [Ca2+]I were measured using a Flex Station 3 (Molecular Devices) with FLIPR fluorescence signals at excitation and emission wavelengths of 485 nm and 525 nm, respectively, every 5 s for 300 s. The fluorescence intensity immediately before administration was expressed as 100%. In addition, changes in [Ca2+]i with and without Aβo were also measured in the absence of extracellular ionized calcium . To confirm Ca2+ influx through calcium channels by Aβo exposure, L-type/T-type calcium-channel antagonist (nicardipine: 10 µM), N-type calcium-channel antagonist (PD173212: 1 µM), or T-type highly selective calcium-channel antagonist (NNC 55-0396: 8 µM) was pretreated 5 min before Aβo addition and changes in [Ca2+]i were measured.i homeostasis (GT863 improved the cell viability of SH-SY5Y cells injured by Aβo, which include HMW Aβo, mainly composed of protofibrils. GT863 inhibited plasma-membrane phospholipid peroxidation, reduced excessive depolarization, and stabilized the plasma membrane against Aβo exposure-induced plasma-membrane injury. Furthermore, GT863 inhibited excessive Caeostasis . The exa


Scientific Reportshttps://doi.org/10.1038/s41598-022-23030-1, published online 01 November 2022Correction to: The original version of this Article contained an error in Figure 2b, where “kg ha-1” was incorrectly given as “kg ha-2” in Figure 2b, unit of horizontal axis. The original Figure The original Article has been corrected.


The octahedral SnCl62– dianion is almost undistorted. The crystal structure can be seen as an arrangement of alternating organic and inorganic layers with little support from C—H⋯Cl—Sn contacts.The asymmetric unit of the title compound, (C a]pyridin-2-ium) hexachloridostannate(IV), (C8H9N2)2[SnCl6], crystallizes in the monoclinic space group P21/n with the asymmetric unit containing an Sn0.5Cl3 fragment  to 2.4881 (8) Å and the cis Cl—Sn—Cl angles approaching 90°. In the crystal, π-stacked chains of cations and loosely packed SnCl62– dianions form separate sheets alternating parallel to (101). Most of the numerous C—H⋯Cl—Sn contacts between the organic and inorganic counterparts with the H⋯Cl distances above the van der Waals contact limit of 2.85 Å are considered a result of crystal packing.The hybrid salt bis(2-methylimidazo[1,5- One necessary component of the reaction is acid, which is introduced as a hydrohalide adduct of the amine  with L+ II to SnIV appeared unavoidable leading to the formation of ubiquitous hexachloridostannate(IV) dianion. Herein, the synthesis, structural analysis and spectroscopic characterization of (I)Pursuing our research on hybrid halometalates incorporating substituted imidazo, crystallizes in the monoclinic space group P21/n. The asymmetric unit consists of an Sn0.5Cl3 fragment 2[ZnCl4] 2[CdCl4] °. The octahedral SnCl62– dianion in (I)cis Cl—Sn—Cl angles approaching 90°  Å  Å, results in a closest separation of 7.7926 (1) Å between the metal atoms. The hybrid salt lacks classical hydrogen-bonding interactions but shows a variety of C—H⋯Cl—Sn contacts between the organic and inorganic counterparts  Fig. 2. In the  Å Fig. 3. The pais Table 2, a featu4.et al., 2016a]pyridinium cations gave 53 salts with about a half (23) reported by our research group. The latter comprise organic–inorganic hybrids with the L+ cation or its derivatives pyridin-2-ium and 2,2′-bis] counterbalanced by transition and main-group  halometalates. The other compounds in the CSD with cations similar to L+ are mostly organic salts with the imidazopyridinium core having various substituents in the rings. Perchlorate NAKNET imidazopyridin-2-ium] dichlorogold tetrachlorogold -1H-imidazol-3-ium] hexachloridostannate(IV), in space group P21/n, with layers formed by isolated [SnCl6]2– octahedra and (C6H9N2)+ organic cations, which propagate along the a-axis direction at y = 0 and y = 1/2 3NH2·HBr  was added to the warm formaldehyde solution prepared by dissolving paraform  in boiling deionized water (10 ml) in a 50 ml conical flask. The solution was stirred vigorously for 1 h at room temperature and filtered. On the following day, 2-PCA  was introduced into the flask under stirring, followed by the addition of SnCl2·2H2O  dissolved in ethanol (10 ml) in 30 min. The solution was kept magnetically stirred at room temperature for another hour, then filtered to remove Sn(OH)2 and allowed to evaporate. It was diluted with methanol (5 ml) since it was thickening. Pale-yellow needles of (I)iPrOH (5 ml). The crystals were filtered off, washed with diethyl ether and dried in air. Yield: 12% (based on Sn). FT–IR : 3422br, 3122vs, 3092vs, 3050vs, 3014, 2956, 2914, 1654, 1566, 1544, 1454, 1374, 1352, 1328, 1258, 1222, 1148vs, 1130, 1038, 986, 920, 789vs, 764, 742, 666, 624vs, 498, 468, 434. 1H NMR : δ (ppm) 9.83 , 8.69 , 8.24 , 7.82 , 7.22 , 7.13 , 4.27 . Analysis calculated for C16H18N4SnCl6 (597.73): C 32.15; H 3.04; N 9.37%. Found: C 32.40; H 2.88; N 9.19%.Synthesis of [6.Uiso(H) = 1.2UeqC for CH, C—H = 0.98 Å, Uiso(H) = 1.5UeqC for CH3). Anisotropic displacement parameters were employed for the non-hydrogen atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023000324/pk2675sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989023000324/pk2675Isup2.hklStructure factors: contains datablock(s) I. DOI: 2235690CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


This sandwich electrochemical immunosensor also exhibited high selectivity, reproducibility and stability.Avian influenza virus H9 subtype (AIV H9) has contributed to enormous economic losses. Effective diagnosis is key to controlling the spread of AIV H9. In this study, a nonenzymatic highly electrocatalytic material was prepared using chitosan (Chi)-modified graphene sheet (GS)-functionalized Au/Pt nanoparticles (GS-Chi-Au/Pt), followed by the construction of a novel enzyme-free sandwich electrochemical immunosensor for the detection of AIV H9 using GS-Chi-Au/Pt and graphene–chitosan (GS-Chi) nanocomposites as a nonenzymatic highly electrocatalytic material and a substrate material to immobilize capture antibodies , respectively. GS, which has a large specific surface area and many accessible active sites, permitted multiple Au/Pt nanoparticles to be attached to its surface, resulting in substantially improved conductivity and catalytic ability. Au/Pt nanoparticles can provide modified