Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen  CRIS) domain . Diverse) domain ,16. Thes is the causative agent of tuberculosis (TB), which imposes a huge burden on human health and on developing economies, with 10 million new infections and 1.6 million deaths in 2017 . The majority of Mtb strains encode a complete CRISPR system (Mtb strain H37Rv (Mtb strains and have been used for many years as a strain typing (spoligotyping) tool , the organization is identical with the exception of a truncating mutation in the csm6 gene (Mtb is a rare example of an isolated type III effector and associated adaptation system.in H37Rv . CRISPR ng) tool , suggesty Figure . In MycoMtb CRISPR system, although recent work suggests it can provide modest immunity against MGE  unless stated otherwise. Protein concentrations were determined by UV quantitation  using calculated extinction coefficients  unless stated otherwise.Enzymes were purchased from Thermo Scientific, BioLine, Promega or New England Biolabs and used according to manufacturer's instructions. Oligonucleotides  and synt6-fusion proteins, Mtb cas10 (csm1), cas11 (csm2), cas7 (csm3), cas5 (csm4), csm5 and csm6 were digested with NcoI and XhoI and ligated into the corresponding sites of the expression vector pEHisTEV , csm2 and csm5 were cloned into pACYCDuet-1 , csm4 was cloned into pEHisTEV  and csm3 was inserted into pEHisTEV  by restriction enzyme cloning. These constructs were used as templates for assembly of M. tuberculosis CRISPR genes, including ribosome binding sites, into MultiColi™  acceptor and donor vectors by sequence- and ligation-independent cloning (SLIC) as per manufacturer's instructions. Briefly, 6-csm4, csm2His and cas10 were introduced into the acceptor pACE; csm3, csm5 were cloned into pDK; Mca csm2 was inserted into pDC using restriction enzyme cloning . The acceptor and donor plasmids were then recombined using Cre recombinase to give pCsm1–5. To obtain variant Csm complexes, His8-V5-csm4 (from pEHisV5TEV-Csm4), Mca csm2 (from pACYCDuet-Mca_Csm2) and cas10 (csm1) were introduced into pACE by SLIC; and csm3, csm5 were inserted into the donor plasmid pDK. Primer-directed mutagenesis was carried out on the assembled acceptor and donor plasmids, before Cre recombination to give pCsm1–5_C3 (Csm3 D35A) and pCsm1–5_Cy (Csm1 D630A/D631A). The identity of all donor and acceptor plasmids was verified by sequencing, and the final, recombined construct was analysed by restriction digest.csm6 (pACYCDuet-1). The pACE assembly was as for pCsm1–5_C3. Csm3, csm5 and csm6 were introduced into pDK; for the ΔCsm6 plasmid, the donor plasmid en route to pCsm1–5 was used. Mutagenesis was carried out as before. All constructs were verified by sequencing. Suitable acceptor and donor plasmid combinations were then assembled by Cre recombination to give pCsm1–6, pCsm1–5_ΔCsm6, pCsm1–6_C3, pCsm1–6_Cy and pCsm1–6_HD (The same templates as for construction of pCsm1–5_C3 were used with the addition of sm1–6_HD . The conaraC and pBAD region was polymerase chain reaction (PCR)-amplified from pBADHisTEV  using primer pair ara-AvrII-R/F and cloned into the XbaI and NdeI sites of pRSFDuet™-1 to give pRSFara. The tetracycline resistance gene was PCR-amplified from the MultiColi™ vector pACE2 using the TetR primer pair; the pRSFara-backbone lacking its resistance gene was PCR-amplified using primer pair RSFara. The two PCR products were digested with SphI and BamHI and joined through ligation to give pRAT. The pUC19 MCS / lacZα gene was introduced into pRAT by megaprimer PCR (primer pair lacZ-pRAT) to give pRAT-Target  derivative with tetracycline resistance was constructed as follows. The T-Target .6-tagged Cas6 was expressed in BL21 (DE3) grown in LB containing 50 μg ml-1 kanamycin and 50 μg ml−1 chloramphenicol. An overnight culture was diluted 100-fold into fresh medium and grown at 37°C, 180 rpm to an OD600 of 0.4. The temperature was lowered to 16°C, and incubation continued until the OD600 reached 0.8. Production was induced with 50 μM IPTG, and the cultures were continued for a further 20 h. Cells were harvested by centrifugation. The pellet was resuspended in 50 mM Tris–HCl, 250 mM NaCl, 10 mM imidazole, 10% glycerol, pH 7.5, sonicated and the cell debris removed by centrifugation. The cleared lysate was loaded onto a pre-equilibrated HisTrap Crude FF  column, washed with 50 mM Tris–HCl, 250 mM NaCl, 30 mM imidazole, 10% glycerol, pH 7.5 and eluted in a gradient to 50 mM Tris, 250 mM NaCl, 500 mM imidazole, 10% glycerol, pH 7.5. Cas6-containing fractions were pooled and further purified by gel filtration  in 50 mM Tris–HCl, 250 mM NaCl, 10% glycerol, pH 7.5. Cas6 and Cas6 H47N were concentrated as required using an Amicon Ultra centrifugal filter .C-terminally HisEscherichia coli C43(DE3) cells were co-transformed with the MultiColi™ construct pCsm1–5  and pCRISPR. Overnight cultures were diluted 100-fold into LB containing 100 μg ml−1 ampicillin and 50 μg ml−1 spectinomycin, incubated at 37°C, 180 rpm until the OD600 reached 0.8. After induction with 100 μM IPTG, incubation was continued at 25°C for 5 h. Cells were harvested by centrifugation and pellets stored at −20°C. Cells were resuspended in lysis buffer  and lysed by sonication. The cleared lysate was loaded onto HisPur Ni-NTA resin (Thermo Scientific), washed with lysis buffer and eluted with lysis buffer containing 250 mM imidazole. Csm complex-containing fractions were diluted 2-fold with lysis buffer to prevent aggregation, pooled, then dialysed at 4°C overnight in the presence of 0.3 mg ml−1 TEV protease against lysis buffer. The protein solution was passed through His-Pur Ni-NTA resin a second time, and the flow-through was concentrated using an Amicon Ultracentrifugal filter  and further purified by gel filtration  using 20 mM Tris–HCl, 250 mM NaCl, 10% glycerol, pH 7.5 as mobile phase. When necessary, the Csm complex was further purified on MonoS 4.6/100 PE  in 20 mM MES, 50 mM NaCl, pH 6 and eluted using a salt gradient to 1 M NaCl. Single-use aliquots were flash-frozen and stored at −80°C.Mtb Csm6 and TsuCsx1 were expressed in C43 cells. Cells were grown for 4 h at 25°C before harvest by centrifugation at 4000 rpm at 4°C for 15 min (JLA 8.1 rotor). Cells were lysed in buffer containing 50 mM Tris–HCl pH 8.0, 0.5 M NaCl, 10 mM imidazole and 10% glycerol supplemented with 1 mg/ml chicken egg lysozyme (Sigma-Aldrich) and one ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablet (Roche) by sonicating six times for 1 min with 1 min rest intervals on ice. Cell lysate was then ultracentrifugated at 40000 rpm (70 Ti rotor) at 4°C for 45 min and loaded onto a 5 ml HisTrap FF Crude column equilibrated with wash buffer containing 50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 30 mM imidazole and 10% glycerol. After washing unbound protein with 20 CV wash buffer, recombinant Csm6 or TsuCsx1 was eluted with a linear gradient of wash buffer supplemented with 0.5 M imidazole across 16 CV, holding at 10% for 2 CV, 20% for 4 CV and 50% for 3 CV. Csm6 could not be concentrated and was therefore further purified using MonoQ  anion exchange chromatography, diluting protein from nickel affinity fractions directly in buffer containing 20 mM Tris-HCl pH 7.5 and 50 mM NaCl and eluting protein across a linear gradient of buffer containing 20 mM Tris-HCl pH 7.5 and 1M NaCl. For TsuCsx1, nickel affinity fractions containing the protein were concentrated using a 10 kDa MWCO ultracentrifugal concentrator, and further purified by size exclusion chromatography  in buffer containing 20 mM Tris-HCl pH 8.0, 0.5 M NaCl and 1 mM DTT. All proteins were aliquoted, flash frozen with liquid nitrogen, and stored at −80°C.4 and cA6 were purchased from BIOLOG Life Science Institute .cA32P]-ATP  using polynucleotide kinase (Thermo Scientific). Oligonucleotides were quantified spectophotometrically using calculated extinction coefficients . RNA ladders were obtained by alkaline hydrolysis .All RNA and DNA oligonucleotides as well as 6-FAM™-labelled RNA substrates were purchased from Integrated DNA Technologies . For nuclease assays, labelled oligonucleotides were gel purified as described previously . Duplexe−1 SUPERase•In™ (Thermo Scientific), pH 7.5. Reactions were initiated by addition of 5′ 6-FAM™-labelled mtbRepeat_Cas6 RNA , 50 mM Tris–HCl, 50 mM NaCl, pH 8.0, and incubating at 30°C for up to 2 h. The reaction was stopped by phenol–chloroform–isoamyl alcohol extraction to remove proteins. P1.A26 RNA , 50 mM Tris–HCl, 50 mM NaCl, pH 8.0. The solution was incubated at 30°C for 90 min, and the reaction stopped by phenol–chloroform extraction. Products were analysed as described above.To test for DNase activity of the Csm complex, 25 nM 5′-2, 0.1 U μl−1 SUPERase•In™ (Thermo Scientific), 50 mM Tris–HCl, 50 mM NaCl, pH 8.0, and incubating at 30°C for up to 2 h. The reaction was stoped by phenol–chloroform–isoamyl alcohol extraction. Reaction mixtures that were used for Csm6 or Tsu Csx1 activation assays were further extracted with chloroform–isoamyl alcohol to remove residual phenol. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis was performed on a Thermo Scientific Velos Pro instrument equipped with HESI source and Dionex UltiMate 3000 chromatography system. Samples were desalted on C18 cartridges (Harvard Apparatus). Compounds were separated on a Kinetex 2.6 μm EVO C18 column  using the following gradient of acetonitrile (B) against 20 mM ammonium bicarbonate (A): 0–2 min 2% B, 2–10 min 2–8% B, 10–11 min 8–95% B, 11–14 min 95% B, 14–15 min 95–2% B, 15–20 min 2% B at a flow rate of 350 μl min−1 and column temperature of 40°C. UV data were recorded at 254 nm. Mass data were acquired on the FT mass analyser in positive ion mode with scan range m/z 200–2000 at a resolution of 30 000.Cyclic oligoadenylate production was triggered by adding 2 μM crude target RNA to 0.8 μM Csm, 1 mM adenosine triphosphate (ATP), 5 mM MgCll-glutamate, pH 6.5. The reaction was started by addition of synthetic cA6 or 0.1 volumes protein-free reaction mixture from the cyclic oligoadenylate production above. The reaction was stopped after 45 min at 37°C by phenol–chloroform extraction. Products were analysed by 20% denaturing polyacrylamide gel electrophoresis.Csm6 (125 nM dimer) was incubated with 25 nM 5′-radiolabelled P1.A26 RNA substrate together with 2 μM cold RNA in 50 mM MES, 100 mM potassium 4, 1 μM cA6 or 1 μl Csm-derived cOA (see above) in pH 7.5 buffer containing 20 mM Tris–HCl pH 7.5, 150 mM NaCl and 1 mM DTT in a 10 μl reaction volume at 35°C. Reactions were stopped at 1, 5, 15 or 30 min by the addition of a reaction volume equivalent of phenol–chloroform and extracted products were analysed by denaturing gel electrophoresis as described above.Csx1 (500 nM dimer) was incubated with 50 nM 5′-radiolabelled A1 RNA substrate and 1 μM cAE. coli C43(DE3). Plasmids were maintained by selection with 100 μg ml−1 ampicillin and 50 g ml−1 spectinomycin. Competent cells were prepared by diluting an overnight culture 50-fold into fresh, selective LB medium; the culture was incubated at 37°C, 220 rpm until the OD600 reached 0.8–1.0. Cells were collected by centrifugation and the pellet resuspended in an equal volume of 60 mM CaCl2, 25 mM MES, pH 5.8, 5 mM MgCl2, 5 mM MnCl2. Following incubation on ice for 1 h, cells were collected and resuspended in 0.1 volumes of the same buffer containing 10% glycerol. Aliquots (100 μl) were stored at −80°C. Target or control plasmid  were added to the competent cells, incubated on ice for 30 min and transformed by heat shock. LB medium (0.5 ml) was added and the transformation mixture incubated with shaking for 2.5 h before collecting the cells and resuspending in 100 μl LB. A total of 5 μl of a 10-fold serial dilution were applied in duplicate to LB agar plates supplemented with 100 μg ml−1 ampicillin and 50 g ml−1 spectinomycin when selecting for recipients only; transformants were selected on LB agar containing 100 μg ml−1 ampicillin, 50 g ml−1 spectinomycin, 25 μg ml−1 tetracycline, 0.2% (w/v) β-lactose and 0.2% (w/v) l-arabinose. Arabinose was omitted when transcriptional induction of target was not required. Plates were incubated at 37°C for 16–18 h. Colonies were counted manually and corrected for dilution and volume to obtain colony-forming units (cfu) ml−1, statistical analysis was performed with RStudio . The experiment was performed as two independent experiments with two biological replicates and at least two technical replicates.pCsm1–6 and pCRISPR were co-transformed into Mtb type III-A effector complex was described very early in the classification of CRISPR systems .Mtb Csm simultaneously using the MultiColi™ system (Geneva Biotech), along with a compatible pCDFDuet-1 derivative expressing an Mtb mini-CRISPR and the Cas6 enzyme  subunit, it was noted that all subunits except Csm2 could be identified. Coupled with the observation that Csm2 was insoluble in isolation, we hypothesized that heterologous expression of this subunit was a barrier to successful expression of the Csm complex. We therefore added a gene encoding the Csm2 homologue from Mycobacterium canettii (Mca) to the Csm expression vector  variant  and a Csm3 D35A variant  with the characteristic 6 nt spacing, consistent with other type III effectors . We also detected substantial amounts of the corresponding linear OA triphosphates that are likely to be intermediates of cOA biosynthesis. When the same analysis was performed on metabolites produced by the Csm Csm3 D35A complex, a significant shift in cOA profile towards larger ring sizes was observed.Most type III CRISPR systems are closely associated with a gene encoding a Csm6 or Csx1 enzyme, which is activated by cOA generated by the Cas10 cyclase domain on target RNA binding ,12 and r6 or cA4  control DNA did not activate Csm6, and neither did an RNA matching the 5′-handle of the crRNA . The variant Csm with a mutation that inactivates the cyclase domain (Cy), as expected, did not activate Csm6, whereas the variant deficient in target RNA cleavage (C3) was, as expected, proficient in Csm6 activation, confirming previous findings for a variety of type III systems  portion of the lacZα gene. We constructed plasmid pRAT, which combines the origin of replication of pRSF-Duet-1 (Novagen), compatible with both Csm/CRISPR constructs, with tetracycline resistance and an arabinose-inducible MCS, and introduced pUC19 lacZα, containing the target sequence, into pRAT to give pRAT-Target. Plasmid maps are shown in We next set out to test if the Csm complex was active ive host . We testpCsm1–6, . pCsm1–6E. coli C43 strain harbouring the complete Mtb Csm interference module, was transformed with 100 ng target plasmid pRAT-Target or with pRAT as negative control. Serial dilutions of the transformation mixture were applied to plates with three different conditions  into E. coli C43 as before. Constructs where csm6 had been omitted (pCsm1–5_ ΔCsm6) or that carried inactivating mutations in the Csm3 subunit (pCsm1–6_ C3), the cyclase (pCsm1–6_ Cy) or the HD domain (pCsm1–6_ HD) were also constructed and introduced into E. coli together with pCRISPR_TetR. As a control, empty backbone (pCsm_Control) was co-transformed with pCDF-Duet-1. Cells were transformed with pRAT, and a 10-fold serial dilution was plated selecting for recipients (selection for pCsm and pCRISPR_TetR only) and for transformants .Thus far, the observed plasmid immunity was less efficient by one to three orders of magnitude compared to those in other type III systems ,33,35,36E. coli.We observed three orders of magnitude fewer transformants for the full WT Csm module (including Csm6) compared to the empty vector control Figure . As expeMtb Csm complex generates a range of cOA species from cA3-cA6 is consistent with previous findings for type III systems  was tested against a labelled RNA substrate in the absence and presence of cOA species . As before, E. coli C43 harbouring pCsm1–5_tsuCsx1 and pCRISPR, were transformed with the target plasmid (pRAT-Target) and pRAT as the control lacking the target sequence. The 10-fold serial dilutions were applied to selective, inducing plates to determine the number of transformants; the cell count for recipients was carried out as before. We observed similar levels of plasmid immunity for the cognate Mtb Csm6 and the heterologous TsuCsx1 systems  CRISPR system, and the variability of CRISPR loci in mycobacteria (spoligotyping) has been utilized to classify isolates for over 20 years. Despite this, little has been reported on the characterization of the Mtb CRISPR system, particularly in vitro. Initial work on reconstituting the Mtb Csm complex in E. coli encountered problems due to poor solubility of the Csm2 (Cas11) subunit. We surmounted this hurdle by swapping the problematic Mtb csm2 gene with that from the closely related species Mycobacterium cannetti, allowing expression of Mtb Csm complex that is functional both in vitro and in vivo. Such an approach may find utility in other circumstances when studying multi-protein complexes.Mtb Csm complex, we could dissect the contributions of the various enzymatic activities of this complex to CRISPR-based immunity. We could detect no strong evidence for target RNA dependent DNA cleavage by the HD nuclease domain of the Mtb Csm complex  of type III-A systems with ancillary ribonucleases activated by cA4. All type III systems studied to date have the capability of making a range of cyclic oligoadenylates from cA3-cA6  viral anti-CRISPRs which disrupt the cOA signalling pathway.Based on the data collated in Table  cA3-cA6 ,40. As w3 molecules, which are often the most abundant species synthesized, at least in vitro  synthesized by the Csm complex should not be discounted as dead-end side products. NanoRNAs have been shown capable of priming transcription initiation in bacteria  for cAin vitro ,12. Giveproteins . Recent proteins ,42 make leotides ,44. Furtbacteria . Recentlrculosis . Deletio4 have been identified in archaea (4 in their CARF domain, acting as self-limiting ribonucleases (6 degradation enzymes, but the most obvious candidates are the CARF domains of Csm6 family proteins. Alternatively, given the larger size and increased flexibility of cA6 compared to cA4, the former may be degraded effectively by promiscuous ribonucleases in the cell.Finally, there is the requirement to re-set the cell to a ground-state when infection has been cleared, which probably requires degradation of the cOA generated during infection. Specialized ‘ring nucleases’ that degrade cA archaea , and recucleases . There igkz676_Supplemental_FileClick here for additional data file.


