The eukaryotic genome is functionally organized into domains of transcriptionally active euchromatin and domains of highly compact transcriptionally silent heterochromatin. Heterochromatin is constitutively assembled at repetitive elements that include the telomeres and centromeres. The histone code model proposes that HP1α forms and maintains these domains of heterochromatin through the interaction of its chromodomain with trimethylated lysine 9 of histone 3, although this interaction is not the sole determinant. We show here that the unstructured hinge domain, necessary for the targeting of HP1α to constitutive heterochromatin, recognizes parallel G-quadruplex (G4) assemblies formed by the TElomeric Repeat-containing RNA (TERRA) transcribed from the telomere. This provides a mechanism by which TERRA can lead to the enrichment of HP1α at telomeres to maintain heterochromatin. Furthermore, we show that HP1α binds with a faster association rate to DNA G4s of parallel topology compared to antiparallel G4s that bind slowly or not at all. Such G4–DNAs are found in the regulatory regions of several oncogenes. This implicates specific non-canonical nucleic acid structures as determinants of HP1α function and thus RNA and DNA G4s need to be considered as contributors to chromatin domain organization and the epigenome. Within the confines of the nucleus, genomic DNA is packaged with histone proteins to create highly folded yet dynamic chromatin fibres. At the most basic level DNA is wrapped 1.67 times around an octamer of four core histones to form a nucleosome ,3.Members of the Heterochromatin Protein 1 (HP1) family are essential architectural proteins that establish and maintain heterochromatin ,4,5. MamIn vitro the hinge domain binds nucleic acids with a preference for RNA, within this hinge region three basic residues (104–106) are required as their mutation to alanine disrupts this interaction with RNA  . Althougwith RNA ,16. The with RNA . Notablywith RNA . The inthromatin . This raeraction ,16.n terminating in a 3′ overhang  that form in the complementary C-rich strands, exist in vivo and contribute to genome function  by heating to 90°C for 5 min, cooling gradually to room temperature, while i-motifs and double-stranded DNA were annealed in 1× Interaction Buffer 2 , by heating to 90°C for 5 min and gradually cooling to room temperature. Experiments with folded G4 oligonucleotides were performed with 1× IB1 while experiments with folded i-motif oligonucleotides and double stranded DNA used 1× IB2.G4-forming oligonucleotides were annealed in 1× Interaction Buffer 1  and expression induced with 0.4 mM IPTG . After lysis, hexahistidine-HP1 paralogs were purified using the AKTA Prime Plus  chromatography system and a Ni-NTA IMAC column by elution with 500 mM imidazole, concentrated using Vivaspin 20 (5 kDa MWCO) ultrafiltration devices  before passing through a Superdex 75 10/300 GL  size exclusion chromatography column, and finally re-concentrated as performed previously.For protein expression, the hexahistidine-HP1 expression vectors were transformed into 2PO4/ Na2HPO4, pH 8, or pH 7 for i-motifs) at 4°C. The gel was rinsed with H2O, stained with 0.35% Stains-All (Merck) in 50% formamide for 15 min, de-stained in H2O, and then imaged.Folded oligonucleotides (0.15 nmols) were separated on a 20% polyacrylamide gel in 0.5× TBE/0.5× IB1  mixed with 0.3 nmols TERRA45 in 1× IB1 were separated on a 5% polyacrylamide gel in 0.5× TBE/0.5× IB1 at 4°C. The gel was then stained with QC Colloidal Coomassie Stain  for 18 h at room temperature, de-stained with HBinding reactions were performed in a final volume of 20 μl using 10 pmol of 3′ TAMRA-labeled TERRA45  spectra were recorded using a Chirascan instrument .Three scans were gathered over wavelengths ranging from 200 to 350 nm in a 0.1-cm path length cell at the standard sensitivity, data pitch 1 nm, continuous scanning mode, response 0.25 s, and bandwidth 1 nm. 1× IB was scanned as a buffer sample and its spectrum subtracted from the average of three scans for each sample. Each CD spectrum was smoothed by averaging 10 neighbor points using software provided by Applied Photophysics Ltd.Biolayer interferometry (BLI) using a BLItz system  was used to examine the binding of HP1 to a range of oligonucleotides as indicated at room temperature.KD calculations.Ni-NTA biosensors (ForteBio) were hydrated with 1× IB, and 4 μl of 100 μg/ml his-tagged HP1α, HP1α 3K-A, HP1β or HP1γ was used to load the Ni-NTA biosensor for 5 min to reach ∼4 nm of signal. The biosensor was then washed with 1× IB. The association step was performed either using 500 nM RNA or DNA oligonucleotides prepared in 1× IB or just 1× IB (reference) for 5 min, then the dissociation step was performed using 1× IB for 5 min. Reference runs were subtracted from test runs to account for dissociation of protein. All oligonucleotides were tested for interaction with Ni-NTA biosensor tips prior to experiments. BLItz Pro 1.2 software was used for curve fitting and KD were obtained by titrating five or more concentrations of oligonucleotide against HP1α.For TERRA96, TERRA45, TERRA22, c-myc2, src2, and src16, values of 2.NIH3T3 cells  were routinely cultured in Dulbecco's Modified Eagle Medium (DMEM)  supplemented with 10% calf serum and 1% penicillin/streptomycin  at 37°C and 5% CO2, Roche cOmplete protease inhibitor), before permeabilization in 0.5% Triton X-100/CSK buffer for 5 min. Cells were washed again with CSK buffer, then incubated for 15 min at room temperature with 2.5 μM of the indicated oligonucleotides in CSK buffer containing 1 U/μl SUPERaseIn RNase Inhibitor (ThermoFisher Scientific). After further washing with CSK buffer, cells were fixed with 2% paraformaldehyde/1× DPBS and processed for indirect immunofluorescence using an HP1α antibody  and anti-rabbit Alexa 647 secondary antibody . Cells were then mounted in Slowfade Diamond Antifade Mountant with DAPI (ThermoFisher Scientific) on a microscope slide and imaged using a Leica SP5 DM6000B Scanning Confocal Microscope equipped with 63×/1.40 objective lens. Probes were excited with 405 and 633 nm excitation lasers, running LAS X software . All samples were imaged under identical conditions and digitally processed for presentation with Affinity Designer v1.6.1 .Asynchronously growing mouse NIH3T3 fibroblasts were washed with 1× Dulbecco’s Phosphate-Buffered Saline , then CSK buffer , 45 nucleotides (TERRA45) or 22 nucleotides (TERRA22) from the telomeric RNA UUAGGG repeat sequence (see in vitro findings (KD) of 2.5 nM was determined for the interaction between HP1α and TERRA96  was performed where purified his-tagged HP1α was immobilized on nickel sensor tips and the tips then immersed in a Kence see  in agreefindings , nor wasfindings . Using B96 Table , while tly Table .+ solution containing TERRA96 . With tTo test if the binding of HP1α to TERRA requires formation of the G4 structure, HP1α was immobilized on nickel sensor tips and then immersed in solutions of either TERRA45 or mutTERRA45, a 45-mer RNA in which four central GGG-tracts of TERRA45 were replaced with GUG . These sSince TERRA as well as telomeric DNA can readily form G4 species, to determine if the specificity of the HP1α interaction is dependent on the nucleic acid forming a G4 assembly, gTEL45, a 45-nucleotide G-rich telomere repeat DNA oligonucleotide was tested for its interaction with HP1α Figure . HoweverKD among Oligos A-G  and did not bind to HP1α. In this regard, Oligo G behaves in a similar manner to gTEL45. Unstructured single-stranded Thy45, consisting of 45 thymidines, also did not interact with HP1α. Therefore, DNA topology and the length of loops in G4 assemblies are key in determining the interaction of HP1α with G4-forming single-stranded nucleic acids.To further check if it is G4 topology that determines the interaction between HP1α and G4s, we tested several DNA oligonucleotides derived from the 45-mer telomeric repeat in which the length of loop regions varied but the number of G-tracts (eight GGG motifs) was the same . In comparison, G4-forming sequences from the SRC coding region that have features of an anti-parallel topology  with HP1α in comparison with antiparallel G4s that bind very slowly or not at all. Therefore, from the kinetic perspective there is a clear preference for a specific G4 topology. Another non-canonical quadruplex structure, the i-motif, features diagonal loops and thus resembles anti-parallel rather than parallel G4 topology. This suggests that the loop orientation in the G4 structure is vital for its interaction with HP1α. Also required for this interaction is the positive charge patch within the hinge region of HP1α, presumably due to electrostatic interactions with the negative loops of G4s. Long loops in G4s contribute to slower koff from HP1α as evident from the series of Oligos A-F. In addition, mutation of the three lysines in the hinge not only disrupts HP1α/G4 interactions but also increases the migration of the HP1α mutant during native PAGE relative to wild-type HP1α. This suggests that the mutation alters not only the charge but also the flexibility of the hinge to prevent the interaction with G4s. Presumably, the hinge becomes more structured upon binding the parallel G4 assembly and it will be of interest to determine how this interaction affects the behavior of the structured chromodomain and chromoshadow domains within chromatin.The main topological feature that differentiates parallel and anti-parallel G4 species is the position of loops that connect adjacent and opposite DNA or RNA strands in the structure ,54. In pKD values of TERRA96 and TERRA45 with HP1α, being 2.5 and 74.4 nM, respectively. Since kon values are similar, the difference in KD lies in the very different koff values . Unlike the BLI binding profile of TERRA45/96, the association of TERRA22 with HP1α is slow, and its release from the protein is faster than for TERRA45, which contributed to a high KD value of 940 nM for TERRA22. Consistent with previously reported mass-spectrometry results (KD values. Such a situation can be realized in vivo as HP1α is known to form higher order oligomers in a manner that is central to the formation and regulation of heterochromatin (There is a significant difference in  results , TERRA22 results ,55 and b results . In TERR results . Such lohromatin ,62.1 of the cell cycle would ensure an enrichment of HP1α at the telomere (in situ competition assay, where the localization of HP1α within heterochromatin foci was disrupted by the presence of parallel G4s. An analogous mechanism appears to maintain pericentromeric heterochromatin where the RNA transcribed from major satellite repeat DNA mediates targeting of HP1α to this domain (The high affinity of HP1α for the G4 topology formed by TERRA96 provides a mechanism by which the non-coding transcript can promote heterochromatin formation at telomeres Figure . The acctelomere , promotitelomere . That G4s domain . Althougs domain , the seckon values observed here for the interaction of HP1α with G4s of various topologies indicate that HP1α would preferentially interact with G4s formed during cell cycle progression of parallel rather than antiparallel topology (in vivo (To date, the formation of G4 species within genomic DNA has been associated with regions of euchromatin suggesting that heterochromatin suppresses G4 formation . In humatopology . The dem(in vivo ,67–70. T(in vivo .in situ. Therefore, these G4 assemblies whether present in non-coding RNA or genomic DNA may serve as signposts for HP1α-mediated functions. These findings also provide further evidence that a ‘G4 genome’ plays a role in the regulation of the functional organization of the genome (In conclusion, our findings show that the HP1α paralog, a major determinant of constitutive heterochromatin, has a faster association rate with G4 species folded into a parallel rather than anti-parallel topology. Notably, an excess of these G4 assemblies can compete HP1α from heterochromatin e genome .The authors declare that the main data supporting the findings of this study are available within the article and its Extended data files. Extra data are available from the corresponding author upon request.gkz1138_Supplemental_FileClick here for additional data file.