active sites for avian influenza virus H9-polyclonal antibody (AIV H9/PAb) immobilization as signal labels. Upon establishing the electrocatalytic activity of Au/Pt nanoparticles on graphene towards hydrogen peroxide (HThe online version contains supplementary material available at 10.1186/s11671-022-03747-8. Since avian influenza virus H9 subtype (AIV H9) was isolated from affected chickens in 1994, AIV H9 has become widespread in poultry . AIV H9 2O2, and thus, Au and Pt nanoparticles have been used to electrochemically catalyse the reduction of H2O2 and increase the sensitivity of electrochemical immunosensors  was used as a model complex, and the diffusion coefficient D was 7.6 × 10–6 cm2 s−1. In Eq.  ure Ret) , 38. The and Ket , as foll64−/3−. The Ket values were calculated to be 6.62 × 10–5 cm s−1, 73.69 × 10–5 cm s−1, 4.92 × 10–5 cm s−1, 3.87 × 10–5 cm s−1 and 2.18 × 10–5 cm s−1 for GCE, GS-Chi-GCE, AIV H9/MAbs-GS-Chi-GCE, BSA-AIV H9/MAbs-GS-Chi-GCE and AIV H9-BSA-AIV H9/MAbs-GS-Chi-GCE, respectively.A corresponds to the geometrical area of the electrode surface, while redox is the concentration of Fe(CN)64−/3−, while the peak current of GS-Chi-GCE increased substantially due to the large electron transfer-promoting effect and excellent conductivity of GS. After AIV H9/MAbs, BSA and 106.37 EID50/mL AIV H9 were immobilized on the modified electrode, the relevant peak current decreased in turn. The CV results were consistent with the EIS results, indicating the successful construction of the AIV H9 immunosensor.In addition, cyclic voltammetry (CV) is an effective technique for evaluating the successful modification of electrodes. The results are shown in Fig. 6.37 EID50/mL AIV H9 and then incubated with the two different types of labels. Finally, amperometric i–t measurements were performed in electrolyte (pH 7.0) with 10 mmol/L H2O2. As expected, the immunosensor using GS-Chi-Au/Pt-H9/PAbs (curve b) as the label displayed a much higher amperometric change than that using GS-Chi-H9/PAbs (curve c), GS-Chi-Au-H9/PAbs (curve d), Chi-Au/Pt-H9/PAbs (curve e) and GS-Chi-Pt-H9/PAbs (curve f) as the labels. The high sensitivity was mainly attributed to the large specific surface area, enhanced electrical conductivity and fast electron transport of GS and the excellent catalytic activity of Au/Pt. When no AIV H9 was immobilized on the electrode, the peak current was similar to curve a after the immunosensor was reacted with GS-Chi-Au/Pt-H9/PAbs (curve g), indicating that our developed immunosensor has good selectivity.Amperometric i–t curve measurements were employed to investigate the electrocatalytic performance of the proposed immunosensor. The results are shown in Fig. 2O2 concentration; (g) incubation time of AIV H9; and (h) incubation time of GS-Chi-Au/Pt-AIV H9/PAbs bioconjugates. Respective descriptions and figures of optimization methods are provided in Electronic Supporting Material. Briefly, the following experimental conditions were observed to produce the best results: (a) the best concentration of GS-Chi was 1.0 mg/mL; (b) best concentration of AIV H9/MAbs was 10 µg/mL; (c) best concentration of AIV H9/PAbs was 0.5 µg/mL; (d) best ratio of Au, Pt and GS was 2:2:20 (Au (0.1 mg/mL), Pt (0.1 mg/mL) and GS (1 mg/mL)); (e) best pH value of the electrolyte was 7.0; (f) best concentration of H2O2 was 10 mM; (g) best incubation time of AIV H9 was 40 min; and (h) best incubation time of GS-Chi-Au/Pt-AIV H9/PAbs bioconjugates was 60 min.The following parameters were optimized: (a) GS-Chi concentration; (b) AIV H9/MAb concentration; (c) AIV H9/PAb concentration; (d) ratio of Au, Pt and GS; (e) pH value of the electrolyte; (f) H2O2 to detect AIV H9 at a concentration ranging from 106.37 EID50 mL−1 to 101.37 EID50 mL−1. As shown in Fig. 50 mL−1 + 0.1936, with a correlation coefficient of R = 0.9932 and a low detection limit of 100.82 EID50 mL−1 (S/N = 3). In addition, the analytical performance of this immunosensor was superior to that of the previous methods developed for the detection of AIV H9  GS-Chi used as a matrix not only had remarkable electroconductivity that promoted electron transfer towards the surface of the electrode but also provided a large specific surface area to firmly bind AIV H9/PAbs, which improved the antigen–antibody reaction to detect AIV H9.Under the optimized reaction conditions, the detection capability of the proposed electrochemical immunosensor was determined by constructing an amperometric i–t curve at an initial potential of − 0.4 V in 10 mL of electrolyte (pH 7.0) containing 10 mM H5.61 EID50 mL−1), H2 (106.29 EID50 mL−1), H3 (107.43 EID50 mL−1), H4 (105.45 EID50 mL−1), H5 (128 HAUs), H6 (106.19 EID50 mL−1), H7 (256 HAUs), H8 (106.23 EID50 mL−1), H10 (105.73 EID50 mL−1), H11 (106.47 EID50 mL−1), H12 (107.12 EID50 mL−1), H13 (105.37 EID50 mL−1), H14 (106.72 EID50 mL−1), H15 (105.81 EID50 mL−1), H16 (105.63 EID50 mL−1), BSA (1.0 μg/mL), glucose (1.0 μg/mL) and vitamin C (1.0 μg/mL), were used in this work to evaluate the selectivity of the developed immunosensor. The results are shown in Fig. 4.37 EID50 mL−1) with other possible interfering substances were similar to those of AIV H9 (104.37 EID50 mL−1) alone, while nonspecific substances and blank solution showed similar current responses, indicating that the proposed electrochemical immunosensor had high selectivity for AIV H9 detection.The selectivity of the immunosensor played a crucial role in detecting target samples without separation. Several interfering substances, including AIV subtype H1  and GS-Chi-Au/Pt-AIV H9/PAbs bioconjugates. As shown in Fig. The long-term stability of the fabricated electrochemical immunosensor was further investigated by storing BSA-AIV H9/MAbs-GS-Chi-GCE at 4 °C when not in use and then successively incubating it with AIV H9  was added to the three AIV H9-positive samples , followed by detection using the fabricated electrochemical immunosensor according to the steps listed above in “n = 5), indicating that the developed immunosensor was feasible for AIV H9 detection in clinical samples.Standard spiking was also employed to evaluate the accuracy and practical applicability of the developed immunosensor. A series of different concentrations of AIV H9  with high electrocatalytic activity towards HAdditional file 1. Appendix A. Supplementary data.


