Drosophila orthologs of the auxiliary ESCRTs Chmp5 and Ist1, DChmp5 and DIst1, which belong to the two auxiliary sub-complexes. While each single null mutant displayed mild defects in development, the Dist1 Dchmp5 double mutant displayed a severe defect, indicating that the two genes act synergistically, but in separate pathways. Moreover, the presented results indicate that the auxiliary ESCRTs provide robustness against cold during development of diverse poikilothermic organisms, probably by preventing the accumulation of the ESCRT-III core component Shrub on the endosomal membrane.The ESCRT pathway, comprising the in sequence acting ESCRT-0, -I, -II, -III and Vps4 complexes, conducts the abscission of membranes away from the cytosol. Whereas the components of the central ESCRT-III core complex have been thoroughly investigated, the function of the components of the associated two auxiliary ESCRT sub-complexes are not well-understood in metazoans, especially at the organismal level. We here present the developmental analysis of the  Summary: The analysis of Chmp5 and Ist1, which belong to the two ESCRT auxiliary sub-complexes in Drosophila, suggests that these ESCRT proteins provide robustness against cold in diverse poikilothermic organisms. Degradation of transmembrane proteins (TMPs) is initiated by their endocytosis from the plasma membrane into early endosomes  family that includes all ESCRT-III core components  machinery. Five ESCRT complexes [ESCRT-0 to ESCRT-III and the vacuolar protein sorting 4 (Vps4) complex] act one after the other to first concentrate ubiquitylated cargo in budding zones (ESCRT-0 till -II) and then invaginate the membrane spot as an ILV (ESCRT-III and Vps4) . CHMP4/Vmponents . Shrub imponents . Core ES pathway .Ustilago maydis  binds to Doa4-independent degradation 2 (Did2) to facilitate the recruitment of Vps4 to the assembled ESCRT-III complex , while Vo maydis .Drosophila, loss of Dchmp5 function in the follicle epithelium results in elongated stalks caused by a degradation defect of Notch and an over-activation of JAK/STAT signalling . We found that mutants of both genes show a cold-sensitive phenotype. Our findings reveal an unexpected role for the auxiliary ESCRTs during development of Drosophila, which is to neutralise the deleterious accumulation of Shrub at the endosomal membrane at cold temperatures. The cold sensitivity was also observed for CHMP5/Vps60 mutants of the phytopathogenic fungus U.maydis. Thus, it appears that the auxiliary ESCRT complexes confer robustness to the ESCRT mediated process against cold in several poikilothermic eukaryotic organisms.Here, we report the analysis of the function of the two auxiliary ESCRT sub-complexes in Dchmp5 were available in Drosophila melanogaster at the start of our analysis, we generated alleles by imprecise P-element excision of G4547P(EP)CG6259, which is inserted in the second intron of Dchmp5 (CG6259)  A. Subseq deleted A. The pr deleted . Complem9.2Dchmp5 homozygosity caused pupal lethality at 25°C and 29°C d08528-f02548  resembled that of early third instar discs of wild-type flies, since only one instead of two ring-like domains in the proximal wing anlage were present (vgQE) enhancer, was also similar to that of early third instar wing imaginal discs of the wild type , in which the ild type G–I. In aild type J,L. The ild type K,M. The ctivated N,O. Ectosc cells . The sim9.2Dchmp5 mutant clones, shifted to 18°C throughout the third instar larval stage, for further analysis of the Dchmp5 mutant endosomal phenotype. We found that TMPs, such as Notch and Delta accumulated in large vesicles in cells of 9.2Dchmp5 mutant clones . In accordance with a previous report, Shrub was detectable throughout the cytosol in a punctate distribution without any obvious association with Notch-positive endosomes in wild-type disc cells . As observed for Snf7 in yeast, Shrub accumulated on endosomes in Vps4 depleted disc cells , the overall appearance of perature A–F. Howeperature G. The dipe cells I.Fig. 49.2Dchmp5 mutant MVBs . Surprisingly, the MVBs of mutant cells contained significantly more dense material in their lumen, suggesting that ILV formation is slightly enhanced rather than reduced . Judged by the normal localisation of the zonula adherens (ZA) core component DE-Cadherin (DE-Cad), as well as Discs large (Dlg), which localises to the basolateral membrane domain, no polarity defect was detected . In addition, the appearance of the ZA and septate junctions (SJs) looked normal in the TEM, even if the flies were constantly raised at 18°C .The loss of function of ESCRT-I, -II and -III core components results in the loss of epithelial polarity of the imaginal disc cells. We therefore monitored the polarity of shifted Fig. S3A). HA-DChmp5 was able to suppress the endosomal phenotype observed in 9.2Dchmp5 mutant cell clones , indicating that the construct is functional. Similar to Shrub, HA-DChmp5 was evenly distributed throughout the cytosol with no obvious association with endosomes . Overexpression of HA-DChmp5 in the posterior compartment with engrailedGAL4  , or strong ubiquitous overexpression with armGAL4 (not shown), had no detectable effect on wing imaginal disc morphology, size of the Notch-positive endosomes or the activity of the Notch pathway, measured by expression of Wg .We generated and expressed a HA-tagged version of DChmp5 with the Gal4 system to monitor its subcellular localisation and effects of DChmp5 overexpression on endosome maturation  rescued 9.2Dchmp5 mutants to the adult stage  through imprecise excision of the P-element insertion G6550P(EP)CG10103, which is inserted in the 5′ UTR of CG10103 . We obtained 51Dist1, an allele where 972 bp of the coding region including the transcriptional start is deleted . Therefore, 51Dist1 is probably a null allele. Homozygous 51Dist1 flies developed into sterile adults at 29°C (both sexes), but died as pharate adults if raised at 18°C . At 25°C, loss of function of 51Dist1 was semi-lethal. No obvious pattern defects were observed at any temperature. Hence, loss of Dist1 function also caused a cold-sensitive phenotype.To inactivate the other auxiliary ESCRT sub-complex, consisting of Ist1 and Did2, we generated alleles of Fig. S4C–F). Clonal analysis revealed that 51Dist1 mutant cells had normal Notch- and Rab7-positive endosomes whose size and appearance were comparable to that of their wild-type neighbours . The distribution of Shrub was normal in cells of mutant wing imaginal discs . TEM analysis revealed that the size and morphology of 51Dist1 mutant MVBs was normal . Hence, the phenotype of 51Dist1 mutants is considerably weaker than that of 9.2Dchmp5 mutants.We did not observe ectopic activation of the Notch pathway  induction of clones 3 days after egg laying (ael), followed by a shift from 25°C to 29°C for 2 days . Hence, the concomitant loss of function of Dist1 and Dchmp5 results in a failure to remove Shrub from the membrane, suggesting that the function of Vps4 is impaired. In further support of this notion, it has been reported that clones mutant for vps4 also bulge out of the disc epithelium as observed here for the double mutant clones  Indand Rab7 A–F. Impot clones . Interes51 Dchmp59.2Dist1 double mutant clones bulged out of the imaginal disc tissue enabled us to unambiguously identify and analyse these clones in the TEM . It is possible that polarity defects would be present at lower temperatures, if the double mutant cells would survive.In contrast to loss of ESCRT-III function, we found that the apico-basal polarity in the obtained double mutant cells was not affected. The ZA component DE-Cad, as well as the baso-lateral membrane component Dlg were localised normally and the ZA had a normal appearance in the TEM  in the wing imaginal disc. To test whether Myc-Shrub is functional, we induced the wing . In agrent cells E,I. The Saccharomycescerevisiae (www.yeastgenome.org). We therefore generated vps60 (Dchmp5 ortholog) and ist1 mutants in a related well-studied microorganism, the phytopathogen U. maydis  rescues Dchmp5 mutants completely and Dchmp5 Dist1 double mutant to a considerable extent , 9.2 FRT2ADchmp5 (this work), 51 Dchmp59.2 FRT2ADist (this work), 08lgd (4-1 FRTG13shrub (I3vps20 (UAS Myc-shrub (UAS shrub-GFP and UAS shrub-RNAi (UAS vps4-RNAi (VDRC #35126), UAS Vps4-DN-Myc , UAS HA-Dchmp5 (this work). Others: Gbe+Su(H)-lacZ (NRE-pGR (wg-lacZ (hedgehogGAL4 (engrailedGAL4 (BL), tubulinGAL80ts (BL #7018), vgQE . For the generation of mutant alleles, we used for 51Dist1: 1 w*; P(EP)CG10103G6550/TM3, Sb1 Ser1y (BL #27211) and for 9.2Dchmp5: 1 w*; P(EP)CG6259G4547/TM3, Sb1 Ser1y (BL #27160). Oregon R served as wild type. BL=Bloomington Stock Center; VDRC (Vienna Drosophila Resource Center).The following fly stocks were used during this analysis. Mutants: ), lgd08 , shrub4-1 FRTG13 , vps20I3-lacZ , NRE-pGRCG6259 and G6550P(EP)CG10103, respectively. The created mutations were sequenced to define the breakpoints and deletion .The alleles UAS HA-Dchmp5 we used the Dchmp5 cDNA  as a template and fused a SalI restriction site, HA-tag and short linker encoding three glycines N-terminally and a KpnI restriction site C-terminally to the Dchmp5 cDNA by PCR using following oligonucleotides: GGAAGTCGAC ATGTACCCGTACGATGTTCCTGACTATGCGGGCGGAGGGATGAATCGCCTTTTTGGTCGTG and GTCCGGTACCCTATGTGGTCTTCAGCGATGT. This DNA fragment was cloned into the multiple cloning site of pUAST-attB using the added SalI and KpnI restriction sites. pUAST-attB-HA-Dchmp5 was inserted into the genomic landing site attP51C , mouse anti-Nextra (Notch extracellular domain) C458.2H (1:100), mouse anti-Delta C594.9B (1:100), mouse anti-Discs large 4F3 (1:500) and rat anti-DE-Cadherin DCAD2 (1:50). Rabbit anti-Rab7 (1:50) , rabbit Images of wing imaginal discs were obtained at a Zeiss AxioImager Z1 microscope including a Zeiss Apotome and using Zeiss Axiovision. Larvae were imaged at a Zeiss Stemi 2000-C stereomicroscope.E. coli strain C41 (Overexpress) and purified using glutathione Sepharose 4B medium . The verified Shrub was injected into rabbits . The obtained Shrub antiserum was affinity purified and verified by western blotting .GST-tagged Shrub was expressed in the Wing discs were fixed in 2.5% glutaraldehyde, washed in 100 mM phosphate buffer and postfixed in 2% osmium tetroxide in phosphate buffer for 1 h on ice. After contrasting en bloc in 2% uranyl acetate, the specimens were dehydrated in ethanol and embedded in araldite using acetone as an intermediate solvent. Thin sections were stained with 2% uranyl acetate and lead citrate. Sections were observed under an EM 902 (Zeiss) microscope at 80 KV.For the statistical analysis of MVBs their perimeter was measured using ImageSP (TRS & SysProg). The obtained data were statistically analysed using Microsoft Excel and GraphPad Prism 7.0d.To quantify the ILV content, electron dense material within the lumen of photographed MVBs was measured by utilizing a self-written macro for the image processing software ‘Fiji’ . This anE. coli K-12 derivative Top10 (Life Technologies) was used. Transformation and cultivation of E. coli were performed according to standard protocols. Growth conditions and source of antibiotics for U. maydis are described elsewhere (Tables S1–S4). For ectopic expression of Dchmp5, the gene construct was inserted into the ipS-locus. All homologous recombination events were analysed via Southern blot (a1b1) served as template.For cloning, lsewhere . For strern blot . For all600=0.5. For each strain, 50 µl of the overnight culture were serially diluted four times 1:5 with sterile H2O to a final OD600=0.0008. 2 µl of each dilution step were spotted onto culture medium plates supplemented with 1% glucose and incubated either over night at 28°C or for nine days at 10°C. Colony growth was documented with a LAS4000 imaging system .Cultures were grown using conditions described elsewhere . Culture