However, they have not been investigated during LDLT. Acute respiratory distress syndrome (ARDS) after living-donor liver transplantation (LDLT) is not uncommon, but it lacks the biomarkers for early detection. Club cell protein 16 (CC16), high-motility group box 1 protein (HMGB1), interleukin-1β, and IL-10 levels were measured at the pretransplant state, 30 minutes after reperfusion, postoperative day 1 (POD1), and POD3. ARDS was diagnosed according to the 2012 Berlin definition. Seventy-three consecutive recipients undergoing LDLT were enrolled and received the same perioperative care plan. Perioperative serum CC16, HMGB1, IL-1β, and IL-10 were not associated with ARDS after LDLT. Of the 73 recipients, 13 developed ARDS with significantly longer durations of mechanical ventilation and intensive care unit stay. Serum CC16 levels on POD1 increased significantly from the pretransplant state in the ARDS group but not in the non-ARDS group. Pretransplant serum CC16 levels were also higher in the ARDS group. The area under the receiver operating characteristic curves for POD1 serum CC16 levels used to discriminate ARDS was 0.803 . By comparison, HMGB1, IL-1NCT01936545, 27 August 2013. The higher pretransplant serum CC16 level and its increased level on POD1 were associated with the development of early ARDS after LDLT. This trial is registered with  Early acute respiratory distress syndrome (ARDS) in the first 72 hours after living-donor liver transplantation (LDLT) is not uncommon , 2 becauClub cell protein 16 (CC16) is a lung secretory protein  with antβ (IL-1β) : 22.9 ± 10.4 vs. 15.5 ± 8.7 ng.mL−1; T2 [POD1]: 27.5 ± 9.6 vs. 16.3 ± 9.4 ng.mL−1; T3 [POD3]: 23.2 ± 11.4 vs. 14.2 ± 10.5 ng.mL−1; T4 [POD3]: 14.2 ± 10.5 vs. 23.2 ± 11.4 ng.mL−1; all p < 0.05; ANOVA results showed that serum CC16 levels were significantly different between patients in the ARDS group and those in the non-ARDS group , the non-ARDS group had a significant increase in IL-10  values. By contrast, there was no significant serum IL-1β change in both the ARDS and non-ARDS groups  in recipients with ARDS is similar to those previously reported in ARDS patients [We found that only patients in the ARDS group had increased serum CC16 levels early in the morning of POD1. Because LDLT is associated with a significant amount of intraoperative bleeding and transfusion, particularly when red blood cells and plasma are transfused, it leads to liver ischemia-reperfusion injury, which increases the levels of multiple inflammatory mediators that become active in the lungs , 28. SubκB , 30 may patients . In ARDSpatients . Our resIn addition to the postoperative increase in serum CC16, we found that pretransplant serum CC16 levels were higher in the ARDS group than in the non-ARDS group. Shah et al. suggested that pretransplant CC16 levels are associated with preexisting epithelial injury before LT, which increases the likelihood of postoperative graft failure . Our resA transient increase in IL-10 after hepatic reperfusion was observed in our patients, which is compatible with the finding of another study  that repβ was associated with the early ARDS after LDLT in our patients. However, an increasing trend of HMGB1 after hepatic reperfusion was observed, which was not identified in IL-1β. This is compatible with the findings of previous studies that have reported that HMGB1 is associated with liver ischemia-reperfusion injury in an experimental model [Although studies have reported that HMGB1 is a pertinent biomarker in the development of lung injury in experimental models , 39 as wal model  and is aal model . This reAlthough this is the first study to report the possible role of serum CC16 levels in ARDS after LDLT, it is limited by its modest sample size. Large-scale validation is necessary to identify more potential variables.In conclusion, our study showed that serum CC16 levels increased early in the morning of POD1 in recipients who developed ARDS but not in those without ARDS. Moreover, higher pretransplant serum CC16 levels were noted in recipients who developed ARDS, which correlated with the pretransplant severity of liver cirrhosis. Therefore, monitoring serum CC16 may provide information for predicting early ARDS after LDLT.


Mph1 kinase phosphorylates the kinetochore component KNL1Spc105/Spc7 on conserved MELT motifs to recruit Bub3-Bub1 complexes [Mph1 then phosphorylates Bub1, which strengthens its interaction with Mad1-Mad2 complexes to produce a signaling platform [Spc105/Spc7 can be bypassed; simply inducing heterodimers of Mps1Mph1 kinase and Bub1 is sufficient to trigger metaphase arrest that is dependent on Mad1, Mad2, and Mad3. We use this to dissect the domains of Bub1 necessary for arrest, highlighting the need for Bub1-CD1, which binds Mad1 [Mph1 kinase, enabling efficient generation of MCC complexes.The spindle checkpoint monitors kinetochore-microtubule interactions and generates a “wait anaphase” delay when any defects are apparent , 2, 3. Tomplexes , 5, 6 viomplexes . Mps1Mphplatform , 10. Theplatform , 11, 12.platform , 16, 17.nds Mad1 , and Bubnds Mad1 , 19. We   •Heterodimers of Mps1 and Bub1 generate robust spindle checkpoint arrest in yeasts•This arrest is independent of kinetochores but requires Bub1-CD1 and the Bub1-TPR•The Bub1-TPR is both necessary and sufficient for Mad3 interaction and recruitment•Recombinant fission yeast Bub1-TPR and Mad3 form a stable complex S. pombe Bub1-TPR domain is both necessary and sufficient to bind and recruit Mad3, enabling efficient generation of MCC complexes.Leontiou et al. demonstrate that heterodimers of Mps1 and Bub1 are sufficient to trigger checkpoint-dependent metaphase arrest in both budding and fission yeast; kinetochores and KNL1 can be bypassed. The  KNL1 is sufficient to generate spindle checkpoint arrest in budding yeast [Mps1 kinase and Spc7KNL1 kinetochore protein [C. elegans [Forced heterodimerization of Mps1 kinase and Spc105ng yeast . We rece protein . However elegans . It has  elegans ; it is t elegans , 10, 24  elegans , 26, 27.bub1-3A allele [Rapamycin can be used to force heterodimerization of two proteins in an inducible fashion. We fused FKBP12 to Bub1 and FKBP12-rapamycin binding (FRB) to Mps1 A and tesA allele ). bub1-3Spc105/Spc7 [spc105-15 allele, which abrogates kinetochore structure-function and is unable to recruit checkpoint proteins to kinetochores at its restrictive temperature [Mps1-Spc7KNL1 signaling scaffold is not important; arrests were generated independent of kinetochore, spindle pole, and nuclear envelope enrichment. spc105-15 mutation did abrogate nocodazole-induced checkpoint arrest, where unattached kinetochores generate the mitotic checkpoint complex (MCC). These budding yeast experiments lead to a model in which Mps1 kinase first phosphorylates Spc105 to produce a binding site for Bub3-Bub1 complexes and then phosphorylates Bub1 to recruit Mad1-2 complexes. Significant overexpression of Mps1 kinase has long been known to be sufficient to checkpoint-arrest yeast cells [GAL-MPS1 bypasses the need for Spc105 phosphorylation for either checkpoint activation or Bub1-Mad1 complex formation  array on chromosome 1. The Mph1 fusion lacks its N terminus, which would otherwise target the fusion protein to endogenous kinetochores  in SynCheck Mph1Mps1-Spc7KNL1-arrested cells but found that this Cut7 kinesin-dependent localization of Mad1 is not necessary for arrest [mad1-ΔCC cells in which no spindle pole localization . Log phase cells were washed in –thiamine medium to induce TetR-Mph1 expression and then grown overnight before cytological analysis and quantitation of their arrest. Mps1 induction, ∼60% of cells had arrested with short metaphase spindles, and this fraction rose to ∼80% after 16 h. This was true for the FL Bub1 fusion and the one lacking the Bub1 kinase domain, but not the other Bub1 constructs. This experiment demonstrates that Bub1 kinase activity is not necessary for Mph1-Bub1 SynCheck arrest and highlights the importance of the N-terminal TPR domain in addition to CD1. We note that, unlike other domains or motifs that are frequently lost or duplicated during MadBub specialization, the TPR domain is conserved in essentially all Bub1-, BubR1-, and Mad3-related proteins [Bub1 has undergone at least 16 independent occurrences of gene duplication, followed by sub-specialization . In humaproteins .Figure 3Mps1-Bub1 SynCheck, we used the bub1-CD1 mutant, which contains mutations in the conserved region of Bub1 that is phosphorylated and then binds Mad1 [bub1-CD1 mutation completely abolished mitotic arrest. We also analyzed complexes formed in Mph1-Bub1 SynCheck arrested cells by immunoprecipitating Bub1 and looking for associated Mad1. Phosphorylated Bub1 CD1 recruits Mad1 to kinetochores, and these proteins have been shown to bind directly in budding yeast reconstitution experiments . To testnds Mad1 . Figure KNL1 function. To confirm this in fission yeast, we carried out two further experiments. Bub3 targets Bub1 to kinetochores, but they only interact with Spc7 after it has been phosphorylated by Mph1 [KNL1 prevents this interaction with Bub3 complexes [spc7-12A cells. In addition, Bub1-Mph1 arrests in the complete absence of Bub3  TPR domains in vitro and found that, unlike the fission yeast domains, they do not form stable heterodimers . This is not surprising because the vertebrate Bub proteins have evolved a distinct heterodimerization domain [We also tested recombinant human (Bub1 and BubR1) and n domain , and alln domain . One hasn domain , 50, a pn domain . Dissectkevin.hardwick@ed.ac.uk).Further information and requests for resources and reagents, such as plasmids and yeast strains, should be directed to and will be fulfilled by the Lead Contact, Kevin Hardwick  were grown in LB broth and induced at 14°C overnight for expression of recombinant TPR proteins.Bub1-FKBP12 and Mps1-FRB strains were constructed by PCR-based integration of the tags using Euroscarf plasmids P30578 (pFA6A-FRB-KanMX6) and P30583 (pFA6A-2xFKBP12-HIS3MX6). Genomic integrations were verified by marker counter-selection or PCR-based analysis. 