A series of 18 cocrystals were obtained through the combination of the heterocyclic molecules imidazolidine-2-thione, 2-mercaptobenzimidazole, 2-mercapto-5-methylbenzimidazole, 2-mercaptobenzoxazole, and 2-mercaptobenzothiazole with the common halogen-bond donors 1,2-, 1,3-, and 1,4-diiodotetrafluorobenzene, 1,3,5-trifluorotriiodobenzene, and tetraiodoethylene. A rich series of hydrogen-, halogen-, and chalcogen-bonding interactions were observed. i.e. ribbons). Lacking the second N—H group, the thiazole and oxazole hydrogen bonding resulted in only dimeric pairs. C—I⋯S and C—I⋯I halogen bonding, as well as C=S⋯I chalcogen bonding, served to consolidate the packing by linking the hydrogen-bonding ribbons or dimeric pairs.Through the combination of heterocyclic thiones with variation in the identity of the heterocyclic elements, namely, imidazolidine-2-thione, 2-mercaptobenzimidazole, 2-mercapto-5-methylbenzimidazole, 2-mercaptobenzoxazole, and 2-mercaptobenzothiazole with the common halogen-bond donors 1,2-, 1,3-, and 1,4-diiodotetrafluorobenzene, 1,3,5-trifluorotriiodobenzene, and tetraiodoethylene, a series of 18 new crystalline structures were characterized. In most cases, N—H⋯S hydrogen bonding was observed, with these interactions in imidazole-containing structures typically resulting in two-dimensional motifs ( This diverse pool of substrates yielded structures with the crystal packing dominated by N—H⋯S hydrogen bonding, leading to thioamide dimers, with longer-range packing motifs created through C—I⋯S and C—I⋯I halogen bonding, as well as the occasional C=S⋯I chalcogen bond.Our group has recently been interested in the role of the S atom in I⋯S halogen- and chalcogen-bonding interactions as a crystal design tool, as well as their roles in the formation of deep eutectic solvents derived from halogen bonding (Peloquin Kα radiation (λ = 0.71073 Å) on a Bruker D8 Venture diffractometer with an Incoatec Iµs microfocus source and a Photon 2 detector.For single-crystal X-ray analysis, crystals were mounted on low background cryogenic loops using paratone oil. Data were collected using Mo Cocrystals were synthesized using imidazolidine-2-thione , 2-mercaptobenzimidazole , 2-mercapto-5-methylbenzimidazole , 2-mercaptobenzoxazole , 2-mercaptobenzothiazole , 1,2-diiodotetrafluorobenzene , 1,3-diiodotetrafluorobenzene , 1,4-tetrafluorobenzene , 1,3,5-trifluoro-2,4,6-triiodobenzene , and tetraiodoethylene . Solvents were obtained from Fisher Scientific. All materials were used as received without further purification. Crystals were formed by slow evaporation under ambient conditions (20–23 °C). Methanol was utilized for the majority of cocrystal preparations; however, if this was not successful, acetone or ethyl acetate was utilized.IT)· were obtained after 3 d.Imidazolidine-2-thione  and 1,3-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Methanol (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless needle-like crystals of 2(IT)· were obtained after 4 d.Imidazolidine-2-thione  and 1,3,5-trifluoro-2,4,6-triiodobenzene  were weighed into a 20 ml glass vial. Methanol (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless needle-like crystals of (MBZIM)·3 were obtained after 4 d.2-Mercaptobenzimidazole  and 1,3-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Methanol (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless needle-like crystals of 4(MBZIM)· were obtained after 3 d.2-Mercaptobenzimidazole  and 1,4-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Methanol (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless plate-like crystals of (MBZIM)·(TIE) were obtained after 7 d.2-Mercaptobenzimidazole  and tetraiodoethylene  were weighed into a 20 ml glass vial. Ethyl acetate (15 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless tabular crystals of (MMBZIM)· were obtained after 3 d.2-Mercapto-5-methylbenzimidazole  and 1,2-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Methanol (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless columnar crystals of (MMBZIM)··2(2OH) were obtained after 3 d.2-Mercapto-5-methylbenzimidazole  and 1,4-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Methanol (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless plate-like crystals of 2(MMBZIM)· were obtained after 4 d.2-Mercapto-5-methylbenzimidazole  and 1,3,5-trifluoro-2,4,6-triiodobenzene  were weighed into a 20 ml glass vial. Methanol (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless needle-like crystals of (MBZOX)· were obtained after 3 d.2-Mercaptobenzoxazole  and 1,2-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Methanol (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless needle-like crystals of (MBZOX)· were obtained after 2 d.2-Mercaptobenzoxazole  and 1,3-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Acetone (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless columnar crystals of (MBZOX)· were obtained after 2 d.2-Mercaptobenzoxazole  and 1,4-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Acetone (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless columnar crystals of 2(MBZOX)· were obtained after 1 d.2-Mercaptobenzoxazole  and 1,3,5-trifluoro-2,4,6-triiodobenzene  were weighed into a 20 ml glass vial. Acetone (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless columnar crystals of (MBZTH)·4 were obtained after 3 d.2-mercaptobenzothiazole  and 1,2-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Methanol (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless plate-like crystals of 3(MBZTH)· were obtained after 3 d.2-Mercaptobenzothiazole  and 1,3-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Methanol (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless plate-like crystals of (MBZTH)·2 were obtained after 4 d.2-Mercaptobenzothiazole  and 1,3-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Methanol (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless tabular crystals of (MBZTH)· were obtained after 2 d.2-Mercaptobenzothiazole  and 1,4-diiodotetrafluorobenzene  were weighed into a 20 ml glass vial. Acetone (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless needle-like crystals of 2(MBZTH)· were obtained after 2 d.2-Mercaptobenzothiazole  and 1,3,5-trifluoro-2,4,6-triiodobenzene  were weighed into a 20 ml glass vial. Acetone (10 ml) was added and the mixture was stirred until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless tabular crystals of (MBZTH)·(TIE) were obtained after 5 d.2-Mercaptobenzothiazole  and tetraiodoethylene  were weighed into a 20 ml glass vial. Methanol (15 ml) was added and the mixture was stirred with gentle heating until a clear solution was obtained. The solvent was allowed to evaporate slowly and colorless block-like crystals of (Uiso(H) = 1.5Ueq(C) for methyl H atoms, and C—H = 0.95 Å and Uiso(H) = 1.2Ueq(C) for other H atoms. H atoms on heteroatoms were located in difference Fourier maps and refined isotropically, utilizing appropriate restraints [N—H = 0.86 (2) Å] where necessary to maintain chemically reasonable geometries. The H atoms of the water molecule in 2(MMBZIM)··2(2OH) were modeled in a disordered arrangement due to symmetry considerations.Crystal data, data collection and structure refinement details are summarized in Table 1IT)·, which was refined in the orthorhombic space group Pbcn with two unique molecules of IT and one molecule of 4DIB1,3-F in the asymmetric unit · and (IT)·2 cocrystals, respectively · ·, was refined in the orthorhombic space group Pbca with one molecule each of IT and 3I3B1,3,5-F in the asymmetric unit. This structure represents the only example within this study without N—H⋯S hydrogen bonding  yielded three new structures dominated by co-operative hydrogen and halogen bonding ·3 was obtained in the triclinic space group PMBZIM and three molecules of 4DIB1,3-F in the asymmetric