We observed a synergistic potentiation of osteoclast progenitor cell differentiation and formation of osteoclasts when DEX was added to either D3- or PTH-treated mouse bone marrow cell (BMC) cultures. Cotreatment of DEX with D3 or PTH increased gene encoding calcitonin receptor , acid phosphatase 5, tartrate resistant (Acp5), cathepsin K (Ctsk), and TNF superfamily member 11 (Tnfsf11) mRNA, receptor activator of NF-κB ligand protein (RANKL), numbers of osteoclasts on plastic, and pit formation and release of C-terminal fragment of type I collagen from cells cultured on bone slices. Enhanced RANKL protein expression caused by D3 and DEX was absent in BMC from mice in which the GC receptor (GR) was deleted in stromal cells/osteoblasts. Synergistic interactions between DEX and D3 on RANKL and osteoclast formation were present in BMC from mice with attenuated GR dimerization. These data demonstrate that the GR cooperates with D3 and PTH signaling, causing massive osteoclastogenesis, which may explain the rapid bone loss observed with high dosages of GC treatment.—Conaway, H. H., Henning, P., Lie, A., Tuckermann, J., Lerner, U. H. Glucocorticoids employ the monomeric glucocorticoid receptor to potentiate vitamin D3 and parathyroid hormone–induced osteoclastogenesis.Glucocorticoid (GC) therapy decreases bone mass and increases the risk of fractures. We investigated interactions between the GC dexamethasone (DEX) and the bone resorptive agents 1,25(OH) Glucocorticoids (GCs) are used clinically for their anti-inflammatory and immunosuppressive functions. The long-term use of GCs, or excess of endogenous GCs due to disease in the adrenal gland or because of enhanced ACTH stimulation from the pituitary gland, are associated with detrimental effects in many organs and tissues, including bone and muscles . A commo6Independent of the underlying disease, GC-induced bone loss results in an initial rapid loss of BMD and an increased fracture risk as early as 3–6 mo after initiation of therapy. A lower rate of bone loss is observed after 1 yr . The ear121620Calcr) and acid phosphatase 5, tartrate resistant , both well-recognized markers of mature osteoclasts, and cathepsin K (Ctsk), which is involved in degradation of bone extracellular matrix.Osteoclasts are multinucleated cells derived from mononucleated progenitor cells in the myeloid cell lineage . The proTnfrsf11b; mRNA (encoding OPG)] (29Tnfsf11) mRNA (encoding RANKL) expression ] , 29–31 a OPG)] 29, 32. It pression , 30, 31.al bones . A decreal bones . The facal bones .ex vivo experiments using neonatal mouse calvarial bones, we have observed that addition of dexamethasone (DEX) or hydrocortisone to parathyroid hormone (PTH) or 1,25(OH)2-vitamin D3 (D3) can synergistically potentiate calcium release and bone matrix degradation from parietal, cortical bone , recombinant mouse M-CSF, recombinant extracellular domain of mouse RANKL , recombinant RANK/TNFRSF11A , and the ELISA kits for mouse RANKL and mouse OPG were purchased from R&D Systems . The kit for leukocyte acid phosphatase staining, Sigma 104 phosphatase substrate, Trizol, DEX, hydrocortisone, prednisolone, and RU38486 were from MilliporeSigma ; α-modification of minimum essential medium (α-MEM), and fetal calf serum were from Thermo Fisher Scientific ; Thermo Sequenase- II DYEnamic ET Terminator Cycle Sequencing Kit was from Amersham ; and oligonucleotide primers were from Thermo Fisher Scientific. HotStar Taq Polymerase Kit, QiaQuick PCR Purification Kit and RNeasy Mini Kit were from Qiagen ; DNA-Free and RNAqueous–4PCR Kit were obtained from Ambion ; First-Strand cDNA Synthesis Kit and the PCR Core Kit were from Hoffmann-La Roche . Fluorescent-labeled probes (reporter fluorescent dye VIC at the 5′end and quencher fluorescent dye Tamra at the 3′end), TaqMan Universal PCR Master Mix, and the kits for real-time quantitative PCR were from Thermo Fisher Scientific; culture dishes, multiwell plates, and glass chamber slides were from Thermo Fisher Scientific; suspension culture dishes from Corning ; bone slices and CrossLabs for Culture ELISA (CTX) were from Immunodiagnostic Systems ; and the mouse bone marrow stromal cell line ST-2 was from Riken BioResource Research Center Cell Bank . D3 was a kind gift from Hoffmann-La Roche or purchased from MilliporeSigma. D3, DEX, hydrocortisone, prednisolone, and RU38486 were dissolved in ethanol. All other compounds were dissolved either in PBS or culture medium.Mouse OPG fused to human IgGdimGR mice and their corresponding wild-type mice have been previously described and were backcrossed to the FVB/N background as described in Rauch et al. , incubated overnight in α-MEM/10% FBS, and subsequently cultured in the same medium with or without hormones and test substances, with concentrations as indicated in legends to figures, for 7–9 d, with medium changed every third day. After this interval, the cells were fixed with acetone in citrate buffer/3% formaldehyde and stained for TRAP. TRAP-positive cells with 3 or more nuclei were considered osteoclasts, and the number of multinucleated osteoclasts was counted (TRAP+ MuOCL). In parallel experiments, cells were harvested for gene expression analyses.Femurs and tibiae from 5- to 7-wk-old male mice were dissected and cleaned from adhering tissues. BMCs were seeded in 48 or 96 multiwells  were placed in 96 multiwell plates and 50 μl α-MEM/10% FBS was added, and bone slices were incubated for 15 min at 37°C. Then, 50 μl α-MEM/10% FBS containing 4 × 106 cells/cm2) were seeded in 25-cm2 flasks in α-MEM/10% FBS and cultured for 2 wk with medium changed every 3 d. After the 2-wk period, cells attached to the bottom of the flasks were detached with trypsin and seeded (2 × 104 cells/cm2) as bone marrow stromal cells in 48 multiwell plates and incubated with or without D3 and DEX. After 24 or 48 h, RNA was isolated for gene expression analyses. The ST-2 cells were seeded in 24 multiwell plates at a density of 2 × 104 cells/cm2 and incubated in α-MEM/10% FBS overnight. Following incubation, medium with and without test substances was added and the cells incubated for 24 h for subsequent gene expression analyses.BMCs Real-time quantitative PCR analyses were performed using the TaqMan Universal PCR Master Mix and a sequence detection system . All genes were analyzed using primers and probes that have been previously specified .6 cells/cm2. The cells were allowed to settle overnight before medium was changed, then cultured for an additional 6 d, with a change of medium after 3 d. After the 6-d culture, cells were washed in PBS and lysed in Triton X-100 (0.2% in H2O). Following centrifugation, supernatant was collected and analyzed for TRAP activity using para-nitrophenyl phosphate as substrate at pH 4.9, in the presence of tartrate (0.17 M). The activity of the enzyme was assessed as the OD405 of liberated para-nitrophenol. Enzyme assays were performed under conditions in which the reactions were proportional to the amount of enzyme and reaction time.BMCs were isolated, as described above, and seeded in 24- or 48-well plates at a cell density of 10BMC were cultured in 24 multiwell plates for 6 or 12 d in the absence and presence of hormones and test substances. Measurements of OPG and RANKL protein synthesis were assessed by analyzing the levels of OPG and RANKL in BMC and in culture medium using commercially available ELISAs. At the end of the incubations, proteins were extracted using 0.2% Triton X-100 for 24 h at room temperature, and the BMC extracts and media were analyzed by ELISA according to the manufacturer’s protocol.post hoc Bonferroni’s, or where appropriate, Dunnett’s T3 test or the independent samples Student’s t test . All experiments have been performed at least twice with comparable results, and all data are presented as the means ± sem.All statistical analyses were performed using 1-way ANOVA with Levene’s homogenicity test, and −8 M) to mouse BMCs resulted in enhanced formation of TRAP+MuOCL, which was maximal at d 5–7 and thereafter declined (−7 M) alone marginally enhanced osteoclastogenesis, and no TRAP+MuOCL were observed in untreated controls (unpublished results). When DEX was added to D3-stimulated cultures, a synergistically enhanced number of TRAP+MuOCL was observed at d 6–8  and hydrocortisone (10−6 M) (+MuOCL (The strong potentiation of TRAP(10−6 M) A. Simila (+MuOCL B.+MuOCL formation was observed in cells cotreated with D3 (10−8 M) and DEX (10−7 M)  D, left.  on bone D, right. on bone E. In D3- on bone E. In agr on bone F.−8 M) and DEX (10−7 M) resulted in a robust synergistic enhancement at d 6 of Calcr mRNA , Acp5 (encoding TRAP) and Ctsk (encoding cathepsin K) expression  than either that of Acp5 or Ctsk . The synergistic up-regulation of the expression of Calcr and Acp5 mRNA was concentration dependent with substantial effects at 10−9 M and higher concentrations of DEX  caused a 6-fold stimulation of Tnfsf11 mRNA (encoding RANKL) in the BMC cultures at d 3 and 6 (−7 M) did not cause any effect by itself, but cotreatment with D3 and DEX caused robust, 19- and 24-fold enhancements of Tnfsf11 mRNA at d 3 and 6, respectively. D3, and cotreatment with D3 and DEX, robustly decreased the time-dependent increase of Tnfrsf11b mRNA (encoding OPG) seen in control cultures over time (−7 M) with D3 caused a slight increase of Tnfsf11 mRNA at 6 h and a strong induction at 24 h .The interaction between GCs and D3 in the BMC cultures may either occur at the level of RANKL- and OPG-expressing stromal cells, at the level of osteoclast progenitors, or may be due to effects mediated by other hematopoetic cells present. Therefore, it was evaluated if DEX affected D3 expression of RANKL and OPG. D3 (10ver time B. In sep at 24 h A. The exwith DEX B. Synergtrations C. The st−8 M) 1.5-fold, but no effect by DEX (10−7 M) was observed (RANKL protein in cell lysates from BMC was slightly increased by D3 (10observed D. Cotreaobserved E. OPG probserved F. OPG probserved G.−7 M) on TRAP+MuOCL formation was inhibited by OPG (300 ng/ml) and by soluble RANK (1 μg/ml) (Acp5 and Ctsk induced by D3+DEX (Tnfsf11 or Tnfrsf11b mRNA expression (The stimulatory effect by cotreatment with D3 and DEX (101 μg/ml) H, I. OPGy D3+DEX J, K withpression L, M.Csf1; encoding CSF1 or M-CSF) mRNA was strongly inhibited by D3 as well as by D3+DEX (The expression of colony-stimulating factor 1 (y D3+DEX C.Nr3c1 (encoding GR) was not significantly regulated by D3, DEX, or by cotreatment with D3+DEX (Vdr) was also not regulated by D3 and DEX nor by cotreatment with D3+DEX (The mRNA expression of h D3+DEX A. The vih D3+DEX B.−8 M) stimulation of the number of TRAP+MuOCL formation was enhanced by costimulation with DEX (10−7 M) (+MuOCL was observed in unstimulated or DEX-stimulated cultures (unpublished results). The TRAP+MuOCL in PTH+DEX-treated cultures were substantially larger than those in PTH-stimulated BMC cultures  plus PTH (10−8 M) further enhanced TRAP+MuOCL formation   was not significantly regulated by PTH, DEX, or by cotreatment with PTH and DEX (Pthr1) mRNA expression, but neither PTH nor PTH+DEX significantly affected Pthr1 mRNA expression , which was potentiated by cotreatment with DEX (10−7 M) in purified primary bone marrow stromal cells  caused a robust, concentration-dependent stimulation of Tnfsf11 mRNA (−10 M D3. Cotreatment of the ST-2 cells with DEX (10−7 M) synergistically potentiated Tnfsf11 mRNA at all concentrations of D3. D3 inhibited Tnfrsf11b mRNA in a concentration-dependent manner at and above 10−10 M in the ST-2 cells (−7 M) robustly decreased Tnfrsf11b mRNA and slightly enhanced the inhibitory effect by D3 at different concentrations  in ST-2 cells was concentration dependent with the EC50 at ∼3 × 10−9 M DEX (−8 M) on Tnfrsf11b mRNA was not further enhanced by DEX at different concentrations  on D3 (10−8 M) that stimulated formation of TRAP+MuOCL in BMC cultures was abolished by the GR antagonist RU 384 (10−6 M), with no effect observed by the antagonist on TRAP+MuOCL formation induced by only D3 (−7 M) (The potentiation by DEX (10 only D3 B. Additi3 (−7 M) C.