3xGFP and 3xFlag strains were constructed by PCR-based integration of tags at the endogenous locus , and subA BUB1 endogenous replacement construct was generated by inserting Bub1 sequences into a HIS3 integrating vector. First, an existing SPC105 integrating vector pSB1332  was muta600 of 0.2-0.5 then arrested by alpha-factor treatment (1 ug/mL) for 2-3 hours. G1 arrest was verified by microscopy. Arrested cells were pelleted and resuspended twice in media lacking alpha-factor. Cells were then washed into fresh media to start the time course and rapamycin or nocodazole was added. Media with 2% galactose was used for galactose induction, and 37° media was used to initiate temperature shifts during alpha-factor washout. Fresh alpha-factor was added to cells once rebudding was visible to ensure cells only cycled through mitosis once. At each time point, 1 mL of culture was briefly centrifuged to pellet the cells, which were then snap-frozen in liquid nitrogen. Pellets were resuspended in SDS sample buffer containing PMSF , lysed by bead beating, and analyzed by western blotting. anti-Pgk1 was purchased from Invitrogen and anti-myc 9E10 was purchased from Covance.Cells were cultured at ambient temperature to OD(303-678) was amplified from genomic DNA (strain from Silke Hauf) and inserted into tTetR-pHFF81C using Gateway recombination [The rTetR was amplified out from pAK2 , digested with NheI and AseI, and inserted into a pHFF81C vector (gift from Ken Sawin) digested with NheI and NdeI. Mph1bination .FL, Bub1Δkinase, Bub1TPR, Bub1N terminus, Bub1CD1mut and Bub1ΔTPR) were then inserted into this vector by Gateway recombination. Gateway cloning was performed using kits  obtained from Invitrogen in accordance with the manufacturer’s instructions.The pRAD15 (gift from Robin Allshire) was amplified using phosphorylated primers before DpnI digestion and re-ligation to re-create the vector with NheI and XhoI sites. These sites were then used to insert a PCR fragment NLS-rTetR-mCherry-FLAG-ccdB into pRAD15. Padh15-NLS-rTetR-mCherry-2xFLAG-ccdB was then amplified out and subsequently joined to a PCR fragment containing the pLYS1U backbone (gift from Jonathan Millar) using KpnI and XhoI to form pLYS1U-Padh15-NLS-rTetR-mCherry-2xFLAG-ccdB (backbone plasmids were constructed by Ivan Yuan ). Bub1 fpMad1ΔCC-hyg. To GFP tag the N terminus of full length Mad1 the endogenous promoter was replaced by Padh21 and GFP sequences inserted before 1kb of mad1WT coding sequence as described above, using Sal1 and EcoRV digested vector and Gibson assembly. The assembled sequences were amplified by PCR and transformed into fission yeast.To truncate Mad1 expressed from its endogenous promoter, 762bp containing the promoter region, 325bp of flanking sequence upstream of this and 566bp of mad1 coding sequence, excluding the first 500bp of mad1, were amplified from genomic DNA. The hygromycin resistance cassette was amplified from pFA6hphMX. The flanking sequence was digested with KpnI and Sal1 and cloned into pBluescript, then the resulting vector was digested with Sal1 and EcoRV and the remaining fragments were assembled by Gibson Assembly (NEB) in the following order; hygR, promoter then coding region, to give 303-678 was amplified from a pDONR 201 plasmid containing Mph1303-678. 3xHA was amplified from a plasmid from the Allshire lab containing codon optimized PYL-3xHA. ABI was amplified from a pMT_CID_ABI_VS_H vector from the Heun lab. These PCR fragments were Dpn1 treated and assembled into a Sma1-digested and antarctic phosphatase treated, gel purified pRad41 yeast expression vector by Gibson assembly.Mph1adh21-NLS-rTetR-mCherry-2xFLAG-Spc71-666  LIC vector. The vector was digested with SspI enzyme and both the vector and the PCR products were run on an agarose gel. The fragments were gel purified and the exonuclease reaction performed using T4 DNA polymerase. The PCR fragments were mixed with the vector and transformed in XL1 Blue E.coli cells according to standard protocol. Colonies were screened by PCR and then sequenced. Correct plasmids were then transformed into Arctic cells DE3 to induce expression of the proteins.Bub1cdc25-22 block, 250 mM ABA stock (Sigma Aldrich A1049) was added to cultures 5 min after release to achieve a final concentration of 250 μM (unless otherwise stated). Samples were collected every 15min and fixed with methanol.Cells were grown at 25°C for 1-2 days on YES  plates. They were then pre-cultured in 10 mL of liquid YES containing amino acid supplements at 25°C over the day and inoculated into a larger culture of YES overnight. The following day, log phase cultures were shifted to 36°C for 3.5 h to block in G2. After this, cultures were cooled quickly in iced water to rapidly shift them back to 25°C and release them from the G2 block. For the synthetic arrest assay following a For the synthetic arrest: fission yeast cells were first grown on YES (rich) plates at 32°C overnight. The following morning, cells were transferred to 10 mL of liquid PMG medium containing 15 μM thiamine and were incubated at 30°C with shaking. After seven hours, cells were harvested by spinning at 6,000 RPM for 2.5 min and washed 3 times with fresh PMG (containing supplements). The cells were transferred to fresh PMG (without thiamine) containing 10 μM anhydrotetracycline , and then incubated at 30oC for 12, 14, 16 and 18hrs. Depending on the experiment the next morning the cells were harvested by spinning at 6,000 RPM for 2.5 min. The cells were washed with 1ml of clear PMG (without glucose) and harvested by spinning at 6,000 RPM for 1 min. The supernatant was removed  and 6-10 μL of cells was deposited on a glass slide and covered with a glass coverslip.Fixed or live cells were imaged immediately using a 100x oil immersion lens and a Zeiss Axiovert 200M microscope (Carl Zeiss Ltd.), equipped with a CoolSnap CCD camera (Photometrics) and Slidebook 5.0 software . Typical acquisition settings: 300 ms exposure (FITC & TRITC), 2x binning, Z series over 3 mm range in 0.5 mm steps (7 planes).2, 1mM EGTA, 10% Glycerol, 0.1% Triton X-100, 1mM Na3VO4, 10 μg/mL CLAAPE , 1mM Pefabloc, 0.01mM microcystin). Approximately 1g of powder were resuspended in 1ml of lysis buffer. The cells were lysed by sonication . After sonication the samples were transferred in 1.5 mL tubes and the cell debris was pelleted using centrifugation . The lysate was then incubated with anti-GFP or anti-Flag-coupled Dynabeads (Invitrogen) for 15-20 minutes at 4°C. The beads were washed four times with wash buffer  and once with PBS+0.0001% Tween 20. The proteins were eluted from the beads by adding 2X sample buffer containing DTT. The samples were incubated with sample buffer at room temperature for 15 minutes, then run on an SDS-PAGE gel.Fission yeast cells were grown at 30°C overnight in 1.5 l of PMG. For synthetic arrest experiments, the mitotically arrested cells were harvested after 16hrs of Mph1 induction. The cells were harvested by centrifugation at 4000 rpm at 4°C, for 10 minutes (using a Beckman centrifuge). Pelleted cells were frozen into small sized drops using liquid nitrogen and immediately processed to lysis or stored at - 80°C until further use. The cells were ground manually using a mortar and pestle. Yeast powders were resuspended in lysis buffer containing 50mM HEPES pH7.6, 75mM KCl, 1mM MgCl600 of approximately 1 at ambient temperature, then induced with galactose addition. Cells were harvested after 2 hours and frozen in liquid nitrogen. Cells were lysed by bead beating or by freezer milling in Buffer H  containing protease inhibitors  and phosphatase inhibitors . Lysate was clarified by high-speed centrifugation for 30 min at 4°. Supernatant was collected and immunoprecipitated with anti-Flag  or anti-GFP (Living Colors) conjugated Protein G Dynabeads (Invitrogen) at 4° for 3 hours. Beads were then washed 5 times with lysis buffer (omitting inhibitors and DTT after the first three washes), then eluted with SDS sample buffer with 5% β-mercaptoethanol for immunoblot analysis.For budding yeast Co-IPs, cells were grown to ODS.pombe His-GFP-Mad3TPR and His-Bub1TPR were purified using immobilised ion metal affinity chromatography (IMAC). Lysates from 5g bacteria were incubated (agitation/rotation) for 30-60 min (at 4°C) with 5 mL of cobalt resin. The beads were pre-equilibrated in lysis buffer lacking protease inhibitors. After incubation, the beads were transferred to a Biorad column and washed with 20 column volumes of wash buffer. Proteins were eluted from the beads using the lysis buffer, without protein inhibitors, containing 250mM imidazole. Peak fractions were dialysed overnight .Cell pellets were thawed on ice and resuspended in lysis buffer containing 50mM Tris pH8.0, 500mM NaCl, 10% glycerol, 10mM imidazole, 5mM β-mercaptoethanol, EDTA-free inhibitor tablet , 1mM Pefabloc. The cells were lysed by sonication . To remove the cell debris, lysed cells were centrifuged at 20,000 rpm, for 30-45 min, at 4°C. Thereafter, the lysate was filtered through a 0.45 um syringe. Both After dialysis, the protein concentrations were measured using a NanoDrop. The proteins were concentrated using 10kDa cut off Vivaspin concentrators at 4500 rpm at 4°C. Samples were then injected (500 μL of concentrated sample) into a Superdex 200 increase 10/300  equilibrated with 50mM Tris pH 8.0, 150mM NaCl, 5% glycerol and 2mM DTT. The buffer was filtered and the gas was removed prior to use. Fractions were analyzed on SDS–PAGE and stained with Coomassie blue.SynCheck arrest experiments were repeated at least three times, scoring at least 200 cells per strain for each time-point, and the data plotted as the mean ± SD, using GraphPad Prism software. Details of the number of experimental repeats, number of cells analyzed, and the relevant statistics are detailed in the figure legends.This study did not generate or analyze datasets or code.