unit. In this structure, hydrogen bonding between thiourea molecules contributes to the formation of ribbons propagating along the a axis · · cocrystal, refined in the monoclinic space group P21/c, with one molecule each of both MBZIM and 4DIB1,4-F in the asymmetric unit. Just as in 4(MBZIM)·3, the structure of (MBZIM)· consists of ribbons of MBZIM molecules propagating in the c direction, formed through thiourea hydrogen bonding  often enables structural motifs that are different from the typical aromatic halogen-bond donors. The cocrystal (MBZIM)·(TIE) was refined in the orthorhombic space group Pnma, with one molecule each of MBZIM and TIE in the asymmetric unit. As in the previous examples, molecules of MBZIM form infinite ribbons through thiourea hydrogen bonding  Å] to reinforce the framework.With four I atoms available, tetraiodoethylene , induces significant changes to the overall hydrogen- and halogen-bonding motifs. The structural literature of this substrate is limited, having only been characterized by single-crystal X-ray diffraction when acting as a ligand for transition metals coordinating through its S atom ·, was refined in the triclinic space group PMMBZIM and 4DIB1,2-F in the asymmetric unit · and most of the cocrystals in the present study ··2(2OH) crystallizes in the triclinic space group PMMBZIM and 2OH, as well as half a molecule of 4DIB1,4-F, in the asymmetric unit. All attempts to obtain an nonhydrated cocrystal with 4DIB1,4-F were unsuccessful, suggesting the packing arrangement formed strictly by halogen bonding contains small but meaningful voids that must be occupied by the water molecule. Discrete hydrogen-bonded dimers are again observed by hydrogen bonding of the thioamides (Table 9MMBZIM)·, with two halogen bonds to each S atom, 2(MMBZIM)··2(2OH) utilizes one C—I⋯S halogen bond and one O—H⋯S hydrogen bond at each S atom. It is the halogen bonding that contributes to the formation of infinite chains by linking the discrete dimers. The water molecule also acts as an N—H⋯O hydrogen-bond acceptor from the N atom that does not participate in thioamide hydrogen bonding and so is an intermediate linker facilitating the formation of an expanded thioamide ribbon motif.Isolated as a hydrated cocrystal from adventitious water, 2·, refined in the monoclinic space group P21/c with one unique molecule of each component in the asymmetric unit. The overall packing motif in this structure is strikingly similar to that in (MMBZIM)·. Two molecules of MMBZIM form dimeric pairs through hydrogen bonding of the thioamides · to (MMBZIM)·, it may be that the C—F⋯I contact is merely coincident within the motif formed by the N—H⋯S and C—I⋯S interactions.Finally, the combination of  Table 10. The remMBZIM, substituting one secondary N atom for an O atom in 2-mercaptobenzoxazole (MBZOX) allows for the study of the structural motifs when only dimers can form through hydrogen bonding · was refined in the monoclinic space group P21/n, with one unique molecule each of MBZOX and 4DIB1,2-F in the asymmetric unit. Here, a hydrogen-bonding thioamide dimer is formed ·. The pattern of interactions in (MBZOX)·, which crystallizes in the monoclinic space group P21/c, with one molecule each of MBZOX and 4DIB1,3-F in the asymmetric unit, is more complex. Thioamide hydrogen-bonding dimers are once again observed ·, which was refined in the monoclinic space group C2/c, with one molecule of MBZOX and one-half of a molecule of 4DIB1,4-F, the packing motif is more reminiscent of its MMBZIM analogue. Thioamide hydrogen-bonding dimers are linked into chains through C—I⋯S halogen bonding ·, was refined in the monoclinic space group P21/c, with one molecule each of MBZOX and 3I3B1,3,5-F in the asymmetric unit. Much of the packing is similar to (MBZOX)·, with thioamide hydrogen-bonding dimers stacking in the b direction ·4, crystallized in the triclinic space group PMBZTH and four molecules of 4DIB1,2-F in the asymmetric unit. Thioamide dimers stack along the a axis ·, refined in the triclinic space group PMBZTH and 4DIB1,3-F within the asymmetric unit, is similar to that of (MMBZIM)· and (MMBZIM)·. Thioamide hydrogen-bonding dimers ·2 contains two unique molecules of MBZTH and four molecules of 4DIB1,3-F. In this case, the thioamide hydrogen-bonding dimers ·, refined in the monoclinic space group P21/n, with one complete molecule of MBZTH and one-half of a molecule of 4DIB1,4-F in the asymmetric unit, is similar to that of 2(MBZOX)·. Thioamide hydrogen-bonding dimers · was obtained in the monoclinic space group P21/c, with one unique molecule each of both MBZTH and 3I3B1,3,5-F in the asymmetric unit. The primary packing motif is similar to that of (MBZTH)·2, with the thioamide hydrogen-bonding dimers (Table 19MBZTH)·(TIE) crystallized in the triclinic space group PMBZTH and two unique half molecules of TIE in the asymmetric unit. Thioamide hydrogen-bonding dimers  extended some structures into three-dimensional frameworks. The 10.1107/S2053229622009548/qw3002sup1.cifCrystal structure: contains datablock(s) 2IT_13F4DIB, IT_135F3I3B, 4MBZIM_313F4DIB, MBZIM_14F4DIB, MBZIM_TIE, MMBZIM_12F4DIB, 2MMBZIM_14F4DIB_2H2O, MMBZIM_135F3I3B, MBZOX_12F4DIB, MBZOX_13F4DIB, 2MBZOX_14F4DIB, MBZOX_135F3I3B, 3MBZTH_412F4DIB, MBZTH_13F4DIB, MBZTH_213F4DIB, 2MBZTH_14F4DIB, MBZTH_135F3I3B, MBZTH_TIE, global. DOI: 10.1107/S2053229622009548/qw30022IT_13F4DIBsup2.hklStructure factors: contains datablock(s) 2IT_13F4DIB. DOI: 10.1107/S2053229622009548/qw3002IT_135F3I3Bsup3.hklStructure factors: contains datablock(s) IT_135F3I3B. DOI: 10.1107/S2053229622009548/qw30024MBZIM_313F4DIBsup4.hklStructure factors: contains datablock(s) 4MBZIM_313F4DIB. DOI: 10.1107/S2053229622009548/qw3002MBZIM_14F4DIBsup5.hklStructure factors: contains datablock(s) MBZIM_14F4DIB. DOI: 10.1107/S2053229622009548/qw3002MBZIM_TIEsup6.hklStructure factors: contains datablock(s) MBZIM_TIE. DOI: 10.1107/S2053229622009548/qw3002MMBZIM_12F4DIBsup7.hklStructure factors: contains datablock(s) MMBZIM_12F4DIB. DOI: 10.1107/S2053229622009548/qw30022MMBZIM_14F4DIB_2H2Osup8.hklStructure factors: contains datablock(s) 2MMBZIM_14F4DIB_2H2O. DOI: 10.1107/S2053229622009548/qw3002MMBZIM_135F3I3Bsup9.hklStructure factors: contains datablock(s) MMBZIM_135F3I3B. DOI: 10.1107/S2053229622009548/qw3002MBZOX_12F4DIBsup10.hklStructure factors: contains datablock(s) MBZOX_12F4DIB. DOI: 10.1107/S2053229622009548/qw3002MBZOX_13F4DIBsup11.hklStructure factors: contains datablock(s) MBZOX_13F4DIB. DOI: 10.1107/S2053229622009548/qw30022MBZOX_14F4DIBsup12.hklStructure factors: contains datablock(s) 2MBZOX_14F4DIB. DOI: 10.1107/S2053229622009548/qw3002MBZOX_135F3I3Bsup13.hklStructure factors: contains datablock(s) MBZOX_135F3I3B. DOI: 10.1107/S2053229622009548/qw30023MBZTH_412F4DIBsup14.hklStructure factors: contains datablock(s) 3MBZTH_412F4DIB. DOI: 10.1107/S2053229622009548/qw3002MBZTH_13F4DIBsup15.hklStructure factors: contains datablock(s) MBZTH_13F4DIB. DOI: 10.1107/S2053229622009548/qw3002MBZTH_213F4DIBsup16.hklStructure factors: contains datablock(s) MBZTH_213F4DIB. DOI: 10.1107/S2053229622009548/qw30022MBZTH_14F4DIBsup17.hklStructure factors: contains datablock(s) 2MBZTH_14F4DIB. DOI: 10.1107/S2053229622009548/qw3002MBZTH_135F3I3Bsup18.hklStructure factors: contains datablock(s) MBZTH_135F3I3B. DOI: 10.1107/S2053229622009548/qw3002MBZTH_TIEsup19.hklStructure factors: contains datablock(s) MBZTH_TIE. DOI: Click here for additional data file.10.1107/S2053229622009548/qw30022IT_13F4DIBsup20.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002IT_135F3I3Bsup21.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw30024MBZIM_313F4DIBsup22.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002MBZIM_14F4DIBsup23.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002MBZIM_TIEsup24.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002MMBZIM_12F4DIBsup25.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw30022MMBZIM_14F4DIB_2H2Osup26.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002MMBZIM_135F3I3Bsup27.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002MBZOX_12F4DIBsup28.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002MBZOX_13F4DIBsup29.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw30022MBZOX_14F4DIBsup30.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002MBZOX_135F3I3Bsup31.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw30023MBZTH_412F4DIBsup32.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002MBZTH_13F4DIBsup33.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002MBZTH_213F4DIBsup34.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw30022MBZTH_14F4DIBsup35.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002MBZTH_135F3I3Bsup36.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229622009548/qw3002MBZTH_TIEsup37.cmlSupporting information file. DOI: 2194060, 2194061, 2194062, 2194063, 2194064, 2194065, 2194066, 2194067, 2194068, 2194069, 2194070, 2194071, 2194072, 2194073, 2194074, 2194075, 2194076, 2194077CCDC references:


In this study, we used clickable ε-PαL and ε-OβL derivatives carrying a C-terminal azide group, which were enzymatically produced and then conjugated with a fluorescent dye to analyze subcellular localization. Interestingly, fluorescent proteins conjugated with the clickable ε-PαL or ε-OβL were also internalized into cells and diffused throughout the cytosol. Notably, a Cre recombinase conjugate with ε-PαL entered cells and mediated the Cre/loxP recombination, and ε-PαL was found to deliver a full-length IgG antibody to the cytosol and nucleus.Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. Although the biological significance of polycationic homopoly(amino acid)s remains unclear, increasing attention has recently been focused on their potential use to achieve cellular internalization. Here, for the first time, we provide direct evidence that two representative bacterial polycationic isopeptides, ε-poly- Bacteria-derived polycationic homopoly(amino acid)s are reported for the first time to directly penetrate cell membrane and deliver proteins of interest into cells, representing a new type of cell penetrating peptides for intracellular delivery. In addition to these linear polymers characterized by the isopeptide backbones, two branched polymers consisting of repeated dipeptide chains have also been found in bacteria: multi-l-arginyl-poly-l-aspartic acid 10 and multi-l-diaminopropionyl-poly-l-diaminopropionic acid11. Among these eight homopoly(amino acid)s, γ-poly-l/d-glutamic acid is the only example of a naturally occurring polyanionic isopeptide. The others are polycationic, and there is only limited knowledge about the antimicrobial activities of the linear polycationic isopeptides. Although the biological significance of the homopoly(amino acid)s mentioned above has largely eluded researchers, their polyionic properties and polyamide structures are currently an area of focus in biological material science.Homopoly(amino acid)s, which are rare in nature, are produced by bacteria as secondary metabolites Fig. . To date15. Although amphipathic and hydrophobic CPPs are also known, polycationic CPPs are frequently employed as vehicles to deliver biological macromolecules (cargoes) into mammalian cells14. The internalization routes of cationic CPPs are themselves broadly divided into energy-independent direct penetration and energy-dependent endocytosis/macropinocytosis15; in both routes the CPP bindings to negatively charged cell-membrane components (such as heparan sulfate proteoglycans) are an essential trigger for the internalization events. In contrast to the direct penetration routes, the cargoes taken up by endocytosis/macropinocytosis must escape from endosomes to the cytosol to avoid degradation, reach their molecular targets, and exert their biological activities. Importantly, CPPs carrying a macromolecule cargo such as a protein usually enter cells only by an endocytotic/macropinocytotic route17. Therefore, recent works have focused on rationally designed synthetic CPPs to obtain more efficient internalizations, both in order to facilitate the direct penetrations or endosomal escapes of the CPP-protein conjugates and to confer resistance to proteolytic degradation21. In addition to these recent landmark gateways, different approaches to simplify various aspects of the methodology are expected to boost practical intracellular delivery of biological macromolecules. Furthermore, the harmful effects from the polycationic features remain a critical issue to be solved in canonical CPPs with eupeptide structure.Unlike the isopeptides with unknown function, standard peptides  with arginine- and/or lysine-rich sequences  are currently attracting interest for their cell-penetrating activities in mammalian cells due to their polycationic features at physiological pH; such eupeptides are called cell-penetrating peptides (CPPs)1)  is much higher than those of canonical CPPs.In this context, a bacterium-origin polycationic polymer, ε-PαL (1) Fig. , has gar1, the polycationic lysine-isopeptide structures are also found in streptothricin (ST) antibiotics  but l-β-lysine (l-βLys): namely, ε-OβL (2). STs show antimicrobial activities and cytotoxicity to mammalian cells. Their molecular targets in prokaryotic cells are the ribosomes, in order to inhibit protein biosynthesis, but their cytotoxic mechanism in eukaryotic cells remains unclear. These harmful effects are boosted by the ε-OβL pendant chain (1–7 mer). In fact, STs with a longer ε-OβL pendant chain  show greater cytotoxicity. Therefore, it has been hypothesized that the ε-OβL chain behaves like a CPP in order to internalize the ST core structure (ST-F) to the cells.In addition to ics Fig. ; however1 and 2 are nonribosomally synthesized by disparate mechanisms  into cells, which could dramatically transform therapeutic approaches and foster experimental breakthroughs in molecular biology.Our previous studies demonstrated that the isopeptide polymer structures in C-terminus of 1 is in demand to decorate materials without any additional electrostatic counterparts; this approach does not eliminate the polycationic feature of 1. However, there have been quite a few successful chemical approaches to solve this issue. In contrast, it has been reported that an ε-PαL-producing bacterium, Streptomyces albulus NBRC14147, can produce ε-PαL ester derivatives when the culture medium is supplemented with short-chain polyols32. Our present study also confirmed the production of an ester derivative in the culture medium containing 0.2% (w/v) glycerol; the C-terminus of 1 was esterified with glycerol, producing ε-PαL-glycerol (3) . As expected, 0.2% (w/v) supplements of these two alcohols resulted in the production of the corresponding ester derivatives  and ε-PαL-PEG (6), in the reaction mixture containing glycerol and PEG, respectively , was purified from the culture broth and used for further experiments after confirming its chemical structure by NMR analysis  derivative carrying a dibenzocyclooctyne (DBCO) group by click reaction  (Supplementary Table 10)  Fig.  was then10  under typical cell culture conditions with serum. Before imaging, cells were washed with heparin, which is highly negatively charged and should strip 10 electrostatically associated with the plasma membrane surface. The cells were then fixed with 4% paraformaldehyde14. In the incubation at 37 °C for 60 min, CM demonstrated that 10 was internalized into cells and diffused throughout the cytosol. The observed green-fluorescent image indicated the direct translocation route for 10 because there were no punctate signals suggestive of endosomes. Moreover, diffuse signals of 10 were also observed on intracellular uptake at 4 °C, a temperature at which energy-dependent endocytosis was