Background and Purpose: Store-operated calcium entry (SOCE) is an important physiological phenomenon that extensively mediates intracellular calcium ion (Ca2+) load. It has been previously found in myocytes isolated from neonatal or diseased hearts. We aimed to determine its existence, molecular nature in undiseased hearts and its potential arrhythmogenic implications under hyperactive conditions.Experimental Approach: Ventricular myocytes isolated from adult FVB mice were studied by using Ca2+ imaging and whole-cell perforated patch-clamp recording. In addition, lead II ECGs were recorded in isolated Langendorff-perfused mice hearts. Functional TRPC channel antibodies and inhibitors, and TRPC6 activator hyperforin were used.Key Results: In this study, we demonstrate the existence and contribution of SOCE in normal adult mouse cardiac myocytes. For an apparent SOCE activation, complete depletion of sarcoplasmic reticulum (SR) Ca2+ by employing both caffeine (10 mM) and thapsigargin (1 μM) or cyclopiazonic acid (10 μM) was required. Consistent with the notion that SOCE may be mediated by heteromultimeric TRPC channels, SOCEs observed from those myocytes were significantly reduced by the pretreatment with anti-TRPC1, 3, and 6 antibodies as well as by gadolinium, a non-selective TRPC channel blocker. In addition, we showed that SOCE may regulate spontaneous SR Ca2+ release, Ca2+ waves, and triggered activities which may manifest cardiac arrhythmias. Since the spontaneous depolarization in membrane potential preceded the elevation of intracellular Ca2+, an inward membrane current presumably via TRPC channels was considered as the predominant cause of cellular arrhythmias. The selective TRPC6 activator hyperforin (0.1–10 μM) significantly facilitated the SOCE, SOCE-mediated inward current, and calcium load in the ventricular myocytes. ECG recording further demonstrated the proarrhythmic effects of hyperforin in ex vivo mouse hearts.Conclusion and Implications: We suggest that SOCE, which is at least partially mediated by TRPC channels, exists in adult mouse ventricular myocytes. TRPC channels and SOCE mechanism may be involved in cardiac arrhythmogenesis via promotion of spontaneous Ca2+ waves and triggered activities under hyperactivated conditions. TRPC cheostasis . The parproaches . Recent proaches , 2009. F2+ entry . Recent 2+ entry , Ca2+ pax injury , as wellx injury . Hyperfolear yet . It has lear yet . TherefoIn the present study, we demonstrate the presence of SOCE in adult mouse ventricular myocytes, and elucidate the possible roles of SOCE/TRPCs in promoting cardiac arrhythmias via facilitating the generation of inward current and calcium load. Our findings support a missing link between upregulated SOCE/TRPCs activities, especially TRPC6, to lethal arrhythmias in the heart.2+ concentration ([Ca2+]) upon restoration of external calcium load (1.8 mM)  antibodies for TRPC1, 3 and 6 were a generous gift from Dr. Robert M. Graham . These antibodies recognize the putative pore-forming region of mouse TRPC channels specifically, and pre-incubation with anti-TRPC6 antibody could effectively block the rise of intracellular Ca(1.8 mM) . The SER2+-free Tyrode’s solution containing 1.0 mg/mL collagenase  and 0.1 mg/mL thermolysin (Sigma) for 15 min. The hearts were removed from the perfusion apparatus after washing out the enzyme solution, the left ventricle were gently teased apart with forceps in a petri dish and the myocytes were filtered through a nylon mesh. The Ca2+ concentration was gradually increased to 1.0 mM, and the cells were stored at room temperature and used within 8 h . Briefly, hearts were removed from mice anesthetized with overdosed isoflurane, and were perfused retrogradely at 37°C in Langendorff fashion with nominally Cathin 8 h .2+ indicator Fluo-4 AM by incubating them for ∼30 min in bath solution containing 4 μM Fluo-4 AM (Molecular Probes), after which the cells were washed and placed in a heated chamber on an inverted microscope. Fluo-4 fluorescence was excited at ∼485 nm and the emission was measured at ∼530 nm using a Nikon Eclipse TE200 inverted microscope with a Fluor x40 oil objective lens . The fluorescence signals were recorded using an Andor Ixon charge-coupled device (CCD) camera (Andor Technology) operated with Imaging Workbench software (INDEC BioSystems) at 50 frames per second with a spatial resolution of 500 × 400 pixels. Fluorescence intensity was measured as the ratio of the fluorescence (F) over the basal diastolic fluorescence (0F). The spontaneous waves were quantified by counting the wave frequency (per minute) appeared after a 3-min perfusion of HF 0.1 μM, compared to the control state.Myocytes were loaded with the Ca2-phosphocreatine, 0.05 cAMP, pH 7.2 with KOH. The cells were superfused with Tyrode’s solution containing (in mM): 136 NaCl, 4.0 KCl, 0.33 Na2PO4, 1.0 CaCl2, 1 MgCl2, 10 glucose and 10 HEPES, pH 7.4 adjusted with NaOH. APs were elicited with 2-ms, 2- to 4-nA square pulses at a PCL of 6 s.Myocytes were patch-clamped using the whole-cell configuration of the perforated patch-clamp technique (240 μg/ml amphotericin B). Voltage or current signals were measured with a MultiClamp 700A patch-clamp amplifier controlled by a personal computer using a Digidata 1322 acquisition board driven by pCLAMP 10 software . For AP recordings, patch pipettes (resistance 2–5 MΩ) were filled with internal solution containing (in mM): 110 K-aspartate, 30 KCl, 5 NaCl, 10 HEPES, 0.1 EGTA, 5 MgATP, 5 Na+, L-type Ca2+, Na+-Ca2+ exchange currents were previously blocked with a K+-free extracellular solution with (in mM) 140 NaCl, 1.0 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, 5.5 glucose, 0.01 Nifedipine, 0.002 SEA400 and 5 HEPES ( pH 7.4 adjusted with NaOH), and a pipette solution containing (in mM) 90 Cs-aspartate, 30 CsCl, 20 traethylammonium chloride (TEA-Cl), 2 MgCl2, 5 Tris-ATP, 0.1 Li2-GTP, 5 EGTA, 2 CaCl2 and 5 HEPES (pH adjusted to 7.2 with CsOH)[17]. All patch clamp and Ca2+ imaging experiments were performed at 37°C.The whole-cell I–V relationship was measured by applying ramp pulses from -110 to +50 mV and the holding potential was -80 mV. K1-S2 arrhythmia induction protocol at twice the pacing threshold intensity was adapted from 1), 3 extra stimuli (S2) with a coupling cycle length of 50, 40, or 30 ms, respectively, were introduced. Each of these 3 sets of stimulation were repeated 3 times. Arrhythmias will be categorized into 5 groups and assigned the following points: 0 points, no arrhythmia; 1 point, 1–3 premature ventricular complexes (PVCs); 2 points, non-sustained VT ; 3 points, sustained VT (>10 consecutive PVCs); and 4 points, VF/SCD ((Pseudo-)Lead II ECGs were recorded in isolated Langendorff-perfused hearts. A pair of Ag-AgCl electrodes were placed close to the apex of the left ventricle as well as at the right atrial appendage to obtain ECG signals at a sampling rate of 1 kHz. Two additional platinum electrodes were placed on the free wall of the right ventricle for stimulation using a stimulator (Grass) triggered by a custom-designed computer program. To induce ventricular arrhythmias, a standard S, VF/SCD .t-tests or ANOVA analysis, with P < 0.05 considered significant.Data are presented as mean ± SEM, and at least 3 animals were used for each group. Statistical significance was assessed using paired, unpaired Student’s 2+. The changes of Ca2+ level (reflected by Fluo-4 fluorescence intensity) were measured by increasing extracellular Ca2+ concentration ([Ca2+]) from 0 to 1 mM  was observed when [Ca2+] was changed from 0 to 1 mM. In order to maximally/completely deplete SR Ca2+, in addition to CPA, we also employed 10 mM caffeine to fully open RyR. As a result, a much larger elevation of Ca2+ level was induced when [Ca2+] was changed from 0 to 1 mM . As shown in Figure 0 = 2.7 ± 0.7) was markedly higher than that after Tha/CPA only , suggesting the existence of SOCE in adult cardiac myocytes, and a maximal SOCE activation requires the complete depletion of SR Ca2+. This SOCE was effectively blocked by SOCE/TRPC blockers gadolinium  and ML-9 , but not by Na+/Ca2+ exchanger (NCX) inhibitor SEA0400  + caffeine (10 mM). Control myocytes without pretreatment with antibodies exhibited a peak SOCE amplitude of F/F0 = 2.4 ± 0.2, n = 39, whereas all three TRPC1, 3 or 6 antibodies significantly inhibited the peak of SOCE to F/F0 = 1.4 ± 0.2 (n = 10), 1.2 ± 0.2 (n = 8), 1.1 ± 0.1 (n = 8), respectively . Similar inhibitory effects to 1.2 ± 0.1 (n = 5) by Gd3+ or to 1.2 ± 0.1 (n = 6) by the TRPC3 blocker Pyr3 pre-perfused in the bath for 10–15 min were also observed   antibodies for TRPC1, 3 and 6 were added, respectively, to pretreat the myocytes for 30 min before SOCE was evaluated following the standard protocol with complete depletion of SR Ca0 = 1.5 ± 0.1, n = 26). Hyperforin further potentiated the SOCE level in a concentration-dependent manner in adult mouse ventricular myocytes. The amplitudes (F/F0) of SOCE in the presence of hyperforin at 0.1, 1, and 10 μM were, 1.9 ± 0.1 (n = 15), 2.4 ± 0.2 (n = 8) and 3.7 ± 0.4 (n = 7), respectively , which were all significantly higher than the control SOCE value before hyperforin application.Furthermore, the participation of TRPC6 in SOCE was confirmed by using various concentrations of hyperforin, a potent activator of TRPC6 . As show2+ transients and Ca2+ waves. Ca2+ transients were measured by pacing myocytes continuously at a PCL of 2 s. As shown in a representative trace  by 0.1 μM hyperforin, and from 2.0 ± 0.2 to 2.4 ± 0.1  by 1 μM hyperforin. To exclude the possibility that the enhancement of Ca2+ transients might be due to the potentiation any other voltage-gated Ca influx mechanisms, we then used a different protocol in which the cells were kept quiescent when they were perfused with hyperforin (0.1 or 1 μM) for 1 min  as indicated by the arrow in each panel). We found that Ca2+ transient amplitude was enhanced by hyperforin treatment even the cells remained in resting condition. Ca2+ transient amplitudes increased from 1.8 ± 0.2 to 2.1 ± 0.3  by 0.1 μM hyperforin and from 2.4 ± 0.5 to 3.0 ± 0.4  by 1 μM hyperforin . These data suggest that hyperforin promoted Ca2+ entry through sarcolemmal membrane and increase of cellular/SR Ca2+ load, unlikely via voltage-gated Ca2+ channels.Next, we evaluated how hyperforin-activated TRPC may affect Cae Figure , the ampM Figure  and 1 μMM Figure . Summarin Figure   without being stimulated. As shown in Figure 2+ waves were observed under baseline in normal cells (2+ waves were significantly facilitated by hyperforin (0.1 μM) with a frequency increase from 22 ± 3/min under control condition to 38 ± 4/min after hyperforin treatment . Taken together, the results shown in Figures 2+ depletion condition and TRPC6 channels independent of SR Ca2+ depletion.Next, we further assessed the effect of hyperforin on spontaneous Caal cells . These sr [Ca2+]  mM withoOCE) after SR Ca2+ depletion was subsequently measured. As described in the Method section, K+, Na+, L-type Ca, and Na+-Ca2+ exchange currents were pre-blocked. Whole-cell currents were recorded under the voltage clamp condition using a ramp protocol from -110 to +50 mV, and the inward current amplitudes at -110 mV were evaluated. As shown in time course and representative traces . As summarized in Figure n = 12 each, ∗p < 0.01) in the inward current, which was attenuated by the application of 1 mM Gd3+ . The I/V relationship revealed a reversal potential around 0 mV, indicating its non-selective property, which is