The cytotoxic activities of the natural precursor 1 and these three derivatives have been measured as IC50 inhibitory growth. The diphenylphosphinyl derivative 4 showed a significant cytotoxic activity on two breast cancer cell lines, namely MCF-7 and MDA-MB-231, with IC50 values of 55.5 and 62.7 [µM], respectively. These results suggest that the kind of structural modifications introduced to synthesize compound 4 represent a promising way to enhance the cytotoxic activity of boldine derivatives.2,9-Dimethoxymethylboldine ( Stock solutions of compounds were prepared in ethanol and the final concentration of this solvent was kept constant at 1%. Control cultures received 1% ethanol alone.The experimental cell cultures were obtained from the American Type Culture Collection . MCF-7 and MDA-MB-231 cells were grown in DMEM-F12 medium containing 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin and 1 mM glutamine. DHF dermal human fibroblast cells were grown in DMEM-F12 medium containing 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin and 1 mM glutamine. Cells were seeded into 96 well microtiter plates in 100 µL at plating density of 3 × 10et al. [3 cells per well of a 96-well, flat-bottomed 200 μL microplate. Cells were incubated at 37 °C in a humidified 5% CO2 / 95% air mixture and treated with the compounds at different concentrations for 72 hours. At the end of the drug exposure, cells were fixed with 50% trichloroacetic acid (TCA) at 4 °C . After washing with distilled water, cells were stained with 0.1% sulforhodamine B, dissolved in 1% acetic acid (50 µL/well) for 30 min, and subsequently washed with 1% acetic acid to remove unbound stain. Protein-bound stain was solubilised with 100 µL of 10 mM unbuffered Tris base, and the cell density was determined using a fluorescence plate reader (wavelength 540 nm). Values shown are the % viability vs. ctrl + SD, with three independent experiments in triplicate.The sulforhodamine B assay was used according to the method of Skehan et al.  with somet al. . Briefly4, may be attributed to the enhanced lipophilicity as well as an increasing intercalating behavior. This in vitro inhibitory effect in the growth of two breast cancer lines cells opens a promising way to introduce structural modifications in this natural product which enhances its biological activity.The derivatization of boldine with a phosphinyl group, introduces a distinct capability as a potential drug for breast cancer treatment which is not found in the boldine itself nor in 2,9-dimethoxy-methylboldine or 2,9-dimethoxymethyl-3-bromoboldine. This capability, due to the phosphinyl group in


BCR-ABL1 isoform and a novel BCR-ABL1 mutation, p.Y440C. The latter is in the C-terminal lobe of the kinase domain, which likely induces deviations in the protein structure and activity and destabilizes its inactive conformation. The treatment was substituted by bosutinib, which binds to the active conformation of the protein, prior to allogeneic bone marrow transplant to overcome the lack of a complete response to dasatinib. These findings strengthen the importance of BCR-ABL1 mutational screening in Ph+ patients, particularly for those who do not achieve complete molecular remission.Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) represents the most common genetic subtype of adult ALL (20%–30%) and accounts for approximately 50% of all cases in the elderly. It has been considered the subgroup of ALL with the worst outcome. The introduction of tyrosine kinase inhibitors (TKIs) allows complete hematologic remission virtually in all patients, with improved disease-free survival and overall survival. Nevertheless, the emergence of resistant mutations in BCR-ABL1 may require different TKI strategies to overcome the patient’s resistance and disease relapse. Here, we report a Ph+B-ALL case with persistent minimal residual disease (MRD) after treatment with dasatinib. The patient expressed the P190 Acute lymphoblastic leukemia (ALL) is a clonal disease in which immature lymphoid cells accumulate mainly in the bone marrow and peripheral blood. The disease is very heterogenous, comprising several hematological, cytogenetic and molecular groups. The Philadelphia chromosome (Ph) is the most frequent genetic abnormality in adult ALL (Ph+ ALL), representing 20%–30% of the B-lineage cases (B-ALL) but only 5% of the pediatric cases. Ph+ ALL frequency increases with age, accounting for approximately 50% of all cases in the elderly.2BCR and ABL1. In Ph+ ALL, the most frequently expressed isoform of BCR-ABL1, accounting for 70% of the cases, is the P190, in which exon 1 of BCR is fused to exon 2 of ABL1 (e1a2).BCR-ABL1. The transforming potential of the P190 isoform is believed to result from the partial loss of BCR domains, namely, the guanine exchange factor/dbl-like domain that mediates the communication with several Ras proteins, which are crucial for the regulation of signaling pathways and processes, such as cell proliferation, differentiation, adhesion, apoptosis and migration.The translocation that characterizes the Ph chromosome, tq34;q11), involves the fusion of the following two genes: 4;q11, inABL1 domain of the chimeric transcript. Indeed, BCR-ABL1 variations are one of the mechanisms that lead to TKI resistance, may be due to pre-existent mutations (primary resistance accounts for one-third of the patients) or mutations that arise from TKI treatment pressure (acquired resistance).15In chronic myeloid leukemia (CML), in which all patients test positive for BCR-ABL1, tyrosine kinase inhibitor (TKI) therapy revolutionized prognosis. The same applies for Ph+ALL, in which complete remission can be achieved in 95% of the patients.BCR-ABL1 isoform and a novel BCR-ABL1 mutation in the region that encodes for the C-terminal lobe domain of the protein.Herein, we describe a Ph+ B-ALL patient expressing the P190−4 for BCR-ABL1 transcripts , characterized as Ph+ by cytogenetics  showed a point mutation in the ABL1 domain of the fusion transcript, c.1319A.G, which lies in the region that translates for the C-terminal lobe of the kinase domain, p.Tyr440Cys , and as an effort to achieve molecular remission, the TKI regimen was changed to bosutinib in post-induction therapy. After 7 weeks on bosutinib, asatinib . NeverthTKI regimens have greatly improved the therapeutic outcome of patients with Ph+ ALL. However, each individual’s sensitivity to each TKI must be considered for a successful therapeutic outcome. Similar to what is recommended for CML, Ph+ ALL patients who do not respond to one of the available TKIs should undergo mutational screening for an assessment of markers of drug resistance mechanisms.12BCR-ABL1 nucleotide substitution, c.1319A.G (codon TAC.TGC), results in the amino acid alteration, p.Tyr440Cys , according to the manufacturer’s instructions. A total of 100 nuclei were scored at diagnosis. This analysis was carried out using a fluorescence microscope , and image capture was performed using Isis FISH Imaging System V 5.7.1 .2, 10 mM NaCl and 10 mM Tris-HCl; pH 7) and centrifuged at 400× g for 5 minutes. The procedure was repeated to lyse residual red blood cells. White blood cell pellet was subjected to total RNA extraction using SV Total RNA Isolation System , according to the manufacturer’s instructions. RNA was resuspended in nuclease-free water and stored at −80°C until use. RNA concentration and purity were determined by UV spectrophotometry. Total RNA extracted (100 ng) was reverse transcribed into cDNA, using the NZY M-MuLV First-Strand cDNA Synthesis kit .RNA was extracted from bone marrow white blood cells. Briefly, 5 mL of sample was mixed with 10 mL of red blood cell lysis solution , 2.5 mM MgCl2, 0.15× Hi-Specific additive (Bioline), 200 µM dNTPs, 400 nM primers and 0.02 U/µL BIOTAQ DNA polymerase (Bioline). Outer PCR was performed using 3 µL of cDNA in a 50 µL reaction, under the following conditions: initial denaturation at 95°C for 5 minutes; 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1.5 minutes; and a final extension step at 72°C for 10 minutes. Inner PCR was performed using 1 µL of outer PCR product in a 50 µL reaction, under the following conditions: initial denaturation at 95°C for 5 minutes; 30 cycles of 94°C for 15 seconds, 55°C for 30 seconds, 72°C for 1 minute; and a final extension step at 72°C for 10 minutes. A control PCR, for the amplification of ABL1, was also performed using primers depicted in Nested-PCR amplification of BCR-ABL1 e1a2 transcript was performed in bone marrow samples using a Corbett Rotor-Gene 6000 thermal cycler  and NZY qPCR Green Master Mix (Nzytech). The reaction mixture was prepared in a final volume of 10 µL with 1 µL of cDNA and 100 nM of each primer: outer PCR primers for BCR-ABL1 transcript identification or ABL1 control primers, specified in −ΔΔCt), in which relative gene expression is given by quantification of the gene of interest (BCR-ABL1) normalized to internal control gene (ABL1), relative to control condition (diagnostics sample).Quantitative monitoring of g; the supernatant was removed; and the dry pellet was resuspended in 100 µL of TE buffer. DNA concentration and purity were determined by UV spectrophotometry. DNA was stored at 4°C until use.Buccal epithelial cells were collected from the subject of this study by twirling a swab on the inner cheeks for 1 minute. The swab was separated from the stick and inserted into a microtube containing 440 µL of lysis buffer . The tube was vortexed for several minutes to promote the release of most epithelial cells. The swab was removed, and 20 µL of proteinase K  was mixed with the cell suspension and incubated for 10 minutes at 50°C. After adding 40 µL of 5 M NaCl, 500 µL of isopropanol was gently mixed with the DNA-containing solution and allowed to stand for 5 minutes at room temperature. The sample was centrifuged for 5 minutes at 12,000× ABL1 exon 8 of patient’s epithelial cells was performed using the following primers: forward 5′-CTCAAATAATCCTCCCACTTCA and reverse 5′-CCTGGAATGCCCACATATAC. PCR mixture components included 1× NH4 reaction buffer (Bioline), 2.5 mM MgCl2, 0.15× Hi-Spec additive (Bioline), 200 µM dNTPs, 400 nM primers, 0.02 U/µL BIOTAQ DNA polymerase (Bioline) and 20 ng of gDNA. PCR conditions included an initial denaturation at 95°C for 5 minutes; 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute; and a final extension step at 72°C for 10 minutes.PCR amplification of BCR-ABL1 e1a2 (GenBank AF113911.1) and ABL1 (GenBank NM005157.5).Basic Local Alignment Search Tool (BLAST), from the National Center for Biotechnology Information (NCBI), was used to align all obtained sequences, via Sanger sequencing, with the following reference sequences: Figure S1BCR-ABL1 e1a2 reference sequence from GenBank AF113911.1 (Seq 2).Alignment of Ph+ ALL patient e1a2 nucleotide sequence (Seq 1) with Note: Arrow indicates the breakpoint in this BCR-ABL1 isoform.Abbreviations: ALL, acute lymphoblastic leukemia; Ph+, Philadelphia chromosome positive.Figure S2DISOclust disorder prediction results.Note: Disorder probability score by residue number in BCR-ABL1 harboring the Y440C on the ABL1 domain.