strongly suppressed. These findings revealed that the cellular internalization of 10 occurred through a direct penetration pathway. However, a low concentration (2 µM) of 10 gave only punctate signals suggestive of endosomes at 37 °C  in hand 36 suppressed these cellular uptakes  was initially employed to assess the cellular internalization of 10 Fig. . HeLa ce °C Fig. . These dntration  µM of 10and Fig. . Therefoake Fig. . Serum iake Fig. , but polake Fig. , strongl °C Fig. . PAO   for 60 min at 37 °C or 4 °C, and then washed and fixed as described above. When the cells were incubated at 37 °C, CM analysis revealed diffuse and vesicular signals  , was chemically derivatized with DBCO-Sulfo-12) Fig. . HeLa ceals Fig. . Incubatals Fig. . While take Fig. . Despite °C Fig. . Endocyt °C Fig. . Unlike 13) Fig.  was hard13) Fig. .Fig. 4Cy12, we performed time-lapse imaging with CM. Notably, 12 showed the formation of fluorescence-enriched spots on the cell membranes in the initial internalization phase 38. ε-PαL (isopeptide) and arginine-rich peptides (eupeptide) should be structurally classified into different classes, but interestingly, ε-PαL can deliver protein cargoes directly and very quickly into cells through NZ. Three direct penetration mechanisms have been proposed in CPPs having α-helix conformations    Fig.  was only12. Time-lapse imaging with optical sectioning microscopy demonstrated that the diffused signal of 12 was continuously detected for at least 12 h (Supplementary Video 15) signals were continuously detected as vesicular forms for up to 12 h   of 16  conjugated with 16) Fig. . We firs 16 Fig. .41. To examine the capability of ε-PαL for antibody internalization, a fluorescently labeled monoclonal anti-α-tubulin antibody (mAbFAM) was conjugated with 8, yielding ε-PαL-mAbFAM (17) , mKO (25 kDa), and Cre recombinase (38 kDa) turned our attention to the delivery of a full-length IgG antibody, which is still challenging due to its high molecular weight (150 kDa)7 kDa, mK17) Fig. . We spec °C Fig. . These fent Fig. . The cyt1) against mammalian cells, while the nontoxicity of 1 in vivo has also been reported so that ε-PαL is used as a natural food preservative in several countries42. However, the in vitro cytotoxicity of 1 in other cell lines has remained unclear. We therefore performed the MTT cell viability assay with 1 in K562, HepG2, and HEK293T cells in addition to HeLa cells, and we confirmed that 1 did not show cytotoxicities in these cell lines  behaves like a CPP to internalize the ST core structure (ST-F) within cells  are often used as mimicking substrates of the aminoacyl substrate tethered to the 4′-PP arm43. However, instead of the aminoacyl-SNACs, we examined whether l-βLys tethered to a chemically synthesized PEG-azide tail   and l-βLys (extending unit) with rORF19 (0.1 mg ml−1) and ATP. Fortunately, the HPLC-ESI-HR-TOF analysis of the reaction mixture showed that rORF19 mediated an ATP-dependent chain elongation (2–4 mer) on the 18 scaffold and produced ε-OβL-PEG-azide (19) , was previously acceptable. Therefore, l-βhLys-PEG-azide (20) was chemically synthesized  the productivities of the elongated isopeptides were improved, and (ii) compound 20 was more consumed by rORF19. Conversely, l-αLys-PEG-azide (22) (Supplementary Table 2-PEG-azide (23) were not accepted as priming units   is mediated by ORF19, a stand-alone adenylation domain of nonribosomal peptide synthetase (NRPS)  Fig.  would be19) Fig. . This fi−1, the reaction mixture produced 21 with longer isopeptide chains (2–9 mer) . The six oligomers with different chain lengths, 21 (2 mer), 21 (3 mer), 21 (4 mer), 21 (5 mer), 21 (6 mer), and 21 (7–13 mer), were then respectively conjugated with FAM by click reaction, producing ε-OβLm-FAM (24) (2 mer), 24 (3 mer), 24 (4 mer), 24 (5 mer), 24 (6 mer), and 24 (7–13 mer) (Supplementary Table 24 (50 µM) at 37 °C and 4 °C for 60 min. CM analysis demonstrated no cell-penetrating activities in the short chains (2 mer and 3 mer)  (Supplementary Table 24 (4–13 mer) was suppressed only by EIPA  into cells.Compound 21 (4–13 mer) by click reaction to examine whether ε-OβL can deliver macromolecules intracellularly (Supplementary Table m-mAG (25)  intracellular delivery was significantly suppressed by PAO and EIPA   Fig.  than 12  °C Fig. . Convers °C Fig. , demonst1 and 2, which had a small cargo , occurred mainly by energy-independent direct penetration. Surprisingly, direct penetrations were also observed in 12 (27 kDa) and 14 (25 kDa), while the canonical CPP (R8) mediated the deliveries of protein cargoes only via endocytosis/macropinocytosis. Considering that 16 (38 kDa) mediated its Cre/loxP recombination in the nucleus and that 17 (150 kDa) escaped from endosomes and then arrived at the nucleus, we conclude that the polycationic conjugation approach with 1 is effective for both the nucleus-targeted delivery and the cytosolic delivery of protein cargoes. Our present study also revealed that the ε-OβL chain of antibiotic STs functions as a CPP to deliver the ST core structure (ST-F) into cells. Furthermore, to our surprise, we found that the conjugation with 2 facilitated endosomal escape of protein cargoes. Although we employed 21 (4–13 mer) in this study, elongation of the isopeptide-chain length could enable direct penetration of protein cargoes.Our explorations showed that the cellular uptakes of 44. CPPs are among the most promising strategies, and CPP-driven technology currently revolves around natural and synthetic eupeptides. Poly-l/d-arginine15, poly-l-histidine45, and poly-l/d-lysine46, which were chemically or genetically produced, have been used as CPPs. To enable the delivery of protein cargoes by these eupeptides, recombinant expression of CPP-cargo fusions at the N- or C-terminus is a straightforward strategy. However, low-level expressions in a host strain are known problems due to the insolubility, toxicity, and truncation of the fusion proteins47. In contrast, the chemical in vitro attachment of a CPP to a protein surface is a promising alternative approach. This bioconjugation process relies on the chemical synthesis of CPPs that often contain a reactive group-end specific to functional groups  on the protein surface. However, the resulting CPP sway arm on the protein body is often biochemically unstable due to proteolytic degradation48. Although the cyclic CPPs  have great potential for overcoming the critical degradation problem49, a much more versatile and facile method without laborious synthesis would expand the application potential of the CPP-driven technology. Therefore, the naturally occurring polycationic isopeptides, 1 and 2, which are resistant to peptidases/proteinases22 and are nontoxic to mammalian cells3, are a promising new class of CPPs. Moreover, their biosynthetic machineries were found to be useful alternatives for installing a clickable group at the C-termini of the polycationic isopeptides.Practical intracellular delivery of biological macromolecules  would dramatically transform therapeutic approaches and foster experimental breakthroughs because numerous potential drug targets  are in the cell interior1 and 2 still remain unclear, like canonical CPPs. However, the protein cargoes conjugated with 1 or 2 were certainly internalized into cells by direct penetration under high concentration conditions (50 μM). Recent studies have focused on rationally designed synthetic CPPs to obtain more efficient internalizations  with low concentration treatments (~2 μM) and to confer resistance to proteolytic degradation51. Concomitantly, these attempts can reduce the amount of synthetic CPPs prepared by advanced chemical synthesis with high cost. In contrast, a cost-effective strategy allows for conducting protein intracellular deliveries under high concentration conditions  preferable for reliable direct penetration uptakes. The bacterial