consistent with TRPC channels as reported by n = 8, ∗p < 0.05 compared to control), which was effectively blocked by 1 mM Gd3+ .The inward current when SOCE occurred  of membrane potential and corresponding elevation of intracellular Ca2+. As shown in the expanded regions  had been removed in this condition when arrhythmias occurred, it seemed that Ca2+ release and NCX current did not play a causal role in spontaneous membrane depolarization, in which case the initiation of intracellular Ca2+ elevation should occur earlier than membrane potential changes. This relation has been well discussed in our previous publication  remained longer compared to control  were used to assess the propensity to develop arrhythmias in each group. As shown in Figures n = 6), which was significantly higher than hearts without treatment with hyperforin .To examine the arrhythmogenic effect of activation of TRPC6 channel by hyperforin at the whole-heart level, hyperforin (0.1 μM) was perfused in 2+ signaling. In addition to replenish the SR stores, its long-term and immediate physiological significances have been profoundly explored in previous studies, includes cell proliferation, apoptosis, muscle contraction and intracellular Ca2+ oscillations   , the rolomyocyte , and theomyocyte .OCE exist in normal adult ventricular myocytes subsequent to a complete depletion of SR Ca2+ storage. SR Ca2+ level can be physiologically reduced following the activation of calcium-releasing signals mediated by IP3R or RyR. Whereas under our experimental condition, specific inhibitors of SR Ca2+ pump  are usually employed to induce SOCE, previous studies seemed to produce only moderate increase in normal adult cardiac myocytes when exposed to CPA or Tha separately (0: over 2.5) compared to that induced only by SERCA inhibitor. This Ca2+ entry can be effectively inhibited by acute application of SOCE blockers Gd3+ and ML-9, but not by the NCX inhibitor SEA0400. We have noticed that neither Gd3+  nor ML-9  is completely selective to TRPC channels. However, our experiments using selective antibodies of TRPC channels provide more convincing evidence for the involvement of TRPC channels.Our present study demonstrated that SOCE and Iparately . Similarparately . Alternaparately . In the 2+ load-independent pathway, e.g., PKA-dependent activation. This issue needs to be further elucidated.It should be pointed out that caffeine also functions as a PDE inhibitor , which i2+ selectivity, the involvement of TRPCs in SOCE is still under debate. For example, the expression of TRPC3 has been examined in DT40 B lympohocytes by two groups individually. One group demonstrated the TRPC3 activation is triggered in response to PLC-γ or PLC-β enzymes, but independent of SR depletion , and it was reported to selectively activate TRPC6 channels. Our data clearly demonstrated that hyperforin at concentrations of 0.1 and 1 μM predominantly potentiated the SOCE and IOCE in adult mouse ventricular myocytes. It also facilitated the spontaneous Ca2+ waves and promoted the calcium transient amplitudes. A previous study has shown that hyperforin exerts complex actions on cortical neurons. In addition to promoting TRPC6 channels in plasma membrane, hyperforin may exert protonophore-like effect, triggering the release of Ca2+ and Zn2+ from mitochondria  in the setting of our experiments, but imply that hyperforin gives rise to Ca2+ most likely via activation of TRPC6 in cardiac myocytes. Our results have demonstrated that although the activation of SOCE requires SR Ca2+ depletion, its potential composing entity, TRPC channels, are able to be activated by HF independent of SR Ca2+ depletion (Figure ex vivo hearts, implying a possible arrhythmogenic role of SOCE/TRPC6 in the whole heart setting. TRPC6 channel was found to be important in the pathologic cardiac remodeling as a key component of a calcium-dependent regulatory loop (Hyperforin is known as an antidepressant compound extracted from St. John’s wort (chondria . Howevern Figure . This isn Figure . We obseory loop . Herein,HW, ZZ, NF, and L-HX performed the experiments and analyzed the data. HW and L-HX conceived and designed the research, interpreted results of experiments, prepared figures, and wrote manuscript. HW, ZZ, NF, and L-HX approved final version of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genotoxic stress triggers a combined action of DNA repair and cell cycle checkpoint pathways. Protein phosphatase 2C delta (referred to as WIP1) is involved in timely inactivation of DNA damage response by suppressing function of p53 and other targets at chromatin. Here we show that WIP1 promotes DNA repair through homologous recombination. Loss or inhibition of WIP1 delayed disappearance of the ionizing radiation-induced 53BP1 foci in S/G2 cells and promoted cell death. We identify breast cancer associated protein 1 (BRCA1) as interactor and substrate of WIP1 and demonstrate that WIP1 activity is needed for correct dynamics of BRCA1 recruitment to chromatin flanking the DNA lesion. In addition, WIP1 dephosphorylates 53BP1 at Threonine 543 that was previously implicated in mediating interaction with RIF1. Finally, we report that inhibition of WIP1 allowed accumulation of DNA damage in S/G2 cells and increased sensitivity of cancer cells to a poly-(ADP-ribose) polymerase inhibitor olaparib. We propose that inhibition of WIP1 may increase sensitivity of BRCA1-proficient cancer cells to olaparib. Cells are constantly challenged with DNA damage that comes both from endogenous and exogenous sources. The most deleterious type of DNA damage is double strand breaks (DSBs) that affect both strands of DNA and if not repaired correctly could lead to chromosomal rearrangements. DSBs are repaired by two major pathways—homologous recombination (HR) and non-homologous end joining (NHEJ). NHEJ operates throughout the cell cycle and results in ligation of two ends of DNA that are not extensively processed . HR is rTwo major factors involved in repair pathway choice are TP53-binding protein 1 (53BP1) and Breast cancer type 1 susceptibility protein (BRCA1) ,12. BRCAMutations in BRCA1 and other DNA repair genes are common cause of cancer but deficient HR can be also exploited as target for cancer therapy . InhibitPPM1D gene and its expression is increasing towards the G2 phase of the cell cycle  supplemented with benzonase (100 U/mL), briefly sonicated and EtBr (50 μg/mL) was added before centrifugation 30 min 4 °C at 20,000 g. GFP-Trap beads  were added to lysate for 1 h before washing 4× with lysis buffer. Alternatively, endogenous WIP1 was immunoprecipitated from U2OS cells by a rabbit affinity-purified antibody generated against human WIP1 and immobilized on protein A/G UltraLink resin . Bound proteins were eluted with 2× loading buffer and analyzed by Western Blotting. HEK293 or U2OS-WIP1-KO cells were transfected with GFP-WIP1 plasmid using polyethylenimine. Cells treated as indicated were extracted in lysis buffer [50 mM Tris pH 8.0, 120 mM NaCl, 1% Tween-20, 0.1% NP-40, 1.0% glycerol, 2 mM EDTA, 3 mM EGTA, 10 mM MgCl4 and 10 μM AlexaFluor 647 azide  for 30 min at RT. Coverslips were incubated with primary antibodies for 2 h in RT, washed 3× in PBS, incubated with secondary antibody for 1 h in RT, washed 3× in PBS, stained with DAPI in PBS for 2 min, washed in dH2O and dried before mounting with Vectashield. Images were acquired using Olympus ScanR system equipped with 40×/1.3 UPLFN or 40×/0.9 objective . Nuclei were segmented based on DAPI intensity and foci were identified using Spot detection module. Total and mean intensities of staining per nucleus were determined. FlowJo v10.6.1 software  was used to determine of intensity or foci number in particular cell population.Cells were seeded on coverslips one day before treatment. Where indicated, cells were pre-extracted 5 min on ice before fixation. Cells were fixed with 4% PFA 15 min RT. Cells were permeabilized using 0.5% Triton-X100 in PBS 5 min and blocked in 1% BSA for 30 min. Where indicated, cells were pulse labeled with EdU 30 min before irradiation and click reaction was performed before primary antibody incubation in 0.1 M Tris pH 8.5, 0.1 M sodium ascorbate, 2 mM CuSOFor cell cycle analysis, cells were incubated with EdU 30 min before harvesting by trypsinization and fixation in 70% EtOH. Cells were permeabilized with 0.5% Triton-X100 in PBS 15 min RT, washed in BSA, click reaction was performed before incubation with primary and secondary antibodies. DAPI was added in final concentration of 5 μg/mL in PBS. Percentage of dead cells was determined 3 days after treatment using Hoechst33258 . To analCells were seeded 1 day before treatment to 96 well plates at 50–500 cells per well, incubated seven days with treatment before resazurin was added in fresh media at final concentration 30 μg/mL . FluoresCells were lysed in 2× lysis buffer, sonicated and protein concentration was determined using BCA protein assay. A total of 20–50 μg lysate was resolved on SDS-PAGE, transferred to nitrocellulose membrane, blocked in 5% milk, incubated with primary and secondary antibodies and developed with ECL.2, 2.5 mM KCl, 0.5 mM DTT) for 5 min, centrifuged, resuspended in buffer A containing EDTA-free protease and phosphatase inhibitors  and dounce homogenized using tight pestle on ice. Nuclei were spun down at 500 g 5 min 4 °C and extracted in buffer C  supplemented with 600 mM KCl on ice for 15 min before centrifugation at max speed 15 min 4 °C. Supernatants were diluted with buffer C to final concentration 150 mM KCl, clarified by centrifugation max speed 15 min 4 °C and stored at −80 °C. For in vitro phosphatase assay 100 μg of nuclear extract was incubated with 250 ng of purified full-length His-WIP1 phosphatase 15 min at 37 °C [U2OS-WIP1-KO cells were treated as indicated and nuclear extracts were prepared using hypotonic lysis as previously described . Brieflyat 37 °C . Where iat 37 °C . Reactio