We determined the effects of diclofenac sodium, octreotide, and their combination on extrapancreatic organ injuries in caerulein-induced acute pancreatitis in mice.  A total of 58 BALB-C male mice (25 g) were divided into seven groups and used to create a caerulein-induced acute pancreatitis model. Diclofenac sodium, octreotide, and their combination were given for treatment of caerulin-induced acute pancreatitis in mice. At the end of the experiment, the lung, liver, kidney, and stomach were removed for histopathologic assessment. P < 0.001, P < 0.001, P < 0.001, P < 0.001, and P = 0.048, respectively); mean congestion, edema, neutrophil inflammation, mononuclear inflammation, and emphysematous change scores in the lung ; mean congestion, edema, and neutrophil inflammation scores in the stomach ; and mean congestion and hydropic degeneration scores in the liver .  Histopathologic investigation revealed a statistically significant difference between the groups in mean congestion, edema, tubular injury, perirenal fat tissue inflammation, and tubular stasis scores in kidney tissue ( Diclofenac sodium alone ameliorates lung edema due to caerulin-induced acute pancreatitis. Acute pancreatitis may cause damage to other organs because of systemic inflammation and cytokines. In particular, severe acute pancreatitis (SAP) causes other organ damage and has a high mortality rate range, 15%–40%) [5%–40% 1. Howevers of SAP .Because of these inflammatory cytokines, SAP causes systemic inflammation that results in damage to other organs, such as the lung, liver, kidney, and stomach , 6, 9–11We obtained approval from the local ethical committee of the Research and Application Center for Experimental Animals at our university. A total of 58 male BALB-C mice (25 g) were divided into four control and three treatment groups. The control groups were group 1, saline (6 mice); group 2, acute pancreatitis ; group 3, octreotide (6 mice); and group 4, diclofenac sodium (6 mice). The treatment groups were group 5, octreotide with acute pancreatitis (9 mice); group 6, diclofenac sodium with acute pancreatitis (10 mice); and group 7, octreotide plus diclofenac sodium with acute pancreatitis (12 mice). The animals were not used in another study before our model, given standard laboratory foods and drinking water, and housed with a 12 : 12 h light/dark cycle at 21°C–24°C. The animals received only water for 12 h before the beginning of the experiment. All experiments were performed under anesthesia, and the mice were scarified 6 h after the last injection.μl saline (intramuscular). Caerulein (50 μg/kg in 50 μl 0.9% NaCl) was injected intraperitoneally seven times at 1 h intervals under anesthesia to induce acute pancreatitis. Octreotide (250 μg/kg) was injected intraperitoneally 1 h after the first caerulein injection and seven times thereafter at 1 h intervals. Diclofenac sodium (15 mg/kg) was given as a single intraperitoneal injection 1 h after the first caerulein injection. All animals were killed by taking 1–2 ml blood from their hearts 6 h after the last caerulein injection, and their lung, liver, kidney, and stomach were removed immediately.The anesthesia protocol consisted of 2.5 mg xylazine plus 7.5 mg ketamine per 100 g body weight in 200 μm sections to obtain slides with lysine, which were stained with hematoxylin–eosin (H&E).All tissues were fixed in 10% formaldehyde solution for 24 h, processed using the autotechnicon instrument , and embedded in paraffin blocks. A microtome was used to cut 5 H&E-stained preparations were evaluated by a single experienced pathologist in a blinded fashion using an Olympus BX51 light microscope. Histopathologic criteria were evaluated for the kidney , lung , stomach , and liver . Scores were graded as grades 0 (no pathology), 1 (mild), 2 (moderate), and 3 (severe).P values from the Kruskal–Wallis test were statistically significant (P < 0.05), Conover's multiple comparison test was used to determine which group differed from others.Data were analyzed using IBM SPSS Statistics version 17.0 software . Descriptive statistics are shown as the median (25th–75th) percentile for ordinal data. Statistical significance in the differences in histopathologic scores among the groups was evaluated with the Kruskal–Wallis test. When P < 0.001, P < 0.001, P < 0.001, P < 0.001, and P = 0.048, respectively; P < 0.001, P < 0.001, P < 0.001, P = 0.030, and P < 0.001, respectively; P = 0.008, P = 0.014, and P < 0.001, respectively; P = 0.029 and P = 0.002, respectively; Histopathologic investigation showed statistically significant differences between the groups in mean congestion, edema, tubular injury, perirenal fat tissue inflammation, and tubular stasis scores in kidney tissue . Comparing group 2 with the other groups, the scores were significantly lower in groups 1, 3, and 4 than in group 2 . There were no statistically significant differences between groups 1, 3, and 4 (P > 0.005). Group 7 (combination group) had higher mean scores than groups 5 and 6, but the difference was not significant (P > 0.05).Conover's multiple comparison test after the Kruskal–Wallis test in multiple comparisons revealed statistically significant differences in mean congestion scores between group 1 and groups 2 and 5–7  but not between group 2 and groups 5–7 (P > 0.05). Comparing group 1 with the other groups showed no significant differences between group 1 and groups 3 and 4 (P > 0.05), but there were significant differences between group 1 and groups 5–7 .There were significant differences between group 2 and groups 1, 3, and 4  but not between group 1 and groups 3 and 4 (P > 0.05). Comparing group 2 and the other groups showed no significant differences between group 2 and groups 3 and 4  or between group 2 and groups 5–7 (P > 0.05).There were significant differences between group 1 and groups 2 and 5–7  but not between group 1 and groups 3 and 4 (P > 0.05). Comparison of group 2 and the other groups showed a significant difference between group 2 and groups 3 and 4  but not between group 2 and groups 5–7 (P > 0.05).Significant differences were found between group 1 and groups 2 and 5–7  but not between group 1 and groups 3, 4, 6, and 7 (P > 0.05). Significant differences also were seen between group 2 and groups 3 and 4  but not between group 2 and groups 5–7 (P > 0.05). We found lower tubular stasis scores in groups 6 and 7 than in group 5, but this was not statistically significant (P > 0.05).Comparing group 1 and the other groups showed significant differences between group 1 and groups 2 and 5  but not between groups 2 and 5 (P = 0.153) nor between groups 1 and 3 (P = 0.126). However, there were significant differences between group 1 and groups 4–7 .Significant differences were found between group 2 and groups 1, 3, 4, 6, and 7 . The lung edema scores in group 6 (diclofenac sodium treatment) were statistically significantly lower than those in groups 5 and 7 . A significant difference also was noted between group 1 and groups 2 and 7 .There were statistically significant differences between group 2 and groups 1, 3, 4, and 6 (P = 0.044). There were no significant differences in other group comparisons (P > 0.05).A significant difference was found only between groups 2 and 7 (P > 0.05). A significant difference was found only between groups 2 and 6 (P = 0.047). No significant difference was noted between group 2 and the other groups (P > 0.05).There was no significant difference between group 1 and all other groups  but not between group 1 and groups 3 and 4 (P > 0.05). Significant differences were noted between groups 2 and 3 (P < 0.001).There were statistically significant differences between group 1 and groups 2 and 5–7 . Comparing group 2 and the other groups revealed significant differences between groups 2 and 3 (P = 0.014) but not in binary group comparison of the treatment groups (P > 0.05).There were significant differences between group 1 and groups 2 and 5–7  but not between group 1 and groups 3–5 and 7 (P > 0.05). Comparing group 2 and the other groups showed significant differences between group 2 and groups 3 and 4 .Significant differences were noted between group 1 and groups 2 and 6 . When we compared group 7 (combined treatment) and groups 5 and 6, group 7 showed statistically significantly higher scores . There was a significant difference between group 1 and groups 2 and 7 .Statistically significant differences were noted between group 2 and groups 1 and 3–6 (P = 0.006) but not between group 1 and the other groups (P > 0.05). However, there were significant differences between group 2 and groups 3–7 .Significant differences were noted between groups 1 and 2  but not between groups 2 and 6 (P > 0.05). Significant differences were also found between groups 1 and 2 and 6  but not between group 1 and the other groups (P > 0.05).There were significant differences between group 2 and groups 3–5 and 7  and 7 (diclofenac sodium plus octreotide combination) were significantly lower than those in group 2 (acute pancreatitis). Group 6 also showed significantly lower lung edema scores than group 2. Therefore, diclofenac sodium as an anti-inflammatory drug ameliorated lung injury in experimental acute pancreatitis. Neutrophil inflammation of the lung in group 7 was significantly lower than that in group 2 in our study. Also, mononuclear inflammation of the lung was only lower in group 6 than in group 2.Dobosz et al.  concludeOur study showed that congestion and edema scores for the stomach in all treatment groups were similar to those of group 2. Although neutrophil inflammation of the stomach in group 7 was similar to that in group 2, it was significantly higher than those in the other treatment groups (groups 5 and 6). We concluded that there might be a counteraction between diclofenac sodium and octreotide.A previous study showed that gadolinium chloride reduced hepatic or systemic cytokine levels and lung injury in experimental pancreatitis . KiyonarIn our study, liver congestion in all treatment groups was significantly lower than that in the acute pancreatitis group. In terms of hydropic degeneration of the liver, the diclofenac sodium treatment group was similar to the acute pancreatitis group and had significantly higher levels than the other treatment groups. However, the octreotide and combination treatment groups showed significantly lower levels than the acute pancreatitis group in terms of hydropic degeneration of the liver. The prognosis of acute pancreatitis is associated with extrapancreatic organ injuries. Also, the treatment of this situation is not clear. We showed the effects of diclofenac sodium, octreotide, and their combination on extrapancreatic organ injuries  in caerulein-induced acute pancreatitis in mice. Our study demonstrated that diclofenac sodium improved experimental acute pancreatitis–associated lung edema. Diclofenac sodium may ameliorate the prognosis of acute pancreatitis by reducing lung edema.In conclusion, diclofenac sodium is a well-known drug that may be a good choice for extrapancreatic organ injuries in cases of acute pancreatitis. Diclofenac sodium improved lung edema–associated experimental acute pancreatitis in our study. Therefore, diclofenac sodium may improve the prognosis of acute pancreatitis by reducing lung edema. Further studies are needed for clarity.