−8 M) and DEX (10−7 M) was abolished by RU 384  GCs do not induce osteoclast formation in BMC cultures but synergistically interact with D3 and PTH to potentiate osteoclast formation, 2) GCs interact with D3 and PTH to increase mRNA and protein expression of RANKL in bone stromal cell, and 3) the effects of GCs are due to GRs present in bone marrow stromal cells expressing Runx2, which act through GR homodimers.Chronic treatment with GCs is associated with decreased bone mass, secondary osteoporosis, and an increased risk for skeletal fractures soon after the onset of treatment. The early loss of bone mass is partially explained by increased bone resorption, whereas the long-term effects are mainly attributed to decreased bone formation . The enh29We initially observed that DEX did not stimulate osteoclast formation in BMC cultures performed either on plastic dishes or on bone slices. This was surprising because GCs have been described to increase the RANKL/OPG ratio by decreasing OPG , 29–32 a29ex vivo in neonatal mouse calvarial bones  indicates that monomeric GR action is mediating this effect, either by binding to GRE half-sites  stimulating RANKL and decreasing OPG formation, 2) synergistically interacting with D3 and PTH to potentiate RANKL formation, and 3) enhancing the bone resorbing effect of mature osteoclasts. In Observations made by us and others in different In summary, we found that osteoclast formation was potentiated when GCs were added with either D3 or PTH to BMC cultures and that this stimulation is due to synergistic potentiation of RANKL formation and dependent on activation of monomeric GRs in bone marrow stromal cells.http://www.fasebj.org to obtain this information.This article includes supplemental data. Please visit Click here for additional data file.


Furthermore, serum 25(OH)D levels were negatively associated with Bone Surface/Bone Volume (BS/BV)  and together with bone CYP27B1 and CYP24A1 mRNA accounted for 10% of the variability of BS/BV (p = 0.001). These data demonstrate that serum 25(OH)D is an independent positive predictor of micro-structural and bone formation measures and may be dependent, in part, on its metabolism within the bone.Vitamin D, along with calcium, is generally considered necessary for bone health and reduction of fractures. However, he effects of improving vitamin D status have not always been observed to improve bone mineral density (BMD). We have investigated whether varying vitamin D status in humans, as measured by serum 25(OH)D levels, relate to micro-structural and histomorphetric measures of bone quality and quantity, rather than density. Intertrochanteric trabecular bone biopsies and serum samples were collected from patients undergoing hip arthroplasty  at Royal Adelaide Hospital. Estimated GFR, serum ionized calcium, alkaline phosphatase, albumin, supplement and medication intake prior to surgery were taken from patient case records. Serum 25(OH)D, 1,25(OH)2D, and parathyroid hormone (PTH) levels were measured by immunoassays. Trabecular bone structural indices were determined by high-resolution micro-CT. Mean wall thickness (MWT) was measured on toluidine blue-stained histological sections. Bone mRNA levels for vitamin D metabolising enzymes CYP27B1 and CYP24A1 were measured by qRT-PCR. While serum 25(OH)D levels did not associate with bone volume/tissue volume (BV/TV%), serum 25(OH)D levels were strongly and independently associated with MWT ( Vitamin D insufficiency and inadequate dietary calcium intake are prevalent worldwide and considered to be a public health issue. Inadequate vitamin D levels have been associated with poor muscle function, increased risk of falls, and osteoporotic fractures. Randomized controlled trials of vitamin D and calcium supplementation show divergent results depending on the clinical outcomes investigated. Studies which have evaluated bone mineral density as an end-point show minimal effect of supplementation  whereas 3 (25(OH)D) and not 1,25-dihydroxyvitamin D3 2D), the active hormone, an apparent conundrum for the endocrine paradigm of vitamin D activity. In fact, the administration of 1,25(OH)2D has been shown to be detrimental for bone health [2D and 25-hydroxyvitamin D 24-hydroxylase (CYP24A1), the enzyme that catalyzes the degradation of 1,25(OH)2D as well as vitamin D receptor (VDR) [2D levels, which provides a physiological context for this clinical observation, i.e., beneficial effect of prohormone 25(OH)D [All current clinical evidence indicates that the beneficial effects of vitamin D on bone are related to the serum levels of the prohormone 25-dihydroxyvitamin De health . Direct or (VDR) . The dem 25(OH)D .2D, 25(OH)D, parathyroid hormone (PTH), bone turnover markers on femoral bone structure in a cross-sectional study of elderly hip fracture patients. We also investigated the contribution of the bone CYP27B1 and CYP24A1 mRNA levels on the role of 25(OH)D in determining bone structure variables. We hypothesize that adequate vitamin D status results in local metabolism of 25(OH)D to 1,25(OH)2D within bone, resulting in measures of improved bone health.The aim of the present study was to investigate the relationships between serum 1,25(OH)Intertrochanteric trabecular bone biopsies, serum and presurgical radiographs were collected from 103 patients  undergoing hip arthroplasty at Royal Adelaide Hospital  between August 2015 and January 2017 due to experiencing a fracture of the neck of femur (NOF). During preoperative preparation, patients or next of kin were asked to participate in this study, and written consent was obtained. Date of surgery, patient age, weight, height, comorbidities, eGFR (MRD-EPI equation), medication history, total plasma levels of ionized calcium (estimated) [2D, intact PTH, osteocalcin and bone-specific alkaline phosphatase.Single blood specimen was collected at the time of surgery in SST tubes (serum separator tubes), centrifuged at 5000 rpm for 10 min to collect serum and stored at −80 °C until required. Serum samples were analyzed by chemiluminescent immunoassay (Diasorin Liaison) for 25(OH)D, 1,25(OH)The trabecular bone biopsies were cut into two halves with one half fixed in formalin  for 7 days at 4 °C followed by storage in 70% ethanol at room temperature until microstructural analyses were conducted. The other half was submerged in RNA later and stored at −80 °C for gene expression analyses.3; range: 291.7–1078.8 mm3); hence, using a uniformly sized region of interest could have potentially oversampled the shorter bone and undersampled the longer bone. Hence, a slice-by-slice analysis tracing approach was adopted to maximize the volume available for analysis which included all trabecular bone and excluded cortical bone and marrow space. Morphometric indices were determined using an adaptive thresholding algorithm with a pre-thresholding of 60 to 255 and a radius of 8. Variables such as bone volume/tissue volume (BV/TV%), trabecular number (Tb.N), and trabecular separation (Tb.Sp) describe trabecular bone quantity or mass. BV/TV% and Tb.N are measures of the amount of trabecular bone within a selected volume of cancellous tissue. However, in addition to the quantity of bone, the trabecular bone quality plays a critical role in determining bone strength and is determined by the three-dimensional orientation and connectivity of individual trabeculae. Trabecular bone quality measures include bone surface/bone volume ratio (BS/BV) and trabecular thickness (Tb.Th). The measurement of BS/BV provides an integrated measure of thickness and texture of the bone.Trabecular bone specimens were assessed for bone mass and bone quality variables with the 1076 Bruker micro-CT imaging system. The X-ray tube was operated at 60 KV and 167 µA with a resolution of 18 µm with a 0.5 mm aluminum filter and exposure time of 1770 ms. The images were reconstructed using the CTAn software  after correcting for smoothing (1), beam hardening (30%) and ring artifact reduction (20). The specimens varied considerable in volume (1093.7 ± 511.2 mmv/v polyethylene glycol (PEG) and stored at room temperature for 10–14 days. Resin embedding then occurred by a solution of 100% MMA, 10% PEG and 0.4% peroxydicarbonate. Biopsies were submerged in 5 mL of this solution and placed in at 37 °C for 24 h to allow the resin to harden. A volume of 5 µm thick decalcified sections were cut and stained by Haematoxylin and Eosin [Trabecular bone biopsies were then processed through 70% to 100% ethanol to dehydrate the sample and then placed in 5 mL solution containing 100% methylmethacrylate (MMA) and 10% nd Eosin . Mean wand Eosin ). Wall tCortical bone measurements: Anterior-Posterior plain radiographs of the contralateral uninjured limb were used to measure average medial (M) and lateral (L) cortical thicknesses and M-L medullary width and femoral width using CARESTREAM software .Total RNA was extracted from the pulverized bone using the TRIZOL reagent , and cDNA samples were synthesized . The mRNA expression of genes of interest was measured by real-time PCR using iQ SYBR Green Supermix , as per the manufacturer’s instructions. Relative gene expression was quantified delta-CT method and normalizing to the expression of β-actin mRNA. The primer sequence for CYP27B1 is TGGCCCAGATCCTAACACATTT (forward) GTCCGGGTCTTGGGTCTAACT (reverse) CYP24A1 CCTGCTGCCAGATTCTCTGGAA (forward) TTGCCATACTTCTTGTGGTACTCC (reverse) and Beta actin CTGCCCTGAGGCACTCTT (forward) AGTTTCGTGGATGCCACAGG (reverse).Statistical analysis was performed using the SPSS statistical Package . Some of the variables were not normally distributed, hence values are expressed as median and interquartile range. Spearman’s correlation coefficient was calculated to determine correlations between structural, metabolic and gene expression variables. The metabolic variables which correlated with structural variables were tested through step-wise multiple linear regression analyses to identify metabolic determinants of bone mass and bone quality variables. Independent samples Kruskal–Wallis test was used to compare bone structural and quality variables between various 25(OH)D intervals. The significance level was set at 0.05.2D  suggesting substrate insufficiency in a significant proportion of the patients. Serum 25(OH)D levels were not associated with eGFR or other measured biochemical parameters. However, serum 1,25(OH)2D levels were determined by a combination of serum 25(OH)D, PTH levels and eGFR   and eGFR and PTH , consistent with a decline in renal function with age resulting in higher PTH values, which contributed to increased serum 1,25(OH)2D levels. Serum osteocalcin, a marker of bone turnover, correlated negatively with eGFR  suggesting that compromised kidney functions in these patients is associated with higher bone turnover.The median serum 25(OH)D levels were 52.7nmol/ L . Serum 2 < 0.01) . Negativr = 0.19, p = 0.06). Neither serum 1,25(OH)2D or PTH correlated with BV/TV%, Tb.N or Tb.Th. Mean wall thickness (MWT), a dynamic marker of completed bone formation, increased consistently and significantly with increasing vitamin D levels   D determined MWT values in a multiple linear regression model , indicative of higher vitamin D levels resulting in more plate-like structures. However, the BS/BV values did not reach statistical significance when divided by serum 25(OH)D intervals . With respect to cortical bone measurements, serum 25(OH)D levels correlated negatively with total femoral diameter  and trend only for medullary width . None of the cortical parameters differ significantly