Here, we study the effect of partial substitution of Fe by Co on the microstructure to gain insight into the mechanism of enhancing magnetic properties of (Nd0.8Pr0.2)2.2Fe14−xCoxB  alloys. Our results show that the substitution Co for Fe changes the magnetic properties obviously by tuning the chemistry and distribution of the intergranular phase between hard magnetic grains. In particular, for (Nd0.8Pr0.2)2.2Fe12Co2B (x = 2) alloy, no obvious intergranular phase is observed. And the through-thickness homogeneity and ultrafine microstructure with an average size of ~25 nm is obtained, which produces maximum product ((BH)max) of 141 kJ/m3, 29% higher than that of quaternary alloy. Our findings provide a new idea to design prospective permanent alloys with increased magnetic properties by tuning the distribution and chemical composition of the intergranular phase.In Nd It is known that rapid solidification by melt spinning is one of the most important methods to produce melt-spun ribbons consisting of randomly oriented Nd2Fe14B grains with an average diameter of less than 100 nm, which are currently used as raw materials for the production of bonded magnets and hot-pressed magnets3. Therefore, optimizing the microstructure of melt-spun ribbons is critical to improving the magnetic properties of Nd-Fe-B PMs. Moreover, in recent years, there has been great effort in developing economically more attractive permanent materials because of the price volatility and limited availability of critical rare earth metals (RE), such as Dy and Nd4. For example, Pathak et al. reported an unexpected increase of the intrinsic coercivity (Hci = 17.7 kOe) of Ce and Co co-doped (Nd0.8Ce0.2)2.4Fe12Co2B melt-spun ribbons at room temperature, which was larger than that of 5.9 wt% Dy containing ribbons5. The segregation of heavy elements at the intergranular phase was speculated to play an important role on the improvement of the coercivity, however, the mechanism of enhancing the coercivity is still veiled.Nd2Fe14B (2:14:1) grains have a strong influence on the coercivity of Nd2Fe14B-based PMs6. Two different types of intergranular phase are observed: i) non-magnetic intergranular phase with large amount of RE (>65 at.%) which improves the coercivity by promoting a magnetic isolation of 2:14:1 grains7; and ii) ferromagnetic intergranular phase containing large fractions of Fe and/or Co more than 65 at.% leads to an improvement of remanence by enhancing the inter-grain coupling8. It is worth noting that the role of Co distribution in both types of intergranular phase is under debate. For non-magnetic intergranular phase of hot-deformed Nd-Fe-B magnets, substitution of Co for Fe led to the formation of a Co-enriched layer at the edges of 2:14:1 grains resulting in a reduction of the magnetocrystalline anisotropy of 2:14:1 phase9, whereas the segregation of Co was found at the interface between the intergranular phase and 2:14:1 phase acting as nucleation sites for reversed magnetic domains10. On the other hand, the enrichment of Co was observed at the magnetic intergranular phase and further promoted by magnetic field annealing, which resulted in an improvement of remanence by strengthening the exchange coupling interaction between hard magnetic grains of Nd-Ce-Fe-Co-B alloys11. Hence, the effect of Co addition on the coercivity needs to be further investigated.It has been found that the composition and distribution of intergranular phase between Nd12. However, achievement of through-thickness homogeneity with an average size less than 50 nm in the ribbon microstructure during melt-spinning is extremely difficult due to some major factors including fabrication conditions and composition of alloys. Our previous work found homogenous microstructure with an average size less than 40 nm through-thickness Ce-Fe-B melt-spun ribbons was obtained by optimizing fabrication parameters (e.g. chamber pressure and wheel speed) during melt-spinning13. Therefore, a refiner microstructure is expected by further adjusting the composition of alloys. Atom probe tomography (APT) can determine quantitatively the local composition distributions in three dimensions with atom resolution and has been used widely to illustrate the distribution of additive element15, microstructural evolution17 and the role of intergranular phase19 in magnetic ribbons. For example, Cu and Nb addition is reported to decrease the grain size effectively resulting in an improvement of magnetic properties of nanocomposite alloys15. It is found by APT technique that Y-containing intergranular phase contributes to the enhanced domain wall pinning at the grain boundary in (Nd0.5Y0.25Dy0.25)1.8Zr0.4Co1.5Fe12.5B ribbons19. In this study, we investigated the microstructure and magnetic properties of (Nd0.8Pr0.2)2.2Fe14−xCoxB  melt-spun ribbons to understand how Co-substitution for Fe could be employed to tailor the magnetic properties by tuning the distribution, type and chemistry of the intergranular phase. Partial substitution of Nd by Pr is chosen because Pr addition can promote uniform distribution of intergranular phase resulting in an increase of magnetic properties in Nd-Fe-B alloys21. The results show that the substitution Co for Fe affects the type, chemistry, and distribution of the intergranular phase. For x = 2 alloy, no obvious intergranular phase is observed, and the through-thickness homogeneity and ultrafine microstructure with an average size of ~25 nm is obtained. It produces maximum product ((BH)max) of 141 kJ/m3, which is 29% higher than that of quaternary alloy.Remanence is another extrinsic magnetic property and it is intimately related to the microstructure of a magnetic material. One effective way of increasing the remanence is to refine the grain size by improving the inter-grain exchange interaction between 2:14:1 grains0.8Pr0.2)2.2Fe14−xCoxB  melt-spun ribbons. The key magnetic property parameters are shown in Table ci) decreases, reaching a minimum at x = 2, and then increases at x = 2.25. In contrast, the remanence (Br) and (BH)max increase, reaching a maximum at x = 2 with a value of 0.91 T and 141 kJ/m3, and then decrease with further Co addition.Figure 0.8Pr0.2)2.2Fe14−xCoxB  melt-spun ribbons is shown in Fig. 22 and given in Table The XRD patterns of the free surface and wheel surface (the surface in contact with the copper wheel) of Nd0.8Pr0.2.2Fe14−x0.8Pr0.2)2.2Fe14−xCoxB  melt-spun ribbons, respectively. It is shown that a homogeneous microstructure through the thickness is observed in all samples. The grain size distribution was determined from the TEM images, see Supplementary Fig. 0.8Pr0.2)2.2Fe12Co2B (x = 2) alloy.Figure 0.8Pr0.2)2.2Fe14B alloy, the high-angle-annular-dark-field (HAADF) images show that 2:14:1 grains are surrounded by thin intergranular layers with bright contrast along the grain boundaries (GBs), marked as arrows 2.2Fe12Co2B (x = 2) alloy indicating no segregation of heavy elements at GBs 2.2Fe14B (x = 0) alloy is shown in Fig. 8 indicating that the intergranular phase is ferromagnetic. Figure 0.8Pr0.2)2.2Fe12Co2B (x = 2) alloy. No segregation of elements is observed at GBs. Figure The atom probe tomography (APT) reconstruction of 2.2Fe14B (x = 0) alloy show that 2:14:1 grains are surrounded by intergranular phase enriched Nd and Pr. A segregation of B is found at the surface between the intergranular phase and 2:14:1 phase. Furthermore, large amount of ferromagnetic element Fe (≈70 at.%) is observed at the intergranular phase, higher than 65 at.% reported by Sepehri-Amin8, and is considered to be ferromagnetic. However, with Co addition for (Nd0.8Pr0.2)2.2Fe11.75Co2.25B (x = 2.25) alloy, besides ferromagnetic intergranular phase, another type of non-ferromagnetic intergranular phase is found. The segregation of Co at the interface between the intergranular phase and 2:14:1 phase is observed. Moreover, Co addition promotes the enrichment of B atoms at the intergranular phase. That is, the enrichment of non-ferromagnetic elements, such as Pr, Nd and B, is found at the intergranular phase, which is helpful to increase the covercivity by weakening the inter-grain coupling of 2:14:1 grains. Therefore, we consider that both increase of Hci  and Br  is due to the role of competition between two types of intergranular phase on the magnetic properties. It is worth noting that no obvious intergranular phase is observed for (Nd0.8Pr0.2)2.2Fe12Co2B (x = 2) alloy. Intergranular phase was reported to retard grain growth resulting in the microstructural refinement23. In our work, however, HAADF image 2.2Fe12Co2B (x = 2) melt-spun ribbons.The microstructure analyses of the 2.2Fe14−xCoxB  melt-spun ribbons in mode of just-focus and under-focus, respectively. All images were observed in the demagnetization state. Big domain size (marked in pink dash) and small domain size (marked in yellow short dash) are both observed in x = 0 alloy  plot26. Here, χirr = dMirr/dH, Mirr is the irreversible magnetization and can be obtained by a dc demagnetization (DCD) experiment as described in ref.27. Figure χirr curves for (Nd0.8Pr0.2)2.2Fe14−xCoxB  melt-spun ribbons. The narrow and intensive peak in χirr curve indicates that each grain couples well with its neighboring grains due to exchange coupling between magnetic phases. The (Nd0.8Pr0.2)2.2Fe12Co2B (x = 2) alloy shows a narrower and higher peak than x = 0 and x = 2.25 alloys suggesting a stronger exchange coupling interaction. Hence, the remanence is enhanced by about 14% from 0.80 T for x = 0 alloy to 0.91 T for x = 2 alloy.An effective method of understanding the exchange coupling interaction is via irreversible magnetic susceptibility 2.2Fe14B (x = 0) alloy, the intergranular phase enriched Nd and Pr between 2:14:1 grains are observed  alloy may due to an enlargement of the undercooling resulting in the increase of the nucleation rate and suppression of the formation of intergranular phase between 2:14:1 grains. With further Co addition (x = 2.25), ferromagnetic and non-ferromagnetic intergranular phases are both observed indicating that increasing Co content stimulates the segregation of Nd and Pr at the intergranular phase.During rapid solidification, achievement of through-thickness homogeneity in the ribbon microstructure appears to be limited by unidirectional heat flow through the solidified portion of the ribbon to the copper wheelsee Fig. . It is d29,*hom is the magnitude of the homogeneous nucleation barrier, and θ is the wetting angle.From the sound fundamental understanding about heterogeneous nucleation, the magnitude of the heterogeneous nucleation barrier is given byθ. Our results show that proper amount of Co addition (x = 2) decreases effectively the grain size suggesting Co addition may reduce θ by enhancing the wettability of liquid metal with the mould surface, which increases the nucleation rate and refines the grain size due to reducing the energy barrier to nucleation. Hence, the ultrafine microstructure with an average size of ~25 nm is obtained, which produces (BH)max of 141 kJ/m3, 29% higher than 101 kJ/m3 of quaternary alloy, and 41% higher than 100 kJ/m3 of (Nd0.8Ce0.2)2.4Fe12Co2B ribbons reported in ref.5. It is worth mentioning that the average Nd2Fe14B grain diameter with 23 nm was observed in Fe82Nd12B6 ribbons30. The remanence with 1.02 T is higher than 0.91 T of x = 2 ribbons, which may ascribe to stronger exchange coupling interaction between Nd2Fe14B grains. Moreover, it is reported that the coercivity with 2021.8 kA/m and (BH)max with 156.2 kJ/m3 is obtained in decoupled Pr15Fe78B7 ribbons and stoichiometric Pr12Fe82B6 ribbons, respectively31. The values of coercivity and (BH)max are both higher than those of our work, which is due to larger anisotropy field of Pr2Fe14B (75 kOe) than 73 kOe of Nd2Fe14B3.That is, the activation barrier for nucleation is dependent on 0.8Pr0.2)2.2Fe14−xCoxB  alloys are investigated. The results show that the substitution Co for Fe can adjust the distribution, type and chemistry of the intergranular phase. Ferromagnetic intergranular phase is found in (Nd0.8Pr0.2)2.2Fe14B (x = 0) alloy, whereas non-ferromagnetic and ferromagnetic intergranular phase are both observed for Co-containing with x = 2.25 alloy. In comparison to the (Nd0.8Pr0.2)2.2Fe14B (x = 0) alloy, both increase of Hci and Br of (Nd0.8Pr0.2)2.2Fe11.75Co2.25B (x = 2.25) alloy is because of the role of competition between two types of intergranular phase on the magnetic properties. For x = 2 alloy, no obvious intergranular phase is observed, and the ultrafine microstructure with an average size of ~25 nm through-thickness homogeneity is obtained. It improves the remanence significantly resulting in an achievement of (BH)max with 141 kJ/m3, 29% higher than that of quaternary alloy without Co addition, and 41% higher than that of (Nd0.8Ce0.2)2.4Fe12Co2B ribbons reported in ref.5.In conclusion, the microstructure and magnetic properties of (Nd0.8Pr0.2)2.2Fe14−xCoxB  were prepared by arc melting the mixture of pure metals Nd (99.5%), Pr (99.5%), Fe (99.5%), Co (99.999%), and a FeB pre-alloy in an argon atmosphere. Each ingot was re-melted four times for homogenization. Ribbons were produced by melt-spinning in an argon atmosphere at a wheel speed of 15 m/s. During melt-spinning, the quenching temperature and chamber pressure were controlled at 1588 K ± 5 K and 0.05 MPa. The nozzle at the bottom of a quartz tube was positioned 8 mm above the copper wheel surface. Magnetic properties were measured using a Quantum Design physical property measurement system (PPMS)  with a maximum magnetic field of 5 T. X-ray diffraction (XRD) patterns were collected using a D/max-2550 diffractometer with Cu Kα radiation . Cross-section samples near the wheel surface and free surface of ribbons for transmission electron microscopy (TEM), and near wheel surface for atom probe tomography (APT) observations were made by a Helios 600i focus ion beam (FIB) system , as shown in Supplementary Figure −8 Pa) on a CAMECA Instruments LEAP_4000X HR local electrode atom probe . The specimens were analyzed in voltage mode with a specimen temperature at 50 K with a target evaporation rate of 1%, and the energy of pulse laser is 60 pJ.Ingots of nominal composition (NdThe effects of Co on the enhancement of magnetic properties by modifying the intergranular phase in Nd-Fe-B alloys