A new family of photochromic diarylethene-based ligands bearing a 2-(imidazol-2-yl)pyridine coordination unit has been developed. Four members of the new family have been synthesized. The photoactive ligands feature non-aromatic ethene bridges , as well as closely spaced photoactive and metal coordination sites aiming a strong impact of photocyclization on the electronic structure of the coordinated metal ion. The ligands with cyclopentenone and cyclohexenone bridges show good cycloreversion quantum yields of 0.20–0.32. The thermal stability of closed-ring isomers reveals half-lives of up to 20 days in solution at room temperature. The ligands were used to explore coordination chemistry with iron(II) targeting photoswitchable spin-crossover complexes. Unexpectedly, dinuclear and tetranuclear iron(II) complexes were obtained, which were thoroughly characterized by X-ray crystallography, magnetic measurements, and Mössbauer spectroscopy. The formation of multinuclear complexes is facilitated by two coordination sites of the diarylethene, acting as a bridging ligand. The bridging nature of the diarylethene in the complexes prevents photocyclization. The compound reveals a crystallographically symmetrical dimeric structure with two iron centers linked by two ligands 6. Each iron ion is coordinated equatorially by the β-keto ester moiety of one molecule 6 and chelated via the imidazole and pyridine groups of another moelcule 6. The distorted octahedral coordination sphere is completed by a H2B(pz)21− anion. Fe–N bond distances of 2.1602(15)–2.1615(15) Å for the pyrazoles and 2.2489(14)–2.2462(14) Å for the imidazole and pyridine moieties are typical for high-spin (HS) iron(II) complexes [trans-effect due to the H2B(pz)21− anion can further elongate that bond. Two iron(II) centers are 8.057 Å apart in the dimer. The ligand 6 appears in the open-ring form and the parallel conformation [2 position twists out of the plane towards the thiophene moiety of another ligand 6.The complex omplexes . Similaromplexes . The two2(H2B(pz)2)2Fe2(B(OMe)3(pz))2(6-H)2] (complex 9). Apparently, this product is the result of destruction of bis(pyrazolyl)borate moieties of complex 8 by methanol. Species 9 crystallizes as red, block-shaped crystals in the triclinic P−1 space group with one molecule in the unit cell. The crystal structure reveals two pairs of differently substituted iron ions connected by a so far unprecedented trimethoxypyrazolylborate bridging group. The periphery Fe ions reveal an octahedral coordination environment with two H2B(pz)21− anions and two methoxy groups of the bridging moiety. The Fe–N bond distances ranging from 2.1361(19) to 2.1631(19) Å in 9 appear similar to those in 8, which confirms a HS-Fe(II) ion. The Fe–O bond lengths vary between 2.1333(15) Å and 2.3110(16) Å. Curiously, the O–B bond distances to the tetrahedral boron ion do not differ in length with 1.453(3) and 1.449(3) Å, respectively.Long time precipitation of crystalline material resulted in a tetranuclear species [Fe6 and by the imidazole and pyridine groups of the second ligand 6. The Fe–O and Fe–N bond lengths to the bridging unit are 2.1687(15) Å and 2.1190(19) Å. The pyridine and imidazole Fe–N bond distances are 2.2467(19) Å and 2.1793(18) Å, which are very similar to those found for the dimer 8. On the contrary, the Fe–O bond distances to the β-keto ester moiety differ much less with 2.0812(15) Å and 2.0098(15) Å. The iron–donor bond distances are in good agreement with an iron(II) HS state. The two iron(II) ions linked by the trimethoxypyrazolylborate group are 5.945 Å apart. The distance between the iron(II) ions connected by ligand 6 in 9 is elongated by 0.244 Å to 8.301 Å compared to 8. The ligand 6 appears also in its open-ring form and parallel conformation.Each of the two interior Fe ions is linked to the bridging unit via its remaining methoxy group and the pyrazole moiety. The octahedral coordination environment is completed with the β-keto ester moiety of one ligand 8 and 9 were used for magnetic susceptibility measurements. Complex 8 shows a nearly constant χT product of 7.63 cm3·mol–1·K above 125 K  ions. By lowering the temperature further, the χT product decreases sharply due to zero-field splitting (ZFS). The rt value of the χT product of 7.63 cm3·mol–1·K is slightly higher than expected spin-only value for two non-interacting HS-Fe(II) ions (χsoT = 6.00 cm3·mol–1·K) due to orbital contribution. The fit for S1 = S2 = 2 spin system  magnetization saturates, reaching a plateau at 8 NAµB, which corresponds to two ferromagnetically coupled HS-Fe(II) ions in the ground state. Zero-field 57Fe Mössbauer spectrometry confirm HS-Fe(II) state at 77 K and 297 K.Microcrystalline samples of ve 125 K . It incrχT product of 9 increases to almost constant value of 14.61 cm3·mol−1·K at rt, which is in good agreement with four uncoupled HS-Fe(II) ions (χsoT = 12.00 cm3·mol–1·K) .The mol–1·K) . A signi8 shows a single major absorption band in the low energy UV region at 346 nm (ε = 6.22 × 104 M−1·cm−1), which appears bathochromically shifted compared to the free ligand. No charge transfer (CT) bands are visible in the spectrum. The low intensity broad shoulder, which spans almost the entire visible region up to 750 nm, can be attributed to d–d transitions  appears sharper compared to 8. The broad shoulder presumably due to d–d transitions also appears contracted, as it disappears at lower wavelengths (<650 nm).The complex 8 and 9 with UV light (λ = 365 nm) in dichloromethane show no appearance of new bands. Prolonged irradiation results in slow photodecomposition. Both 8 and 9 share a common structural motif that prevents photocyclization. Coordination of the β-keto ester moiety and the imidazole/pyrazole moiety to two different iron(II) ions puts the ligand under severe strain. Thus, the structural reorganization necessary to accommodate a new planar structure formed by the photocyclization reaction is impossible without breaking Fe–N or Fe–O bonds.Attempts to induce photocyclization of diarylethenes in complexes 8 and 9 represent cyclic complexes of diarylethenes with Fe(II) ions. In recent years, related derivatives of photochromic diarylethenes [6 featuring two coordination sites in the heteroaryl moiety and bridge provides unique opportunities to construct novel macrocyclic systems. Our future efforts will be concentrated on the synthesis of photoactive complexes based on ligand 6.Note, that the obtained complexes lethenes –51 becamlethenes  and lighlethenes . Ligand 6 readily forms a dimeric complex 8 with “FeII(H2B(pz)2)2”, with its β-keto ester moiety acting as a second coordination site. Slow crystallization yielded a tetranuclear Fe(II) complex 9, where the base dimeric unit is expanded by an unprecedented trimethoxypyrazolylborate bridging group. The photocyclization of iron complexes is inhibited due to the rigid coordination of the imidazole group to the metal ion, which prevents the rotation of the group needed for cyclization.A new class of photochromic diarylethene-based ligands featuring a 2-(imidazol-2-yl)pyridine coordination unit as a heteroaryl moiety has been developed. Four members of the new family have been synthesized. All ligands show good combinations of cyclization/cycloreversion quantum yields, whereas the thermal stability of closed-ring isomers is comparable with those reported for a diarylethene-based ligand with a phenanthroline bridge. Cyclohexenone ligand File 1Experimental details and peripheral discussion.File 21, 6, 8, and 9.X-ray data for compounds


In conclusion, TMAO cannot be considered a direct cause or an exacerbating risk factor of cardiac damage at the cellular level in acute conditions.Trimethylamine N-oxide (TMAO) is an organic compound derived from dietary choline and L-carnitine. It behaves as an osmolyte, a protein stabilizer, and an electron acceptor, showing different biological functions in different animals. Recent works point out that, in humans, high circulating levels of TMAO are related to the progression of atherosclerosis and other cardiovascular diseases. However, studies on a direct role of TMAO in cardiomyocyte parameters are still limited. The purpose of this work is to study the effects of TMAO on isolated adult rat cardiomyocytes. TMAO in both 100 µM and 10 mM concentrations, from 1 to 24 h of treatment, does not affect cell viability, sarcomere length, intracellular ROS, and mitochondrial membrane potential. Furthermore, the simultaneous treatment with TMAO and known cardiac insults, such as H Investigations were focused on cell viability after TMAO or TMAO and stressors co-treatment, assessing cell morphology and functionality with α-actinin staining and specific probes that measure oxidative stress status and mitochondrial membrane potential.Trimethylamine N-oxide (TMAO) is an amine oxide directly introduced through diet or synthetized from its precursors, primarily L-carnitine and choline, that are transformed into trimethylamine (TMA) by the gut microbiota. Once absorbed, TMA in most mammals is oxidized by hepatic FMOs to form TMAO which enters the systemic circulation. Several studies illustrate different biological functions of TMAO in other animals. In elasmobranchs and deep-sea fishes, it acts as an osmolyte able to counteract either osmotic or hydrostatic pressure. It is a protein stabilizer preserving protein folding and it also acts as an electron acceptor balancing oxidative stress ,2. TMAO clerosis . Furtherclerosis . Finally2O2 50 μM, and H2O2 50 μM + TMAO 100 μM. After 1 h or 24 h of treatment, cardiomyocytes were labeled with propidium iodide (PI) and marked nuclei of suffering cells were detected by confocal microscopy at 568 nm. Concentrations used were taken from the literature: TMAO 100 µM is recognized as a marker of cardiovascular risk, TMAO 10 mM is over the physiological range and was tested here to detect any effect induced by high concentrations of the compound [2O2, used here as a positive control, had effects only after 24 h. Simultaneous treatment with H2O2 and TMAO did not improve or worsen the effect of the stressor on cell viability ; H2O2 + TMAO: 1.33 ± 1.33, n = 3, 41 cells (*** p < 0.001)). In order to investigate the effect of TMAO on cell viability, cardiomyocytes were treated with TMAO 100 μM, TMAO 10 mM, Hcompound . As show2O2 50 μM used as a positive control caused cardiomyocyte shrinkage, a condition that was not improved or worsened by the simultaneous treatment with TMAO. In cardiomyocytes treated with DOX 1 µM for 24 h, no sarcomere length variations were observed, because the DOX treatment in our model was designed to induce mild damage preceding cell shortening. Even so, TMAO 100 µM did not modify DOX-treated cardiomyocytes ; H2O2 + TMAO: 1.28 ± 0.03, n = 3, 24 cells (*** p < 0.001); DOX: 1.62 ± 0.02, n = 3, 16 cells; DOX + TMAO: 1.65 ± 0.01, n = 3, 15 cells).To evaluate if TMAO was able to alter sarcomere structures after 24 h of treatment, sarcomere length was measured in α-actinin-stained cardiomyocytes. As shown in n = 3, 34 cells; TMAO 10 mM: 1.32 ± 0.23, n = 3, 40 cells, vs. CTRL; 3 h—TMAO 100 µM: 0.96 ± 0.05, n = 3, 45 cells; TMAO 10 mM: 1.15 ± 0.09, n = 3, 52 cells, vs. CTRL; 24 h—TMAO 100 µM: 1.18 ± 0.04, n = 5, 40 cells; TMAO 10 mM: 1.24 ± 0.16, n = 3, 52 cells; DOX: 1.33 ± 0.11, n = 4, 21 cells (** p < 0.01); DOX+TMAO: 1.27 ± 0.01, n = 6, 31 cells (*** p < 0.001), vs. CTRL).In order to determine a variation in total ROS produced after treatment with TMAO for 1 h, 3 h, or 24 h, cells were labeled with the DCF-DA probe and its fluorescence was quantified and related to the control. As shown in n = 5, 65 cells; TMAO 10 mM: 1.11 ± 0.11, n = 3, 49 cells, vs. CTRL. 3 h—TMAO 100 µM: 1.02 ± 0.06, n = 3, 26 cells; TMAO 10 mM: 0.88 ± 0.05, n = 4, 39 cells, vs. CTRL; 24 h—TMAO 100 µM: 1.08 ± 0.11, n = 3, 21 cells; TMAO 10 mM: 0.95 ± 0.15, n = 3, 54 cells; DOX: 0.73 ± 0.02, n = 3, 24 cells (** p < 0.01); DOX + TMAO: 0.69 ± 0.05, n = 3, 22 cells (** p < 0.01), vs. CTRL).To investigate the potential metabolic damage induced by TMAO, cardiomyocytes treated with TMAO 100 µM and 10 mM for 1 h, 3 h, or 24 h were labeled with the JC-1 probe. 