across vitamin D intervals D levels did not correlate with the bone structure variables of BV/TV%, Tb.Th and Tb.N by either linear analyses nor when serum 25(OH)D levels were grouped by intervals of 25(OH)D adequacy ranging from deplete to replete A–C. HoweD levels . MWT tre = 0.12) . Further 0.0001) . Althougon model . Bone suntervals E. Neithentervals F,H. Neitp = 0.02) and CYP27B1 mRNA negatively; as a trend, only (p = 0.1) contributed to the serum 25(OH)D determination of BS/BV D with either MWT or BS/BV, multiple linear regression analyses were carried out to investigate the variance accounted by these measures. Bone CYP27B1 and CYP24A1 mRNA levels did not contribute to the serum 25(OH)D determination of MWT . Howeverof BS/BV .2D synthesis and for intestinal calcium absorption [Vitamin D deficiency, as measured by serum 25(OH)D levels, has been shown to be associated with increased risk of hip fracture in the elderly . Despitesorption . In clinsorption  and the 2D synthesis. The association between eGFR and circulating 1,25(OH)2D levels in this elderly cohort is consistent with the role renal function has in determining vitamin D status [2D levels are determined by eGFR, in addition to PTH and 25(OH)D levels, no bone parameters were determined by serum 1,25(OH)2D levels, suggesting that changes to 25(OH)D levels were more important to determine MWT and BS/BV.An incomplete understanding of the mechanistic actions of how vitamin D, in particular 25(OH)D, mediates positive effects on the skeleton also contributes to the uncertainty for the need to recommend levels of serum 25(OH)D, level above which is required for renal 1,25(OH)D status . Interesp = 0.021), when we adjusted for multiple comparisons, the significance was (p = 0.2), perhaps due to smaller number of participants per group. BS/BV, an indicator of texture and strength of bone, was negatively associated with 25(OH)D levels suggesting that patients with adequate vitamin D status had plate-like structure consistent with higher bone strength. Significantly lower BS/BV and Tb.Pf values were reported in a recent clinical study examining the effect of bisphosphonate treatment on femoral head trabecular bone micro-architecture in non-treated fracture controls as compared to hip-fracture patients. Authors concluded that lower values for both the variables suggest preserved connectivity of trabecular bone in the bisphosphonate group. More importantly, Tb.Pf and BS/BV were strongly correlated with measures of bone strength [The strong positive relationship between serum 25(OH)D levels and MWT suggests that osteoblastic bone formation was improved in the presence of higher serum 25(OH)D levels. Serum 25(OH)D levels above 50 nmol/L significantly increase MWT. While it is possible that serum 25(OH)D levels of 70 nmol/L and above also increase MWT group 2D [2D from 25(OH)D via CYP27B1 activity, which directly promotes mineralization [We have previously proposed, using animal and cell culture studies, that the direct supply of 25(OH)D to bone is required for local 1,25(OH)ynthesis ,17 and cynthesis  and resoynthesis ,20 proce25(OH)2D . We have25(OH)2D . Furtherlization . While tlization .2D levels to improve bone health.In conclusion, we have demonstrated that higher serum 25(OH)D levels are associated with improved bone formation. The association of serum 25(OH)D levels with bone quality variables of MWT and BS/BV, rather than bone mass variables, suggests that determining the efficacy of vitamin D supplementation on bone health should take in consideration measures of bone quality beyond bone mineral density. Furthermore, higher 25(OH)D along with higher expression of bone CYP27B1 and lower expression of CYP24A1 is associated with stronger plate-like structure and supports the paradigm of bone supply of 1,25(OH)


The Focal Adhesion Targeting (FAT) domain of Focal Adhesion Kinase (FAK) is a promising drug target since FAK is overexpressed in many malignancies and promotes cancer cell metastasis. The FAT domain serves as a scaffolding protein, and its interaction with the protein paxillin localizes FAK to focal adhesions. Various studies have highlighted the importance of FAT-paxillin binding in tumor growth, cell invasion, and metastasis. Targeting this interaction through high-throughput screening (HTS) provides a challenge due to the large and complex binding interface. In this report, we describe a novel approach to targeting FAT through fragment-based drug discovery (FBDD). We developed two fragment-based screening assays—a primary SPR assay and a secondary heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) assay. For SPR, we designed an AviTag construct, optimized SPR buffer conditions, and created mutant controls. For NMR, resonance backbone assignments of the human FAT domain were obtained for the HSQC assay. A 189-compound fragment library from Enamine was screened through our primary SPR assay to demonstrate the feasibility of a FAT-FBDD pipeline, with 19 initial hit compounds. A final total of 11 validated hits were identified after secondary screening on NMR. This screening pipeline is the first FBDD screen of the FAT domain reported and represents a valid method for further drug discovery efforts on this difficult target. Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor tyrosine kinase overexpressed at the protein and mRNA levels in over 80% of solid malignant tumors . NumerouAn alternative drug-targeting approach is focusing on the FAT domain, a four-helical bundle, which has been shown to be involved in multiple protein-protein interactions at the focal adhesion site . FAK locFragment-based drug discovery (FBDD) is an alternative approach that can be utilized in properly targeting PPIs ,28,29, iIn this paper, we report the development and optimization of a primary surface plasmon resonance (SPR) assay and a secondary heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) assay for the detection of fragment binders to the FAT-Paxillin binding interface. We show the development of several FAT constructs, including two single mutants and a double mutant, as controls for the SPR assay. We show the optimization of the SPR assay by testing different buffers and their effect on LD2 binding. We also report backbone resonance assignments of the human FAT domain (S892–H1052) sequence for HSQC-NMR studies. Finally, we screen a pilot (189 compound) fragment library through SPR and validate the hits through HSQC-NMR with a final hit count of 11.Fragment-based screening requires very sensitive technologies to detect the weaker binding of fragments. Surface plasmon resonance has the capacity and sensitivity to detect such binding, making it a suitable platform to develop a fragment screening assay . To deveeq at 80 RU, potentially attributable to an increased solubility of LD2. The optimal and final buffer chosen for all future studies was 100 mM Tris pH 8.0, 200 mM NaCl, 0.5% Tween-20, and 5% DMSO (Buffer 6).Buffer conditions are critical for SPR-based assays. Inadequate buffers can damage immobilized protein, promote analyte insolubility, and produce inaccurate responses. To optimize the buffer conditions, biotinylated AviTag FAT was immobilized onto a single channel on the SADH SPR chip, and LD2 (50 µM) binding to FAT was tested in various buffer conditions A. BufferD from kinetic fitting of a single injection of an automatic concentration gradient  HSQC are exchange broadened beyond detection for residues S889, S978, N991, K1002, T1010, S1011, L1012, K1018, L1021, and K1044, indicating the presence of internal motional modes with correlation times in the µs to ms range. The location of α-helices based on chemical shift index (CSI) and characteristic sequential and medium range NOEs are in very good agreement with X-ray crystal structure . Bu. Bu36]. The 29 available compounds, which includes the 19 curated SPR hits, were run at 1 mM through HSQC-NMR as a secondary assay to validate the hit identification from SPR. Four representative HSQC-NMR overlays are presented in FAK is critical in cancer development due to its function as a central component to multiple oncogenic signaling pathways ,5,6,7. TD around the same range as FCI. This switch to OneStep allowed us to conserve reagents and time while providing a relatively accurate estimate for KD compared to a fixed concentration injection. We then optimized buffer conditions, and interestingly, Buffer 3 containing Triton X-100 demonstrated non-ideal binding kinetics and non-specific binding with no LD2 present. We hypothesize that the structure of Triton X-100 is conducive to non-specific interactions with our immobilized AviTag FAT construct, making Tween-20 the better detergent to use. Intriguingly, Buffer 6 containing 5% DMSO had the highest binding response of all buffers tested. We speculate the higher DMSO concentration allows for greater LD2 solubility promoting more binding interactions. Following the mutant study with LD2, results showed that LD2 has a higher selectivity ratio for L994E (3) than I936A (1.5) indicating preferential binding of LD2 to the 2-3 binding site over the helix 1-4 site.To develop a SPR-based FAT FBDD screening platform, we first designed an AviTag construct of FAT and adopted the OneStep injection method. The comparison of OneStep to FCI validated the OneStep injection, with a KTo further validate any fragment hit from SPR, a secondary HSQC-NMR assay was developed, this provided an orthogonal fragment screening platform for FAT drug discovery. In the LD2 titration on our HSQC-NMR assay, the helix 2-3 binding site has almost twice the amount of CSPs over the helix 1-4 binding site further strengthening the argument that the 2-3 site has a higher affinity for LD2. The backbone resonance assignments acquired are also the first reported on human FAK. The FAT construct used had an extra 30 amino acids on the N-terminal from previously determined avian FAT constructs used for resonance assignments, making it the largest FAT construct to have backbone assignments reported. NMR also demonstrated that FAT forms a dimer in solution consistent with a previously reported crystal structure (PDB ID: 1K04) . The dimD > 100 µM with a good majority being in the low mM range. These low affinity fragments still produce a detectable response on SPR with binding curves capable of being fit through a kinetic model. Through HSQC-NMR, CSPs of low affinity compounds are still measurable. Such high sensitivity assays are required to properly detect fragment hits. Remarkably, a couple fragment hits exhibit similar binding trends to LD2, where they have a higher selectivity ratio for L994E over I936A, indicating preferential binding to the 2-3 site, such as compounds 033 and compounds 009 shown in The results of our fragment library screen demonstrate the sensitivity of both assays. Due to their small inherent size, fragments typically have weaker binding affinities. As shown in Hit selection was based purely on the binding curve to the WT, however selectivity ratio and binding affinity to the mutants are key in prioritization of the fragment hits. Certain fragment hits such as compound 168 demonstrate higher selectivity ratios to the single mutant than to WT, perhaps representative of significant conformational changes in the double mutant protein. Other fragments like compound 141 have selectivity ratios less than one for the single mutants, which could suggest allosteric binding to FAT. However, due to the weak estimated binding affinity of this compound (>8 mM), it was difficult to observe CSPs in HSQC NMR and thus could not identify an alternative binding site. While allosteric binding could be a potential approach to inhibiting the FAT-Paxillin interaction, direct competitors will be prioritized in future studies making selectivity ratio key for compound selection and iterative drug