H2A.Z variant has emerged as a critical player in regulating plant responses to environment; however, the mechanism by which H2A.Z mediates this regulation remains unclear. In Arabidopsis, H2A.Z has been proposed to have opposite effects on transcription depending on its localization within the gene. These opposite roles have been assigned by correlating gene expression and H2A.Z enrichment analyses but without considering the impact of possible H2A.Z post-translational modifications. Here, we show that H2A.Z can be monoubiquitinated by the PRC1 components AtBMI1A/B/C. The incorporation of this modification is required for H2A.Z-mediated transcriptional repression through a mechanism that does not require PRC2 activity. Our data suggest that the dual role of H2A.Z in regulating gene expression depends on the modification that it carries, while the levels of H2A.Z within genes depend on the transcriptional activity. Arabidopsis H2A.Z plays an important role in regulating gene expression in response to stressors; however, the underlying mechanism is still puzzling. Here, the authors show that monoubiquitination of H2A.Z by AtBMI1 is required for H2A.Z-mediated transcriptional repression. According to this, H2A.Z variant has emerged as a critical player in regulating plant responses to environment. Several reports have demonstrated that Arabidopsis H2A.Z plays a role in regulating gene expression in response to temperature fluctuations, drought, pathogen infection, and nutrient stress7. Nevertheless, in addition to its role in transcriptional regulation, H2A.Z in plants has been related to other processes, such as DNA repair, somatic homologous recombination, and replication origin specification8.Eukaryotic organisms must respond to environmental changes in order to survive. This response involves changes in gene expression in which chromatin structure plays a central role. Chromatin organization can be altered through the incorporation of histone variants that, unlike canonical histones, occurs independently of replication. This different mechanism of deposition makes them adaptable to respond to environmental stimuli12, but it is also found further within gene bodies13. H2A.Z has been proposed to have either a promoting or repressive effect on transcription depending on its localization within the gene. The presence of H2A.Z in +1 nucleosome has been associated with transcriptional activation, while its presence in nucleosomes further within gene bodies is associated with transcriptional repression and positively correlated with gene responsiveness13. In contrast, a recent report showed that H2A.Z enrichment at +1 nucleosome is required to repress gene expression14; therefore, the role of H2A.Z is still puzzling.H2A.Z in Arabidopsis, as in other organisms, frequently localizes immediately downstream transcription start sites, co-localizing with +1 nucleosome16. The incorporation of this modification is required for H2A.Z-mediated transcriptional repression. Moreover, our data indicate that more than H2A.Z incorporation H2A.Z monoubiquitination is what is decisive for H2A.Z-mediated repression.A reason for these conflicting results could be that the incorporation of H2A.Z has been examined as a whole without considering posttranscriptional modifications, which provides an incomplete picture to understand the role of this histone variant. Here, we show that H2A.Z can be monoubiquitinated by the PRC1 components AtBMI1A/B/CHTA8, HTA9, and HTA1117. Expression analysis of the three genes in wild-type Col-0 (WT) seedlings at 7 days after germination (DAG) showed that HTA9 displayed the highest expression levels while HTA8 the lowest , which is involved in the incorporation of H2A.Z8. Accordingly, the levels of the flowering-promoter FLOWERING LOCUS T (FT) are increased in hta9hta11 and those of the flowering-repressor FLOWERING LOCUS C (FLC) are reduced2.In Arabidopsis, there are three functional genes encoding H2A.Z, hta9hta11 histone extracts at 7 DAG using anti-HTA9 antibody not only confirmed reduced levels for HTA9-predicted band in hta9hta11  under the control of CaMV35s promoter  analysis of WT and a11 Fig.  but alsoa11 Fig. , indicatter Fig. . Anti-HTter Fig. , confirm19. This modification occurs mostly at lysine (K) 120 and to a lesser extent at K121 and K12518. Thus we compared the protein sequences of HTA9 and human H2A.Z and selected K129 and K132 as possible target lysine/s for monoubiquitination in Arabidopsis    marks have been detected in mammalian embryonic stem cells at bivalent genes but they decrease upon differentiationly) Fig. . These mly) Fig. , which s_RR Fig. , which wels Fig. ; howevercue Fig. . Since tnts Fig. . We foun20; however, the PRC1 monoubiquitin ligase module in Arabidopsis, which comprises one AtBMI1 (AtBMI1A/B/C) and one AtRING1 (AtRING1A/B) protein22, has so far only been involved in canonical H2A lysine 121 monoubiquitination (H2AK121ub)21. This activity is required to maintain gene repression throughout plant development24. atbmi1a/b/c mutant remains in an embryo maturating-like phase due to misexpression of the embryonic program after germination25 30, in the repression of around 5000 genes31. In fact, PRC2 requires the presence of H2AK121ub marks to incorporate H3K27me3 at these genes31. Accordingly, atbmi1a/b/c mutant displays strongly reduced levels of H2AK121ub and H3K27me331. Therefore, to investigate whether AtBMI1 activity is involved in H2A.Z monoubiquitination, we compared the levels of modified HTA9 in WT and atbmi1a/b/c  mediates H2A and H2A.Z monoubiquitinationb/c Fig.  and we fb/c Fig.  and thatb/c Fig. . Furtherted Fig. . All tog32 ; however, the number of downregulated genes in hta9hta11/FLAG-HTA9_N was higher than in hta9hta11/FLAG-HTA9_RR . Consistent with this, we found that a high percentage of the genes downregulated in hta9hta11 recovered their WT expression levels in both hta9hta11/FLAG-HTA9_N and hta9hta11/FLAG-HTA9_RR plants  in WT seedlings31 4 , in which H3K27me3 deposition is abolished genome wide28, and in hta9hta11, but we found that only a 10% of the genes in each set overlapped in this comparison , and the little overlap found between the upregulated genes in hta9hta11 and fie mutants, it seems that, despite H2A.Z may co-localize with H3K27me3 at a subset of targets and may modulate the H3K27me3 levels, the transcriptional-repressive effect of H2A.Zub is not dependent on PRC2 activity.We next wondered whether the combined action of H2A.Zub and H3K27me3 is required to regulate the expression of H2A.Z/H3K27me3_WT genes. Therefore, we compared the genes upregulated in the PRC2 mutant son Fig. . Therefo13. In line with this, we found a negative correlation between gene expression levels and H2A.Z enrichment along genes  were similarly incorporated into genes whose expression is recovered or partially recovered in one or in the two lines , in which the incorporation of H2A.Z has been shown to have a promoting and a repressive effect in transcription, respectively41. Consistent with this, WT-like expression levels of FLC were recovered in both lines ) or downregulated (ZINC TRANSPORTER 5 (ZIP5); FRUCTOSE-BISPHOSPHATE ALDOLASE 2 (FBA2)) to different extents in hta9hta11 and also ACTIN 7 (ACT7) as a negative control for H2A.Z incorporation  function, which is one of the PRC2 H3K27 trimethyltransferases in Arabidopsishta9hta11 or pie1 and fie or clf mutants (our data and ref. 39) suggests that H3K27me3, although may have a reinforcing effect, is dispensable for H2A.Z-mediated repression. In agreement with this, a recent report showed that, despite the transcriptional activation of anthocyanin biosynthesis genes in loss of H2A.Z mutants is associated with reduced levels of H2A.Z and H3K27me3, the reduced levels of H3K27me3 are not associated with changes in the expression of these genes46, supporting a central role of H2A.Z in the transcriptional repression of these genes.In any case, while the number of genes commonly upregulated in loss of H2A.Z and PRC1 mutants clearly suggests a direct connection between H2A.Z regulation and PRC1 activity, the small number of commonly upregulated genes in hta9hta11 chromatin, the transcriptional repressive effect of H2A.Z was significantly recovered only when FLAG-HTA9_N was incorporated, supporting that H2A.Z monoubiquitination, more than H2A.Z incorporation, is what is decisive for H2A.Z-mediated repression. In addition, our results support that the different levels of H2A.Z found at repressed and activated genes are consequence of the transcriptional activity, which is in agreement with recent results in animals47. Nonetheless, besides transcriptional regulation, H2A.Z is involved in other processes such as DNA repair and somatic homologous recombination8, thus it might be possible that the incorporation and/or distribution of this histone variant follow different rules depending on the process in which is involved.Interestingly, despite FLAG-HTA9_N and FLAG-HTA9_RR being similarly incorporated into In summary, based on our and other recent findings we propose a model in which H2A.Z monoubiquitination plays a crucial role in transcriptional repression and, most probably, H2A.Z acetylation in transcriptional activation, while the different levels of H2A.Z along the genes may be a consequence of gene activity rather than an active mechanism to establish repression Fig. .Arabidopsis thaliana Col-0 wild type (WT), hta9–1hta11–2 (hta9hta11)2, atbmi1a/b/c25, atbmi1a/b weak21 mutants and transgenic plants harboring the different constructs were grown under long day conditions (16 h light and 8 h dark) at 21 °C on MS agar plates containing 1.5% sucrose and 0.8% agar for 7 days. MS agar plates were supplemented with hygromycin (20 µg ml−1) for selection of transgenic plants.Arabidopsis native (N) or mutated HTA9 cDNA, in which K129 or both K129 and K132 were replaced by arginine (R), , was N-terminally epitope tagged with FLAG and introduced into a modified pCAMBIA 1302 vector (pCAMBIA 130 N)48 under the control of the CaMV35s promoter. For the incorporation of the FLAG-tag and C-terminal mutations, we designed specific primers that are indicated in Supplementary Table Agrobacterium strain GV3101 by the freeze–thaw method49 and subsequently transformed into WT, hta9hta11, and atbmi1a/b/c+/− mutant plants by the floral-dip method50.ACTIN2 was used as endogenous