2O2 or doxorubicin, tested for up to 24 h of treatment. Taken together, these results suggest that TMAO should not be considered a primary cause of acute cardiac damage and that the molecule could not revert or worsen existing risk factors of cardiac damage.This study provides novel insights into the physiological role of TMAO in isolated adult rat cardiomyocytes. Our findings do not show effects of TMAO on cell viability, sarcomere length, ROS production, and mitochondrial membrane potential within the range of concentration used. Moreover, we demonstrate that TMAO does not exacerbate or counteract the effect of known insults, such as HIn the last few years, many studies suggest a strong relationship between diet, gut microbiota, and cardiovascular diseases . In partExperiments have mainly now focused on the role of TMAO in damaging endothelial cells. It has been described as upregulating cellular senescence, thereby reducing cell proliferation, increasing the expression of senescence markers, such as p53 and p21, and impairing cell migration . HyperteOnly a few studies are centered on the direct effect of the molecule on cardiac cells; in particular, they focus on the impairment of mitochondrial metabolism in the heart and underline TMAO as an agent that increases the severity of cardiovascular events or that enhances the progression of cardiovascular diseases . Savi anThis scenario presents TMAO as either a marker or a direct agent involved in vascular and cardiac outcomes, but recent papers seem to oppose this point of view, highlighting uncertainty about the causative relation between TMAO and CVD . The funThe present study fits into this debate and the results presented agree with other works supporting TMAO as a non-damaging factor. In fact, it is well known that the loss of vital cardiac cells is a damaging condition that hampers primarily the functionality of the heart and has several aggravating responses also in peripheral tissues. Our first investigations underline no toxic effect in cardiomyocytes exposed to high concentrations of TMAO, highlighting the result that the molecule is not involved in inducing cardiac tissue cell loss, and no alterations of cardiac structure emerge from the evaluation of sarcomere length and cytoskeletal organization. Oxidative stress could be considered one of the causative factors of senescence in cells and one of the promoters of cardiometabolic reorganization in response to injury. With respect to TMAO as a possible inducer of ROS rising, both in a cytoplasmic and a mitochondrial environment, we show no variation in ROS production even after 24 h of treatment and we detect no depolarization of mitochondrial membrane potential, underlining the result of no direct influence by the molecule in inducing cardiac cell senescence.Experiments were performed on female adult rats which were allowed ad libitum access to tap water and standard rodent diet. The animals received human care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health , and in accordance with Italian law . The scientific project was supervised and approved by the Italian Ministry of Health, Rome, and by the ethical committee of the University of Torino . Rats were anaesthetized by i.p. injection of tiletamine  and sacrificed by stunning and cervical dislocation.2, 5.5 D-glucose, 5 HEPES, 2 CaCl2, pH adjusted to 7.34 with NaOH. Ca2+ free Tyrode solution containing (in mM): 154 NaCl, 4 KCl, 1 MgCl2, 5.5 D-glucose, 5 HEPES, 10 2,3-Butanedione monoxime, 5 taurine, pH 7.34. All drug-containing solutions were prepared fresh before the experiments and the Tyrode solutions were oxygenated (O2 100%) before each experiment. Unless otherwise specified, all reagents for cell isolation and experiments were purchased from Sigma-Aldrich .Tyrode standard solution containing (in mM): 154 NaCl, 4 KCl, 1 MgCl2+ free Tyrode solution, and cannulated via the aorta. All the following operations were carried on under a laminar flow hood. The heart was perfused at a constant flow rate of 10 mL/min with Ca2+ free Tyrode solution (37 °C) with a peristaltic pump for approximately 5 min to wash away the blood and then with 10 mL of Ca2+ free Tyrode supplemented with collagenase (0.3 mg/mL) and protease (0.02 mg/mL). Hearts were then perfused and enzymatically dissociated with 20 mL of Ca2+ free Tyrode containing 50 µM CaCl2 and the same enzymatic concentration as before. Atria and ventricles were then separated and the ventricles were cut in small pieces and shaken for 10 min in 20 mL of Ca2+ free Tyrode solution in the presence of 50 µM CaCl2, collagenase, and protease. Calcium ion concentration was slowly increased to 0.8 mM. Cardiomyocytes were then plated on glass cover slips or glass bottom dishes , both treated with laminin to allow cell adhesion.Isolated cardiomyocytes were obtained from the hearts of adult rats (200–300 g body weight) according to the previously described method . Brieflyhttps://imagej.nih.gov/ij/) and cell viability was calculated as percentage of /total cells.Cell viability was evaluated by propidium iodide (PI) staining on glass bottom dishes for adherent cells. At the end of the treatments, cells were incubated with PI  for 5 min in the dark. Nuclei of suffering cells were detected with confocal microscopy using an Olympus Fluoview 200 microscope  at 568 nm (magnification 20×). Merged images were created with ImageJ  in PBS and stained for 24 h at +4 °C with a mouse monoclonal anti-α-actinin primary antibody . Cover slides were washed twice with PBS and incubated 1 h at room temperature with the secondary antibody . After two washes in PBS, coverslips were mounted on standard slides with DABCO and observed after 24 h under a confocal microscope. Confocal fluorimetric measurements were acquired using a Leica SP2 laser scanning confocal system , equipped with a 40× water-immersion objective. Image processing and analysis were performed with ImageJ software. Sarcomere length was evaluated measuring the distance between Z lanes in n = 10 sarcomeres/cell.Cardiomyocytes on glass coverslips were stimulated with TMAO and with HProduction of ROS was evaluated by fluorescence microscopy using a 2′-7′-dichlorofluorescein diacetate probe (DCF-DA). After adhesion on glass bottom dishes, DCF-DA solution (5 µg/mL) was added to each dish 30 min prior to the end of the treatment, then the cells were washed with standard Tyrode solution. Fluorescence images at 488 nm were acquired using an Olympus Fluoview 200 microscope (magnification 60×). Fluorescence variations were calculated with the definition and measurement of regions of interest (ROIs) using ImageJ software and expressed as relative Medium Fluorescence Index (MFI) compared to control, fixed at 1.Mitochondrial membrane potential was evaluated by staining cardiomyocytes with the dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1). JC-1 solution (10 µM) was added to each dish 30 min prior to the end of the treatment, then the cells were washed with standard Tyrode solution. Fluorescence images at 488 nm and 568 nm were acquired using an Olympus Fluoview 200 microscope (magnification 60×). Amounts of the monomeric form of the dye were quantified using the red/green fluorescence ratio in the ROIs using ImageJ software and expressed as folds towards control, fixed at 1.p < 0.05 were regarded as statistically significant.All data are expressed as mean ± standard error of the mean. For differences between mean values, Bonferroni’s multiple comparisons test was performed. Differences with In summary, this study demonstrates that TMAO is not directly involved in causing or exacerbating cardiac damage in an acute stress model . However


Vitamin D not only promotes Ca homeostasis, but it also inhibits bone resorption and promotes osteogenesis, thus playing a role in the maintenance of normal bone metabolism. Because 1α,25D3 plays an important role in osteogenesis, vitamin D formulations, such as alfacalcidol (ALF) and eldecalcitol (ELD), are used for treating osteoporosis. While it is known that, in contrast to ALF, ELD is an active ligand that directly acts on bone, the reason for its superior osteogenesis effects is unknown. Cyp27b1-knockout mice (Cyp27b1–/–mice) are congenitally deficient in 1α,25D3 and exhibit marked hypocalcemia and high parathyroid hormone levels, resulting in osteodystrophy involving bone hypocalcification and growth plate cartilage hypertrophy. However, because the vitamin D receptor is expressed normally in Cyp27b1–/–mice, they respond normally to 1α,25D3. Accordingly, in Cyp27b1–/–mice, the pharmacological effects of exogenously administered active vitamin D derivatives can be analyzed without being affected by 1α,25D3. We used Cyp27b1–/–mice to characterize and clarify the superior osteogenic effects of ELD on the bone in comparison with ALF. The results indicated that compared to ALF, ELD strongly induces ECaC2, calbindin-D9k, and CYP24A1 in the duodenum, promoting Ca absorption and decreasing the plasma concentration of 1α,25D3, resulting in improved osteogenesis. Because bone morphological measurements demonstrated that ELD has stronger effects on bone calcification, trabecular formation, and cancellous bone density than ALF, ELD appears to be a more effective therapeutic agent for treating postmenopausal osteoporosis, in which cancellous bone density decreases markedly. By using Cyp27b1–/–mice, this study was the first to succeed in clarifying the osteogenic effect of ELD without any influence of endogenous 1α,25D3. Furthermore, ELD more strongly enhanced bone mineralization, trabecular proliferation, and cancellous bone density than did ALF. Thus, ELD is expected to show an effect on postmenopausal osteoporosis, in which cancellous bone mineral density decreases markedly. In the future, this study may enable the development of next-generation active vitamin D derivatives with higher affinity for bone than ELD.Calcium (Ca) absorption from the intestinal tract is promoted by active vitamin D (1α,25D Next, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1) in the kidneys catalyzes 1α-hydroxylation to form 1α,25D3. Meanwhile, 25D3 undergoes 24-hydroxylation by 25-hydroxyvitamin D 24-hydroxylase (CYP24A1) to be metabolized into 24,25-dihydroxyvitamin D3 .Vitamin D3 is transported in the plasma when bound with vitamin D-binding protein (DBP) to reach target tissues , a vitamin D-deficient feed, supplemented with 2.4 IU/g of vitamin D3 (Diet11+D). The feed was manufactured by CLEA Japan at our request. Medium-chain triglycerides, 1α-hydroxyvitamin D3 (ALF), and 2β-hydroxypropoxy-1α,25-dihydroxyvitamin D3 (ELD) were supplied by Chugai Pharmaceutical Co., Ltd. . The ATDC5 cells used in this study are cartilage cells isolated from mouse teratocarcinoma . Organic solvents of HPLC grade were purchased from Nacalai Tesque . Other chemicals were highest-grade commercial chemicals.Heterozygous  methods . Male anAll cell experimental protocols were performed in accordance with the Guidelines for Cell Experiments at Kobe Pharmaceutical University and were approved by The Ethics Committee of Kobe Pharmaceutical University, Kobe Japan. All animal experimental protocols were performed in accordance with the Guidelines for Animal Experiments at Kobe Pharmaceutical University and were approved by The Animal Research and Ethics Committee of Kobe Pharmaceutical University, Kobe Japan. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.All mice were weaned at 3 weeks of age. After weaning, they were allowed to freely feed on a diet of solid food (Diet 11+D) and deionized water. The mice were raised in an enclosure with controlled temperature and humidity . The mice were raised until 9 weeks of age (administration period: 6 weeks). Body weight was measured once per week starting directly after weaning .ALF and ELD dosing solutions were formulated by dissolving 100 μL of ethanol containing 10 μg/mL of ALF or ELD into 7.9 mL of a medium-chain triglyceride solution. The vehicle dosing solution was prepared similarly using 100 μL of ethanol solution. The solutions were administered orally 3 times per week to achieve a dosage of 0.25 μg/kg body weight.Plasma was collected weekly in heparin-coated capillary tubes  from the caudal vein, under anesthesia. The plasma Ca concentration was measured using a Ca measurement kit . At 9 weeks of age, cardiac plasma was collected, and serum was separated by centrifugation. The plasma PTH concentration was measured using a mouse PTH measurement kit .9k , mouse Cyp2r1 , mouse Cyp24a1 , and mouse β-actin  as a control. Primer specificity was evaluated by electrophoresis of the PCR product.Total RNA was isolated from the mouse duodenum and liver using Isogen , in accordance with the manufacturer’s protocol. First-strand cDNA was generated using AMV reverse transcriptase . PCRs were conducted using a SYBR Green Core Reagent Kit  on a CFX96 Real-time PCR System , according to the manufacturer’s protocol. Primers were designed to target mouse epithelial calcium channel 2 (ECaC2) , mouse calbindin-DMice were anesthetized and then euthanized by means of cardiac plasma collection. The femurs and tibias were collected. SOFTEX soft X-ray imaging equipment  was used to acquire soft X-ray images of the femurs and tibias.Parameters related to bone structure, bone shape, and bone resorption were analyzed in non-decalcified specimens of femurs obtained from female mice after 6 weeks of administrations. Total bone density, cortical bone density, and cancellous bone density of the femoral metaphysis, as well as cortical bone density and cortical bone thickness in the diaphysis were measured by peripheral quantitative computed tomography (pQCT). Horizontal, vertical and twisting bone strength in the femur were measured by pQCT. Two-dimensional (2D) and three-dimensional (3D) structural analyses of femoral trabeculae were conducted by micro-CT. The pQCT and micro CT analyses were performed at Kureha Special Laboratory .Bone-tissue sections were prepared by Kureha Special Laboratory. Bone calcification was assessed by von Kossa staining, cartilage formation by toluidine blue staining, and osteoid formation by Villanueva staining of the bone specimens. The bone sections were observed by bright-field microscopy using an all-in-one fluorescence microscope . To analyze bone formation speed by calcein double labeling, mice were administered 0.1 mL of calcein dosing solution per 10 g of body weight 4 days prior to and 1 day prior to tissue collection. The specimens were observed under the BZ-8000 microscope using an excitation wavelength of 480 nm and emission wavelength of 505 nm.2. The medium was replaced every other day.ATDC5 cells were maintained in Dulbecco’s modified Eagle’s and Ham-F12 [DMEM/F12]  supplemented with 1% penicillin, 1% streptomycin, and 10% fetal bovine serum  at 37°C in a humidified atmosphere with 5% CO−8 M sodium selenite , and 10 μg/ml insulin  in 96-well plates (1 × 104 cells/well). In addition, 10−7–10−10 M 1α,25D3, ALF, ELD, or 10−6–10−9 M 25D3 was added. The media were changed every other day. Differentiation of ATDC5 cells was monitored using the cell counting reagent WST-1 . Following addition of WST-1 reagent, the optical density at 450 nm was read.ATDC5 cells reached confluence 3 days after seeding. At day 5, cells were exposed to chondrogenic medium consisting of DMEM/Ham’s F-12 (1:1) containing 5% FBS, 10 μg/ml human transferrin , 3 × 10−7–10−10 M 1α,25D3, ALF, ELD, or 10−6–10−9 M 25D3 using the method described above. The cells were rinsed with PBS and fixed in 95% methanol for 20 min, and then stained with 0.1% alcian blue 8GX  in 0.1 M HCl overnight. The stained cells were rinsed with PBS three times and observed by microscopy.ATDC5 cells were plated in 12-multiwell plates and cultured in chondrogenic medium with 105) were suspended in 2 mL of medium and transfected with 0.5 μg of pGVB2-rat Cyp24a1 luciferase reporter vector  containing a rat Cyp24a1 gene promoter including two VDREs and 0.25 μg of pRL-CMV vector (Toyo Ink) as an internal control, using Tfx-50 reagent . The cells were incubated with 10−7–10−10 M 1α,25D3, ALF, ELD, or 10−6–10−9 M 25D3 for 48 h. Next, the binding capacity to VDR was examined by using the VDR-GAL4 one-hybrid luciferase system. ATDC5 cells (2 × 105) were suspended in 2 mL of medium and transfected with 0.5 μg of pM-human VDR incorporating whole human VDR cDNA upstream of the DNA-binding domain of the GAL4 gene, which is a transcriptional regulator in yeast, 0.5 μg of pGVP2-GAL2 containing the SV40 promoter incorporating the binding site of rat GAL4 upstream of the firefly luciferase gene, and 0.25 μg of pRL-CMV vector  as an internal control, using Tfx-50 reagent (Promega). The cells were incubated with 10−9 M 1α,25D3, ALF, ELD, or 10−7 M 25D3 and 0, 5, or 25 μg/mL DBP for 48 h. The luciferase activities of the cell lysates were measured with a luciferase assay kit (Toyo Ink), according to the manufacturer’s instructions. Transactivation measured as luciferase activity was standardized to the luciferase activity of the same cells determined with the Sea Pansy luciferase assay system as a control (Toyo Ink).To asses the activity via the VDRE, ATDC5 cells  duplex oligoribonucleotide of the reported mouse megalin mRNA sequence. Alexa Fluor-labeled siRNA  was used as control siRNA. ATDC5 cells were transfected with 50 pmol of each siRNA with the use of Lipofectamine RNAiMAX (Thermo Fisher Scientific) in 1 mL of Opti-MEM serum-free medium (Thermo Fisher Scientific), in accordance with the manufacturer’s instructions. Megalin mRNA was quantified by real-time PCR as described above. The obtained megalin-knockdown cells transfected with 0.5 μg of pGVB2-rat Cyp24a1 vector and 0.25 μg of pRL-CMV vector using Tfx-50 reagent. The cells were incubated with 10−9 M 1α,25D3, ALF, ELD, or 10−7 M 25D3 for 48 h. To assay transcriptional activity, the luciferase assay described above was used.Stealth RNAi t-test or Dunnett’s test and Tukey–Kramer's honestly significant difference (HSD) test. P < 0.05 was considered significant.Data are expressed as the mean ± SEM. The number of specimens is n = 5. Means were compared using an unpaired Student’s Cyp27b1+/+ and Cyp27b1–/–mice that were administered ALF, ELD, or vehicle for 6 weeks after weaning. From five weeks of age, Cyp27b1–/–mice exhibited significantly lower body weight than the Cyp27b1+/+ mice. In contrast, in Cyp27b1–/–mice administered ALF or ELD, body weight increased to levels similar to those observed in Cyp27b1+/+ mice , having a 3-hydroxypropoxy group at the 2α position, is more difficult for CYP24A1 to metabolize than 1α,25D3 [Cyp27b1+/+ mice indicated that ELD can promote Ca absorption and improve osteogenesis in patients with osteoporosis. By using Cyp27b1–/–mice in our experiment, we were able to demonstrate that ELD exhibits Ca metabolism-regulating effects without being influenced by endogenous 1α,25D3.It has been reported that ELD reduces plasma 1α,25D animals . This ap+/+ mice , it appepromoter , it appe 1α,25D3 . Therefo 1α,25D3 , which m3 in terms of increasing lumbar vertebrae and femur bone density, and inhibiting bone resorption [3 concentration [3 to analyze the actions of ELD alone. Therefore, in order to clarify the actions of ELD alone on bone, excluding the effects of 1α,25D3, we analyzed the osteogenic actions of ALF and ELD in Cyp27b1–/–mice. Externally, the femurs and tibias in vehicle-treated Cyp27b1–/–mice were observed to have hypertrophy in the epiphyseal region and reduced bone length. However, the administration of ALF or ELD improved these abnormalities to a state similar to that observed in Cyp27b1+/+ mice. Bone samples of Cyp27b1–/–mice exhibited hypocalcification, hypertrophy and irregularity in the growth-plate cartilage, and increased osteoid formation, as well as poor osteogenesis speed and clear lesions, indicating imperfect osteogenesis. These abnormalities were completely restored by ALF and ELD administration, and osteogenesis occurred at the level observed in Cyp27b1+/+ mice. Osteogenic abnormalities involving bone length and bone density in Cyp27b1–/–mice can be sufficiently improved with Ca supplementation [Cyp27b1+/+ mice due to ALF or ELD administration indicates that not only Ca metabolism was maintained, but 1α,25D3 was metabolized and generated from ALF as well as the direct actions of ELD on the bone. Previously, it has been reported that when ATDC5 cells, which can differentiate into cartilage cells, are cultured to differentiate into cartilage cells, the addition of 1α,25D3 markedly inhibits such differentiation [3, but also ALF and ELD, ATDC5 cell proliferation and differentiation were inhibited in a dose-dependent manner Cyp27b1+/inuously –19. Prevntiation . Therefontiation . Accordintiation . By usinCyp27b1–/–mice treated with vehicle, the administration of ALF and ELD allowed these animals to maintain bone strength at a level similar to that of Cyp27b1+/+ mice Click here for additional data file.