discovery.Notably, all fragment binders showed CSPs in the same region of the 2-3 binding site on their HSQC maps. Residues L959, E956, and K955 consistently show CSPs on fragment hits, however other residues on a different region of the helix 2-3 site such as L964 and A966 show none. Our NMR assay can be used to perform a second site screen where we search for binders that produce CSPs on this secondary region within the helix 2-3 site. If such compounds are found, linking of fragment hits from both regions in the helix 2-3 site could produce a more potent inhibitor.To conclude, we were able to successfully develop two orthogonal fragment screening assays in SPR and NMR and screened a small fragment library as a pilot, representing the first FBDD screen against the FAT domain. Furthermore, we obtained nearly complete backbone resonance assignments for the human FAT domain encompassing residues S892-H1052 from full length FAK. The pilot screening of this fragment library allows for a great starting point in the FBDD pipeline. Out of the 189 fragments screened, a final 11 compounds are suitable starting points for further optimization through medicinal chemistry. Our next steps will be a fragment screen with a larger fragment library to potentially identify new fragments with alternative chemotypes and different binding sites. Also, we intend to grow out the fragments by SAR to enhance the binding affinity to FAT. Furthermore, we will explore X-ray crystallography studies to identify the three-dimensional conformation of our fragment hits in the binding pocket. Using HSQC-NMR, we can perform second site screening of the helix 2-3 binding site to find linking partners to our current fragment hits . The neaOligonucleotides (forward 5′-GCTACCATGGGCAGCAGCCATCATCATCATCATCACAG-CAGCGGCCTGGTGCCGCGCGGCAGCGGCCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAACATATGCTCG-3′ and reverse 5′-CGAGCATATGTTCGTGCCATTC-GATTTTCTGAGCCTCGAAGATGTCGTTCAGGCCGCTGCCGCGCGGCACCAGGCCGCTGCTGTGATGATGATGATGATGGCTGCTGCCCATGGTAGC-3′) containing the AviTag sequence (GLNDIFEAQKIQWHE) , a hexahmaxR is the highest possible SPR response given by an analyte binding to an immobilized protein. proteinR is the response change attributed to the immobilization of the ligand. rM are the molecular masses of analyte and protein. proteinV is the number of binding sites per protein available for analyte binding. Through this equation, by aiming for an maxR of around 150 RU, the above immobilization levels were derived for peptide and fragment studies. All samples tested were diluted in Buffer 6 injected using the OneStep gradient injection method at a flow rate of 150 µL/min. 3% sucrose was utilized as a bulk standard control for OneStep injection and a DMSO calibration curve was performed using a concentration range of 3.5% to 6.5% DMSO. Fragments were tested for solubility at 1 mM in the above buffer by centrifugation. SPR data was processed through Qdat software (ForteBio) allowing for the normalization of the baseline prior to injection, alignment of the channels, subtraction of the reference channel, and blank subtraction. Kinetic data were fitted to a pseudo-first order 1:1 interaction binding model to calculate DK. Mutant selectivity ratios were calculated by the following equation, where eqR represents level of response achieved at binding equilibrium.SPR binding studies were performed on a ForteBio Pioneer FE (Fragment Edition) SPR system utilizing a SADH Streptavidin-coated Dextran Hydrogel biosensor . SPR running buffer was optimized and the final buffer chosen was 100 mM Tris-HCl, 200 mM NaCl, 0.05% Tween-20 (Buffer 6). Each biotinylated Avitag-FAT construct to be tested was diluted in Buffer 6 and injected at 10 µL/min to achieve approximately 1000 RU of immobilized protein for peptide studies during optimization and 7000 RU for fragment screening. Channel 2 was always left empty as the reference control. Immobilization levels were decided based on the following equation:DK is the equilibrium dissociation constant.Ligand efficiency (LE) of binding fragments was calculated by the following equation, where R is the ideal gas constant, T is the analysis temperature in Kelvin, HAC is the integer value of non-hydrogen (heavy) atoms in the molecule, and 15N]-labeled FAT, encompassing residues S892-H1052, is expressed using minimal media with enhanced M9 salts: . Purified 15N labeled FAT was buffered exchanged into thrombin cleavage buffer . Buffer exchanged FAT was incubated with 3 mL of Thrombin beads  at a concentration of 1 mg/mL. The thrombin reaction proceeds shaking for 6 h at room temperature. Thrombin beads were removed through centrifugation at 500 g for 2 min. Supernatant was collected and incubated with Ni-NTA beads to remove uncleaved protein and HisTag remnants. Cleaved protein was gel filtered through a HiLoad Superdex 75 pg 26/60 column into NMR buffer . -labeled 19 kDa FAT-892 (comprising residues Ser892 to His1052 of full-length protein) was prepared similarly at 0.5 mM concentration in 90% H2O/10% D2O, 100 mM Tris pH 8.0, 100 mM NaCl, 2 mM DTT, 50 µM DSS and 0.02 % sodium azide. An isotropic overall rotational correlation time of about ~16.5 ns was inferred from average backbone 15N spin relaxation times -NOESY-[NH/CH] (τmix = 70 ms). Side-chain 1Hα, 1Hβ assignments were obtained from 3D HBHA(CO)NH. The location of α-helices was identified based on a consensus chemical shift index (CSI) [NMR experiments needed for backbone assignments were acquired at 37 °C on Agilent  DD2 600 MHz and Varian INOVA 750 MHz spectrometers  equipped with cryogenic and room temperature ROSA 6.6  and analROSA 6.6  and CARAROSA 6.6 . 1H chembackbone  and 13Cβex (CSI)  and patt15N FAT concentration of 100 µM. Fragments were screened at a concentration of 1 mM with a DMSO concentration of 1%. LD2 titration reported in 1H and 15N, respectively. Each 2D experiment was collected with spectral widths of 14.03 and 30.0 ppm, 2048 and 256 points respectively, and 16 scans per FID averages [Two-dimensional HSQC-NMR samples are all prepared at a averages ,47,48,49Mutation primers for L994E (5’-CTTGTTGATGAGCTCACCCTCGTCAGAGTTCAATAGC-TTC-3’ and 5’-GAAGCTATTGAACTCTGACGAGGGTGAGCTCATCAACAAG-3’), I936A (5’-GATTTTACTGGACATCTCGGCGACAGCTTTCACCAGGCCC-3’ and 5’-GGGCCTGG-TGAAAGCTGTCGCCGAGATGTCCAGTAAAATC-3’), and I936E (5’-GGATTTTACTGGA-CATCTCCTCGACAGCTTTCACCAGGCCC-3’ and 5’-GGGCCT-GGTGAAAGCTGTCGAG-GAGATGTCCAGTAAAATCC-3’) were designed through Agilent Technologies . The Agilent QuikChange Lightning kit was used to complete the mutations. Mutations were verified through sequencing and each new construct was purified and biotinylated in the same way as the wild type construct.


CDKN2A/ARF/CDKN2B genes encoded at chromosome 9p21 have been described in both pediatric and adult acute lymphoblastic leukemia (ALL), but their prognostic value remains controversial, with limited data on adult T-ALL. Here, we investigated the presence of homozygous and heterozygous deletions of the CDKN2A/ARF and CDKN2B genes in 64 adult T-ALL patients enrolled in two consecutive trials from the Spanish PETHEMA group. Alterations in CDKN2A/ARF/CDKN2B were detected in 35/64 patients (55%). Most of them consisted of 9p21 losses involving homozygous deletions of the CDKNA/ARF gene (26/64), as confirmed by single nucleotide polymorphism (SNP) arrays and interphase fluorescence in situ hybridization (iFISH). Deletions involving the CDKN2A/ARF/CDKN2B locus correlated with a higher frequency of cortical T cell phenotype and a better clearance of minimal residual disease (MRD) after induction therapy. Moreover, the combination of an altered copy-number-value (CNV) involving the CDKN2A/ARF/CDKN2B gene locus and undetectable MRD (≤ 0.01%) values allowed the identification of a subset of T-ALL with better overall survival in the absence of hematopoietic stem cell transplantation.Recurrent deletions of the The online version of this article (10.1186/s13045-018-0639-8) contains supplementary material, which is available to authorized users. At present, treatment response based on minimal residual disease (MRD), monitoring for early and accurate identification of high-risk patients in whom treatment might be intensified, represents a milestone in virtually all childhood and adult acute lymphoblastic leukemia (ALL) trials , 2. DespCDKN2A/ARF and CDKN2B genes in a cohort of 64 adult T-ALL patients enrolled in two consecutive Spanish PETHEMA  trials  at chromosome 9p21 which involved the CDKN2A/ARF/CDKN2B genes and cortical T-ALL  ) vs. only 36% [8–64%]), for the remaining patients; (p = 0.05), when the OS was censored at transplantation  when patients’ follow-up was not censored at transplantation  analysis based on the MRD data  and treaion Fig. ; of noteion Fig. .Fig. 1PrCDKN2B gene , but not the CDKN2A/ARF gene deletions, conferred a better prognosis in terms of OS   deletions of the Additional file 1:Table S1. Frequency and type of CDKN2A/ARF/CDKN2B gene deletions as detected by qPCR in adult T-ALL (n = 64). Table S2. Comparison between the CNA status of the CDKN2A/ARF and CDKN2B genes in adult T-ALL as assessed by qPCR, SNP-array and iFISH techniques. Table S3. Adult T-ALL: prognostic factors for overall survival. Table S4. Adult T-ALL patient characteristics at diagnosis and follow-up. Table S5. (A) RCN values obtained for the CDKN2A/ARF and CDKN2B genes in selected samples with a 100% blast cell content. (B). Most robust cut-off values to distinguish between normal, heterozygous and homozygous genotypes. The mean and standard deviation (SD) of the values obtained in panel A are indicated for each genotype. Figure S1. Prognostic impact of the CDKN2B gene CNA status on overall survival of adult T-ALL patients (n = 62). In panel A all CDKN2B gene deletions were analyzed together, while in panel B bi-allelic and mono-allelic CDKN2B gene deletions were separately considered. Figure S2. Flowchart summarizing the HR-20011 PETHEMA treatment protocol, including the time points at which MRD assessment was performed (highlighted in red). Figure S3. Calibration curves used to calculate RCN values according to the different percentage contamination of the sample by normal (i.e. non-blastic) cells. In panel A, a pure (100% blasts) homozygous sample was mixed with different amounts of normal (2 N) DNA, as shown on the x-axis. RCN values are shown on the y-axis. In panel B a pure (100%) heterozygous sample was mixed with different amounts of normal (2 N) DNA, as shown on the x-axis; RCN values are depicted on the y-axis. (PDF 274 kb)