control. Primers used are specified in Supplementary Table Total RNA was extracted from 10 seedlings at 12 DAG using the RNeasy Plant Mini Kit (Qiagen) and on-column DNase treatment (Qiagen) was performed to remove any DNA contamination. cDNAs were reverse-transcribed from total RNAs with the QuantiTect Reverse Transcription Kit (Qiagen). For reverse transcription–PCR analyses, hta9hta11/FLAG-HTA9_N, or hta9hta11/FLAG-HTA9_RR seedlings at 7 DAG was collected and fixed in 1% Formaldehyde. Fixed chromatin was extracted and fragmented using a Bioruptor® Pico (Diagenode) in fragments of 200–500 bp. Disrupted chromatin was resuspended in IP buffer  and incubated with anti-H3 antibody  or anti-HTA9 antibody  at 4 °C overnight with gentle rocking. Protein A magnetic beads (Diagenode kch-802–660) were added and incubated for 4 h at 4 °C with gentle rocking to collect immunocomplexes. After 3–4 washes with IP buffer, immunocomplexes were eluted in Laemmli buffer and boiled for 10 min to analyze by WB.One gram of WT, 2SO4 to obtain a histone-enriched extract. The extracted proteins were precipitated with 20% trichloroacetic acid and then washed 3 times with acetone, air-dried, and re-suspended in urea 3 M. Laemmli buffer was added to the samples and boiled for 10 min to analyze by WB.Nuclei from 1 g of 7 day-old seedlings were extracted and treated overnight with 0.4 N H25, 1:2000), and anti-ubiquitin ; horseradish peroxidase-conjugated goat anti-rabbit  and rabbit anti-mouse (Sigma A9044) were used as secondary antibodies at 1/10,000 and 1/20,000 dilutions, respectively. Chemiluminescence detection was performed with ECL Prime Western Blotting Detection Reagent  following the manufacturer’s instructions.Proteins were separated on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene difluoride membrane  by semi-dry blotting in 25 mM Tris–HCl, 192 mM glycine, and 10% methanol. The following primary antibodies were used: anti-HTA9 , anti-H4 , anti-H3K27me3  anti-AtH2AK121ub . The TruSeq RNA Sample Prep Kit v2 Illumina was used for library preparation following the manufacturer’s recommendations. Sequencing of RNA libraries was carried out with the Illumina HiSeq 2500 sequencer, yielding an average of approximately 35–40 million 100-nucleotide-long paired-end reads for each sample. The high quality of each sample was verified using the software package FASTQC. Read mapping to the A. thaliana TAIR10 reference genome and transcript assembly were performed with the software tools HISAT2 and StringTie51. The number of genes scored as present in at least one of our samples was 23,486, representing around 70% of Arabidopsis nuclear genes. DEGs were selected using the Bioconductor R packages Ballgown51 and LIMMA52. Gene expression was measured in FPKM (fragments per kilobase of exon and million mapped reads). DEGs were selected according to a log2-fold change cut-off >|1| in the different comparisons and a p value <0.05 according to a moderated t test.RNA-seq was performed in two biological replicates for WT, 31. For H2A.Z localization in WT seedlings at 10 DAG, we used previously published data (accession number GSE96834)32.ChIP-seq data for the localization of H3K27me3 and H2AK121ub in WT seedlings at 7 DAG were generated and analyzed previously (accession number GSE89358)53 and peak calling was carried out with the software package MACS254. All nuclear genes were included in the analysis. Metagene plots were generated with a custom R script based on the R Bioconductor package ChIPeakAnno55 and A thaliana genome database R package, TxDb.Athaliana.Biomart.plantsmart2856. Processing of the BAM files were performed using the software package BEDTools57 to obtain RPKM (reads per kilo base per million mapped reads) normalized data.Read mapping to the TAIR10 reference genome was performed using bowtieACTIN7 or Histone H4. qPCR data are shown as the means of two to four biological replicates. Primers used for ChIP-qPCR are listed in Supplementary Table ChIP experiments were performed on 1 g of 7-day-old whole seedlings fixed in 1% Formaldehyde. Chromatin was extracted from fixed tissue and fragmented using a Bioruptor® Pico (Diagenode) in fragments of 200–500 bp. The sheared chromatin was immunoprecipitated overnight using the following antibodies and dilutions: Anti-H3K27me3 , anti-HTA9  or anti-H4 . Immunocomplexes were captures using Protein A Sepharose beads CL-4B . After washing the Protein-A beads, chromatin was eluted and the crosslinking was reversed overnight at 65 °C. The DNA from the immunoprecipitated chromatin was treated with RNase and proteinase K and purified by phenol–chloroform extraction followed by ethanol precipitation. For ChIP-qPCR, amplification was performed using the SensiFAST™ SYBR® & Fluorescein Kit (Bioline) and iQ5 Biorad system. Results are given as the percentage of input normalized to Further information on research design is available in the Supplementary informationPeer ReviewReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Source Data


In vivo investigations of the direct impact of intermittent hypoxia are frequently hampered by multiple co-morbidities of patients. Therefore, cell culture experiments are important model systems to elucidate molecular mechanisms that are involved in the cellular response to alternating oxygen conditions and could represent future targets for tailored therapies. In this study, we focused on mouse lung endothelial cells as a first frontier to encounter altered oxygen due to disturbances in airway or lung function, that play an important role in the development of secondary diseases like vascular disease and pulmonary hypertension. We analyzed key markers for endothelial function including cell adhesion molecules, molecules involved in regulation of fibrinolysis, hemostasis, redox balance, and regulators of gene expression like miRNAs. Results show that short-time exposure to intermittent hypoxia has little impact on vitality and health of cells. At early timepoints and up to 24 h, many endothelial markers are unchanged in their expression and some indicators of injury are even downregulated. However, in the long-term, multiple signaling pathways are activated, that ultimately result in cellular inflammation, oxidative stress, and apoptosis.Intermittent hypoxia is a major factor in clinical conditions like the obstructive sleep apnea syndrome or the cyclic recruitment and derecruitment of atelectasis in acute respiratory distress syndrome and positive pressure mechanical ventilation.  Special issues of the pulmonary vascular bed are the maintenance of a low vascular resistance, despite the exposure to mechanical stress, and to the highest O2 tension in the body  of approximately 40 mmHg, derived from the right ventricle passes the precapillary pulmonary circulation and blood with a pO2 of approximately 100 mmHg in the postcapillary segment returns to the left atrium of the heart. The ultrastructure of endothelial cells in the different segments varies according to the respective function. Under hypoxic conditions, the pulmonary vasculature undergoes a meanwhile broadly investigated hypoxia-induced remodeling including vasoconstriction that might lead in its chronic form to pulmonary hypertension , but is also encountered in cyclic recruitment and derecruitment of atelectasis (CA) during positive pressure mechanical ventilation. OSAS is r system .In our study, we aimed to analyze cell responses of endothelial cells from mouse lung to short-term and chronic intermittent hypoxia with regard to cell growth, gene expression of key endothelial markers, redox systems, and regulatory molecules (miRNAs). We show that depending on exposure time, intermittent hypoxia can have both, protective and injurious effects on lung endothelial cells.Isolation of endothelial cells from mouse lungs was performed after animals have been humanely killed according to the current legislation . Permission for these experiments was granted by the local animal ethics committee at Medical University Vienna and the Federal Ministry for Science, Research and Economy of Austria (GZ: BMWFW-66.009/0089-WF/v/3b/2016).Mouse primary lung endothelial cells were isolated from enzymatically digested lung tissue from adult C57B/L6 mice by magnetic separation, according to the method described in 2 conditions supplied by premixed gas bottles: (1) 21% O2 – 5% CO2 – 74% N2; (2) 0% O2 – 5% CO2 – 95% N2; (3) 10% O2 – 5% CO2 – 85% N2; (4) 0–21% O2 oscillations – 5% CO2 – rest N2 with a frequency of six oscillations per hour . Equal amounts of proteins as analyzed by BCA protein assay  were loaded on an 8, 10, or 12% SDS-PAGE gel. After separation, proteins were semidry-blotted onto nitrocellulose membranes. Specific bands were detected with the following antibodies: anti-ICAM1 ; anti-VCAM1 ; anti-p65NFκB ; anti-SOD2 ; anti-TXN1 ; anti-HMGB1 ; and anti-ß-actin . For analysis of phosphorylated ERK1/2 cell lysates were prepared in the presence of a protease and phosphatase inhibitor mini tablet . The primary antibodies were as follows: anti-phospho ERK1/2  and anti-ERK1/2 . After incubation with primary antibodies overnight at 4°C, the washed membranes were incubated with the respective HRP-coupled secondary antibodies for 45 min at room temperature. The chemiluminescence reaction was developed with Westernbright Sirius reagent  and analyzed on a Vilber Lourmat  imaging system.For miRNA expression profiling we used the miScript miRNA PCR Array Mouse miFinder . For information on the full list of tested miRNAs, see the Supplementary Figure 2) were expressed as “fold change” and were analyzed by the comparative Ct (ΔΔCt) method.Changes in gene expressions under different exposure conditions  in DMSO (5.7 mg/ml stock solution) at a final concentration of 0.57 mg/ml and incubated at room temperature for 1 h on a rotator. Dishes were coated with biotinylated gelatin at a concentration of 0.25 mg/ml (50 μl/well) overnight at 4°C. Plates were washed two times with 200 μl PBS and seeded with 3 × 104 cells/well in full serum-containing M199 medium. Cells were grown in a normal incubator 72 h prior to gas exposure. After 4, 24, and 72 h of exposure to gas conditions as described above, FITC-avidin (Thermo Fisher Scientific cat.nr. 434411) was added to the culture medium at a final concentration of 25 μg/ml for 3 min. Unbound FITC-avidin was washed out with 2 × 200 μl PBS. Finally, 100 μl PBS were added and fluorescence was measured on a Victor3 Plate reader  using Ex = 485 nm and Em = 535 nm; 0.1 s, at the bottom.We used a method described by n = 6 replicates and were repeated three times, each with a new cell preparation. Exceptions are oxidative stress and miRNA arrays as well as Western blot experiments with n = 3. Mean values in the text are given as mean ± standard deviation (SD). We used SPSS® software (SPSS Version 24) for statistical analysis. Statistic tests for comparison of multiple groups , moderate/intermediate hypoxia , anoxia , intermittent hypoxia/O2 oscillations  at a frequency of six cycles per hour. At different timepoints, we evaluated cell growth, cytokine release, gene and protein expression, and monolayer permeability. For permeability assays, cells were plated in 96-well plates as described in the Materials and Methods section and exposed to the same gas conditions.Primary mouse lung endothelial cells were isolated, grown for 2 weeks and repurified by magnetic separation in order to prevent contamination with other cell types. Cells were plated at a density of 1–2 × 103 under normoxia, 90.4 ± 13.9 × 103 