polycationic isopeptides, 1 and 2, are intriguing new tools with this potential. Given the proof of these cell-penetrating activities, we propose the name polycationic isopeptides entering cells (PIECEs) for 1 and 2. Although they are the first examples of bacterial PIECEs, further investigations of the cell-penetrating activities in bacterial polycationic isopeptides, such as γ-poly-l/d-diaminobutyric acid5, β-poly-l-diaminopropionic acid6, ε-poly-l-β-lysine7, cyanophycin10, and multi-l-diaminopropionyl-poly-l-diaminopropionic acid , were purchased from Tokyo Chemical Industry (Japan). β-hLys was purchased from Watanabe Chemical (Japan). DBCO-PEG4-5/6-FAM was purchased from Jena Bioscience (Germany). DBCO-Sulfo-N-hydroxysuccinimidyl ester (DBCO-Sulfo-NHS) was purchased from BROADPHARM (USA). FAM-NHS ester was purchased from ANASPEC (USA). R8-azide (9) and R8-FAM (11) were purchased from Eurofins Genomics (Japan). The endocytosis/macropinocytosis inhibitors, i.e., phenylarsine oxide (PAO), Dynasore, monodansylcadaverine (MDC), and ethylisopropyl amiloride (EIPA), were purchased from Fujifilm Wako Pure Chemical (Japan), Adipogen Life Sciences (USA), Sigma-Aldrich (USA), and TOCRIS bioscience (USA), respectively. All other chemicals used were of analytical grade.Short-chain polyols, 2-propyn-1-ol, 3-buten-1-ol, tetraethylene glycol (PEG), triethylene glycol mono(2-propynyl) ether , 11-azido-3,6,9-trioxaundecanol (PEG-azide), and 11-azido-3,6,9-trioxaundecan-1-amine  was used to overexpress mAG, mKO, and Cre recombinase. Human cervical cancer cells (HeLa), human embryonic kidney cells (HEK293T), human hepatocellular carcinoma cells (HepG2), and human acute myelocytic leukemia cells (K562) were purchased from RIKEN BioResource Research Center (Japan). Human neuroblastoma cells (SH-SY5Y) and human dermal fibroblast cells (HDF) were purchased from the American Type Culture Collection (USA) and PromCell (Germany), respectively. The cell lines  authenticated by RIKEN BioResource Research Center (Japan) were not further validated. The cell lines, SH-SY5Y and HDF, were not authenticated. We purchased the cell lines  tested for mycoplasma contamination by RIKEN BioResource Research Center (Japan), but SH-SY5Y and HDF were not tested for mycoplasma contamination.52 was used as a PCR template to amplify the Cre recombinase gene.Two plasmids, pmAG1-S1 and pmKO1-MC1 , were used as the PCR templates to amplify the mAG and mKO genes, and the pmKO1-MC1 plasmid was also used to construct the Cre activity reporter plasmid. pKU470 (a gift from Dr. Haruo Ikeda)S. albulus NBRC14147 was grown in M3G medium53 for 1 day at 28 °C. When the pH of the culture broth reached around 4, 0.2% (w/v) of the short-chain polyol  was added to the culture medium. After culturing for 2 days at 28 °C, the resulting ε-PαL ester derivative in the culture broth was analyzed by HPLC-HR-ESI-MS (see below).54, and then further purified by preparative HPLC using a reversed-phase column  at 35 °C at a flow rate of 7 ml min−1 and with a linear gradient of acetonitrile in water in 0.1% (v/v) HFBA run over 30 min (20–50% [v/v] acetonitrile for 30 min). Fractions were collected and monitored by an ultraviolet (UV) detector at 210 nm. The fraction containing the purified ε-PαL ester derivatives was lyophilized to give a white powder (approximately 300 mg). The amounts of ε-PαL and the ε-PαL ester derivatives produced in the supernatant were determined according to the method reported by Itzhaki55.The ε-PαL ester derivatives were purified from the culture broth (1000 ml) using ion complex formation as described in our previous study56, the recombinant Pls (rPls) was purified from an overexpressing Streptomyces strain. We performed enzyme reactions with glycerol and PEG to determine whether the ε-PαL ester derivatives were produced by Pls. The reaction mixture containing 100 mM N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS)-NaOH (pH 8.5), 1 mM l-αLys, 5 mM MgCl2, 5 mM ATP, 1 mM dithiothreitol (DTT), 20% (w/v) glycerol, 0.2% (w/v) NP-40, and 300 µg ml−1 rPls was incubated with and without 1% (w/v) PEG at 25 °C for 10 min. The reaction mixture containing ε-PαL-glycerol (3) or ε-PαL-PEG (6) was then analyzed by HPLC-HR-ESI-MS (see below).Using the method described in our previous studyE. coli BL21(DE3) for expression as C-terminally 6×His-tagged fusion proteins. The recombinant proteins (enzymes) were purified using standard protocols with nickel-nitriloacetic acid (Ni-NTA) Sepharose (Qiagen) and then used for further experiments.The following three sets of PCR primers (Supplementary Table 10): ε-PαL-PEG-azide  was added to 50 ml of 80% (v/v) DMSO. Next, DBCO-PEG4-5/6-FAM (27.5 µmol) was added and the mixture was stirred for 3 h at 25 °C. ε-PαL-FAM (10) in the reaction mixture was analyzed by HPLC-HR-ESI-MS and was then purified by preparative HPLC as described above. The fractions were collected and monitored by an ultraviolet (UV) detector at 254 nm. The fraction containing the purified 10 was lyophilized to give a yellow powder (approximately 50 mg).ε-PαL-FAM (12), ε-PαL-mKO (14), and ε-PαL-Cre (16): The recombinant proteins (enzymes) (5 µmol) were added to 50 ml of 100 mM NaHCO3, along with DBCO-Sulfo-NHS ester (10 µmol). After stirring for 4 h at 4 °C, the reaction mixtures were dialyzed with 20 mM sodium phosphate buffer (pH 6.0), and 8 (5.5 µmol) was added to 50 ml of the dialyzed solution and stirred for 4 h at 4 °C. Additionally, 12 and 14 were further purified by ion-exchange chromatography using a HiTrap SP HP column (Cytiva) on the AKTA system  after confirming the productions of 12, 14, and 16 by HPLC-HR-ESI-MS analysis (see below). Buffer A  and Buffer B  were used as eluents. Flow rate: 3 ml min−1; gradient: 0–20 column volume (CV) , 20–30 CV . Fractions were collected and monitored by a UV detector at 280 nm. The fractions containing the purified proteins were concentrated by ultrafiltration  and then used for further experiments. ε-PαL-Cre (16) was used for further experiments without the purification by ion-exchange chromatography.ε-PαL-mAG (13) and R8-mKO (15): The recombinant mAG and mKO (0.5 µmol) were added to 5 ml of 100 mM NaHCO3, along with DBCO-Sulfo-NHS ester (1 µmol). After stirring for 4 h at 4 °C, the reaction mixtures were dialyzed with 20 mM sodium phosphate buffer (pH6.0), and R8-azide  was added to 5 ml of the dialyzed solution and stirred for 4 h at 4 °C. The resulting conjugates, 13 and 15, were further purified by ion-exchange chromatography using a HiTrap SP HP column (Cytiva) on the AKTA system  as described above, after confirming the productions of 13 and 15 by HPLC-HR-ESI-MS analysis (see below). The fractions containing 13 and 15 were concentrated by ultrafiltration (Vivaspin 6) and then used for further experiments.R8-mAG (r gene) . After stirring at 37 °C for 30 min, DBCO-Sulfo-NHS ester (4 µmol) was added to the reaction mixture and stirred at 37 °C for 30 min. The reaction mixtures were dialyzed with 20 mM sodium phosphate buffer (pH 6.0), and 8 (2 µmol) was added to 10 ml of the dialyzed solution and stirred for 4 h at 4 °C. Further, 17 in the reaction mixture was concentrated by ultrafiltration (Vivaspin 6) and used for further experiments after confirming the production of 17 by HPLC-HR-ESI-MS analysis.Anti-α-tubulin antibody   was added to 10 ml of 100 mM NaHCO2. The cell lines and corresponding media were as follows: HeLa, Dulbecco’s modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS); HEK293T, DMEM with 10% FBS; HepG2, DMEM with 10% FBS; HDF, DMEM with 10% FBS; SH-SY5Y, DMEM/Ham’s F-12 with 10% FBS; and K562, RPMI1640 with 10% FBS. All media contain 100 U/ml penicillin and 100 µg/ml streptomycin.Cell lines were grown at 37 °C in a humidified atmosphere with 5% CO10–17, 24, or 25) was added to the cells and then incubated at 4 °C or 37 °C for 60 min or 120 min after washing the