The proportion of macrolide-resistant isolates increased with increasing penicillin MICs; even isolates with reduced penicillin susceptibility (MIC = 0.06 μg/ml) were much more likely to be macrolide-resistant than basally penicillin-susceptible isolates (MIC < 0.03 μg/ml). The contribution of recombination to strain diversification was assessed through quantitating 35B/CC558-specific bioinformatic pipeline features among non-CC558 CCs and determining the sizes of gene replacements. Although IPD has decreased greatly and stabilized in the post-PCV13 era, the species continually generates recombinants that adapt to selective pressures exerted by vaccines and antimicrobials. These data serve as a baseline for monitoring future changes within each invasive serotype.Invasive pneumococcal disease (IPD) has greatly decreased since implementation in the U.S. of the 7 valent conjugate vaccine (PCV7) in 2000 and 13 valent conjugate vaccine (PCV13) in 2010. We used whole genome sequencing (WGS) to predict phenotypic traits  and determine multilocus genotypes from 5334 isolates (~90% of cases) recovered during 2015–2016 through Active Bacterial Core surveillance. We identified 44 serotypes; 26 accounted for 98% of the isolates. PCV13 serotypes (inclusive of serotype 6C) accounted for 1503 (28.2%) isolates, with serotype 3 most common , while serotypes 1 and 5 were undetected. Of 305 isolates from children <5 yrs, 60 (19.7%) were of PCV13 serotypes 19A, 19F, 3, 6B, and 23F . We quantitated MLST-based lineages first detected during the post-PCV era (since 2002) that potentially arose through serotype-switching. The 7 predominant emergent post-PCV strain complexes included 23B/CC338, 15BC/CC3280, 19A/CC244, 4/CC439, 15A/CC156, 35B/CC156, and 15BC/CC156. These strains accounted for 332 isolates  and were more frequently observed in children <5 yrs . Fifty-seven categories of recently emerged (in the post PCV7 period) putative serotype-switch variants were identified, accounting for 402 isolates. Many of these putative switch variants represented newly emerged resistant strains. Penicillin-nonsusceptibility (MICs > 0.12 μg/ml) was found among 22.4% (1193/5334) isolates, with higher penicillin MICs (2–8 μg/ml) found in 8.0% (425/5334) of isolates that were primarily  serotypes 35B and 19A. Most  penicillin-nonsusceptible isolates were macrolide-resistant, 410 (34.4%) of which were  The two major emphases of IPD strain surveillance, identification of serotype distributions and antimicrobial resistance phenotype, have not changed over several decades. The distributions of these two basic pneumococcal strain features informs strategic formulation of next-generation vaccines, evaluation of current vaccines, and establishment of appropriate antibiotic usage for clinical cases. Pneumococcal conjugate vaccines (PCVs) are very effective in reducing incidence of PCV-type IPD , circumstantial data indicates that serotype-switch strains arising through recombinational replacement of the Here we reveal distributions of serotypes and key antimicrobial phenotypes for invasive disease isolates identified through routine surveillance in 2015–2016. In addition, we depict basic strain structures of each individual serotype and quantitate strain complexes that have only become apparent within the post-PCV period.https://www.cdc.gov/abcs/reports-findings/survreports/spneu16.html. WGS accessions are only provided for the 5212 isolates that yielded high quality sequencing metrics  during 2015–2016. ABCs is an active population and laboratory-based system which covers a population of approximately 32.2 million individuals. ABCs areas, methods, and key surveillance data through 2016 are described at Streptococcus pneumoniae strains were cultured on Trypticase soy agar supplemented with 5% sheep blood and incubated overnight at 37°C in 5% CO2. Genomic DNA for short-read WGS was extracted manually using a modified QIAamp DNA mini kit protocol . Nucleic acid concentration was quantified by an Invitrogen Qubit assay  and samples were sheared using a Covaris M220 ultrasonicator  programmed to generate 500-bp fragments. Libraries were constructed on the SciCloneG3  using a TruSeq DNA PCR-Free HT library preparation kit with 96 dual indices  and quantified by a KAPA qPCR library quantification method . WGS was generated employing two MiSeq instruments and the MiSeq v2 500 cycle kit (Illumina Inc).Genomic DNA preparation, library construction, whole genome sequencing (WGS), and bioinformatics pipeline features have been previously described  equates to an MIC of ≥2 μg/ml, with intermediate resistance (penI) at 0.12–1 μg/ml and reduced susceptibility at 0.06 μg/ml. All year 2015 isolates were also subjected to conventional broth dilution testing as previously described  to which we added the previously described 3 digit PBP type  was used to read map genomes against the corresponding reference sequence and to build a core SNP alignment. Isolates that had under 90% coverage of the reference sequence were excluded from the analysis. Trees were built using RAxML with ascertainment correction. PlotTree (https://github.com/katholt/plotTree) was used for the visualization of metadata in trees.For phylogenetic analysis of strain sets, a reference sequence was selected for the major ST from the year 2015 dataset. Snippy  features characteristic of 35B/CC558 strains were measured through comparison with the 35B/ST558 reference sequence from isolate 20154871  clonal complex was also predominant for the remaining isolates from all ages  , clindamycin-resistant (cliR), penicillin-susceptible (penS) phenotype, while 22F, 12F, and 33F comprised the majority of the eryR, penS phenotype . In contrast, 120/768 (15.6%) of the penI and 379/425 (89.2%) of the penR isolates were PI-1+ with the addition of isolates that were both PI-1+ and PI-2+ (p < 0.0001).Serotypes had widely varying proportions of different combined resistance phenotypes to penicillin, erythromycin, and clindamycin (includes both constitutive and inducible resistance), primarily inferred by the presence of specific PBP types, e Figure . Pilus de Figure , howevery Figure . Of the Figures ermB, although 7 isolates (4 different serotypes) contained the 23S rRNA A2061G substitution conferring this resistance trait  coincided with additional resistance features besides beta lactam resistance (shown for macrolide and clindamycin-resistance). For example, this is evident within serotypes 3  set of 116 serotype 19A, 19F, and 3  strains   sTable .The majority of isolates  were basally susceptible to penicillin (MIC < 0.03 μg/ml), with an additional 135 isolates that were susceptible but with reduced susceptibility (MIC = 0.06 μg/ml) sTable .folA and/or folP  was found in 1005 of the 5334 isolates (18.8%) due to mutations within .0 μg/ml The sections that follow provide general depictions of clonal complexes, resistance features, and recently documented (2002 or later) putative serotype-switch variants within each serotype. sTable We quantitated strain complexes only detected in the post-PCV era (2002 or later) that potentially arose through serotype-switching events, as documented within the global MLST database and in our own surveillance records. We identified 57 categories that were defined by serotype/CC combinations  with no documented record prior to 2002 and they accounted for 404 (7.7%) of the 5334 isolates , 4 (28 isolates), 3 (11 isolates), 6C (4 isolates), 19F (3 isolates), 6A (2 isolates), and 6B (1 isolate). The number of PCV13 serotype switch progeny is inflated substantially by the number of 19A/ST695 (CC244) isolates Figure , even thThe most promiscuous serotype recipient CCs, accounting for 6-7 different putative progeny serotypes, were CCs 156 (7 serotypes), 63 (7 serotypes), and 338 (6 serotypes). CC338 accounted for the most putative progeny, primarily through the rapid post-PCV emergence of 23B/CC338 (23B/ST1373)  in 15 genetic exchanges and as a genetic recipient (of cps6B) in a single exchange, resulting in the indicated progeny strains shown in the right column. Each exchange is defined by 1–3 specific double crossover events deduced in 1–25 progeny isolates.There were numerous examples of apparent hybrid strains that exhibited typing pipeline features highly associated with two different strains of known lineages . The fifth serotype switch event involved a 35B/CC558 cps35B donor and a 15A/ST11818 recipient. It is interesting that there were 4 15A/ST11818 isolates recovered, 3 of which shared the common 15A/CC63 PBP type 24-31-114. The PBP type of the progeny 35B/ST11818 (4-31-114) and its cps35B region sequence are consistent with a gene replacement event effecting a serotype switch event and a change in PBP type. Here, a single chromosomal fragment carrying cps35B and the 3′ 544 codons of pbp1a (encompassing PBP subtype 1a-4) from a 35B/CC558 strain replaced the corresponding region from a 15A/ST11818/PBP type 24-31-114 strain.There were 5 clear instances of serotype switching events involving a 35B/CC558 parental donor strain, involving 5 different donor serotype lineages. The most important serotype 35B switch variant, accounting for 25 isolates, arose from a single event where the 9V/ST156 ts Table  and was ts Table . The sects Table , resultecps6B locus donor and a 35B/ST558 recipient strain (last row). Comparison of the cps locus from this recombinant to a cps6B reference locus (CR931639) and the 35B/ST558 cps35B reference revealed potential crossover sites corresponding to bases 2321 (within wzg) and 16033 (immediately downstream of 4 gene rhamnose biosynthetic gene cluster) of the cps6B reference, with 99.2% sequence identity shared within the 13,743 bp overlap of these two cps6B loci. As expected, sequences upstream and downstream of the cps6B locus that encompassed the full-length pbp2x and pbp1a genes in the recombinant strain were highly similar to the 35B/ST558 recipient reference strain .A sixth serotype switch depicted in Table cps switches, but introduced changes in MLST and/or PBP loci that were observed in the progeny. Except for entry 10, where the short length (565 bp) of the pbp2b putative replacement fragment does not provide strong association with a 35B/CC558 donor, these putative recombination events appeared non-subjective, involving replacement fragments of 1424 bp - > 26,001 bp that closely matched the 35B/ST558 reference. Four of these hybrid 15A/CC63 strains (entries 6-9) had higher predicted MICs for beta lactams due to the introduced changes in PBP type. Replacement of 2- 3 PBP markers resulted in a change from the median 15A/CC63 pen MIC from 0.25 to 2 μg/ml (entries 6 and 7). The predicted change from PBP type 13-115-73 (the closest matching PBP type among our 15A isolates) to 13-7-73 results in change of pen MIC from 1 to 2 μg/ml. All 3 33F hybrid progeny appeared to result from an identical strain mixing that effected 3 large and unlinked gene replacements within a 33F/ST10491 recipient (entry 12 in Table The remaining 9 entries in Table Another independent serotype 35B switch variant described in Table ermB expression combined with reduced penicillin-susceptibility due to a first step PBP-2x substitution (0.06 μg/ml indicated), coincides with the finer-detailed phylogeny shown sFigure Despite being a PCV13 serotype, serotype 3 was the major IPD serotype overall during 2015–2016. It is interesting that the clade centering upon ST180-2 coincides with the recently described globally emergent CC180 clades I-β and II  that we have found within ABCs isolates was from 2 19F/ST271 isolates recovered during 2004 and 2012 (data not shown).Three serotype switch variant lineages (11 isolates) were putatively derived from 33F/ST2705, 19F/ST271, and 11A/ST62 recipient strains (sTable mef + lineages (196 isolates) that included the ST7314-focused clade and ST433-2 .www.mlst.net). It is interesting that 23B/ST1373 has been associated with a divergent cps locus  was found in both serotypes (there were 4 23B/ST338-1 isolates and 126 23A/ST338-1 isolates). Phylogenetic analysis was consistent with 23B/ST338-1 arising from at least one serotype switch with a 23A/ST338-1 recipient sFigure  and was ermB-conferred resistance to erythromycin and clindamycin) , which corresponds to increased antibiotic resistance in the ST338-3 focused clade (higher penicillin MIC of 0.5 μg/ml and ) Figure .There was one penicillin-nonsusceptible (PBP type 0-0-1) variant ST10492) of the major 33F/CC100 lineage within serotype 23A (sTable  of the mermB+ isolates was a SLV (ST2543) of the major 15A/ST63 (sTable One PBP variant (ST66-3) was comprised of 5 penicillin-nonsusceptible isolates. One of the 7  a SLV ST543 of thwciG35B-encoded acetyltransferase gene , almost all of which occurred within single isolates, possibly indicates the lack of a selective advantage for this serologic variant of 35B.The serotype 35B isolates include the recently described 35B serologic subtype 35D caused by mutations within the mef.The proportion of penicillin-nonsusceptible 35B ABCs isolates has steadily increased throughout most of the PCV era  , comprised of intermediately penicillin-resistant, s Figure  that ares Figure . Nine CCmef+ ST156-1 putative switch variant was also observed . Single switch variants were also observed of penI ST338 (major 23A MLST) and penR, mef+, ST11576 (SLV of the major 23F genotype ST81) (sTable A recent putative serotype switch variant within CC156 was comprised of 39 antibiotic-susceptible ST3811/ST3811-1 isolates that included 4 of the 19 21%) pediatric isolates  CC100 isolates were also observed. Six 3 locus variants  of ST62 indicating possible common ancestry with the major 11A/CC62 were evident.CC100/33F included 192 mef+ isolates, with 3 ST4179 penR (pen MIC = 4 μg/ml) isolates. Remaining 19A CCs were previously described .The four outlier isolates of the three largely susceptible CCs included a penI, eryR serotype derivative (single locus variant) of a serogroup 6 lineage  of serotype 6C, were grouped together Figure . The arrTwo different CC62 clades were resolved through eBURST, with the major set primarily  positive for the PI2 pilus backbone protein gene  that are grouped together Figure  since thmef+), CC63 , and CC81 .An additional 31 isolates were of potential recent serotype-switch CCs  of the pediatric isolates. There was a single serotype 10A switch variant with a putative serogroup 6 recipient .There were 19 meningitis case isolates among the 96 isolates  of the major serogroup 23A/23B CC439 has abruptly emerged  pediatric isolates) recovered during 1999 from a surveillance population of about 18.6 million people  isolates consisted of 3 unrelated STs. There was a single penR, eryR ST2011 isolate. ST2011 is linked with 10 NT isolates and 2 serogroup 6 isolates in the global MLST database, all recovered during 1999-2007).This initial depiction of our first 2 years of WGS-based ABCs strain surveillance provides an encompassing data framework for comparison with future strain surveillance. Here WGS has allowed us a closer glimpse into the active process of strain mixing through recombination. The amount of recombination deduced from simply observing 35B/CC558 markers distributed among non serotype 35B strains suggests a species continually engineering and presenting new experimental strains for “testing” the property of fitness. Here we assume that a basic level of fitness is described by the emergence of a strain in our IPD surveillance to the level of detection. For this to have occurred, we assume that the strain was generated and carried within the upper respiratory reservoir for some period of time. For this reason we view with concern the two 3/ST271 isolates described in this study . This highly resistant strain combines the current most successful invasive serotype with the most successful invasive clonal complex of the decade before PCV13 implementation  that have only recently shown successful emergence, serotype switching has been a key mechanism in shaping the pneumococcal population structure long before PCV introduction  strains have emerged faster than strains that are resistant to one of these antibiotics alone  and each genotype constitutes a distinct highly related phylogenetic cluster sFigure . It is sFor unknown reasons, pan-susceptible serotype 19F/CC251 strains have declined more slowly than other 19F CCs that were abundant before PCVs and actually increased in IPD slightly in 2016 relative to 2015. It might prove important to examine such strains in PCV13-based opsonophagocytosis assays to evaluate whether antibodies generated in response to type 19F antigen in the vaccine have functionality against these specific type 19F strains. Serotype 6C is an example where such investigation proved important. Originally mistyped as serotype 6A, 6C proved to be a distinct serotype not targeted by PCV7 that differed within its primary repeating unit structure (Park et al., Our primary objective was to provide a meaningful descriptive account of IPD isolates recovered through population-based surveillance in the post-PCV13 era. We hope that these strain data will support a wide array of studies to assist in prevention efforts and contribute to our basic understanding of IPD strains.The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.All authors reviewed the manuscript and provided constructive feedback. BB wrote the study, performed many of the analyses provided, and guided bioinformatics pipeline development. SC performed, directed, and evaluated all whole genome sequencing data. BM developed the bioinformatics pipeline and provided periodic updates. YL provided the bioinformatics approach for prediction of β-lactam MICs conferred by newly encountered PBP types. ZL, TT, and HW performed whole genome sequencing. LM oversaw all laboratory operations. JR provided phylogenetic analysis for selected serotypes. RG maintained typing antisera as well as performed conventional testing of selected isolates. TP assisted in the writing and provided population-based disease rates for the ABCs data.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.


Bacillus cereus, an opportunistic human pathogen, utilizes the autoinducing PapR peptide signal that mediates the activation of the pleiotropic virulence regulator PlcR. A set of synthetic 7-mer PapR-derived peptides  have been shown to inhibit efficiently the PlcR regulon activity and the production of virulence factors, reflected by a loss in hemolytic activity without affecting bacterial growth. Interestingly, these first potent synthetic inhibitors involved D-amino acid or alanine replacements of three amino acids; proline, glutamic acid, and phenylalanine of the heptapeptide PapR. To better understand the role of these three positions in PlcR activity, we report herein the second generation design, synthesis, and characterization of PapR7-derived combinations, alternate double and triple alanine and D-amino acids replacement at these positions. Our findings generate a new set of non-native PapR7-derived peptides that inhibit the PlcR regulon activity and the production of virulence factors. Using the amino acids substitution strategy, we revealed the role of proline and glutamic acid on PlcR regulon activation. Moreover, we demonstrated that the D-Glutamic acid substitution was crucial for the design of stronger PlcR antagonists. These peptides represent potent synthetic inhibitors of B. cereus QS and constitute new and readily accessible chemical tools for the study of the PlcR system. Our method might be applied to other quorum sensing systems to design new anti-virulence agents.The quorum sensing (QS) system of  N-acyl homoserine lactones, while QS in Gram-positive species mainly relies on the secretion of auto-inducing oligopeptides to bind and activate their cognate quorum sensors. In the past decade, a rapid increase of interest in bacterial quorum sensing peptides (QSPs) has emerged. Therefore, new QSPs databases are being established to provide chemical structures overview, microbial origin and functionality responses of these QS-derived signaling peptides (Quorum sensing (QS) is a cell-cell communication mechanism used to coordinate bacterial group behaviors  by assessing cell density through the production, secretion, and detection of small signaling molecules . Gram-nepeptides .Enterococcus faecalis  or in the cytoplasm of the bacterial cell. In the latter case, the quorum-sensing regulators are controlled by direct interaction with a internalized signaling peptide . They hafaecalis , the Rapsubtilis , the traectively  and the ptococci  and predlivarius . The las. cereus  and aimRost cell .Staphylococcus, which uses a peptide-thiolactone as the extracellular signal, the design of molecules modulating QS systems in Gram-positive bacteria has been poorly explored  at the end of the exponential growth stage. PapR7 is imported by the oligopeptide permease system  was published  the central proline residue may be required for the PapR peptides to fit into the binding groove on PlcR and c) the glutamic acid of the FEF PapR7 core motif may function to selectively allow PapR to bind PlcR by ionic interactions with Lys87 and 89. In a follow up study in 2013, Grenha and co-workers, have determined the crystal structure of the ternary complex DNA-PlcR-PapR7. It has been reported that both PapR7 phenylalanine residues are located in hydrophobic pockets and the only specific interactions are made between the glutamate of PapR7 and residues Lys89, Gln237, and Tyr275 of PlcR.The structural and molecular basis for the activation of PlcR by PapR has been the focus of several studies, which have revealed interesting insights on the PlcR – PapR interactions. The PlcR – PapR relationship has been shown to be strain specific; comparison of the amino acid sequences of PlcR and PapR from 29 different strains demonstrated the existence of four classes (I to IV) of PlcR – PapR pairs, defining four distinct pherotypes in the onserved . In 2007ublished . Accorditivation . Howeverfor PlcR . In 2008und PlcR . StructuB. cereus. Thus, we recently studied the PlcR – PapR activation in B. cereus and B. thuringiensis at the molecular level. We designed, synthesized and characterized synthetic PapR 7-mer derived peptides to determine the contribution of each residue within PlcR – PapR7 interactions. Our findings reveal the first set of non-native peptides that can repress the PlcR regulon and thus relevant virulence factors. Moreover, we could demonstrate that the repression is mediated by QS and regulation of PlcR expression without affecting bacterial growth  glutamic acid (E) or phenylalanine (F) of the heptapeptide PapR (ADLPFEF). To better understand the role of these three crucial positions in PlcR activation, we report herein the second generation design, synthesis, and characterization of PapR7-derived combinations, alternate double and triple alanine and D-amino acids replacement at these positions. We propose this systematic replacement approach to elucidate other quorum quenching agents in Gram-positive bacteria.Binding of PapR to PlcR is essential to trigger QS-mediated functions in l growth . InteresB. thuringiensis 407 CryplcA′Z (Bt A’Z) and the PapR null-mutant 407 CrypapR plcA’Z (Bt ΔpapR A’Z) strains, containing a transcriptional fusion between the promoter of plcA and the lacZ reporter gene , microwave irradiation, procedures on Rink Amide resin  in SPE polypropylene Single-Fritted tubes. The Fmoc-protecting group was then removed by treating the resin with 20% (v/v) piperidine diluted in dimethylformamide (DMF) followed by heating to 80°C in the microwave  with stirring. To couple each amino-acid, Fmoc-protected amino acids , were dissolved in DMF and mixed with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate  and diisopropylethylamine . The solution was allowed to pre-activate for 5 min before being added to the resin, and heated to 70°C in a multimode microwave  with stirring. After each coupling/deprotection cycle the resin was drained and washed with DMF (3 × 5 mL). Once peptide synthesis was completed, the peptide was cleaved from the resin, by mixing the resin with 3 mL cleavage cocktail of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), and 2.5% deionized water for 3 h with agitation. The peptide mixture was precipitated from the TFA solution by the addition of cold ether and collected by centrifugation (Eppendorf R5810 8000 rpm for 10 min). The ether was then removed, and the peptide was dried under a stream of nitrogen, and lyophilized, before high-performance liquid chromatography (HPLC) purification.