under moderate hypoxia, 89.3 ± 15.6 × 103 under anoxia, and 89.5 ± 9.5 × 103 under intermittent hypoxia. At 24 h, cell numbers were 85.4 ± 9.0 × 103 under normoxia, 80.0 ± 12.7 × 103 under moderate hypoxia, 45.6 ± 15.1 × 103 under anoxia, and 71.8 ± 7.6 × 103 under intermittent hypoxia indicating a loss of 47% for anoxia compared to normoxia (p < 0.0001). At 72 h, cell numbers were 91.0 ± 5.6 × 103 under normoxia, 101.0 ± 9.8 × 103 under moderate hypoxia, 8.0 ± 9.8 × 103 under anoxia, and 50.5 ± 6.5 × 103 under intermittent hypoxia indicating a gross loss of living cells under anoxia and a reduction by 45% under intermittent hypoxia (p < 0.001 versus normoxia). Figure As a measure for living cell mass, we used trypan blue staining and counted white cells/well in a Neubauer Chamber Figure . After 42 conditions . Normalized MIP2 levels after 24 h were 125 ± 139 pg under normoxia, 128 ± 78 pg under moderate hypoxia, 71 ± 34 pg under anoxia, and 58 ± 8 pg under intermittent hypoxia. After 72 h, MIP2 levels had increased to 229 ± 135 pg under normoxia, 246 ± 87 pg under moderate hypoxia, 1907 ± 377 pg under anoxia , but only 269 ± 34 pg under intermittent hypoxia.We measured the release of the chemokines/cytokines (C-X-C motif) ligand 1 , macrophage inflammatory protein 2 (MIP2), and interleukin 6 (IL6) into the culture supernatants under all four different Os Figure . Normalip = 0.043).After 24 h, IL6 levels were 72 ± 19 pg under normoxia, 79 ± 21 pg under moderate hypoxia, 58 ± 24 pg under anoxia, and 84 ± 14 pg under intermittent hypoxia. After 72 h, IL-6 levels increased to 176 ± 28 pg under normoxia, 313 ± 171 pg under moderate hypoxia, 749 ± 493 pg under anoxia, and 447 ± 72 pg under intermittent hypoxia. Therefore, IL6 levels were increased by 78% under moderate hypoxia, 325% with high variation (due to the low number of remaining cells) under anoxia, and 154% under intermittent hypoxia compared to normoxia. The value of intermittent hypoxia reached statistical significance (p < 0.01) and 0.5 ± 0.08 under intermittent hypoxia. Fold change after 24 h was 0.8 ± 0.1 under moderate hypoxia, +9.4 ± 4.2 under anoxia (p < 0.0001) and 0.9 ± 0.2 under intermittent hypoxia. After 72 h, VEGF fold regulation compared to normoxia was +1.4 ± 0.1 under moderate hypoxia, +9.8 ± 4.1 (p < 0.0001) under anoxia, and +1.61 ± 0.1-fold under intermittent hypoxia.We further assessed VEGF and TNFα expression as cellular mRNA levels by qRT-PCR, as secretion into the cell culture supernatant was not detectable under basal conditions Figure . Using np < 0.0001), +17.5 ± 10 with high variation under anoxia (p < 0.0001), and +2.2 ± 1.0 under intermittent hypoxia.TNFα fold regulation after 4 h was +1.4 ± 0.5 under moderate hypoxia, 0.8 ± 0.7 under anoxia, and 0.7 ± 0.3 under intermittent hypoxia. At 24 h fold change was 0.9 ± 0.4 under moderate hypoxia, 0.6 ± 0.3 under anoxia, and +1.2 ± 0.8 under intermittent hypoxia. After 72 h, TNFα fold regulation compared to normoxia was +11.3 ± 2.2 under moderate hypoxia (p < 0.0001). Similarly, VCAM1 had a slight downward trend at 4 h under intermittent hypoxia, was significantly down regulated after 24 h of anoxia  and unchanged under intermittent hypoxia, was still downregulated at 72 h of anoxia , but was to be upregulated under intermittent hypoxia after 72 h . p-Selectin expression slightly increased at 4 h under moderate hypoxia , was especially low under severe hypoxia 0.3 ± 0.04-fold expression after 24 h (p < 0.001) and 0.2 ± 0.8-fold expression (p < 0.0001) after 72-h compared to normoxia, but its level was not or only slightly changed under intermittent hypoxia at all time points. NOS3 expression was statistically unchanged after 4 and 24 h under moderate and intermittent hypoxia. A strong effect was observed under severe hypoxia, with upregulation at 4 h  and downregulation at 72 h . Intermittent hypoxia induced a down regulation or trend at 4 h intermittent hypoxia for uPA, tPA, and PAI  and a slight upregulation of uPA , tPA , and PAI  after 72 h intermittent hypoxia. Endothelial activation results in the release of the Weibel-Palade bodies, which are storage granules of endothelial cells containing as major components p-selectin, vWF, and angiopoietin-2 (ANGPT2). vWF mRNA was downregulated after 4 h intermittent hypoxia  and slightly upregulated after 24 h  and after 72 h of intermittent hypoxia . ANGPT2 was upregulated under severe hypoxia , but with a trend downregulated after 4 h , 24 h , and with a trend upregulated after 72 h of intermittent hypoxia . The inflammatory transcription factor NFκB subunit p65 showed no change at 4 h of all conditions, showed a trend toward upregulation under all hypoxic conditions at 24 and 72 h with high variation, but the strongest and statistically significant increase was observed with 72 h of intermittent hypoxia . The cellular stress protein heme oxygenase-1 (HMOX1) was strongly upregulated under severe hypoxia . Intermittent hypoxia induced a trend to down-regulation of HMOX1 after 4 h , 24 h , and no change after 72 h. Receptor for advanced glycation endproducts (RAGE) had a downward trend at 4 h intermittent hypoxia , was upregulated after 24 h under anoxia , and had also a slight upward trend after 24 and 72 h under intermittent hypoxia. Finally, the renin-angiotensin-system (RAS) representative angiotensin-converting enzyme 1 (ACE1) did not show any major changes apart from anoxia, where it was upregulated .We analyzed relative gene transcript levels compared to the normoxic condition after 4, 24, and 72 h for cell adhesion molecules involved in the inflammatory response, members of the fibrinolytic system, stress response genes, and other markers for lung endothelial function Figure . While Ip = 0.03), the superoxide dismutases 2 and 3 were reduced by 5.9 and 5.5-fold (both p < 0.001), respectively, thioredoxin 1 was reduced by 7.3-fold (p = 0.012), and the related thioredoxin reductase 3 was even reduced by 9.1-fold (p = 0.01) , mmu-miR-322-5p was reduced by 36.5-fold (p = 0.048) and mmu-miR-218-5p was reduced by 19.8-fold (p = 0.004) compared to normoxia, while no significant change was observed under moderate hypoxia for these miRNAs  and intermittent hypoxia (0–21% O2) was reflected by an increase of the phosphorylation state of (Thr202/Tyr204). Phosphorylated ERK  increased threefold to a plateau level at 24 h under sustained hypoxia, while intermittent hypoxia continuously increased phosphorylated ERK until 72 h to reach approximately ninefold of baseline levels.Activation of the MAPkinase ERK1/2 by sustained moderate hypoxia (10% Op < 0.05). At 24 h the permeability had markedly increased under severe hypoxia . At 54 h the permeability had further increased under severe hypoxia and also reached a significant level under intermittent hypoxia .Confluent cultures of mouse lung endothelial cells were exposed to normoxia, moderate hypoxia, anoxia and intermittent hypoxia and after 4, 24, and 54 h the accessibility of the coated gelatine for a fluorophore-labeled ligand given to the medium was assessed by reading the emitted fluorescent light from the bottom of the plate  exposure to moderate frequency intermittent hypoxia is seemingly well tolerated without significant loss in vitality or major occurrence of indicators for injury. On the contrary, chronic exposure to IH elaborates multiple ways of inflammatory and injurious processes, every single one of those being capable of inducing cellular damage.Cell growth and secreted factors represent the overall outcome parameter for the sum of molecular events: While 24 h of intermittent hypoxia is depicted by normal cell growth and absence of significant increase in inflammatory cytokines, 72 h of intermittent hypoxia reveals the detrimental effects: cell numbers are reduced by around 45% and a significant release of inflammatory cytokines like IL6 and CXCL1 is detected. Further markers of inflammatory processes are the increase of cell adhesion molecules ICAM1 and VCAM1 after 72 h. Again, these markers are not elevated after 24 h, but on the contrary are even down-regulated at earlier timepoints. Components of the fibrinolytic system uPA, tPA, and PAI-1 are changed after 72 h of intermittent hypoxia, resulting in an upregulation of uPA and tPA and PAI1. Plasminogen activators might have beneficial effects on fibrin deposition and associated inflammation, but on the other side tPA knockout mice have revealed especially positive effects with regard to decreased vascular permeability and improved lung function including pulmonary artery pressure, airway resistance, and pulmonary compliance .An overactive renin-angiotensin-system (RAS) in the lung can boost the inflammatory factors ICAM, VCAM, TNFα, and IL6 and can have profibrotic effects . In our in vitro  RAGE that becomes activated due to tissue damage and endothelial activation . RAGE hain vitro . After 4in vitro . Under cin vitro . RAGE acFinally, the cellular stress protein HMOX1 is already especially upregulated under severe hypoxia at early timepoints. HMOX1 is frequently regarded as a target for treatment of inflammatory conditions, as this enzyme degrades heme to biologically active end products that affect apoptosis and inflammation . We obseS-glutathionylation of proteins (frequently under conditions of oxidative stress), thus ultimately leading to functional changes. One important target is IKKβ, a kinase involved in the activation of the proinflammatory NFκB pathway. Sufficient catalytic activity of Gstp1 is vital for repressing NFκB. Downregulation of Gstp1 leads to reduced S-glutathionylated IKKβ and an increased nuclear translocation of NFκB with a concomitant increase of inflammatory cytokines  and enhanced protein synthesis. Calcium-dependent kinases (CaMKs) additionally phosphorylate the co-activator p300 in order to enhance HIF1α transcriptional activity . Barrier function was assessed by transendothelial electrical resistance (TEER) measurements as opposed to transmigration of a labeled avidin molecule in our assay. These factors might explain differences in the time onset of barrier dysfunction.Monolayer permeability as measured with our functional assay will be a surrogate of many factors including subtle intracellular changes up to major events like cell death. The effect of intermittent hypoxia on different organ systems has been observed in several animal experiments, sometimes with contradictory outcome. The available literature, however, draws its conclusions from diverse model systems especially with regard to intermittent hypoxia regimen. Generally, it can be assumed, that lower frequency and mild hypoxia as well as acute incidences more likely lead to adaptation of the organism and beneficial outcomes than high frequency oscillations with severe hypoxia and chronic exposure . The ben2). A major limitation of our study is the use of mixed lung endothelial cell types, a draw-back, that cannot be easily circumvented, as not special cell markers are known, that can distinguish between different phenotypes of endothelial cells in the lung. Also, cell culture media components might influence the expression of some endothelial markers, as for example described for heparin, ECGF and PAI expression -proteomic, epigenetic, and systems biology methodology are still desirable goals.PW, KM, VT, and KK designed the study. PW, LS-G, and VT performed the study. MW and IL provided the expertise in cell culture and lung endothelium. PW, VT, TB, and KK analyzed the data. VT and KK wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.