cells with the medium. The cells were then washed with PBS, 100 µg ml−1 heparin in PBS, and then two more times with PBS. We added 4% paraformaldehyde in PBS to fix the cells, and incubated them at 25 °C for 30 min. 5 µg ml−1 wheat germ agglutinin (WGA)/Alexa594 (or Alexa488) in PBS was added and incubated at 25 °C for 10 min to stain the cell membrane after washing the cells thrice with PBS. Then, the cells were treated with ProLongTM Gold antifade reagent with DAPI . The cells were observed by using an LSM 900 Confocal Laser Scanning Microscope (ZEISS).Cells were seeded into each well of an 8-well chamber slide glass  and were incubated at 37 °C for 24 h. A fresh medium containing a sample  was added to the cells and then incubated at 4 °C or 37 °C for 5–60 min. The cells were then washed with PBS, followed by 100 µg ml−1 heparin in PBS, and then twice with PBS. 300 μl of lysis buffer  was added to the cells for cell lysis. The resulting cell lysates were centrifuged for 30 min , and the fluorescence (485 ex/535 em) of the supernatants was measured with a fluorescent plate reader .HeLa cells were seeded into each well of a 12-well plate  and were incubated for 24 h at 37 °C in DMEM containing 10% FBS. Next, a fresh medium containing a sample  or R8-mKO (15) was added, and the time-lapse images of the cells were captured every 1 min for 30 min by an LSM 900 Confocal Laser Scanning Microscope (ZEISS).HeLa cells were seeded in a 35 mm glass-bottom dish  and were incubated in DMEM containing 10% FBS for 24 h at 37 °C. Next, a fresh warmed (37 °C) medium containing 50 µM of ε-PαL-mAG (12) or R8-mKO (15) was added, and the cells were incubated at 37 °C for 60 min. The cells were then washed with PBS, followed by 100 µg ml−1 heparin in PBS, and then two more times with PBS. Finally, fresh DMEM containing 10% FBS was added and incubated at 37 °C. The time-lapse images were captured every 10 min for 12 h by optical sectioning microscopy  to track 12 or 15 after the cellular uptakes.HeLa cells were seeded in a 35 mm glass-bottom dish  and were incubated at 37 °C in DMEM containing 10% FBS for 24 h. Next, a fresh medium containing 50 µM of ε-PαL-mAG (16) at 37 °C for 60 min. The cells were then washed with PBS, followed by 100 µg ml−1 heparin in PBS, and then two more times with PBS. Finally, fresh DMEM containing 10% FBS was added and incubated at 37 °C for 24 h to express a fluorescent protein. The live-cell imaging was captured by fluorescence microscopy (Leica DMi8).HEK293T cells were transfected with the plasmid pmKO_loxP_mAG using FuGENE Transfection (Promega) according to the manufacturer’s specifications. The cells were washed with DMEM medium and then treated with 50 µM of ε-PαL-Cre -l-β-Lysine ·DCHA, which had been synthesized as described in the Supplementary Note according to the methods previously described57, was acid extracted with AcOEt/10% citric acid to remove DCHA. NH2-PEG-azide (0.55 mmol) and DMT-MM (1.5 mmol) were added to an MeOH solution (10 ml) of the free (Boc)2-l-βLys, and the mixture was stirred at room temperature overnight. The residue was dissolved into Et2O (10 ml), washed with brine, and dried (Na2SO4) after a concentration in vacuo. A crude (Boc)2-l-βLys-PEG-azide thus obtained by evaporation was added to 4N HCl/AcOEt (20 ml), and the mixture was stirred at room temperature for 3 h. After evaporation, HCl remained in the mixture was further removed by azeotropic evaporation with toluene/MeOH. The residue was dissolved in water and lyophilized, affording l-βLys-PEG-azide (18)·HCl as a colorless oil . l-βhLys-PEG-azide (20)·HCl (38% yield) and l-αLys-PEG-azide (22)·HCl (81% yield) were synthesized from l-βhLys and l-αLys, respectively, by bis-Boc derivatization, condensation with NH2-PEG-azide, and subsequent deprotection. Their NMR data are summarized in Supplementary Tables 3, the recombinant ORF19 (rORF19) was purified from an overexpressing E. coli strain. A reaction mixture (100 μl) consisting of 50 mM TAPS (pH 9.0), 18% (v/v) glycerol, 5 mM ATP, 5 mM MgCl2, 5 mM l-βLys, 1 mM acceptor substrate , and 0.1 mg ml−1 rORF19 was incubated for 1 h at 30 °C. The reaction was quenched by heating at 100 °C for 5 min, the mixture was centrifuged, and the supernatant was analyzed by HPLC-HR-ESI-MS (see below).According to the method described in our previous study2, 5 mM l-βLys, 1 mM 20, and 1 mg ml−1 rORF19 was incubated at 30 °C overnight  was purified by a disposable ion-exchange column . The mixture (20 ml) was added to 80 ml of 20% (v/v) aqueous methanol and applied to a column of InertSep MC-2  aqueous methanol) after adjusting the pH of the reaction mixture to 7 by HCl. The product  was eluted with 10 ml of 1 M NH4OH after the column had been washed with 50 ml of 20% (v/v) aqueous methanol. The elution fraction was lyophilized and then used as an acceptor substrate for the 2nd round enzyme reaction. The 2nd round enzyme reaction mixture (20 ml) consisting of 50 mM TAPS (pH 9.0), 18% (v/v) glycerol, 5 mM ATP, 5 mM MgCl2, 5 mM l-βLys, 1 mM short-chain 21 (2–9 mer), and 1 mg ml−1 rORF19 was incubated overnight at 30 °C (Supplementary Table 21 (2–13 mer), was purified by InertSep MC-2 and then further purified by preparative HPLC using a reversed-phase column  at 40 °C at a flow rate of 7 ml min−1 and with a linear gradient of acetonitrile in water in 0.1% (v/v) HFBA run over 36 min [20–38% (v/v) acetonitrile for 36 min]. Fractions were collected and monitored by an ultraviolet (UV) detector at 210 nm. The fraction containing the purified 21 was lyophilized and used for further experiments.A reaction mixture (20 ml) consisting of 50 mM TAPS (pH 9.0), 18% (v/v) glycerol, 5 mM ATP, 5 mM MgClm-FAM (24) and ε-OβLm-mAG (25) were synthesized by the same procedure as used for the synthesis of 10 and 12 (see above).ε-OβL1), ε-PαL-glycerol (3), ε-PαL-alkyne (4), ε-PαL-alkene (5), ε-PαL-PEG (6), ε-PαL-PEG-alkyne (7), ε-PαL-PEG-azide (8), and ε-PαL-FAM (10) were analyzed by HPLC-HR-ESI-MS  using a reversed-phase column  at 40 °C at a flow rate of 0.3 ml min−1 and with a three-step linear gradient of acetonitrile (ACN) in water in 0.1% (v/v) heptafluorobutyric acid (HFBA)  run over 30 min .ε-PαL (12), R8-mAG (13), ε-PαL-mKO (14), R8-mKO (15), and ε-PαL-Cre (16) were analyzed by HPLC-HR-ESI-MS (maXis plus) using a reversed-phase column  at 70 °C at a flow rate of 0.3 ml min−1 and with a linear gradient of ACN in water in 0.1% (v/v) trifluoroacetic acid (TFA) run over 30 min .ε-PαL-mAG (l-βLys-PEG-azide (18), l-βhLys-PEG-azide (20), and l-αLys-PEG-azide (22) were analyzed by HPLC-HR-ESI-MS (maXis plus) using a reversed-phase column  at 40 °C at a flow rate of 0.3 ml min−1 and with a three-step linear gradient of ACN in water in 0.1% (v/v) HFBA run over 30 min .1H- and 13C-NMR spectra were recorded at 600 and 150 MHz, respectively, using a JNM-ECA 600 NMR (JEOL). One- and two-dimensional experiments (double quantum Filtered-correlation spectroscopy [DQF-COSY] and constant time-heteronuclear multi-bond connectivity [CT-HMBC]) were performed at ambient temperature. The samples were dissolved in D2O.p-value was  < 0.05. No statical methods were used to predetermine sample size. Where statical analyses were performed, the sample sizes are listed in the corresponding figure legends. All experiments presented in the manuscript were reproducible. The results are presented as the mean ± standard deviation (s.d.) (n = 3). Information regarding number of biological and technical replicates is reported for individual experiments in the figure legends.All statistical analyses were performed using GraphPad Prism software . Differences were considered significant when the calculated Further information on research design is available in the Peer Review FileSupplymentary informationDescription of Additional Supplementary DataSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Reporting Summary-New