–1 for semi-preparative separations and 1 mL min–1 for analytical separations; mobile phase A = 18 MΩ water + 0.1% TFA; mobile phase B = ACN. Purities were determined by integration of peaks with UV detection at 220 nm using a linear gradient (first prep 5% B → 65% B over 60 min and second prep 26% B → 36% B over 20 min). The purity of the tested peptides was determined using a linear gradient (5% B → 65% B over 60 min). MALDI-TOF spectrometry  was used to validate the synthesized peptides molecular weight (Crude peptides were purified and characterized with Reverse-Phase (RP)-HPLC. The crude peptides were diluted to a final concentration of 10 mg/ml in a solution of 20% acetonitrile (ACN) in water (v/v) or dimethyl sulfoxide (DMSO). A semi-preparative Phenomenex Kinetex C18  was used for preparative RP-HPLC work. An analytical Phenomenex Gemini C18 column  was used for analytical RP-HPLC work . Standarr weight . The pur–3 in modified LB to a final volume of 1 liter and incubated at 37°C with shaking (200 rpm) until onset of the stationary phase of bacterial growth (OD600 3 ± 0.5). Various concentrations of synthetic peptides were added to 2 ml aliquots of culture, which were incubated for 1 h before centrifugation  and quantification of β-galactosidase.Bt ΔpapR A’Z cells were grown overnight in LB medium with selective antibiotic. The cells were diluted 10–3 in modified LB to a final volume of 1 L and incubated at 37°C with shaking (200 rpm) until the end of the lag or late-exponential of bacterial growth . Different concentrations of synthetic peptides were added to 2 ml aliquots of culture and incubated for various times (1–24 h) before centrifugation  and quantification of β-galactosidase activity.Bt A’Z cells were grown overnight in LB medium. The cells were diluted 10600 was measured and β-galactosidase activity was assayed. The final results were reported as percentage of activation, which is the ratio between the Miller units obtained after addition of the PapR7 analogs. In Bt ΔpapR A’Z strain, the plcA promoter activity was very low and considered as a baseline. In Bt A’Z strain, the untreated bacteria were considered as 100% of activation and the results were normalized accordingly. Each assay was repeated at least three times.β-galactosidase activity was measured as described previously , with miB. cereus ATCC 14579 cells were grown overnight in LB medium. The cells were diluted 10–3 in modified LB to a final volume of 1 liter and incubated at 37°C with shaking (200 rpm) until the end of the lag phase of bacterial growth (OD600 0.1 ± 0.03). Different concentrations of synthetic peptides were added to 2 ml aliquots of culture and incubated for 2.5 h before centrifugation , separation and filtration (0.2 μm filter) of the supernatants of the treated cultures. Analyses of hemolytic activity were conducted as previously described using human red blood cells  and two D-amino acid substitutions (PapR7 – dE6 and dF7) showed great reduction of PlcR regulon expression and virulence factor secretion , glutamic acid (Glu6) and phenylalanine (Phe7) through systematic single or/and multiple amino acid substitution strategy. We designed, synthesized and purified a second generation set of PapRresidues .7-derived peptide combinations for its ability to modulate the expression of the PlcR regulon using B. thuringiensis 407 CryB. cereus group. This strain cured of its plasmid is acrystalliferous and shows high phylogenic similarity with the B. cereus reference strain ATCC 14579  and PapR-null mutant B. thuringiensis 407papR plcA′Z (Bt ΔpapR A’Z). Both reporter strains contain a transcriptional fusion between the plcA promoter region and the lacZ gene. plcA is a member of the PlcR regulon and its expression directly reflects the activity of PlcR. The activity of each PapR7-derived peptide combination was evaluated and compared to the previously described five B. cereus inhibitory synthetics PapR7-derived peptides ; single, double and triple. Similar to our first-generation single replacement inhibitors, none of the new PapR7-derived peptide combinations were capable of activating the PlcR regulon. These findings revealed that any alanine and D-amino acid replacements, at the positions Pro4, Glu6 and Phe7 of PapR7 derivatives, are critical for PlcR regulon activation. These derivatives can therefore be classified as potential candidates for the development of potent second-generation PlcR inhibitors.We first conducted an initial screening of all the analogs at high peptide concentration (5 μM) in order to evaluate their ability to activate the PlcR regulon to a level comparable to the synthetic PapR peptide . Both fi600 of 1.8 ± 0.1). As shown in 7 derivatives activities were classified by D-amino acid enantiomer or Ala replacements at either of the three PapR7 crucial positions . The previously reported inhibitors, PapR7-P4A; E6A; F7A; dE6 and dF7, were also included for comparison. Peptides PapR7 – P4A:E6A, P4A:E6A:F7A and P4A:E6A:dF7 did not show any reduction in plcA’Z activity. PapR7 – E6A:F7A, E6A:dF7, P4A:F7A, P4A:dE6, P4A:dF7, PapR7 – P4A:dE6:dF7 and P4A:dE6:F7A were able to reduce plcA’Z activity by approximately 40%. We identified two candidate peptides that inhibited plcA’Z activation when added at late-exponential phase. Indeed, PapR7 – dE6:dF7 and dE6:F7A reduced plcA’Z activity by 71 and 65 %, respectively, similarly to the inhibitory activity of their parent reporter inhibitors PapR7 – dE6, dF7 and F7A . To confirm PapR7 – dE6:dF7 and dE6:F7A potent inhibition, we repeated the experiment with several concentrations of these inhibitors in order to determine their IC50 values  for reducing the activation of the PlcR regulon in late-exponential phase of bacterial growth  did not reduce the PlcR regulon expression even in low bacterial density. Importantly, the bacterial growth was not affected by the addition of all the examined peptides (data not shown).We have previously reported that inhibition through PapRependent . To examer 2.5 h  and afteer 2.5 h . As has nditions . We obse to 24 h . Combini7- P4A:E6A, P4A:E6A:F7A and P4A:E6A:dF7), seven variants of PapR7 containing multiple replacements combinations with median to high inhibitory activities  and two very potent inhibitors that abolish activation of plcA’lacZ (PapR7 – dE6:dF7 and dE6:F7A) and compete with endogenous PapR. These two peptides are proposed as quorum quenchers that do not affect the bacterial growth but inhibit the expression of the PlcR regulon.Throughout these competition studies, we identified a group of three non-inhibitory peptidic combinations , we observed great activity also for PapR7 – P4A:dE6:dF7 and P4A:dE6:F7A analogs. Regard to PapR7 – dE6:dF7 and dE6:F7A derivatives, the relative IC50 value trends were highly similar to those in the lacZ-reporter assays results  slightly improved its IC50 , while alanine substituted at position Phe7 (PapR7 –dE6:F7A) did not show any effect. PapR7– P4A:dE6:dF7 and P4A:dE6:F7A exhibited lower IC50 values compared to their parent single inhibitors PapR7– P4A, dF7 and F7A but a higher IC50 value compared to the parent peptide PapR7–dE6  are located in hydrophobic pockets  reduced hemolysis of red blood cells by 60%. However, these replacements combined with D-Phenylalanine yielded analogs (PapR7 – E6A:7dF and P4A:7dF) with stronger antagonistic activities (approximately 83% inhibition). These results indicate that the inclusion of D-Phenylalanine may contribute to hydrophobic interactions with PlcR by preserving the aromatic ring side chain interaction.Crystal structure of PapR pockets , and inv of PlcR . Replaci7 – 6dE:F7A and 6dE:dF7 displayed similar inhibitory effect, regardless of other modifications  in position Phe7 (D-amino (PapR7 – E6A:F7A and E6A:dF7). PapR7 – P4A:6dE:F7A and P4A:6dE:dF7 fully prevented hemolysis of red blood cells, however, replacing only Glu6 with alanine yielded two non-inhibitory peptidic combinations PapR7- P4A:E6A:F7A and P4A:E6A:dF7 as was supported by IC50 values , with D-glutamic acid yielded two potent PlcR antagonists; PapR7- P4A:6dE:F7A and P4A:6dE:dF7. Overall, these three sets of new PapR7-derived peptide combinations support previous published studies and reveal the important role of three crucial positions at designing potent PlcR antagoinsts; Pro4, Phe7 and especially Glu6 that may function to selectively allow PapR, but not other similar autoinducers, to bind PlcR.Interestingly, in an earlier study  the authB. cereus, highlighting this system as an attractive target for an alternative treatment to prevent infection. We have previously reported the first five potent synthetic peptidic inhibitors of B. cereus PlcR-PapR QS system  and two D-amino acid substitutions (PapR7 – dE6 and dF7). We concluded that the critical residues for PapR7 –PlcR interaction and PlcR activation were proline, glutamic acid and phenylalanine. To further understand their role in PlcR activity, a new set of PapR7 analogs with double and triple alanine and D-amino acid replacements at these positions were designed and synthesized. Multiple amino acid substitutions revealed that any replacement at these positions Pro4, Glu6 and Phe7 of PapR7 derivatives, is critical for PlcR regulon activation in the ΔpapR mutant strain. A comprehensive competition study of all PapR7-derived peptides combinations in late-exponential phase identified four promising QS peptidic inhibitors candidates; PapR7 – dE6:dF7 and dE6:F7A and two other analogs with additional alanine substituted PapR7 – P4A:dE6:dF7 and P4A:dE6:F7A, all contained D-Glutamic acid at position 6 of the C-terminus of heptapeptide PapR. The two potent inhibitors PapR7 – dE6:dF7 and dE6:F7A generated similar inhibitory activity as their parent single replacement reported inhibitors PapR7 – dE6, dF7 and F7A with comparable IC50 values ≅ 1–2.6 μM. Our results verified previous reports that inhibition through PapR7 derivatives is cell density dependent . Moreover, by exposing the bacterial cells to these analogs at earlier stage (OD600 0.1 ± 0.03) we discovered a new series of inhibitors . Similar to the parent peptidic inhibitors, we hypothesized that the positive autoregulatory loop was blocked and quorum quenching was achieved throughout growth by the inhibitory multiple combinations PapR7 derivatives.The PapR-PlcR QS system is extensively involved in the pathogenesis of S system ; three iependent . We showB. cereus. The inhibitory PapR7 peptidic analogs identified using the lacZ-reporter assays were even more efficient in reducing the hemolytic activity of wild type B. cereus ATCC 14579.We next used a human red-blood cells hemolytic assay as a direct method to assess a QS-related phenotype linked to virulence in wild-type 7 in PlcR receptor recognition; first, we showed the important role of proline or glutamic acid residues in PapR- PlcR interactions and as key in designing strong inhibitors. Second, we demonstrated that inclusion of D-Phenylalanine at Phe7 contribute to PapR7 derivatives inhibitory activities probably due to its hydrophobic features. Moreover, by interfering this Glu6 specific interactions with PlcR, we found the potential of D-Glutamic substitution at designing potent PlcR antagonist. These findings are consistent with previous study (B. cereus group. Interestingly, while all the PapR sequences from different strains of the B. cereus group showed divergences in their three N-terminal residues, the E6 position was conserved. In the current study we highlighted the precise and unpredictable engineering of natural pheromone in our effort to develop new Quorum Quenching agents, reflecting the trade-off between good peptide binding and lower activation. These new non-native peptides inhibitors may be applied as chemical tools to further study the role of PlcR and other QS in all B. cereus group members. Further, our method of single and multiple amino acid replacements might be applied to other QS system to design new anti-virulence agents.Our findings both corroborate and extend previous observations regarding the role of the PapRus study  which inNo datasets were generated or analyzed for this study.AY, LS, and EM performed the research. DL and ZH analyzed the data and 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.